Joe GN Garcia

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Books

• Yuan, J. X., Garcia, J. G., West, J. B., Hales, C. A., Rich, S., & Archer, S. L. (2011). Textbook of pulmonary vascular disease. Springer US. doi:10.1007/978-0-387-87429-6
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  The Human Pulmonary Circulation: Historical Introduction.- Microcirculation of the Lung: Functional and Anatomic Aspects.- Pulmonary Vascular Development.- Pulmonary Vascular Function.- Pulmonary Vascular Mechanics.- Modeling of the Pulmonary Vasculature.- Metabolic and Clearance Function at the Pulmonary Microvascular Endothelial Surface in Pulmonary Hypertension.- The Pharmacology of the Major Vasoactive Mediators Relevant to the Pathogenesis of Pulmonary Hypertension.- The Normal Fetal and Neonatal Pulmonary Circulation.- Excitation-contraction Coupling and Regulation of Pulmonary Vascular Contractility.- Endothelial Regulation of Pulmonary Vascular Tone.- Acute Lung Injury: The Injured Lung Endothelium, Therapeutic Strategies for Barrier Protection and Vascular Biomarkers.- Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle.- Receptor-mediated Signal Transduction and Cell Signaling.- Role of Calcium as a Second Messenger in Signaling: A Focus on Endothelium.- Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells.- The Chemistry of Biological Gases.- Role of Oxygen-derived Species in the Regulation of Pulmonary Vascular Tone.- Mitochondrial ROS and Redox State in Pulmonary Vascular O2 Sensing.- Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation.- Role of Ca2+ in Vascular Smooth Muscle Gene Expression and Proliferation.- Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells.- The Coagulation Cascade and its Regulation.- Platelets in Pulmonary Vascular Physiology and Pathology.- Lysis and Organization of Pulmonary Thromboemboli.- Interactions of Leukocytes and Coagulation Factors with the Vessel Wall.- Interaction of the Plasminogen System with the Vessel Wall.- Endothelial Apoptosis and Repair in Pulmonary Arterial Hypertension.- Bronchial Arterial Circulation in the Human.- Animal Models of Pulmonary Hypertension.- Transgenic and Gene Targeted Mouse Models for Pulmonary Hypertension.- Animal Models of Increased Lung Vascular Permeability.- Isolation and Culture of Pulmonary Vascular Smooth Muscle and Endothelial Cells.- Conventional Patch Clamp Techniques and High-throughput Patch Clamp Recordings on a Chip for Measuring Ion Channel Activity.- Measurement of Pulmonary Vascular Structure and Pulmonary Blood Distribution by MDCT and MR Imaging Techniques.- Quantification of DNA, RNA and Protein Expression.- Gene Cloning, Transfection and Mutagenesis.- Approaches for Manipulation of Gene Expression.- Bioinformatics, Genomics, and Functional Genomics: Overview.- Genomic Application to Study Pulmonary Hypertension.- Proteomics and Functional Proteomics.- Maintenance, Propagation and Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.- Identification of Adult Stem and Progenitor Cells in the Pulmonary Vasculature.- Differentiation of Embryonic Stem Cells to Vascular Cell Lineages.- Statistics and Clinical Data Analysis: A Reference Guide.- Hypoxic Pulmonary Vasoconstriction.- Pathogenic Roles of Ca2+ and Ion Channels in Hypoxia-mediated Pulmonary Hypertension.- Roles of Endothelium-derived Vasoactive and Mitogenic Factors in the Development of Chronic Hypoxia-mediated Pulmonary Hypertension.- Oxygen-sensitive Transcription Factors and Hypoxia-mediated Pulmonary Hypertension.- Developmental Regulation of Pulmonary Vascular Oxygen Sensing.- Pulmonary Vascular Remodeling by High Oxygen.- Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms.- Carbon Monoxide and Heme Oxygenase in the Regulation of Pulmonary Vascular Function and Structure.- Shear Stress, Cell Signaling and Pulmonary Vascular Remodeling.- Pulmonary Hypertension and the Extracellular Matrix.- Role of Progenitor Cells in Pulmonary Vascular Remodeling.- Receptor Signaling in Pulmonary Arterial Hypertension.- Role of Endothelium in the Development of Pulmonary Hypertension.- Coagulation and the Vessel Wall in Pulmonary Embolism.- Alveolar Epithelial Fluid Transport in Lung Injury.- High Altitude Pulmonary Edema .- Statins and Acute Lung Injury.- Genomics of Acute Lung Injury and Vascular Barrier Dysfunction.- Ventilator-induced Mechanical Stress and Lung Vascular Dysfunction.- Classification of Pulmonary Hypertension: History and Perspectives.- Epidemiology of Pulmonary Arterial Hypertension.- Air Pollution and the Pulmonary Vasculature.- Idiopathic Pulmonary Arterial Hypertension.- Genetics of Familial and Idiopathic Pulmonary Arterial Hypertension.- Pulmonary Arterial Hypertension Related to Scleroderma and Collagen Vascular Diseases.- Pathology and Management of Portopulmonary Hypertension.- Pathobiology and Treatment of Pulmonary Hypertension in HIV Disease.- Pulmonary Arterial Hypertension Secondary to Anorexigens and Other Drugs and Toxins.- Pulmonary Arterial Hypertension Related to Gauchers, Sarcoidosis and Other Disorders.- Pediatric Pulmonary Hypertension: An Integrated View from Pediatric Subspecialists.- Persistent Pulmonary Hypertension of the Newborn: Mechanisms and Treatment.- The Pulmonary Circulation in Congenital Heart Disease.- Pulmonary Hypertension Secondary to Congenital Systemic to Pulmonary (Left-to-Right) Shunts.- Surgical Evaluation of Congenital Heart Disease-associated Pulmonary Hypertension.- Pulmonary Veno-occlusive Disease.- Left Ventricular Diastolic Heart Function and Pulmonary Hypertension.- Pulmonary Hypertension Associated with Chronic Obstructive Pulmonary Diseases.- Pulmonary Hypertension Associated with Interstitial Lung Disease.- High Altitude Pulmonary Hypertension.- Pulmonary Hypertension and Congenital Heart Defects at High Altitude.- Pulmonary Embolism and Deep Vein Thrombosis.- Pulmonary Hypertension Due to Pulmonary Embolism and Thromboembolic Obstruction of Proximal and Distal Pulmonary Arteries.- Risk Factors for Chronic Thromboembolic Pulmonary Hypertension.- Evaluation of Small Vessel Arteriopathy in Chronic Thromboembolic Pulmonary Hypertension.- Hemolytic Anemia Associated Pulmonary Hypertension: Sickle Cell Disease and Thalassemia-associated Pulmonary Hypertension.- Pulmonary Hypertension due to Schistosomiasis.- Pulmonary Hypertension due to Capillary Hemangiomatosis.- Molecular Basis of Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease.- Right Ventricular Dysfunction in Pulmonary Hypertension.- Large Vessel Pulmonary Arteritis.- Tumors of the Pulmonary Vascular Bed.- Cor Pulmonale.- Pregnancy and Contraception in Patients with Pulmonary Arterial Hypertension.- Cardiac Catheterization in the Patient with Pulmonary Hypertension.- Imaging of Pulmonary Vascular Diseases.- Histological and Pathological Diagnosis of Pulmonary Hypertension: Pathological Classification of Pulmonary Vascular Lesions.- Echocardiography in Pulmonary Vascular Disease.- Calcium Channel Blockers in the Treatment of Pulmonary Arterial Hypertension.- Prostacyclin and Prostaglandins.- Endothelin Receptor Antagonists for the Treatment of Pulmonary Arterial Hypertension.- Phosphodiesterase Inhibitors in the Treatment of Pulmonary Hypertension.- Nitric Oxide for Children.- The Serotonin System as a Therapeutic Target in Pulmonary Hypertension.- Combination Therapy for Pulmonary Arterial Hypertension.- Thrombolytic and Anti-coagulant Therapy for Pulmonary Embolism and Chronic Thromboembolic Pulmonary Hypertension.- Nursing Care of Patients with Pulmonary Arterial Hypertension.- Atrial Septostomy.- Evaluation of Patients with Chronic Pulmonary Thromboembolic Pulmonary Hypertension for Pulmonary Endarterectomy.- Pulmonary Endarterectomy.- Evaluation of Patients with Pulmonary Hypertension for Lung Transplantation.- Lung Transplantation for Pulmonary Hypertension.- Living-donor Lower Lobar Lung Transplantation for Pulmonary Arterial Hypertension.- Results of Lung Transplantation.- Index.

Chapters

• Bime, C., Gurguis, C. I., Hecker, L., Hecker, L., Wang, T., Wang, T., Garcia, J. G., Desai, A. A., Desai, A. B., & Desai, A. A. (2017). MicroRNAs in Inflammatory Lung Disease. In Translating MicroRNAs to the Clinic. Academic Press. doi:10.1016/B978-0-12-800553-8.00006-8
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  Inflammatory lung diseases include both acute inflammatory diseases such as acute respiratory distress syndrome (ARDS) and chronic inflammatory diseases including asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis and sarcoidosis. Both acute and chronic inflammatory lung diseases are associated with airway inflammation and are influenced by a combination of environmental, genetic, and epigenetic components. Epigenetic regulation, including microRNA (miRNA), of gene expression contributes to inflammatory lung disease development and severity. With the advancement of “omics” technology, miRNA expression and function in inflammatory lung diseases have been well studied, leading to a clear conclusion that miRNAs are differentially expressed under inflammatory lung disease conditions and contribute to genomic dysregulation in these diseases. Here we summarized the most up-to-date findings of miRNA in the inflammatory lung diseases including asthma, ARDS, COPD, cystic fibrosis, and sarcoidosis, and clinical implications of miRNA in these diseases. miRNAs are valued to be served as biomarkers, diagnostic tools, as well as therapeutic targets of inflammatory lung diseases.
• Casanova, N., Casanova, N., Navarrete, J. R., Quijada, H., Hecker, L., Hecker, L., & Garcia, J. G. (2016). Erratum to: Health Disparities in ARDS. In Health Disparities in ARDS. Springer International Publishing. doi:10.1007/978-3-319-23675-9_17
• Casanova, N., Casanova, N., Navarrete, J. R., Quijada, H., Hecker, L., Hecker, L., & Garcia, J. G. (2016). Health Disparities in Respiratory Medicine. In Health Disparities in ARDS. Springer International Publishing. doi:10.1007/978-3-319-23675-9_7
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  Acute respiratory distress syndrome (ARDS) is a major cause of morbidity and mortality in critically ill patients occurring in the context of sepsis and other systemic inflammatory disorders. Health disparities correlated with socioeconomic status and health care access disproportionately affect racial and ethnic minorities and lead to differences in prevalence and outcomes of ARDS. In addition, the role of genetic and environmental factors in critical illness outcomes has been examined. Multiple studies have highlighted differences in allelic variant frequencies between European descendant and African descent individuals. These allelic variants play an important role in inflammatory and infectious pathways in conditions that predispose to ARDS. In this chapter, we address race and ethnicity in the context of the complex genetic and environmental interactions that influence disparities in outcomes in ARDS in African American and Hispanic/Latino populations, groups most affected by health care disparities. We postulate that multiple factors contribute to poor outcomes in ethnic and racial minorities with ARDS and that precision medicine can be a realistic approach to overcome those disparities.
• Garcia, A., Garcia, A. N., & Garcia, J. G. (2012). Methods to Assess Vascular Permeability During Angiogenic Processes. In The Textbook of Angiogenesis and Lymphangiogenesis: Methods and Applications. Springer, Dordrecht. doi:10.1007/978-94-007-4581-0_4
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  Angiogenesis is a multi-faceted process that is, in part, reliant on dynamic regulation of the endothelium that lines the bodies vasculature. Here we have detailed angiogenic factors VEGF, angiopoietin 1 and 2, sphingosine 1-Phosphate (S1P), and hepatocyte growth factor, all of which act, in part, via alterations of endothelial barrier function. As these alterations in endothelial integrity provide insight into angiogenic responses, we further specify detailed protocols that may be used in its study. These include Transwell Albumin clearance, Evans Blue Albumin Permeability assays, and Trans-endothelial Electrical Resistance.
• Chiang, E. T., Wang, T., & Garcia, J. G. (2011). Acute Lung Injury: The Injured Lung Endothelium, Therapeutic Strategies for Barrier Protection, and Vascular Biomarkers. In Textbook of Pulmonary Vascular Disease.. Springer, Boston, MA. doi:10.1007/978-0-387-87429-6_12
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  The vascular endothelium can be considered as an organ/tissue which comprises a monolayer of endothelial cells which serve as a semipermeable cellular barrier separating the inner space of blood vessels from its surrounding tissue and to control the exchange of fluids and cells between the two compartments. Since the pulmonary circulation receives the entire cardiac output, the large surface area of the lung microvasculature is well suited for sensing mechanical, chemical, and cellular injury by inhaled or circulating substances. This endothelial barrier is dynamically regulated through exposure to these various stimuli of physiological and pathological origin and serves to regulate multiple key biological processes (including lung fluid balance and solute transport between vascular compartments). For example, an increase in vascular permeability is a necessary feature of the body’s defense mechanism to provide injured tissues with access to leucocytes, resulting in tissue edema due to fluid extravasation. However, during conditions of intense lung inflammation such as observed in acute lung injury or its severer form of acute respiratory distress syndrome, the large surface area becomes a liability and provides the opportunity for profound vascular permeability resulting in massive fluid accumulation in the alveolar space and progressively leading to pulmonary failure. Alterations in vascular permeability occur not only in acute inflammatory lung disorders primarily caused by sepsis, pneumonia, and trauma which result in high rates of patient morbidity and mortality, but are an attractive target for therapeutic intervention in subacute lung inflammatory disorders such as ischemia–reperfusion injury, radiation lung injury, and asthma. Thus, understanding the mechanisms of endothelial barrier dysfunction is vital for the management and treatment of key and enigmatic pulmonary disorders.
• Machado, R., & Garcia, J. G. (2011). Genomics of Acute Lung Injury and Vascular Barrier Dysfunction. In Textbook of Pulmonary Vascular Disease. Springer, Boston, MA. doi:10.1007/978-0-387-87429-6_63
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  Acute lung injury (ALI) is a devastating ­syndrome of diffuse alveolar damage that develops via a variety of local and systemic insults such as sepsis, trauma, ­pneumonia, and aspiration. It is interestingly to note that only a subset of individuals exposed to potential ALI-inciting insults develop the disorder and the severity of the disease varies from complete resolution to death. In addition, ALI susceptibility and severity are also affected by ethnicity as evidenced by the higher mortality rates observed in African-American ALI patients compared with other ethnic groups in the USA. Moreover, marked differences in strain-specific ALI responses to inflammatory and injurious agents are observed in preclinical animal models. Together, these observations strongly indicate genetic components to be involved in the pathogenesis of ALI. The identification of genes contributing to ALI would potentially provide a better understanding of ALI pathobiology, yield novel biomarkers, identify individuals or populations at risk, and prove useful for the development of novel and individualized therapies. Genome-wide searches in animal models have identified a number of quantitative trait loci that associate with ALI susceptibility. In this chapter, we utilize a systems biology approach combining cellular signaling pathway analysis with population- based association studies to review established and suspected candidate genes that contribute to dysfunction of endothelial cell barrier integrity and ALI susceptibility.
• Mitra, S., Garcia, J. G., Ma, S. F., & Lloveras, D. T. (2009). Genetic Insights into Endothelial Barrier Regulation in the Acutely Inflamed Lung. In The Pulmonary Endothelium: Function in Health and Disease. John Wiley & Sons, Ltd. doi:10.1002/9780470747490.CH24
• Wolfson, R. K., Lang, G. D., Jacobson, J. R., Garcia, J. G., & Jacobson, J. L. (2009). Therapeutic Strategies to Limit Lung Endothelial Cell Permeability. In The Pulmonary Endothelium: Function in Health and Disease. John Wiley & Sons, Ltd. doi:10.1002/9780470747490.CH21
• Nonas, S., Petrache, I., & Garcia, J. G. (2008). Cigarette Smoking, Emphysema and Lung Endothelium. In Am J Physiol Lung Cell Mol Physiol. Blackwell Publishing Ltd. doi:10.1002/9780470755976.CH27
• Garcia, J. G. (2007). Regulation of Endothelial Barrier Responses and Permeability. In Endothelial Biomedicine. Cambridge University Press. doi:10.1017/CBO9780511546198.114
• Moreno, L., Moreno, L., Dudek, S. M., & Garcia, J. G. (2006). Sphingolipids and lung vascular barrier regulation. In Sphingolipid Biology. Springer Japan. doi:10.1007/4-431-34200-1_31
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  Long thought to function primarily as the structural components of lipid membranes, sphingolipids are now also recognized as vitally important signaling mediators regulating a diverse range of functions. We recently described the potent vascular barrier-regulating properties of one of these sphingolipids, the lipid and angiogenic factor sphingosine 1-phosphate (S1P) (Garcia et al, 2001). Since disruption of vascular barrier integrity commonly occurs in highly morbid inflammatory lung conditions, a better understanding of the mechanism of barrier regulation would have important clinical implications. In this chapter, we provide a brief overview of vascular barrier regulation before detailing the mechanisms underlying potent barrier-enhancing effects of S1P in vitro and in vivo in models of acute lung injury (ALI) syndromes. The potential ramifications of these findings for the development of specific therapeutic interventions for patients with ALI syndromes are then discussed.
• Jacobson, J. R., Garcia, J. G., & Jacobson, J. L. (2005). Pulmonary Vascular Barrier Regulation by Thrombin and Edg Receptors. In Cell Signaling in Vascular Inflammation. Humana Press. doi:10.1007/978-1-59259-909-7_14
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  Lung vascular permeability is directly determined by the integrity of the endothelial cell (EC) monolayer, which involves dynamic regulation by the actomyosin cytoskeleton with subsequent effects on cell—cell and cell—matrix interactions. Although the list of biochemical and biophysical agonists that alter EC barrier regulation is extensive, in this chapter we consider two in particular—the barrier-disruptive serine protease thrombin and barrier-enhancing sphingosine-1-phosphate, an activator of the Edg receptor. Both agonists, via elaborate receptor-specific and tightly orchestrated signaling pathways, offer important insights into lung vascular barrier regulation with respect to the role of individual cellular components and signaling events that target the endothelial cytoskeleton. Additionally, we detail the combined effect of thrombin and cyclic stretch, mechanistically distinct agonists, to provide further insight into lung vascular regulation. Our understanding of EC barrier regulation and lung vascular permeability has advanced remarkably in only a short time and, with the use of newly available technologies, will undoubtedly continue to rapidly evolve.
• Ma, S., Garcia, J. G., & Ye, S. Q. (2004). Gene Expression Profiling in Pulmonary and Systemic Vascular Cells Exposed to Biomechanical Stimuli. In Proteomic and Genomic Analysis of Cardiovascular Disease. Wiley‐VCH Verlag GmbH & Co. KGaA. doi:10.1002/352760152X.CH10
• Garcia, J. G., Aschner, J. L., & Malik, A. B. (1992). Regulation of Thrombin-Induced Endothelial Barrier Dysfunction and Prostaglandin Synthesis. In Thrombin. Springer US. doi:10.1007/978-1-4615-3296-5_11
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  The development of tissue culture techniques which allowed for the successful isolation and maintenance of cultured endothelial cells nearly two decades ago has resulted in an explosion of information which has refuted the previously held perception of vascular endothelium as a passive, inert tissue. In recent years the high metabolic activity of endothelial cells has been documented and an exhaustive list of diverse endothelial cell functions has been recognized as being modulated by interaction with the bioregulatory coagulant protein, thrombin.

Journals/Publications

• Knox, K. S., Casanova, N. G., Nikolich-zugich, J., Knox, K. S., Garcia, J. G., Casanova, N. G., Camp, S. M., & Bime, C. (2021). Strategies to DAMPen COVID-19-mediated lung and systemic inflammation and vascular injury.. Translational research : the journal of laboratory and clinical medicine, 232, 37-48. doi:10.1016/j.trsl.2020.12.008
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  Approximately 15%-20% of patients infected with SARS-CoV-2 coronavirus (COVID-19) progress beyond mild and self-limited disease to require supplemental oxygen for severe pneumonia; 5% of COVID-19-infected patients further develop acute respiratory distress syndrome (ARDS) and multiorgan failure. Despite mortality rates surpassing 40%, key insights into COVID-19-induced ARDS pathology have not been fully elucidated and multiple unmet needs remain. This review focuses on the unmet need for effective therapies that target unchecked innate immunity-driven inflammation which drives unchecked vascular permeability, multiorgan dysfunction and ARDS mortality. Additional unmet needs including the lack of insights into factors predicting pathogenic hyperinflammatory viral host responses, limited approaches to address the vast disease heterogeneity in ARDS, and the absence of clinically-useful ARDS biomarkers. We review unmet needs persisting in COVID-19-induced ARDS in the context of the potential role for damage-associated molecular pattern proteins in lung and systemic hyperinflammatory host responses to SARS-CoV-2 infection that ultimately drive multiorgan dysfunction and ARDS mortality. Insights into promising stratification-enhancing, biomarker-based strategies in COVID-19 and non-COVID ARDS may enable the design of successful clinical trials of promising therapies.
• Nagle, R. B., Marr, K. D., Hernandez-cortes, D., Harryman, W. L., Garcia, J. G., & Cress, A. E. (2021). Cohesive cancer invasion of the biophysical barrier of smooth muscle. Cancer and Metastasis Reviews, 40(1), 205-219. doi:10.1007/s10555-020-09950-2
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  Smooth muscle is found around organs in the digestive, respiratory, and reproductive tracts. Cancers arising in the bladder, prostate, stomach, colon, and other sites progress from low-risk disease to high-risk, lethal metastatic disease characterized by tumor invasion into, within, and through the biophysical barrier of smooth muscle. We consider here the unique biophysical properties of smooth muscle and how cohesive clusters of tumor use mechanosensing cell-cell and cell-ECM (extracellular matrix) adhesion receptors to move through a structured muscle and withstand the biophysical forces to reach distant sites. Understanding integrated mechanosensing features within tumor cluster and smooth muscle and potential triggers within adjacent adipose tissue, such as the unique damage-associated molecular pattern protein (DAMP), eNAMPT (extracellular nicotinamide phosphoribosyltransferase), or visfatin, offers an opportunity to prevent the first steps of invasion and metastasis through the structured muscle.
• Sammani, S., Desai, A. A., Sammani, S., Quijada, H., Mitra, S., Jacobson, J. R., Garcia, J. G., Epshtein, Y., Desai, A. A., Chen, W., & Bandela, M. (2021). UCHL1, a deubiquitinating enzyme, regulates lung endothelial cell permeability in vitro and in vivo.. American journal of physiology. Lung cellular and molecular physiology, 320(4), L497-L507. doi:10.1152/ajplung.00492.2020
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  Increasing evidence suggests an important role for deubiquitinating enzymes (DUBs) in modulating a variety of biological functions and diseases. We previously identified the upregulation of the DUB ubiquitin carboxyl terminal hydrolase 1 (UCHL1) in murine ventilator-induced lung injury (VILI). However, the role of UCHL1 in modulating vascular permeability, a cardinal feature of acute lung injury (ALI) in general, remains unclear. We investigated the role of UCHL1 in pulmonary endothelial cell (EC) barrier function in vitro and in vivo and examined the effects of UCHL1 on VE-cadherin and claudin-5 regulation, important adherens and tight junctional components, respectively. Measurements of transendothelial electrical resistance confirmed decreased barrier enhancement induced by hepatocyte growth factor (HGF) and increased thrombin-induced permeability in both UCHL1-silenced ECs and in ECs pretreated with LDN-57444 (LDN), a pharmacological UCHL1 inhibitor. In addition, UCHL1 knockdown (siRNA) was associated with decreased expression of VE-cadherin and claudin-5, whereas silencing of the transcription factor FoxO1 restored claudin-5 levels. Finally, UCHL1 inhibition in vivo via LDN was associated with increased VILI in a murine model. These findings support a prominent functional role of UCHL1 in regulating lung vascular permeability via alterations in adherens and tight junctions and implicate UCHL1 as an important mediator of ALI.
• Sun, B., Sammani, S., Martin, D., Desai, A. A., Valera, D. G., Sun, X., Sun, B., Song, J. H., Sammani, S., Quijada, H., Oita, R. C., Natarajan, V., Moreno-vinasco, L., Mascarenhas, J. B., Martin, D. R., Liu, Z., Kempf, C. L., Jacobson, J. R., Hernon, V. R., , Garcia, J. G., et al. (2021). Endothelial eNAMPT amplifies pre-clinical acute lung injury: efficacy of an eNAMPT-neutralising monoclonal antibody.. The European respiratory journal, 57(5). doi:10.1183/13993003.02536-2020
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  The severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target..Wild-type C57BL/6J or endothelial cell (EC)-cNAMPT -/- knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced ("one-hit") or a combined LPS/ventilator ("two-hit")-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe (99mTc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo..Immunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-cNAMPT -/- mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models..These findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.
• Sun, X., Sun, B. L., Sammani, S., Garcia, J. G., Dudek, S. M., Camp, S. M., & Bermudez, T. (2021). Genetic and epigenetic regulation of the non-muscle myosin light chain kinase isoform by lung inflammatory factors and mechanical stress.. Clinical science (London, England : 1979), 135(7), 963-977. doi:10.1042/cs20201448
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  The myosin light chain kinase gene, MYLK, encodes three proteins via unique promoters, including the non-muscle isoform of myosin light chain kinase (nmMLCK), a cytoskeletal protein centrally involved in regulation of vascular integrity. As MYLK coding SNPs are associated with severe inflammatory disorders (asthma, acute respiratory distress syndrome (ARDS)), we explored clinically relevant inflammatory stimuli and promoter SNPs in nmMLCK promoter regulation..Full-length or serially deleted MYLK luciferase reporter promoter activities were measured in human lung endothelial cells (ECs). SNP-containing non-muscle MYLK (nmMYLK) DNA fragments were generated and nmMYLK promoter binding by transcription factors (TFs) detected by protein-DNA electrophoretic mobility shift assay (EMSA). Promoter demethylation was evaluated by 5-aza-2'-deoxycytidine (5-Aza). A preclinical mouse model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) was utilized for nmMLCK validation..Lung EC levels of nmMLCK were significantly increased in LPS-challenged mice and LPS, tumor necrosis factor-α (TNF-α), 18% cyclic stretch (CS) and 5-Aza each significantly up-regulated EC nmMYLK promoter activities. EC exposure to FG-4592, a prolyl hydroxylase inhibitor that increases hypoxia-inducible factor (HIF) expression, increased nmMYLK promoter activity, confirmed by HIF1α/HIF2α silencing. nmMYLK promoter deletion studies identified distal inhibitory and proximal enhancing promoter regions as well as mechanical stretch-, LPS- and TNFα-inducible regions. Insertion of ARDS-associated SNPs (rs2700408, rs11714297) significantly increased nmMYLK promoter activity via increased transcription binding (glial cells missing homolog 1 (GCM1) and intestine-specific homeobox (ISX), respectively). Finally, the MYLK rs78755744 SNP (-261G/A), residing within a nmMYLK CpG island, significantly attenuated 5-Aza-induced promoter activity..These findings indicate nmMYLK transcriptional regulation by clinically relevant inflammatory factors and ARDS-associated nmMYLK promoter variants are consistent with nmMLCK as a therapeutic target in severe inflammatory disorders.
• Trakht, I. N., Sturrock, E. D., Popova, I. A., Petukhov, P. A., Lyubimov, A. V., Lubbe, L., Leplina, O. Y., Kalantarov, G. F., Garcia, J. G., F, K. G., Dudek, S. M., Danilov, S. M., & Chernykh, E. R. (2021). Epitope mapping of novel monoclonal antibodies to human angiotensin I-converting enzyme.. Protein science : a publication of the Protein Society, 30(8), 1577-1593. doi:10.1002/pro.4091
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  Angiotensin I-converting enzyme (ACE, CD143) plays a crucial role in blood pressure regulation, vascular remodeling, and immunity. A wide spectrum of mAbs to different epitopes on the N and C domains of human ACE have been generated and used to study different aspects of ACE biology, including establishing a novel approach-conformational fingerprinting. Here we characterized a novel set of 14 mAbs, developed against human seminal fluid ACE. The epitopes for these novel mAbs were defined using recombinant ACE constructs with truncated N and C domains, species cross-reactivity, ACE mutagenesis, and competition with the previously mapped anti-ACE mAbs. Nine mAbs recognized regions on the N domain, and 5 mAbs-on the C domain of ACE. The epitopes for most of these novel mAbs partially overlap with epitopes mapped onto ACE by the previously generated mAbs, whereas mAb 8H1 recognized yet unmapped region on the C domain where three ACE mutations associated with Alzheimer's disease are localized and is a marker for ACE mutation T877M. mAb 2H4 could be considered as a specific marker for ACE in dendritic cells. This novel set of mAbs can identify even subtle changes in human ACE conformation caused by tissue-specific glycosylation of ACE or mutations, and can detect human somatic and testicular ACE in biological fluids and tissues. Furthermore, the high reactivity of these novel mAbs provides an opportunity to study changes in the pattern of ACE expression or glycosylation in different tissues, cells, and diseases, such as sarcoidosis and Alzheimer's disease.
• Valera, D. G., Valdez, M., Sun, X., Song, J. H., Sammani, S., Oita, R. C., Moreno-vinasco, L., Martin, D., Liu, Z., Kempf, C. L., Hernon, V. R., Gregory, T., Garcia, J. G., Garcia, A. N., Cuellar, G., Cress, A. E., Casanova, N. G., Camp, S. M., Burns, K., , Bime, C., et al. (2021). Involvement of eNAMPT/TLR4 signaling in murine radiation pneumonitis: protection by eNAMPT neutralization.. Translational research : the journal of laboratory and clinical medicine. doi:10.1016/j.trsl.2021.06.002
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  Therapeutic strategies to prevent or reduce the severity of radiation pneumonitis are a serious unmet need. We evaluated extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a damage-associated molecular pattern protein (DAMP) and Toll-Like Receptor 4 (TLR4) ligand, as a therapeutic target in murine radiation pneumonitis. Radiation-induced murine and human NAMPT expression was assessed in vitro, in tissues (IHC, biochemistry, imaging), and in plasma. Wild type C57Bl6 mice (WT) and Nampt+/- heterozygous mice were exposed to 20Gy whole thoracic lung irradiation (WTLI) with or without weekly IP injection of IgG1 (control) or an eNAMPT-neutralizing polyclonal (pAb) or monoclonal antibody (mAb). BAL protein/cells and H&E staining were used to generate a WTLI severity score. Differentially-expressed genes (DEGs)/pathways were identified by RNA sequencing and bioinformatic analyses. Radiation exposure increases in vitro NAMPT expression in lung epithelium (NAMPT promoter activity) and NAMPT lung tissue expression in WTLI-exposed mice. Nampt+/- mice and eNAMPT pAb/mAb-treated mice exhibited significant histologic attenuation of WTLI-mediated lung injury with reduced levels of BAL protein and cells, and plasma levels of eNAMPT, IL-6, and IL-1β. Genomic and biochemical studies from WTLI-exposed lung tissues highlighted dysregulation of NFkB/cytokine and MAP kinase signaling pathways which were rectified by eNAMPT mAb treatment. The eNAMPT/TLR4 pathway is essentially involved in radiation pathobiology with eNAMPT neutralization an effective therapeutic strategy to reduce the severity of radiation pneumonitis.
• Zamanian, R. T., Xiao, L., Wilmot, B., Wilkins, M. R., Vanderpool, R. R., Sweatt, A. J., Sun, W., Rhodes, C. J., Oldham, W. M., Nichols, W. C., Modena, B. D., Loscalzo, J., Lindstrom, S., Leopold, J. A., Kelly, R. S., Jain, M., Horn, E. M., Hemnes, A. R., Hall, K. T., , Ghiassian, S. D., et al. (2021). NHLBI-CMREF Workshop Report on Pulmonary Vascular Disease Classification: JACC State-of-the-Art Review.. Journal of the American College of Cardiology, 77(16), 2040-2052. doi:10.1016/j.jacc.2021.02.056
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  The National Heart, Lung, and Blood Institute and the Cardiovascular Medical Research and Education Fund held a workshop on the application of pulmonary vascular disease omics data to the understanding, prevention, and treatment of pulmonary vascular disease. Experts in pulmonary vascular disease, omics, and data analytics met to identify knowledge gaps and formulate ideas for future research priorities in pulmonary vascular disease in line with National Heart, Lung, and Blood Institute Strategic Vision goals. The group identified opportunities to develop analytic approaches to multiomic datasets, to identify molecular pathways in pulmonary vascular disease pathobiology, and to link novel phenotypes to meaningful clinical outcomes. The committee suggested support for interdisciplinary research teams to develop and validate analytic methods, a national effort to coordinate biosamples and data, a consortium of preclinical investigators to expedite target evaluation and drug development, longitudinal assessment of molecular biomarkers in clinical trials, and a task force to develop a master clinical trials protocol for pulmonary vascular disease.
• Zhang, D. D., Wei, Y., Schmidlin, C. J., Ooi, A., Luo, G., Liu, P., Kerimoglu, B., Garcia, J. G., Dodson, M., & Chapman, E. (2021). The NRF2-LOC344887 signaling axis suppresses pulmonary fibrosis.. Redox biology, 38, 101766. doi:10.1016/j.redox.2020.101766
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  Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible disease characterized by an increase in differentiation of fibroblasts to myofibroblasts and excessive accumulation of extracellular matrix in lung tissue. Pharmacological activation of NRF2 has proved to be a valuable antifibrotic approach, however the detailed mechanisms of how NRF2 mediates antifibrotic function remain unclear. In this study, we found that the antifibrotic function of sulforaphane (SFN), an NRF2 activator, was largely dependent on LOC344887, a long noncoding RNA. Two functional AREs were identified in both the promoter and intron 1 of LOC344887, which defines LOC344887 as a novel anti-fibrotic NRF2 target gene. RNA-seq analysis revealed that LOC344887 controls genes and signaling pathways associated with fibrogenesis. Deletion or downregulation of LOC344887 enhanced expression of CDH2/N-cadherin, as well as a number of other fibrotic genes and blunted the antifibrotic effects of SFN. Furthermore, LOC344887-mediated downregulation of fibrotic genes may involve the PI3K-AKT signaling pathway, as pharmacologic inhibition of PI3K activity blocked the effects of LOC344887 knockdown. Our findings demonstrate that NRF2-mediated LOC344887 upregulation contributes to the antifibrotic potential of SFN by repressing the expression of CDH2 and other fibrotic genes, providing novel insight into how NRF2 controls the regulatory networks of IPF. This study provides a better understanding of the molecular mechanisms of NRF2 activators against pulmonary fibrosis and presents a novel therapeutic axis for prevention and intervention of fibrosis-related diseases.
• Zhang, Q., Zhang, D. D., White, E., Wei, Y., Shakya, A., Schmidlin, C. J., Ooi, A., Liu, P., Li, H., Kiela, P. R., Garcia, J. G., Dodson, M., Ding, X., & Chapman, E. (2021). Non-canonical NRF2 activation promotes a pro-diabetic shift in hepatic glucose metabolism.. Molecular metabolism, 51, 101243. doi:10.1016/j.molmet.2021.101243
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  NRF2, a transcription factor that regulates cellular redox and metabolic homeostasis, plays a dual role in human disease. While it is well known that canonical intermittent NRF2 activation protects against diabetes-induced tissue damage, little is known regarding the effects of prolonged non-canonical NRF2 activation in diabetes. The goal of this study was to determine the role and mechanisms of prolonged NRF2 activation in arsenic diabetogenicity..To test this, we utilized an integrated transcriptomic and metabolomic approach to assess diabetogenic changes in the livers of wild type, Nrf2-/-, p62-/-, or Nrf2-/-; p62-/- mice exposed to arsenic in the drinking water for 20 weeks..In contrast to canonical oxidative/electrophilic activation, prolonged non-canonical NRF2 activation via p62-mediated sequestration of KEAP1 increases carbohydrate flux through the polyol pathway, resulting in a pro-diabetic shift in glucose homeostasis. This p62- and NRF2-dependent increase in liver fructose metabolism and gluconeogenesis occurs through the upregulation of four novel NRF2 target genes, ketohexokinase (Khk), sorbitol dehydrogenase (Sord), triokinase/FMN cyclase (Tkfc), and hepatocyte nuclear factor 4 (Hnf4A)..We demonstrate that NRF2 and p62 are essential for arsenic-mediated insulin resistance and glucose intolerance, revealing a pro-diabetic role for prolonged NRF2 activation in arsenic diabetogenesis.
• Zhang, Y., Yan, X., Woolard, T. N., Wisniewski, S. R., Vukmirovic, M., Schupp, J. C., Prasse, A., Morris, A., Moller, D. R., Mihaljinec, A., Maier, L. A., Lynn, H., Leader, J. K., Koth, L. L., Kaminski, N., Hu, B., Hochheiser, H., Herzog, E. L., Gulati, M., , Gibson, K. F., et al. (2021). Transcriptomics of bronchoalveolar lavage cells identifies new molecular endotypes of sarcoidosis.. The European respiratory journal. doi:10.1183/13993003.02950-2020
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  Sarcoidosis is a multisystem granulomatous disease of unknown origin with a variable and often unpredictable course and pattern of organ involvement. In this study we sought to identify specific bronchoalveolar lavage (BAL) cell gene expression patterns indicative of distinct disease phenotypic traits.RNA sequencing by Ion Torrent Proton was performed on BAL cells obtained from 215 well characterised patients with pulmonary sarcoidosis enrolled in the multicenter Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Weighted Gene Co-expression Network Analysis (WGCNA) and non-parametric statistics were used to analyse genome wide BAL transcriptome. Validation of results was performed using a microarray expression data set of an independent sarcoidosis cohort (Freiburg, Germany (n=50)).Our supervised analysis found associations between distinct transcriptional programs and major pulmonary phenotypic manifestations of sarcoidosis including TH1 and TH17 pathways associated with hilar lymphadenopathy, TGFB1 and MTOR signaling with parenchymal involvement, and IL7 and IL2 with airway involvement. Our unsupervised analysis revealed gene modules that uncovered four potential sarcoidosis endotypes including hilar lymphadenopathy with increased acute T cell immune response; extraocular organ involvement with PI3 K activation pathways; chronic and multiorgan disease with increased immune response pathways; and multiorgan with increased IL-1 and IL-18 immune and inflammatory responses. We validated the occurrence of these endotypes using gene expression, pulmonary function tests and cell differentials from Freiburg. Taken together our results identify BAL gene expression programs that characterise major pulmonary sarcoidosis phenotypes and suggest the presence of distinct disease molecular endotypes.
• Zhou, T., Ma, S. F., Knox, K. S., Gonzalez-garay, M. L., Casanova, N. G., Zhou, T., Sweiss, N. J., Noth, I., Ma, S., Lussier, Y. A., Knox, K. S., Gonzalez-garay, M. L., Garcia, J. G., & Casanova, N. G. (2021). MicroRNA and protein-coding gene expression analysis in idiopathic pulmonary fibrosis yields novel biomarker signatures associated to survival.. Translational research : the journal of laboratory and clinical medicine, 228, 1-12. doi:10.1016/j.trsl.2020.07.009
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  Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology that poses significant challenges in early diagnosis and prediction of progression. Analyses of microRNA and gene expression in IPF have yielded potentially predictive information. However, the relationship between microRNA/gene expression and quantitative phenotypic value in IPF remains controversial, as is the added value of this approach to current molecular signatures in IPF. To identify biomarkers predictive of survival in IPF via a microRNA-driven strategy. We profiled microRNA and protein-coding gene expression in peripheral blood mononuclear cells from 70 IPF subjects in a discovery cohort. We linked the microRNA/gene expression level with the quantitative phenotypic variation in IPF, including diffusing capacity of the lung for carbon monoxide and the forced vital capacity percent predicted. In silico analyses of expression profiles and quantitative phenotypic data allowed the generation of 2 sets of IPF molecular signatures (unique for microRNAs and protein-coding genes) that predict IPF survival. Each signature performed well in a validation cohort comprised of IPF patients aggregated from distinct patient populations recruited from different sites. Resampling test suggests that the protein-coding gene based signature is comparable and potentially superior to published IPF prognostic gene signatures. In conclusion, these results highlight the utility of microRNA-driven peripheral blood molecular signatures as valuable and novel biomarkers associated to individuals at high survival risk and for potentially facilitating individualized therapies in this enigmatic disorder.
• Zhou, T., Tang, H., Juneman, E., Fei, Y. D., Desai, A. A., Zhou, T., Yuan, J. X., Xie, A., Tang, H., Shi, G., Parikh, D. S., Machado, R. F., Koren, G., Kittles, R. A., Kim, T. Y., Kanthi, Y., Kansal, M. M., Juneman, E., Indik, J. H., , Hillery, C. A., et al. (2021). IL-18 mediates sickle cell cardiomyopathy and ventricular arrhythmias.. Blood, 137(9), 1208-1218. doi:10.1182/blood.2020005944
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  Previous reports indicate that IL18 is a novel candidate gene for diastolic dysfunction in sickle cell disease (SCD)-related cardiomyopathy. We hypothesize that interleukin-18 (IL-18) mediates the development of cardiomyopathy and ventricular tachycardia (VT) in SCD. Compared with control mice, a humanized mouse model of SCD exhibited increased cardiac fibrosis, prolonged duration of action potential, higher VT inducibility in vivo, higher cardiac NF-κB phosphorylation, and higher circulating IL-18 levels, as well as reduced voltage-gated potassium channel expression, which translates to reduced transient outward potassium current (Ito) in isolated cardiomyocytes. Administering IL-18 to isolated mouse hearts resulted in VT originating from the right ventricle and further reduced Ito in SCD mouse cardiomyocytes. Sustained IL-18 inhibition via IL-18-binding protein resulted in decreased cardiac fibrosis and NF-κB phosphorylation, improved diastolic function, normalized electrical remodeling, and attenuated IL-18-mediated VT in SCD mice. Patients with SCD and either myocardial fibrosis or increased QTc displayed greater IL18 gene expression in peripheral blood mononuclear cells (PBMCs), and QTc was strongly correlated with plasma IL-18 levels. PBMC-derived IL18 gene expression was increased in patients who did not survive compared with those who did. IL-18 is a mediator of sickle cell cardiomyopathy and VT in mice and a novel therapeutic target in patients at risk for sudden death.
• Zhu, Z., Zhang, Z., Yuan, Q., Xin, J., Wang, M., Tejera, P., Su, L., Shen, S., Meyer, N. J., Lin, X., Li, H., Hutchinson, J. N., Garcia, J. G., Du, M., Dong, X., Christie, J. D., Christiani, D. C., Chen, F., & Cai, G. (2021). Integrative omics provide biological and clinical insights into acute respiratory distress syndrome.. Intensive Care Medicine, 1-11. doi:10.1007/s00134-021-06410-5
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  Purpose Acute respiratory distress syndrome (ARDS) is accompanied by a dysfunctional immune-inflammatory response following lung injury, including during coronavirus disease 2019 (COVID-19). Limited causal biomarkers exist for ARDS development. We sought to identify novel genetic susceptibility targets for ARDS to focus further investigation on their biological mechanism and therapeutic potential. Methods Meta-analyses of ARDS genome-wide association studies were performed with 1250 cases and 1583 controls in Europeans, and 387 cases and 387 controls in African Americans. The functionality of novel loci was determined in silico using multiple omics approaches. The causality of 114 factors potentially involved in ARDS development was assessed using Mendelian Randomization analysis. Results There was distinct genetic heterogeneity in ARDS between Europeans and African Americans. rs7967111 at 12p13.2 was functionally associated with ARDS susceptibility in Europeans (odds ratio = 1.38; P = 2.15 × 10-8). Expression of two genes annotated at this locus, BORCS5 and DUSP16, was dynamic but ultimately decreased during ARDS development, as well as downregulated in immune cells alongside COVID-19 severity. Causal inference implied that comorbidity of inflammatory bowel disease and elevated levels of C-reactive protein and interleukin-10 causally increased ARDS risk, while vitamin D supplementation and vasodilator use ameliorated risk. Conclusion Our findings suggest a novel susceptibility locus in ARDS pathophysiology that implicates BORCS5 and DUSP16 as potentially acting in immune-inflammatory processes. This locus warrants further investigation to inform the development of therapeutic targets and clinical care strategies for ARDS, including those induced by COVID-19.
• Casanova, N., Oita, R. C., Ndukum, J., Lynn, H., Garcia, J. G., Casanova, N., Camp, S. M., & Bime, C. (2020). The acute respiratory distress syndrome biomarker pipeline: crippling gaps between discovery and clinical utility.. Translational research : the journal of laboratory and clinical medicine, 226, 105-115. doi:10.1016/j.trsl.2020.06.010
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  Recent innovations in translational research have ushered an exponential increase in the discovery of novel biomarkers, thereby elevating the hope for deeper insights into "personalized" medicine approaches to disease phenotyping and care. However, a critical gap exists between the fast pace of biomarker discovery and the successful translation to clinical use. This gap underscores the fundamental biomarker conundrum across various acute and chronic disorders: how does a biomarker address a specific unmet need? Additionally, the gap highlights the need to shift the paradigm from a focus on biomarker discovery to greater translational impact and the need for a more streamlined drug approval process. The unmet need for biomarkers in acute respiratory distress syndrome (ARDS) is for reliable and validated biomarkers that minimize heterogeneity and allow for stratification of subject selection for enrollment in clinical trials of tailored therapies. This unmet need is particularly highlighted by the ongoing SARS-CoV-2/COVID-19 pandemic. The unprecedented numbers of COVID-19-induced ARDS cases has strained health care systems across the world and exposed the need for biomarkers that would accelerate drug development and the successful phenotyping of COVID-19-infected patients at risk for development of ARDS and ARDS mortality. Accordingly, this review discusses the current state of ARDS biomarkers in the context of the drug development pipeline and highlight gaps between biomarker discovery and clinical implementation while proposing potential paths forward. We discuss potential ARDS biomarkers by category and by context of use, highlighting progress in the development continuum. We conclude by discussing challenges to successful translation of biomarker candidates to clinical impact and proposing possible novel strategies.
• Karnes, J. H., Tang, H., Desai, A. A., Vanderpool, R. R., Zamanian, R. T., Yuan, J. X., Yu, J., Wiener, H. W., Vanderpool, R., Tiwari, H. K., Tang, H., Sweatt, A. J., Steiner, H. E., Schwantes-an, T. H., Rischard, F., Perez, V. D., Pauciulo, M. W., Nichols, W. C., Natarajan, B., , Nair, V., et al. (2020). Genetic Admixture and Survival in Diverse Populations with Pulmonary Arterial Hypertension.. American journal of respiratory and critical care medicine, 201(11), 1407-1415. doi:10.1164/rccm.201907-1447oc
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  Rationale: Limited information is available on racial/ethnic differences in pulmonary arterial hypertension (PAH).Objectives: Determine effects of race/ethnicity and ancestry on mortality and disease outcomes in diverse patients with PAH.Methods: Patients with Group 1 PAH were included from two national registries with genome-wide data and two local cohorts, and further incorporated in a global meta-analysis. Hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated for transplant-free, all-cause mortality in Hispanic patients with non-Hispanic white (NHW) patients as the reference group. Odds ratios (ORs) for inpatient-specific mortality in patients with PAH were also calculated for race/ethnic groups from an additional National Inpatient Sample dataset not included in the meta-analysis.Measurements and Main Results: After covariate adjustment, self-reported Hispanic patients (n = 290) exhibited significantly reduced mortality versus NHW patients (n = 1,970) after global meta-analysis (HR, 0.60 [95% CI, 0.41-0.87]; P = 0.008). Although not significant, increasing Native American genetic ancestry appeared to account for part of the observed mortality benefit (HR, 0.48 [95% CI, 0.23-1.01]; P = 0.053) in the two national registries. Finally, in the National Inpatient Sample, an inpatient mortality benefit was also observed for Hispanic patients (n = 1,524) versus NHW patients (n = 8,829; OR, 0.65 [95% CI, 0.50-0.84]; P = 0.001). An inpatient mortality benefit was observed for Native American patients (n = 185; OR, 0.38 [95% CI, 0.15-0.93]; P = 0.034).Conclusions: This study demonstrates a reproducible survival benefit for Hispanic patients with Group 1 PAH in multiple clinical settings. Our results implicate contributions of genetic ancestry to differential survival in PAH.
• Marciniak, A., Polt, R., Marciniak, A., Garcia, J. G., & Camp, S. M. (2020). In silico Docking Studies of Fingolimod and S1P1 Agonists.. Frontiers in pharmacology, 11, 247. doi:10.3389/fphar.2020.00247
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  The sphingosine-1-phosphate receptor 1 (S1P1), originally the endothelial differentiation gene 1 receptor (EDG-1), is one of five G protein-coupled receptors (GPCRs) S1P1 - 5 that bind to and are activated by sphingosine-1-phosphate (S1P). The lipid S1P is an intermediate in sphingolipid homeostasis, and S1P1 is a major medical target for immune system modulation; agonism of the receptor produces a myriad of biological responses, including endothelial cell barrier integrity, chemotaxis, lymphocyte trafficking/targeting, angiogenesis, as well as regulation of the cardiovascular system. Use of in silico docking simulations on the crystal structure of S1P1 allows for pinpointing the residues within the receptor's active site that actively contribute to the binding of S1P, and point to how these specific interactions can be exploited to design more effective synthetic analogs to specifically target S1P1 in the presence of the closely related receptors S1P2, S1P3, S1P4, and S1P5. We examined the binding properties of the endogenous substrate as well as a selection of synthetic sphingosine-derived S1P1 modulators of S1P1 with in silico docking simulations using the software package Molecular Operating Environment® (MOE®). The modeling studies reveal the relevance of phosphorylation, i.e., the presence of a phosphate or phosphonate moiety within the substrate for successful binding to occur, and indicate which residues are responsible for S1P1 binding of the most prominent sphingosine-1-phosphate receptor (S1PR) modulators, including fingolimod and its structural relatives. Furthermore, trends in steric preferences as for the binding of enantiomers to S1P1 could be observed, facilitating future design of receptor-specific substrates to precisely target the active site of S1P1.
• Oita, R. C., Molugu, T. R., Garcia, J. G., Chawla, U., Camp, S. M., & Brown, M. F. (2020). Nicotinamide phosphoribosyltransferase purification using SUMO expression system.. Analytical biochemistry, 598, 113597. doi:10.1016/j.ab.2020.113597
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  Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the salvage pathway required for nicotinamide adenine dinucleotide synthesis. The secreted NAMPT protein serves as a master regulatory cytokine involved in activation of evolutionarily conserved inflammatory networks. Appreciation of the role of NAMPT as a damage-associated molecular pattern protein (DAMP) has linked its activities to several disorders via Toll-like receptor 4 (TLR4) binding and inflammatory cascade activation. Information is currently lacking concerning the precise mode of the NAMPT protein functionality due to limited availability of purified protein for use in in vitro and in vivo studies. Here we report successful NAMPT expression using the pET-SUMO expression vector in E. coli strain SHuffle containing a hexa-His tag for purification. The Ulp1 protease was used to cleave the SUMO and hexa-His tags, and the protein was purified by immobilized-metal affinity chromatography. The protein yield was ~4 mg/L and initial biophysical characterization of the protein using circular dichroism revealed the secondary structural elements, while dynamic light scattering demonstrated the presence of oligomeric units. The NAMPT-SUMO showed a predominantly dimeric protein with functional enzymatic activity. Finally, we report NAMPT solubilization in n-dodecyl-β-d-maltopyranoside (DDM) detergent in monomeric form, thus enhancing the opportunity for further structural and functional investigations.
• Polt, R., Perez, R. G., Marciniak, A., Garcia, J. G., Garcia, A. N., Dudek, S. M., Chiang, E. T., Camp, S. M., & Bittman, R. (2020). Sphingosine-1-phosphate receptor-independent lung endothelial cell barrier disruption induced by FTY720 regioisomers.. Pulmonary circulation, 10(1). doi:10.1177/2045894020905521
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  Vascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregulation remain unclear, thereby impairing the development of desperately needed effective therapeutics. We have shown that sphingosine-1-phosphate (S1P) and 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) analogues are useful tools for exploring vascular barrier regulation mechanisms..To experimentally define the effects of FTY720 regioisomers on lung endothelial cell barrier regulation..Specific barrier-regulatory receptor and kinase inhibitors were utilized to probe signaling mechanisms involved in FTY720 regioisomer-mediated human lung endothelial cell barrier responses (trans-endothelial electrical resistance, TER). Docking simulations with the S1P1 receptor were performed to further evaluate FTY720 regioisomer signaling..FTY720 regioisomers produced potent endothelial cell barrier disruption reflected by declines in TER alterations. Pharmacologic inhibition of Gi-coupled S1P receptors (S1P1, S1P2, S1P3) failed to alter FTY720 regioisomer-mediated barrier disruption; findings that were corroborated by docking simulations demonstrating FTY720 regiosomers were repelled from S1P1 docking, in contrast to strong S1P1 binding elicited by S1P. Inhibition of either the barrier-disrupting PAR-1 receptor, the VEGF receptor, Rho-kinase, MAPK, NFkB, or PI3K failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption. While FTY720 regioisomers significantly increased protein phosphatase 2 (PP2A) activity, PP2A inhibitors failed to alter FTY720 regioisomer-induced endothelial cell barrier disruption..Together, these results imply a vexing model of pulmonary vascular barrier dysregulation in response to FTY720-related compounds and highlight the need for further insights into mechanisms of vascular integrity required to promote the development of novel therapeutic tools to prevent or reverse the pulmonary vascular leak central to ARDS outcomes.
• Rizzo, A. N., Letsiou, E., Garcia, J. G., Dudek, S. M., Demeritte, R., & Belvitch, P. (2020). Arg mediates LPS-induced disruption of the pulmonary endothelial barrier.. Vascular pharmacology, 128-129, 106677. doi:10.1016/j.vph.2020.106677
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  Acute Respiratory Distress Syndrome (ARDS) is a devastating disease process that involves dysregulated inflammation and decreased alveolar-capillary barrier function. Despite increased understanding of the pathophysiology, no effective targeted therapies exist to treat ARDS. Recent preclinical studies suggest that the multi-tyrosine kinase inhibitor, imatinib, which targets the Abl kinases c-Abl and Arg, has the potential to restore endothelial dysfunction caused by inflammatory agonists. Prior work demonstrates that imatinib attenuates LPS (lipopolysaccharide)-induced vascular leak and inflammation; however, the mechanisms underlying these effects remain incompletely understood. In the current study, we demonstrate that imatinib inhibits LPS-induced increase in the phosphorylation of CrkL, a specific substrate of Abl kinases, in human pulmonary endothelial cells. Specific silencing of Arg, and not c-Abl, attenuated LPS-induced pulmonary vascular permeability as measured by electrical cellular impedance sensing (ECIS) and gap formation assays. In addition, direct activation of Abl family kinases with the small molecule activator DPH resulted in endothelial barrier disruption that was attenuated by Arg siRNA. In complementary studies to characterize the mechanisms by which Arg mediates endothelial barrier function, Arg silencing was found to inhibit LPS-induced disruption of adherens junctions and phosphorylation of myosin light chains (MLC). Overall, these results characterize the mechanisms by which imatinib protects against LPS-induced endothelial barrier disruption and suggest that Arg inhibition may represent a novel strategy to enhance endothelial barrier function.
• Sun, B. L., Casanova, N., Sun, X., Sun, B. L., Oita, R. C., Hernon, V. R., Gregory, T., Garcia, J. G., Garcia, A. N., Cress, A. E., Casanova, N., Camp, S. M., & Algotar, A. M. (2020). Role of secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) in prostate cancer progression: Novel biomarker and therapeutic target.. EBioMedicine, 61, 103059. doi:10.1016/j.ebiom.2020.103059
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  There remains a serious need to prevent the progression of invasive prostate cancer (PCa). We previously showed that secreted extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is a multifunctional innate immunity regulator via TLR4 ligation which has been implicated in PCa progression. Here we investigate the role of eNAMPT as a diagnostic biomarker and therapeutic target in the progression of PCa..Tumor NAMPT expression and plasma eNAMPT level were evaluated in human subjects with various PCa tumor stages and high risk subjects followed-up clinically for PCa. The genetic regulation of NAMPT expression in PCa cells and the role of eNAMPT in PCa invasion were investigated utilizing in vitro and in vivo models..Marked NAMPT expression was detected in human extraprostatic-invasive PCa tissues compared to minimal expression of organ-confined PCa. Plasma eNAMPT levels were significantly elevated in PCa subjects compared to male controls, and significantly greater in subjects with extraprostatic-invasive PCa compared to subjects with organ-confined PCa. Plasma eNAMPT levels showed significant predictive value for diagnosing PCa. NAMPT expression and eNAMPT secretion were highly upregulated in human PCa cells in response to hypoxia-inducible factors and EGF. In vitro cell culture and in vivo preclinical mouse model studies confirmed eNAMPT-mediated enhancement of PCa invasiveness into muscle tissues and dramatic attenuation of PCa invasion by weekly treatment with an eNAMPT-neutralizing polyclonal antibody..This study suggests that eNAMPT is a potential biomarker for PCa, especially invasive PCa. Neutralization of eNAMPT may be an effective therapeutic approach to prevent PCa invasion and progression.
• Sun, B. L., Casanova, N., Tang, H., Sammani, S., Ahmed, M., Desai, A. A., Vanderpool, R. R., Yuan, J. X., Wang, J., Vanderpool, R., Tang, H., Sun, X., Sun, B. L., Sammani, S., Rischard, F., Oita, R. C., Moreno-vinasco, L., Lynn, H., Kempf, C. L., , Gupta, G., et al. (2020). Direct Extracellular NAMPT Involvement in Pulmonary Hypertension and Vascular Remodeling. Transcriptional Regulation by SOX and HIF-2α.. American journal of respiratory cell and molecular biology, 63(1), 92-103. doi:10.1165/rcmb.2019-0164oc
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  We previously demonstrated involvement of NAMPT (nicotinamide phosphoribosyltransferase) in pulmonary arterial hypertension (PAH) and now examine NAMPT regulation and extracellular NAMPT's (eNAMPT's) role in PAH vascular remodeling. NAMPT transcription and protein expression in human lung endothelial cells were assessed in response to PAH-relevant stimuli (PDGF [platelet-derived growth factor], VEGF [vascular endothelial growth factor], TGF-β1 [transforming growth factor-β1], and hypoxia). Endothelial-to-mesenchymal transition was detected by SNAI1 (snail family transcriptional repressor 1) and PECAM1 (platelet endothelial cell adhesion molecule 1) immunofluorescence. An eNAMPT-neutralizing polyclonal antibody was tested in a PAH model of monocrotaline challenge in rats. Plasma eNAMPT concentrations, significantly increased in patients with idiopathic pulmonary arterial hypertension, were highly correlated with indices of PAH severity. eNAMPT increased endothelial-to-mesenchymal transition, and each PAH stimulus significantly increased endothelial cell NAMPT promoter activity involving transcription factors STAT5 (signal transducer and activator of transcription 5), SOX18 (SRY-box transcription factor 18), and SOX17 (SRY-box transcription factor 17), a PAH candidate gene newly defined by genome-wide association study. The hypoxia-induced transcription factor HIF-2α (hypoxia-inducible factor-2α) also potently regulated NAMPT promoter activity, and HIF-2α binding sites were identified between -628 bp and -328 bp. The PHD2 (prolyl hydroxylase domain-containing protein 2) inhibitor FG-4592 significantly increased NAMPT promoter activity and protein expression in an HIF-2α-dependent manner. Finally, the eNAMPT-neutralizing polyclonal antibody significantly reduced monocrotaline-induced vascular remodeling, PAH hemodynamic alterations, and NF-κB activation. eNAMPT is a novel and attractive therapeutic target essential to PAH vascular remodeling.
• Sun, X., Sun, B., Babicheva, A., Vanderpool, R., Oita, R., Casanova, N., Tang, H., Gupta, A., Gupta, G., Rischard, F., Sammani, S., Kempf, C., Moreno-Vinasco, L., Camp, S., Wang, J., Desai, A., Yuan, J., & Garcia, J. G. (2020). Direct Extracellular NAMPT involvement in development of pulmonary hypertension: Transcriptional regulation by HIF2a. American Journal of Respiratory Cell and Molecular Biology.
• Sznajder, J. I., Ridge, K. M., Morales-nebreda, L., Misharin, A. V., Magnani, N. D., Iwai, K., Garcia, J. G., Dada, L. A., Budinger, G. R., & Brazee, P. L. (2020). Linear ubiquitin assembly complex regulates lung epithelial-driven responses during influenza infection.. The Journal of clinical investigation, 130(3), 1301-1314. doi:10.1172/jci128368
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  Influenza A virus (IAV) is among the most common causes of pneumonia-related death worldwide. Pulmonary epithelial cells are the primary target for viral infection and replication and respond by releasing inflammatory mediators that recruit immune cells to mount the host response. Severe lung injury and death during IAV infection result from an exuberant host inflammatory response. The linear ubiquitin assembly complex (LUBAC), composed of SHARPIN, HOIL-1L, and HOIP, is a critical regulator of NF-κB-dependent inflammation. Using mice with lung epithelial-specific deletions of HOIL-1L or HOIP in a model of IAV infection, we provided evidence that, while a reduction in the inflammatory response was beneficial, ablation of the LUBAC-dependent lung epithelial-driven response worsened lung injury and increased mortality. Moreover, we described a mechanism for the upregulation of HOIL-1L in infected and noninfected cells triggered by the activation of type I IFN receptor and mediated by IRF1, which was maladaptive and contributed to hyperinflammation. Thus, we propose that lung epithelial LUBAC acts as a molecular rheostat that could be selectively targeted to modulate the immune response in patients with severe IAV-induced pneumonia.
• Tang, H., Vanderpool, R. R., Desai, A. A., Zou, G., Zhong, N., Zheng, Q., Zhang, Z., Zhang, N., Zhang, J., Yuan, J. X., Yang, K., Wu, X., Wang, J., Vanderpool, R. R., Tang, H., Rischard, F., Makino, A., Lu, W., Liu, S., , Liu, C., et al. (2020). Tetramethylpyrazine: A promising drug for the treatment of pulmonary hypertension.. British journal of pharmacology, 177(12), 2743-2764. doi:10.1111/bph.15000
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  Tetramethylpyrazine (TMP) was originally isolated from the traditional Chinese herb ligusticum and the fermented Japanese food natto and has since been synthesized. TMP has a long history of beneficial effects in the treatment of many cardiovascular diseases. Here we have evaluated the therapeutic effects of TMP on pulmonary hypertension (PH) in animal models and in patients with pulmonary arterial hypertension (PAH) or chronic thromboembolic pulmonary hypertension (CTEPH)..Three well-defined models of PH -chronic hypoxia (10% O2 )-induced PH (HPH), monocrotaline-induced PH (MCT-PH) and Sugen 5416/hypoxia-induced PH (SuHx-PH) - were used in Sprague-Dawley rats, and assessed by echocardiography, along with haemodynamic and histological techniques. Primary cultures of rat distal pulmonary arterial smooth muscle cells (PASMCs) were used to study intracellular calcium levels. Western blots and RT-qPCR assays were also used. In the clinical cohort, patients with PAH or CTEPH were recruited. The effects of TMP were evaluated in all systems..TMP (100 mg·kg-1 ·day-1 ) prevented rats from developing experimental PH and ameliorated three models of established PH: HPH, MCT-PH and SuHx-PH. The therapeutic effects of TMP were accompanied by inhibition of intracellular calcium homeostasis in PASMCs. In a small cohort of patients with PAH or CTEPH, oral administration of TMP (100 mg, t.i.d. for 16 weeks) increased the 6-min walk distance and improved the 1-min heart rate recovery..Our results suggest that TMP is a novel and inexpensive medication for treatment of PH. Clinical trial is registered with www.chictr.org.cn (ChiCTR-IPR-14005379).
• Teran Arce, P. F., Madine, J., Migrino, R., Nussinov, R., Garcia, J. G., Niemiec, M. J., Davies, H. A., Jang, H., & Younger, S. (2019). Medin Oligomer Membrane Pore Formation: A Potential Mechanism of Vascular Dysfunction. Biophysical Journal.
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  Medin, a 50-amino-acid cleavage product of the milk fat globule-EGF factor 8 protein, is one of the most common forms of localized amyloid found in the vasculature of individuals older than 50 years. Medin induces endothelial dysfunction and vascular inflammation, yet despite its prevalence in the human aorta and multiple arterial beds, little is known about the nature of its pathology. Medin oligomers have been implicated in the pathology of aortic aneurysm, aortic dissection, and more recently, vascular dementia. Recent in vitro biomechanical measurements found increased oligomer levels in aneurysm patients with altered aortic wall integrity. Our results suggest an oligomer-mediated toxicity mechanism for medin pathology. Using lipid bilayer electrophysiology, we show that medin oligomers induce ionic membrane permeability by pore formation. Pore activity was primarily observed for preaggregated medin species from the growth-phase and rarely for lag-phase species. Atomic force microscopy (AFM) imaging of medin aggregates at different stages of aggregation revealed the gradual formation of flat domains resembling the morphology of supported lipid bilayers. Transmission electron microscopy images showed the coexistence of compact oligomers, largely consistent with the AFM data, and larger protofibrillar structures. Circular dichroism spectroscopy revealed the presence of largely disordered species and suggested the presence of β-sheets. This observation and the significantly lower thioflavin T fluorescence emitted by medin aggregates compared to amyloid-β fibrils, along with the absence of amyloid fibers in the AFM and transmission electron microscopy images, suggest that medin aggregation into pores follows a nonamyloidogenic pathway. In silico modeling by molecular dynamics simulations provides atomic-level structural detail of medin pores with the CNpNC barrel topology and diameters comparable to values estimated from experimental pore conductances.
• Thistlethwaite, P. A., Tang, H., Simonson, T. S., Shyy, J. Y., Desai, A. A., Yuan, J. X., Xiong, M., Wu, K., Wang, Z., Wang, J., Valdez-jasso, D., Thistlethwaite, P. A., Tang, H., Simonson, T. S., Shyy, J. Y., Rodriguez, M., Rahimi, S., Rahimi, S., Makino, A., , Lin, R., et al. (2020). Endothelial platelet-derived growth factor-mediated activation of smooth muscle platelet-derived growth factor receptors in pulmonary arterial hypertension.. Pulmonary circulation, 10(3), 2045894020948470. doi:10.1177/2045894020948470
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  Platelet-derived growth factor is one of the major growth factors found in human and mammalian serum and tissues. Abnormal activation of platelet-derived growth factor signaling pathway through platelet-derived growth factor receptors may contribute to the development and progression of pulmonary vascular remodeling and obliterative vascular lesions in patients with pulmonary arterial hypertension. In this study, we examined the expression of platelet-derived growth factor receptor isoforms in pulmonary arterial smooth muscle and pulmonary arterial endothelial cells and investigated whether platelet-derived growth factor secreted from pulmonary arterial smooth muscle cell or pulmonary arterial endothelial cell promotes pulmonary arterial smooth muscle cell proliferation. Our results showed that the protein expression of platelet-derived growth factor receptor α and platelet-derived growth factor receptor β in pulmonary arterial smooth muscle cell was upregulated in patients with idiopathic pulmonary arterial hypertension compared to normal subjects. Platelet-derived growth factor activated platelet-derived growth factor receptor α and platelet-derived growth factor receptor β in pulmonary arterial smooth muscle cell, as determined by phosphorylation of platelet-derived growth factor receptor α and platelet-derived growth factor receptor β. The platelet-derived growth factor-mediated activation of platelet-derived growth factor receptor α/platelet-derived growth factor receptor β was enhanced in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell compared to normal cells. Expression level of platelet-derived growth factor-AA and platelet-derived growth factor-BB was greater in the conditioned media collected from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell than from normal pulmonary arterial endothelial cell. Furthermore, incubation of idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell with conditioned culture media from normal pulmonary arterial endothelial cell induced more platelet-derived growth factor receptor α activation than in normal pulmonary arterial smooth muscle cell. Accordingly, the conditioned media from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell resulted in more pulmonary arterial smooth muscle cell proliferation than the media from normal pulmonary arterial endothelial cell. These data indicate that (a) the expression and activity of platelet-derived growth factor receptor are increased in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell compared to normal pulmonary arterial smooth muscle cell, and (b) pulmonary arterial endothelial cell from idiopathic pulmonary arterial hypertension patients secretes higher level of platelet-derived growth factor than pulmonary arterial endothelial cell from normal subjects. The enhanced secretion (and production) of platelet-derived growth factor from idiopathic pulmonary arterial hypertension-pulmonary arterial endothelial cell and upregulated platelet-derived growth factor receptor expression (and function) in idiopathic pulmonary arterial hypertension-pulmonary arterial smooth muscle cell may contribute to enhancing platelet-derived growth factor/platelet-derived growth factor receptor-associated pulmonary vascular remodeling in pulmonary arterial hypertension.
• Vanderpool, R. R., Rischard, F., Ravellette, K. S., Janowski, A. M., & Garcia, J. G. (2020). Abstract 17308: Unsupervised K-means Clustering to Identify Signatures of Right Ventricle Dysfunction in Patients With Pulmonary Hypertension. Circulation, 142. doi:10.1161/circ.142.suppl_3.17308
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  Introduction: Right ventricular (RV) function is a determinant of mortality in patients with pulmonary hypertension but current assessments are based on individual variables. Unsupervised k-means c...
• Vanderpool, R. R., Tang, H., Simonson, T. S., Gonzalez-garay, M. L., Desai, A. A., Yuan, J. X., Wang, J., Vanderpool, R. R., Tang, H., Stearman, R. S., Simonson, T. S., Shi, Y., Sangam, S., Romanoski, C. E., Rischard, F., Qi, X., Makino, A., Kanthi, Y., Gonzalez-garay, M. L., , Geraci, M. W., et al. (2020). Transcriptomic profiles in pulmonary arterial hypertension associate with disease severity and identify novel candidate genes.. Pulmonary circulation, 10(4), 2045894020968531. doi:10.1177/2045894020968531
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  Using RNAseq, we identified a 61 gene-based circulating transcriptomic profile most correlated with four indices of pulmonary arterial hypertension severity. In an independent dataset, 13/61 (21%) genes were differentially expressed in lung tissues of pulmonary arterial hypertension cases versus controls, highlighting potentially novel candidate genes involved in pulmonary arterial hypertension development.
• Vanderpool, R. R., Yuan, J. X., Vizza, C. D., Vanderpool, R., Torre, R., Scoccia, G., Sciomer, S., Rischard, F., Poscia, R., Pezzuto, B., Papa, S., Miotti, C., Manzi, G., Luongo, F., Kubba, S., Garcia, J. G., Fedele, F., Badagliacca, R., & Airhart, S. (2020). Clinical implications of idiopathic pulmonary arterial hypertension phenotypes defined by cluster analysis.. The Journal of heart and lung transplantation : the official publication of the International Society for Heart Transplantation, 39(4), 310-320. doi:10.1016/j.healun.2019.12.012
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  >Despite advances in drug development, life expectancy in idiopathic pulmonary arterial hypertension (IPAH) remains unacceptable. Contemporary IPAH characterization is based on criteria that may not adequately capture disease heterogeneity and may be proposed as a possible explanation for why patient outcome is still unfavorable. The aim of this study was to apply cluster analysis to improve phenotyping of patients with IPAH and analyze long-term clinical outcome of derived clusters..Patients with IPAH from 2 referral centers (n = 252) were evaluated with clinical, hemodynamic, and echocardiographic assessment and cardiopulmonary exercise test. Patients were classified according to cluster analysis and followed for clinical worsening occurrence..The cluster analysis identified 4 IPAH phenotypes. Cluster 1 was characterized by young patients, mild pulmonary hypertension (PH), mild right ventricular (RV) dilation and high oxygen (O2) pulse; Cluster 2 by severe PH and RV dilation and high O2 pulse; and Cluster 3 by male patients, severe PH and RV dilation, and low O2 pulse. Cluster 4 patients were older and overweight, with mild PH and RV dilation and low O2 pulse. After a mean follow-up of 995 ± 623 days, 123 (48.8%) patients had clinical worsening. Cluster 1 patients presented the best prognosis, whereas Cluster 3 had the highest rates of clinical worsening. Compared with Cluster 1, risk of clinical worsening ranged from 4.12 (confidence interval [CI] 1.43-11.92; p = 0.009) for Cluster 4 to 7.38 (CI 2.80-19.40) for Cluster 2 and 13.8 (CI 5.60-34.0; p = 0.0001) for Cluster 3..Cluster analysis of clinical variables identified 4 distinct phenotypes of IPAH. Our findings underscore the high degree of disease heterogeneity that exists within patients with IPAH and the need for advanced clinical testing to define phenotypes to improve treatment strategy decision-making. CONDENSED ABSTRACT Idiopathic pulmonary arterial hypertension (IPAH) characterization is based on criteria that may not adequately capture disease heterogeneity. The aim of this study was to apply cluster analysis to improve phenotyping of IPAH. Patients with IPAH (n = 252) were evaluated with clinical, hemodynamic, and echocardiographic assessment and cardiopulmonary exercise test. Within the umbrella category of IPAH, it was the combination of mean pulmonary arterial pressure, right ventricular size, and oxygen pulse that further stratified patients into novel IPAH phenotypes that significantly associate with clinical worsening. These findings underscore the need for novel multidimensional IPAH phenotyping for improved patient care and trial quality.
• Wang, C., Garcia, J. G., & Cai, H. (2020). More to Add to E-Cigarette Regulations: Unified Approaches.. Chest, 157(4), 771-773. doi:10.1016/j.chest.2019.11.024
• Xu, S., Wang, C., Jiang, J., Huang, K., Garcia, J. G., & Cai, H. L. (2020). Targeting NOX4 alleviates sepsis-induced acute lung injury via attenuation of redox-sensitive activation of CaMKII/ERK1/2/MLCK and endothelial cell barrier dysfunction.. Redox biology, 36, 101638. doi:10.1016/j.redox.2020.101638
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  Increased pulmonary vascular permeability due to endothelial cell (EC) barrier dysfunction is a major pathological feature of acute respiratory distress syndrome/acute lung injury (ARDS/ALI), which is a devastating critical illness with high incidence and excessive mortality. Activation of NADPH oxidase (NOX) induces EC dysfunction via production of reactive oxygen species (ROS). However, the role(s) of NOX isoform(s), and their downstream signaling events, in the development of ARDS/ALI have remained unclear. Cecal Ligation Puncture (CLP) was used to induce preclinical septic ALI in wild-type mice and mice deficient in NOX2 or p47phox, or mice transfected of control siRNA, NOX1 or NOX4 siRNA in vivo. The survival rate of the CLP group at 24 h (26.6%, control siRNA treated) was substantially improved by NOX4 knockdown (52.9%). Mice lacking NOX2 or p47phox, however, had worse outcomes after CLP (survival rates at 0% and 8.3% respectively), whereas NOX1-silenced mice had similar survival rate (30%). NOX4 knockdown attenuated lung ROS production in septic mice, whereas NOX1 knockdown, NOX2 knockout, or p47phox knockout in mice had no effects. In addition, NOX4 knockdown attenuated redox-sensitive activation of the CaMKII/ERK1/2/MLCK pathway, and restored expression of EC tight junction proteins ZO-1 and Occludin to maintain EC barrier integrity. Correspondingly, NOX4 knockdown in cultured human lung microvascular ECs also reduced LPS-induced ROS production, CaMKII/ERK1/2/MLCK activation and EC barrier dysfunction. Scavenging superoxide in vitro and in vivo with TEMPO, or inhibiting CaMKII activation with KN93, had similar effects as NOX4 knockdown in preserving EC barrier dysfunction. In summary, we have identified a novel, selective and causal role of NOX4 (versus other NOX isoforms) in inducing lung EC barrier dysfunction and injury/mortality in a preclinical CLP-induced septic model, which involves redox-sensitive activation of CaMKII/ERK1/2/MLCK pathway. Targeting NOX4 may therefore prove to an innovative therapeutic option that is markedly effective in treating ALI/ARDS.
• Batai, K., Karnes, J. H., Garcia, J. G., Saydain, G., Tang, H., Desai, A. A., Argula, R. G., Yung, D., Wort, S. J., Wilt, J., Wilkins, M. R., White, R. J., Wharton, J., Walter, R. E., Walsworth, A. K., Waisfisz, Q., Ulrich, A., Trembath, R. C., Tregouet, D., , Treacy, C. M., et al. (2019). Genetic determinants of risk in pulmonary arterial hypertension: international genome-wide association studies and meta-analysis.. The Lancet. Respiratory medicine, 7(3), 227-238. doi:10.1016/s2213-2600(18)30409-0
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  Rare genetic variants cause pulmonary arterial hypertension, but the contribution of common genetic variation to disease risk and natural history is poorly characterised. We tested for genome-wide association for pulmonary arterial hypertension in large international cohorts and assessed the contribution of associated regions to outcomes..We did two separate genome-wide association studies (GWAS) and a meta-analysis of pulmonary arterial hypertension. These GWAS used data from four international case-control studies across 11 744 individuals with European ancestry (including 2085 patients). One GWAS used genotypes from 5895 whole-genome sequences and the other GWAS used genotyping array data from an additional 5849 individuals. Cross-validation of loci reaching genome-wide significance was sought by meta-analysis. Conditional analysis corrected for the most significant variants at each locus was used to resolve signals for multiple associations. We functionally annotated associated variants and tested associations with duration of survival. All-cause mortality was the primary endpoint in survival analyses..A locus near SOX17 (rs10103692, odds ratio 1·80 [95% CI 1·55-2·08], p=5·13 × 10-15) and a second locus in HLA-DPA1 and HLA-DPB1 (collectively referred to as HLA-DPA1/DPB1 here; rs2856830, 1·56 [1·42-1·71], p=7·65 × 10-20) within the class II MHC region were associated with pulmonary arterial hypertension. The SOX17 locus had two independent signals associated with pulmonary arterial hypertension (rs13266183, 1·36 [1·25-1·48], p=1·69 × 10-12; and rs10103692). Functional and epigenomic data indicate that the risk variants near SOX17 alter gene regulation via an enhancer active in endothelial cells. Pulmonary arterial hypertension risk variants determined haplotype-specific enhancer activity, and CRISPR-mediated inhibition of the enhancer reduced SOX17 expression. The HLA-DPA1/DPB1 rs2856830 genotype was strongly associated with survival. Median survival from diagnosis in patients with pulmonary arterial hypertension with the C/C homozygous genotype was double (13·50 years [95% CI 12·07 to >13·50]) that of those with the T/T genotype (6·97 years [6·02-8·05]), despite similar baseline disease severity..This is the first study to report that common genetic variation at loci in an enhancer near SOX17 and in HLA-DPA1/DPB1 is associated with pulmonary arterial hypertension. Impairment of SOX17 function might be more common in pulmonary arterial hypertension than suggested by rare mutations in SOX17. Further studies are needed to confirm the association between HLA typing or rs2856830 genotyping and survival, and to determine whether HLA typing or rs2856830 genotyping improves risk stratification in clinical practice or trials..UK NIHR, BHF, UK MRC, Dinosaur Trust, NIH/NHLBI, ERS, EMBO, Wellcome Trust, EU, AHA, ACClinPharm, Netherlands CVRI, Dutch Heart Foundation, Dutch Federation of UMC, Netherlands OHRD and RNAS, German DFG, German BMBF, APH Paris, INSERM, Université Paris-Sud, and French ANR.
• Borbiev, T., Garcia, J. G., & Verin, A. D. (2019). [Role of phosphorylation of myosin and actin-binding proteins in endothelial permeability induced by thrombin]. Bioorganicheskaia khimiia, 29(5), 510-7.
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  The thrombin-induced dysfunction of the barrier function of the blood vessel endothelium, which manifests itself in increased permeability, is largely mediated via the initiation of specific receptors that trigger multiple signaling cascades, including the activation of some protein kinases and the phosphorylation of their cytoskeletal targets. The role of the phosphorylation of myosin and actin-binding proteins in the thrombin-induced permeability of the endothelium and possible mechanisms of the regulation of the endothelium barrier function are discussed.
• Casanova, N., Abraham, I., Oita, R. C., Ndukum, J., Miller, E. J., Mekontso-dessap, A., Lynn, H., Lussier, Y. A., Garcia, J. G., Downs, C. A., Casanova, N., Carter, D., Camp, S. M., Bime, C., & Abraham, I. (2019). Development of a biomarker mortality risk model in acute respiratory distress syndrome.. Critical care (London, England), 23(1), 410. doi:10.1186/s13054-019-2697-x
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  There is a compelling unmet medical need for biomarker-based models to risk-stratify patients with acute respiratory distress syndrome. Effective stratification would optimize participant selection for clinical trial enrollment by focusing on those most likely to benefit from new interventions. Our objective was to develop a prognostic, biomarker-based model for predicting mortality in adult patients with acute respiratory distress syndrome..This is a secondary analysis using a cohort of 252 mechanically ventilated subjects with the diagnosis of acute respiratory distress syndrome. Survival to day 7 with both day 0 (first day of presentation) and day 7 sample availability was required. Blood was collected for biomarker measurements at first presentation to the intensive care unit and on the seventh day. Biomarkers included cytokine-chemokines, dual-functioning cytozymes, and vascular injury markers. Logistic regression, latent class analysis, and classification and regression tree analysis were used to identify the plasma biomarkers most predictive of 28-day ARDS mortality..From eight biologically relevant biomarker candidates, six demonstrated an enhanced capacity to predict mortality at day 0. Latent-class analysis identified two biomarker-based phenotypes. Phenotype A exhibited significantly higher plasma levels of angiopoietin-2, macrophage migration inhibitory factor, interleukin-8, interleukin-1 receptor antagonist, interleukin-6, and extracellular nicotinamide phosphoribosyltransferase (eNAMPT) compared to phenotype B. Mortality at 28 days was significantly higher for phenotype A compared to phenotype B (32% vs 19%, p = 0.04)..An adult biomarker-based risk model reliably identifies ARDS subjects at risk of death within 28 days of hospitalization.
• Casanova, N., Sun, X., Lynn, H., Gonzales-garay, M., Garcia, J. G., Casanova, N., & Bime, C. (2019). Genomic and Genetic Approaches to Deciphering Acute Respiratory Distress Syndrome Risk and Mortality.. Antioxidants & redox signaling, 31(14), 1027-1052. doi:10.1089/ars.2018.7701
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  Significance: Acute respiratory distress syndrome (ARDS) is a severe, highly heterogeneous critical illness with staggering mortality that is influenced by environmental factors, such as mechanical ventilation, and genetic factors. Significant unmet needs in ARDS are addressing the paucity of validated predictive biomarkers for ARDS risk and susceptibility that hamper the conduct of successful clinical trials in ARDS and the complete absence of novel disease-modifying therapeutic strategies. Recent Advances: The current ARDS definition relies on clinical characteristics that fail to capture the diversity of disease pathology, severity, and mortality risk. We undertook a comprehensive survey of the available ARDS literature to identify genes and genetic variants (candidate gene and limited genome-wide association study approaches) implicated in susceptibility to developing ARDS in hopes of uncovering novel biomarkers for ARDS risk and mortality and potentially novel therapeutic targets in ARDS. We further attempted to address the well-known health disparities that exist in susceptibility to and mortality from ARDS. Critical Issues: Bioinformatic analyses identified 201 ARDS candidate genes with pathway analysis indicating a strong predominance in key evolutionarily conserved inflammatory pathways, including reactive oxygen species, innate immunity-related inflammation, and endothelial vascular signaling pathways. Future Directions: Future studies employing a system biology approach that combines clinical characteristics, genomics, transcriptomics, and proteomics may allow for a better definition of biologically relevant pathways and genotype-phenotype connections and result in improved strategies for the sub-phenotyping of diverse ARDS patients via molecular signatures. These efforts should facilitate the potential for successful clinical trials in ARDS and yield a better fundamental understanding of ARDS pathobiology.
• Garcia, A., Casanova, N., Valera, D. G., Oita, R. C., Moreno-vinasco, L., Garcia, J. G., Garcia, A. N., & Casanova, N. (2019). Late Breaking Abstract - Extracellular Nicotinamide Phosphoribosyltransferase (eNAMPT) is a biologic therapeutic target in preclinical and human radiation pneumonitis (RP). European Respiratory Journal. doi:10.1183/13993003.congress-2019.oa1599
• Gardeux, V., Achour, I., Li, J., Maienschein-Cline, M., Li, H., Pesce, L., Parinandi, G., Bahroos, N., Winn, R., Foster, I., Garcia, J. G., & Lussier, Y. A. (2019). 'N-of-1-pathways' unveils personal deregulated mechanisms from a single pair of RNA-Seq samples: towards precision medicine. Journal of the American Medical Informatics Association : JAMIA, 21(6), 1015-25.
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  The emergence of precision medicine allowed the incorporation of individual molecular data into patient care. Indeed, DNA sequencing predicts somatic mutations in individual patients. However, these genetic features overlook dynamic epigenetic and phenotypic response to therapy. Meanwhile, accurate personal transcriptome interpretation remains an unmet challenge. Further, N-of-1 (single-subject) efficacy trials are increasingly pursued, but are underpowered for molecular marker discovery.
• Gonzalez-Garay, M. L., Garcia, J. G., Zhou, T., & Casanova, N. (2019). Low Dose Carbon Monoxide Exposure in Idiopathic Pulmonary Fibrosis Produces a CO Signature Comprised of Oxidative Phosphorylation Genes. Scientific Reports. doi:10.1038/s41598-019-50585-3.
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  We recently reported the results of a multicenter, double-blinded, clinical trial of inhaled CO in patients with idiopathic pulmonary fibrosis (IPF). Despite modest increases in CO blood levels, low dose CO exposure failed to demonstrate a significant effect on the primary study endpoint of changes in serum metalloproteinase-7 (MMP7), or secondary endpoints of physiologic measures, hospitalization, death, or patient-reported outcomes. In the present study, we evaluated the effect of CO exposure (12 week treatment) on genome-wide gene expression in peripheral blood mononuclear cells (PBMC) derived from these IPF study subjects.We identified a clear CO signature dominated by significant dysregulation of genes in the oxidative phosphorylation pathway. These findings suggest this signature may serve as a potential genomic biomarker for CO exposure and perhaps for titration of dosage to allow precision testing of therapies in future low dose CO therapeutic studies in IPF.
• Khalpey, Z., Qu, N., Hemphill, C., Louis, A. V., Ferng, A. S., Son, T. G., Stavoe, K., Penick, K., Tran, P. L., Konhilas, J., Lagrand, D. S., & Garcia, J. G. (2019). Rapid porcine lung decellularization using a novel organ regenerative control acquisition bioreactor. ASAIO journal (American Society for Artificial Internal Organs : 1992), 61(1), 71-7.
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  To regenerate discarded lungs that would not normally be used for transplant, ex vivo reseeding after decellularization may produce organs suitable for clinical transplantation and therefore close the donor gap. Organ regenerative control acquisition (Harvard Biosciences, Holliston, MA), a novel bioreactor system that simulates physiological conditions, was used to evaluate a method of rapid decellularization. Although most current decellularization methods are 24-72 hours, we hypothesized that perfusing porcine lungs with detergents at higher pressures for less time would yield comparable bioscaffolds suitable for future experimentation. Methods involved perfusion of 1% Triton X-100 (Triton) and 0.1% sodium dodecyl sulfate at varied physiological flow rates. Architecture of native and decellularized lungs was analyzed with hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Dry gas and liquid ventilation techniques were introduced. Our 7 hour decellularization procedure removes nuclear material while maintaining architecture. Bioscaffolds have the microarchitecture for reseeding of stem cells. Hematoxylin and eosin staining suggested removal of nuclear material, whereas SEM and TEM imaging demonstrated total removal of cells with structural architecture preserved. This process can lead to clinical implementation, thereby increasing the availability of human lungs for transplantation.
• Miranti, C. K., Arif-tiwari, H., Warfel, N. A., Wang, M., Simpson, S. J., Rubenstein, C. S., Nagle, R. B., Miranti, C. K., Mcgrath, J. E., Marr, K. D., Ingabire, N., Hinton, J. P., Gard, J. M., Garcia, J. G., Cress, A. E., & Arif-tiwari, H. (2019). Gene Editing of α6 Integrin Inhibits Muscle Invasive Networks and Increases Cell-Cell Biophysical Properties in Prostate Cancer.. Cancer research, 79(18), 4703-4714. doi:10.1158/0008-5472.can-19-0868
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  Human prostate cancer confined to the gland is indolent (low-risk), but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding α6β1 integrin was required for smooth muscle invasion both in vitro and in vivo. Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of α6 integrin and E-cadherin in a cell-cell location and α6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing α6 integrin (DU145 α6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo. In contrast, cells without α6 integrin (DU145 α6KO) and cells expressing an integrin mutant (DU145 α6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo, and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 α6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 α6AA cells were two-fold decreased in normalized resistance and were defective in restoring resistance after a 1 μmol/L S1P challenge as compared with the DU145 α6WT cells. The results suggest that gene editing of a specific α6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in prostate cancer. SIGNIFICANCE: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific α6 integrin extracellular region.
• Schipper, D. A., Louis, A. V., Dicken, D. S., Johnson, K., Smolenski, R. T., Black, S. M., Runyan, R., Konhilas, J., Garcia, J. G., & Khalpey, Z. (2019). Improved metabolism and redox state with a novel preservation solution: implications for donor lungs after cardiac death (DCD). Pulmonary circulation, 7(2), 494-504.
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  Lungs donated after cardiac death (DCD) are an underutilized resource for a dwindling donor lung transplant pool. Our study investigates the potential of a novel preservation solution, Somah, to better preserve statically stored DCD lungs, for an extended time period, when compared to low-potassium dextran solution (LPD). We hypothesize that Somah is a metabolically superior organ preservation solution for hypothermic statically stored porcine DCD lungs, possibly improving lung transplant outcomes. Porcine DCD lungs (n = 3 per group) were flushed with and submerged in cold preservation solution. The lungs were stored up to 12 h, and samples were taken from lung tissue and the preservation medium throughout. Metabolomic and redox potential were analyzed using high performance liquid chromatography, mass spectrometry, and RedoxSYS®, comparing substrate and pathway utilization in both preservation solutions. Glutathione reduction was seen in Somah but not in LPD during preservation. Carnitine, carnosine, and n-acetylcarnosine levels were elevated in the Somah medium compared with LPD throughout. Biopsies of Somah exposed lungs demonstrated similar trends after 2 h, up to 12 h. Adenosine gradually decreased in Somah medium over 12 h, but not in LPD. An inversely proportional increase in inosine was found in Somah. Higher oxidative stress levels were measured in LPD. Our study suggests suboptimal metabolic preservation in lungs stored in LPD. LPD had poor antioxidant potential, cytoprotection, and an insufficient redox potential. These findings may have immediate clinical implications for human organs; however, further investigation is needed to evaluate DCD lung preservation in Somah as a viable option for transplant.
• Singleton, P. A., Lingen, M. W., Fekete, M. J., Garcia, J. G., & Moss, J. (2019). Methylnaltrexone inhibits opiate and VEGF-induced angiogenesis: role of receptor transactivation. Microvascular research, 72(1-2), 3-11.
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  Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, agents that inhibit angiogenesis have important therapeutic implications in numerous malignancies. We examined the effects of methylnaltrexone (MNTX), a peripheral mu opioid receptor antagonist, on agonist-induced human EC proliferation and migration, two key components in angiogenesis. Using human dermal microvascular EC, we observed that morphine sulfate (MS), the active metabolite, morphine-6-glucuronide (M6G), DAMGO ([d-Ala(2), N-Me-Phe(4), Gly(5)-ol]enkaphalin) and VEGF induced migration which were inhibited by pretreatment with MNTX at therapeutically relevant concentration (0.1 microM). The biologically inactive metabolite morphine-3-glucuronide (M3G) did not affect EC migration. We next examined the mechanism(s) by which MNTX inhibits opioid and VEGF-induced angiogenesis using human pulmonary microvascular EC. MS and DAMGO induced Src activation which was required for VEGF receptor transactivation and opioid-induced EC proliferation and migration. MNTX inhibited MS, DAMGO and VEGF induced tyrosine phosphorylation (transactivation) of VEGF receptors 1 and 2. Furthermore, MS, DAMGO and VEGF induced RhoA activation which was inhibited by MNTX or VEGF receptor tyrosine kinase inhibition. Finally, MNTX or silencing RhoA expression (siRNA) blocked MS, DAMGO and VEGF-induced EC proliferation and migration. Taken together, these results indicate that MNTX inhibits opioid-induced EC proliferation and migration via inhibition of VEGF receptor phosphorylation/transactivation with subsequent inhibition of RhoA activation. These results suggest that MNTX inhibition of angiogenesis can be a useful therapeutic intervention for cancer treatment.
• Sun, B., Moreno-vinasco, L., Valera, D. G., Valdez, M., Sun, B., Oita, R. C., Moreno-vinasco, L., Garcia, J. G., Garcia, A. N., Cress, A. E., & Carrie, K. (2019). NAMPT Is a Novel Participant and Therapeutic Target in Radiation-Induced Lung Injury (RILI). International Journal of Radiation Oncology Biology Physics, 105(1). doi:10.1164/ajrccm-conference.2019.199.1_meetingabstracts.a7255
• Trakht, I. N., Sotlar, K., Metzger, R., Klieser, E., Garcia, J. G., & Danilov, S. M. (2019). Tissue ACE phenotyping in lung cancer.. PloS one, 14(12), e0226553. doi:10.1371/journal.pone.0226553
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  Pulmonary vascular endothelium is the main metabolic site for Angiotensin I-Converting Enzyme (ACE)-mediated degradation of several biologically-active peptides (angiotensin I, bradykinin, hemo-regulatory peptide Ac-SDKP). Primary lung cancer growth and lung cancer metastases decrease lung vascularity reflected by dramatic decreases in both lung and serum ACE activity. We performed precise ACE phenotyping in tissues from subjects with lung cancer..ACE phenotyping included: 1) ACE immunohistochemistry with specific and well-characterized monoclonal antibodies (mAbs) to ACE; 2) ACE activity measurement with two ACE substrates (HHL, ZPHL); 3) calculation of ACE substrates hydrolysis ratio (ZPHL/HHL ratio); 4) the pattern of mAbs binding to 17 different ACE epitopes to detect changes in ACE conformation induced by tumor growth (conformational ACE fingerprint)..ACE immunostaining was dramatically decreased in lung cancer tissues confirmed by a 3-fold decrease in ACE activity. The conformational fingerprint of ACE from tumor lung tissues differed from normal lung (6/17 mAbs) and reflected primarily higher ACE sialylation. The increase in ZPHL/HHL ratio in lung cancer tissues was consistent with greater conformational changes of ACE. Limited analysis of the conformational ACE fingerprint in normal lung tissue and lung cancer tissue form the same patient suggested a remote effect of tumor tissue on ACE conformation and/or on "field cancerization" in a morphologically-normal lung tissues..Local conformation of ACE is significantly altered in tumor lung tissues and may be detected by conformational fingerprinting of human ACE.
• Vanderpool, R. R., Desai, A. A., Yuan, J. X., Wickstrom, K., Vanderpool, R. R., Rischard, F. P., Puri, R., Osorio, A., Garcia, J. G., Desai, A. A., & Black, S. M. (2019). EXPRESS: Surfing the Right Ventricular Pressure Waveform: Methods to assess Global, Systolic and Diastolic RV Function from a Clinical Right Heart Catheterization.. Pulmonary circulation, 10(1), 2045894019850993. doi:10.1177/2045894019850993
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  Right ventricular (RV) function strongly associates with mortality in patients with pulmonary arterial hypertension (PAH). Current methods to determine RV function require temporal measurements of ...
• Vanderpool, R. R., Vanderpool, R., Rischard, F., Garcia, J. G., & Airhart, S. (2019). Obstructive lung disease and respiratory swings contribute to the misdiagnosis of WSPH group 2 PH. European Respiratory Journal. doi:10.1183/13993003.congress-2019.pa1449
• Varadharaj, S., Watkins, T., Cardounel, A. J., Garcia, J. G., Zweier, J. L., Kuppusamy, P., Natarajan, V., & Parinandi, N. L. (2019). Vitamin C-induced loss of redox-dependent viability in lung microvascular endothelial cells. Antioxidants & redox signaling, 7(1-2), 287-300.
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  Recent clinical trials have shown that vitamin C, at pharmacological concentrations (milligram to approximately gram), upon infusion into circulation, modulates vasodilation and vascular tone in humans. This also results in the elevated concentrations of vitamin C in circulation in the millimolar range. Here, it was hypothesized that vitamin C at pharmacological concentrations (millimolar) would induce oxidative stress and cause loss of redox-dependent cell viability in vascular endothelial cells (ECs). To test the hypothesis, bovine lung microvascular ECs (BLMVECs) in monolayer cultures were exposed to vitamin C (0-10 mM) for different time periods (0-2 h). Electron paramagnetic resonance spectroscopy revealed the intracellular formation of ascorbate free radical in a dose- and time-dependent fashion. Vitamin C also induced formation of intracellular reactive oxygen species in a dose-dependent fashion. It was observed that vitamin C induced morphological alterations and loss of cell viability in a dose- and time-dependent fashion, as measured by light microscopy and Alamar Blue redox cell viability assay, respectively. Vitamin C analogues failed to induce such changes. Vitamin C depleted cellular GSH levels in a dose-dependent fashion, suggesting that vitamin C altered thiol-redox status in BLMVECs. Antioxidants, intracellular iron chelator, and catalase protected cells against vitamin C-induced loss of redox-dependent cell viability, confirming the role of hydrogen peroxide and iron during redox cycling of vitamin C. These results, for the first time in detail, established that vitamin C at pharmacological doses induced oxidative stress and loss of redox-dependent cell viability in microvascular ECs.
• Wang, M., Nagle, R. B., Knudsen, B. S., Hinton, J. P., Gard, J. M., Garcia, J. G., & Cress, A. E. (2019). Integrin α6β4E variant is associated with actin and CD9 structures and modifies the biophysical properties of cell-cell and cell-extracellular matrix interactions.. Molecular biology of the cell, 30(7), 838-850. doi:10.1091/mbc.e18-10-0652
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  Integrin α6β4 is an essential, dynamic adhesion receptor for laminin 332 found on epithelial cells, required for formation of strong cell-extracellular matrix (ECM) adhesion and induced migration, and coordinated by regions of the β4C cytoplasmic domain. β4E, a unique splice variant of β4 expressed in normal tissue, contains a cytoplasmic domain of 231 amino acids with a unique sequence of 114 amino acids instead of β4C's canonical 1089 amino acids. We determined the distribution of α6β4E within normal human glandular epithelium and its regulation and effect on cellular biophysical properties. Canonical α6β4C expressed in all basal cells, as expected, while α6β4E expressed within a subset of luminal cells. α6β4E expression was induced by three-dimensional culture conditions, activated Src, was reversible, and was stabilized by bortezomib, a proteasome inhibitor. α6β4C expressed in all cells during induced migration, whereas α6β4E was restricted to a subset of cells with increased kinetics of cell-cell and cell-ECM resistance properties. Interestingly, α6β4E presented in "ringlike" patterns measuring ∼1.75 × 0.72 microns and containing actin and CD9 at cell-ECM locations. In contrast, α6β4C expressed only within hemidesmosome-like structures containing BP180. Integrin α6β4E is an inducible adhesion isoform in normal epithelial cells that can alter biophysical properties of cell-cell and cell-ECM interactions.
• Wang, T., Brown, M. E., Kelly, G. T., Camp, S. M., Mascarenhas, J. B., Sun, X., Dudek, S. M., & Garcia, J. G. (2019). Myosin light chain kinase ( MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions. Pulmonary circulation, 8(2), 2045894018764171.
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  Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y and Y phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK, the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.
• Wang, T., Shimizu, Y., Wu, X., Kelly, G. T., Xu, X., Wang, L., Qian, Z., Chen, Y., & Garcia, J. G. (2019). Particulate matter disrupts human lung endothelial cell barrier integrity via Rho-dependent pathways. Pulmonary circulation, 7(3), 617-623.
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  Increased exposure to ambient particulate matter (PM) is associated with elevated morbidity and mortality in patients with cardiopulmonary diseases and cancer. We and others have shown that PM induces lung microvascular barrier dysfunction which potentially enhances the systemic toxicity of PM. However, the mechanisms by which PM disrupts vascular endothelial integrity remain incompletely explored. We hypothesize that PM induces endothelial cell (EC) cytoskeleton rearrangement via Rho GTPase-dependent pathways to facilitate vascular hyperpermeability. Fine PM induced time-dependent activation of cytoskeletal machinery with increases in myosin light chain (MLC) phosphorylation and EC barrier disruption measured by transendothelial electrical resistance (TER), events attenuated by the Rho-dependent kinase (ROCK) inhibitor Y-27632 or the reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC). Both Y-27632 and NAC prevented PM-induced stress fiber formation and phospho-MLC accumulation in human lung ECs. PM promotes rapid accumulation of Rho-GTP. This event is attenuated by NAC or knockdown of RhoA (siRNA). Consistent with ROCK activation, PM induced phosphorylation of myosin light chain phosphatase (MYPT) at Thr850, a post-translational modification known to inhibit phosphatase activity. Furthermore, PM activates the guanine nucleotide exchange factor (GEF) for Rho, p115, with p115 translocation to the cell periphery, in a ROS-dependent manner. Together these results demonstrate that fine PM induces EC cytoskeleton rearrangement via Rho-dependent pathways that are dependent upon the generation of oxidative stress. As the disruption of vascular integrity further contributes to cardiopulmonary physiologic derangements, these findings provide pharmacologic targets for prevention of PM-induced cardiopulmonary toxicity.
• Wang, Z., Yang, K., Zheng, Q., Zhang, C., Tang, H., Babicheva, A., Jiang, Q., Li, M., Chen, Y., Carr, S. G., Wu, K., Zhang, Q., Balistrieri, A., Wang, C., Song, S., Ayon, R. J., Desai, A. A., Black, S. M., Garcia, J. G., , Makino, A., et al. (2019). Divergent changes of p53 in pulmonary arterial endothelial and smooth muscle cells involved in the development of pulmonary hypertension. American journal of physiology. Lung cellular and molecular physiology, 316(1), L216-L228.
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  The tumor-suppressive role of p53, a transcription factor that regulates the expression of many genes, has been linked to cell cycle arrest, apoptosis, and senescence. The noncanonical function or the pathogenic role of p53 has more recently been implicated in pulmonary vascular disease. We previously reported that rapid nuclear accumulation of hypoxia-inducible factor (HIF)-1α in pulmonary arterial smooth muscle cells (PASMCs) upregulates transient receptor potential channels and enhances Ca entry to increase cytosolic Ca concentration ([Ca]). Also, we observed differences in HIF-1α/2α expression in PASMCs and pulmonary arterial endothelial cells (PAECs). Here we report that p53 is increased in PAECs, but decreased in PASMCs, isolated from mice with hypoxia-induced pulmonary hypertension (PH) and rats with monocrotaline (MCT)-induced PH (MCT-PH). The increased p53 in PAECs from rats with MCT-PH is associated with an increased ratio of Bax/Bcl-2, while the decreased p53 in PASMCs is associated with an increased HIF-1α. Furthermore, p53 is downregulated in PASMCs isolated from patients with idiopathic pulmonary arterial hypertension compared with PASMCs from normal subjects. Overexpression of p53 in normal PASMCs inhibits store-operated Ca entry (SOCE) induced by passive depletion of intracellularly stored Ca in the sarcoplasmic reticulum, while downregulation of p53 enhances SOCE. These data indicate that differentially regulated expression of p53 and HIF-1α/2α in PASMCs and PAECs and the cross talk between p53 and HIF-1α/2α in PASMCs and PAECs may play an important role in the development of PH via, at least in part, induction of PAEC apoptosis and PASMC proliferation.
• Yao, W., Mu, W., Zeifman, A., Lofti, M., Remillard, C. V., Makino, A., Perkins, D. L., Garcia, J. G., Yuan, J. X., & Zhang, W. (2019). Fenfluramine-induced gene dysregulation in human pulmonary artery smooth muscle and endothelial cells. Pulmonary circulation, 1(3), 405-18.
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  Fenfluramine is prescribed either alone or in combination with phentermine as part of Fen-Phen, an anti-obesity medication. Fenfluramine was withdrawn from the US market in 1997 due to reports of heart valvular disease, pulmonary arterial hypertension, and cardiac fibrosis. Particularly, idiopathic pulmonary arterial hypertension (IPAH), previously referred to as primary pulmonary hypertension (PPH), was found to be associated with the use of Fen-Phen, fenfluramine, and fenfluramine derivatives. The underlying mechanism of fenfluramine-associated pulmonary hypertension is still largely unknown. We reasoned that investigating drug-induced gene dysregulation would enhance our understanding of the fenfluramine-associated pathogenic mechanism of IPAH. Whole-genome gene expression profiles in fenfluramine-treated human pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells (isolated from normal subjects) were compared with baseline expression in untreated cells. Fenfluramine treatment caused dysregulation in a substantial number of genes involved in a variety of pathways and biological processes. In addition to several common pathways and biological processes such as "MAPK signaling pathway," "inflammation response," and "calcium signaling pathway" shared between both cell types, pathways and biological processes such as "blood circulation," "muscle system process," and "immune response" were enriched among the dysregulated genes in PASMC. Pathways and biological processes such as those related to cell cycle, however, were enriched among the dysregulated genes in PAEC, indicating that fenfluramine could affect unique pathways (or differentially) in different types of pulmonary artery cells. While awaiting validation in a larger cohort, these results strongly suggested that fenfluramine could induce significant dysregulation of genes in multiple biological processes and pathways critical for normal pulmonary vascular functions and structure. The transcriptional and posttranscriptional changes in these genes may, therefore, contribute to the pathogenesis of fenfluramine-associated IPAH.
• Bime, C., Pouladi, N., Sammani, S., Batai, K., Casanova, N., Zhou, T., Kempf, C. L., Sun, X., Camp, S. M., Wang, T., Kittles, R. A., Lussier, Y. A., Jones, T. K., Reilly, J. P., Meyer, N. J., Christie, J. D., Karnes, J. H., Gonzalez-Garay, M., Christiani, D. C., , Yates, C. R., et al. (2018). Genome-Wide Association Study in African Americans with Acute Respiratory Distress Syndrome Identifies the Selectin P Ligand Gene as a Risk Factor. American journal of respiratory and critical care medicine, 197(11), 1421-1432.
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  Genetic factors are involved in acute respiratory distress syndrome (ARDS) susceptibility. Identification of novel candidate genes associated with increased risk and severity will improve our understanding of ARDS pathophysiology and enhance efforts to develop novel preventive and therapeutic approaches.
• Gross, C. M., Kellner, M., Wang, T., Lu, Q., Sun, X., Zemskov, E. A., Noonepalle, S., Kangath, A., Kumar, S., Gonzalez-Garay, M., Desai, A. A., Aggarwal, S., Gorshkov, B., Klinger, C., Verin, A. D., Catravas, J. D., Jacobson, J. R., Yuan, J. X., Rafikov, R., , Garcia, J. G., et al. (2018). LPS-induced Acute Lung Injury Involves NF-κB-mediated Downregulation of SOX18. American journal of respiratory cell and molecular biology, 58(5), 614-624.
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  One of the early events in the progression of LPS-mediated acute lung injury in mice is the disruption of the pulmonary endothelial barrier resulting in lung edema. However, the molecular mechanisms by which the endothelial barrier becomes compromised remain unresolved. The SRY (sex-determining region on the Y chromosome)-related high-mobility group box (Sox) group F family member, SOX18, is a barrier-protective protein through its ability to increase the expression of the tight junction protein CLDN5. Thus, the purpose of this study was to determine if downregulation of the SOX18-CLDN5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both SOX18 and CLDN5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar downregulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. SOX18 overexpression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced CLDN5 expression (siRNA) reduced the HLMVEC barrier-protective effects of SOX18 overexpression. The mechanism by which LPS decreases SOX18 expression was identified as transcriptional repression through binding of NF-κB (p65) to a SOX18 promoter sequence located between -1,082 and -1,073 bp with peroxynitrite contributing to LPS-mediated NF-κB activation. We conclude that NF-κB-dependent decreases in the SOX18-CLDN5 axis are essentially involved in the disruption of human endothelial cell barrier integrity associated with LPS-mediated acute lung injury.
• Kurdyukov, S., Eccles, C. A., Desai, A. A., Gonzalez-Garay, M., Yuan, J. X., Garcia, J. G., Rafikova, O., & Rafikov, R. (2018). New cases of Glucose-6-Phosphate Dehydrogenase deficiency in Pulmonary Arterial Hypertension. PloS one, 13(8), e0203493.
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  Pulmonary Arterial Hypertension (PAH) is a fatal disorder with limited treatment options and reduced life expectancy after diagnosis. Complex genetic backgrounds in PAH complicates identification of causative mutations that is essential for an understanding of the disease diagnostics and etiology especially for idiopathic PAH (iPAH). Hemolysis has been implicated as contributing to the pathobiology of PAH. Glucose-6-Phosphate Dehydrogenase (G6PD) expression and activity define erythrocyte's antioxidant capacity, and its decrease contributes to erythrocyte fragility. As G6PD deficiency was previously reported in a limited number of PAH cases, we tested whether iPAH patients exhibit underlying G6PD alterations in erythrocytes. A cohort of 22 PAH patients and 8 non-PAH patients were recruited for this study. DNA isolated from Peripheral Blood Mononuclear Cells (PBMC) was used for detection of mutations in the coding region of the G6PD gene. RNA isolated from PBMC was used for determination of G6PD mRNA expression level. G6PD activity was measured in Red Blood Cell (RBC) pellets. Three patients had missense mutations in G6PD (Val291Met, Asn126Asp, Asp194Glu), however, only one mutation (Val291Met) results in a severe G6PD deficiency. A single patient with mutation (Asn126Asp) showed a 21% decrease in G6PD activity, two subjects showed G6PD deficiency without mutations, and one patient had a decreased level of G6PD mRNA and reduced enzyme levels. This study demonstrates that a moderate decrease in G6PD activity is associated with PAH. Screening for G6PD activity and mutations in the G6PD gene may provide early detection of individuals predisposed to PAH.
• Liu, P., Rojo de la Vega, M., Sammani, S., Mascarenhas, J. B., Kerins, M., Dodson, M., Sun, X., Wang, T., Ooi, A., Garcia, J. G., & Zhang, D. D. (2018). RPA1 binding to NRF2 switches ARE-dependent transcriptional activation to ARE-NRE-dependent repression. Proceedings of the National Academy of Sciences of the United States of America, 115(44), E10352-E10361.
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  NRF2 regulates cellular redox homeostasis, metabolic balance, and proteostasis by forming a dimer with small musculoaponeurotic fibrosarcoma proteins (sMAFs) and binding to antioxidant response elements (AREs) to activate target gene transcription. In contrast, NRF2-ARE-dependent transcriptional repression is unreported. Here, we describe NRF2-mediated gene repression via a specific seven-nucleotide sequence flanking the ARE, which we term the NRF2-replication protein A1 (RPA1) element (NRE). Mechanistically, RPA1 competes with sMAF for NRF2 binding, followed by interaction of NRF2-RPA1 with the ARE-NRE and eduction of promoter activity. Genome-wide in silico and RNA-seq analyses revealed this NRF2-RPA1-ARE-NRE complex mediates negative regulation of many genes with diverse functions, indicating that this mechanism is a fundamental cellular process. Notably, repression of , which encodes the nonmuscle myosin light chain kinase, by the NRF2-RPA1-ARE-NRE complex disrupts vascular integrity in preclinical inflammatory lung injury models, illustrating the translational significance of NRF2-mediated transcriptional repression. Our findings reveal a gene-suppressive function of NRF2 and a subset of negatively regulated NRF2 target genes, underscoring the broad impact of NRF2 in physiological and pathological settings.
• Lynn, H., Sun, X., Ayshiev, D., Siegler, J. H., Rizzo, A. N., Karnes, J. H., Gonzales Garay, M., Wang, T., Casanova, N., Camp, S. M., Ellis, N. A., & Garcia, J. G. (2018). Single nucleotide polymorphisms in the MYLKP1 pseudogene are associated with increased colon cancer risk in African Americans. PloS one, 13(8), e0200916.
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  Pseudogenes are paralogues of functional genes historically viewed as defunct due to either the lack of regulatory elements or the presence of frameshift mutations. Recent evidence, however, suggests that pseudogenes may regulate gene expression, although the functional role of pseudogenes remains largely unknown. We previously reported that MYLKP1, the pseudogene of MYLK that encodes myosin light chain kinase (MLCK), is highly expressed in lung and colon cancer cell lines and tissues but not in normal lung or colon. The MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. In this study, we further validate MYLKP1 as an oncogene via elucidation of the functional role of MYLKP1 genetic variants in colon cancer risk.
• Marciniak, A., Camp, S. M., Garcia, J. G., & Polt, R. (2018). An update on sphingosine-1-phosphate receptor 1 modulators. Bioorganic & medicinal chemistry letters, 28(23-24), 3585-3591.
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  Sphingolipids represent an essential class of lipids found in all eukaryotes, and strongly influence cellular signal transduction. Autoimmune diseases like asthma and multiple sclerosis (MS) are mediated by the sphingosine-1-phosphate receptor 1 (S1P) to express a variety of symptoms and disease patterns. Inspired by its natural substrate, an array of artificial sphingolipid derivatives has been developed to target this specific G protein-coupled receptor (GPCR) in an attempt to suppress autoimmune disorders. FTY720, also known as fingolimod, is the first oral disease-modifying therapy for MS on the market. In pursuit of improved stability, bioavailability, and efficiency, structural analogues of this initial prodrug have emerged over time. This review covers a brief introduction to the sphingolipid metabolism, the mechanism of action on S1P, and an updated overview of synthetic sphingosine S1P agonists.
• Mascarenhas, J. B., Tchourbanov, A. Y., Danilov, S. M., Zhou, T., Wang, T., & Garcia, J. G. (2018). The Splicing Factor hnRNPA1 Regulates Alternate Splicing of the MYLK Gene. American journal of respiratory cell and molecular biology, 58(5), 604-613.
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  Profound lung vascular permeability is a cardinal feature of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI), two syndromes known to centrally involve the nonmuscle isoform of myosin light chain kinase (nmMLCK) in vascular barrier dysregulation. Two main splice variants, nmMLCK1 and nmMLCK2, are well represented in human lung endothelial cells and encoded by MYLK, and they differ only in the presence of exon 11 in nmMLCK1, which contains critical phosphorylation sites (Y and Y) that influence nmMLCK enzymatic activity, cellular translocation, and localization in response to vascular agonists. We recently demonstrated the functional role of SNPs in altering MYLK splicing, and in the present study we sought to identify the role of splicing factors in the generation of nmMLCK1 and nmMLCK2 spliced variants. Using bioinformatic in silico approaches, we identified a putative binding site for heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a recognized splicing factor. We verified hnRNPA1 binding to MYLK by gel shift analyses and that hnRNPA1 gene and protein expression is upregulated in mouse lungs obtained from preclinical models of ARDS and VILI and in human endothelial cells exposed to 18% cyclic stretch, a model that reproduces the excessive mechanical stress observed in VILI. Using an MYLK minigene approach, we established a direct role of hnRNPA1 in MYLK splicing and in the context of 18% cyclic stretch. In summary, these data indicate an important regulatory role for hnRNPA1 in MYLK splicing, and they increase understanding of MYLK splicing in the regulation of lung vascular integrity during acute lung inflammation and excessive mechanical stress, such as that observed in ARDS and VILI.
• Oita, R. C., Camp, S. M., Ma, W., Ceco, E., Harbeck, M., Singleton, P., Messana, J., Sun, X., Wang, T., & Garcia, J. G. (2018). Novel Mechanism for Nicotinamide Phosphoribosyltransferase Inhibition of TNF-α-mediated Apoptosis in Human Lung Endothelial Cells. American journal of respiratory cell and molecular biology, 59(1), 36-44.
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  Nicotinamide phosphoribosyltransferase (NAMPT) exists as both intracellular NAMPT and extracellular NAMPT (eNAMPT) proteins. eNAMPT is secreted into the blood and functions as a cytokine/enzyme (cytozyme) that activates NF-κB signaling via ligation of Toll-like receptor 4 (TLR4), further serving as a biomarker for inflammatory lung disorders such as acute respiratory distress syndrome. In contrast, intracellular NAMPT is involved in nicotinamide mononucleotide synthesis and has been implicated in the regulation of cellular apoptosis, although the exact mechanisms for this regulation are poorly understood. We examined the role of NAMPT in TNF-α-induced human lung endothelial cell (EC) apoptosis and demonstrated that reduced NAMPT expression (siRNA) increases EC susceptibility to TNF-α-induced apoptosis as reflected by PARP-1 cleavage and caspase-3 activation. In contrast, overexpression of NAMPT served to reduce degrees of TNF-α-induced EC apoptosis. Inhibition of nicotinamide mononucleotide synthesis by FK866 (a selective NAMPT enzymatic inhibitor) failed to alter TNF-α-induced human lung EC apoptosis, suggesting that NAMPT-dependent NAD generation is unlikely to be involved in regulation of TNF-α-induced EC apoptosis. We next confirmed that TNF-α-induced EC apoptosis is attributable to NAMPT secretion into the EC culture media and subsequent eNAMPT ligation of TLR4 on the EC membrane surface. Silencing of NAMPT expression, direct neutralization of secreted eNAMPT by an NAMPT-specific polyclonal antibody (preventing TLR4 ligation), or direct TLR4 antagonism all served to significantly increase EC susceptibility to TNF-α-induced EC apoptosis. Together, these studies provide novel insights into NAMPT contributions to lung inflammatory events and to novel mechanisms of EC apoptosis regulation.
• Rhodes, C. J., Batai, K., Bleda, M., Haimel, M., Southgate, L., Germain, M., Pauciulo, M. W., Hadinnapola, C., Aman, J., Girerd, B., Arora, A., Knight, J., Hanscombe, K. B., Karnes, J. H., Kaakinen, M., Gall, H., Ulrich, A., Harbaum, L., Cebola, I., , Ferrer, J., et al. (2018). Genetic determinants of risk in pulmonary arterial hypertension: international genome-wide association studies and meta-analysis. The Lancet. Respiratory medicine.
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  Rare genetic variants cause pulmonary arterial hypertension, but the contribution of common genetic variation to disease risk and natural history is poorly characterised. We tested for genome-wide association for pulmonary arterial hypertension in large international cohorts and assessed the contribution of associated regions to outcomes.
• Rosas, I. O., Goldberg, H. J., Collard, H. R., El-Chemaly, S., Flaherty, K., Hunninghake, G. M., Lasky, J. A., Lederer, D. J., Machado, R., Martinez, F. J., Maurer, R., Teller, D., Noth, I., Peters, E., Raghu, G., Garcia, J. G., & Choi, A. M. (2018). A Phase II Clinical Trial of Low-Dose Inhaled Carbon Monoxide in Idiopathic Pulmonary Fibrosis. Chest, 153(1), 94-104.
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  Preclinical studies have demonstrated that low-dose carbon monoxide (CO) can abrogate experimental lung fibrosis. To test the therapeutic role of inhaled CO, we designed a clinical study in patients with idiopathic pulmonary fibrosis (IPF).
• Siegler, J. H., Garcia, J. G., Wang, T., Casanova, N., Gonzalez-Garay, M. L., Karnes, J. H., Ayshiev, D., Sun, X., & Lynn, H. D. (2018). Single nucleotide polymorphisms (SNPs) in the MYLKP1 pseudogene are associated with increased colon cancer risk in African Americans. PLOS ONE.
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  We previously reported that MYLKP1, the pseudogene of MYLK that encodes myosin light chain kinase (MLCK), is highly expressed in lung and colon cancer cell lines and tissues but not in normal lung or colon. The MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. In this study, we further validate MYLKP1 as an oncogene via elucidation of the functional role of MYLKP1 genetic variants in colon cancer risk
• Siegler, J. H., Siegler, J. H., Siegler, J. H., Garcia, J. G., Garcia, J. G., Garcia, J. G., Wang, T., Wang, T., Wang, T., Casanova, N., Casanova, N., Casanova, N., Gonzalez-Garay, M. L., Gonzalez-Garay, M. L., Gonzalez-Garay, M. L., Karnes, J. H., Karnes, J. H., Karnes, J. H., Ayshiev, D., , Ayshiev, D., et al. (2018). Single nucleotide polymorphisms (SNPs) in the MYLKP1 pseudogene are associated with increased colon cancer risk in African Americans. PLOS ONE, 13, 13(8):e0200916.
• Tang, H., Babicheva, A., McDermott, K. M., Gu, Y., Ayon, R. J., Song, S., Wang, Z., Gupta, A., Zhou, T., Sun, X., Dash, S., Wang, Z., Balistrieri, A., Zheng, Q., Cordery, A. G., Desai, A. A., Rischard, F., Khalpey, Z., Wang, J., , Black, S. M., et al. (2018). Endothelial HIF-2α contributes to severe pulmonary hypertension due to endothelial-to-mesenchymal transition. American journal of physiology. Lung cellular and molecular physiology, 314(2), L256-L275.
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  Pulmonary vascular remodeling characterized by concentric wall thickening and intraluminal obliteration is a major contributor to the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Here we report that increased hypoxia-inducible factor 2α (HIF-2α) in lung vascular endothelial cells (LVECs) under normoxic conditions is involved in the development of pulmonary hypertension (PH) by inducing endothelial-to-mesenchymal transition (EndMT), which subsequently results in vascular remodeling and occlusive lesions. We observed significant EndMT and markedly increased expression of SNAI, an inducer of EndMT, in LVECs from patients with IPAH and animals with experimental PH compared with normal controls. LVECs isolated from IPAH patients had a higher level of HIF-2α than that from normal subjects, whereas HIF-1α was upregulated in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. The increased HIF-2α level, due to downregulated prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase that promotes HIF-2α degradation, was involved in enhanced EndMT and upregulated SNAI1/2 in LVECs from patients with IPAH. Moreover, knockdown of HIF-2α (but not HIF-1α) with siRNA decreases both SNAI1 and SNAI2 expression in IPAH-LVECs. Mice with endothelial cell (EC)-specific knockout (KO) of the PHD2 gene, egln1 (egln1), developed severe PH under normoxic conditions, whereas Snai1/2 and EndMT were increased in LVECs of egln1 mice. EC-specific KO of the HIF-2α gene, hif2a, prevented mice from developing hypoxia-induced PH, whereas EC-specific deletion of the HIF-1α gene, hif1a, or smooth muscle cell (SMC)-specific deletion of hif2a, negligibly affected the development of PH. Also, exposure to hypoxia for 48-72 h increased protein level of HIF-1α in normal human PASMCs and HIF-2α in normal human LVECs. These data indicate that increased HIF-2α in LVECs plays a pathogenic role in the development of severe PH by upregulating SNAI1/2, inducing EndMT, and causing obliterative pulmonary vascular lesions and vascular remodeling.
• Tang, H., Wu, K., Wang, J., Vinjamuri, S., Gu, Y., Song, S., Wang, Z., Zhang, Q., Balistrieri, A., Ayon, R. J., Rischard, F., Vanderpool, R., Chen, J., Zhou, G., Desai, A. A., Black, S. M., Garcia, J. G., Yuan, J. X., & Makino, A. (2018). Pathogenic Role of mTORC1 and mTORC2 in Pulmonary Hypertension. JACC. Basic to translational science, 3(6), 744-762.
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  Concentric lung vascular wall thickening due to enhanced proliferation of pulmonary arterial smooth muscle cells is an important pathological cause for the elevated pulmonary vascular resistance reported in patients with pulmonary arterial hypertension. We identified a differential role of mammalian target of rapamycin (mTOR) complex 1 and complex 2, two functionally distinct mTOR complexes, in the development of pulmonary hypertension (PH). Inhibition of mTOR complex 1 attenuated the development of PH; however, inhibition of mTOR complex 2 caused spontaneous PH, potentially due to up-regulation of platelet-derived growth factor receptors in pulmonary arterial smooth muscle cells, and compromised the therapeutic effect of the mTOR inhibitors on PH. In addition, we describe a promising therapeutic strategy using combination treatment with the mTOR inhibitors and the platelet-derived growth factor receptor inhibitors on PH and right ventricular hypertrophy. The data from this study provide an important mechanism-based perspective for developing novel therapies for patients with pulmonary arterial hypertension and right heart failure.
• Wang, X., Wang, L., Garcia, J. G., Dudek, S. M., Shekhawat, G. S., & Dravid, V. P. (2018). The Significant Role of c-Abl Kinase in Barrier Altering Agonists-mediated Cytoskeletal Biomechanics. Scientific reports, 8(1), 1002.
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  Exploration of human pulmonary artery endothelial cell (EC) as a prototypical biomechanical system has important pathophysiologic relevance because this cell type plays a key role in the development of a wide variety of clinical conditions. The complex hierarchical organization ranging from the molecular scale up to the cellular level has an intimate and intricate relationship to the barrier function between lung tissue and blood. To understand the innate molecule-cell-tissue relationship across varied length-scales, the functional role of c-Abl kinase in the cytoskeletal nano-biomechanics of ECs in response to barrier-altering agonists was investigated using atomic force microscopy. Concurrently, the spatially specific arrangement of cytoskeleton structure and dynamic distribution of critical proteins were examined using scanning electron microscopy and immunofluorescence. Reduction in c-Abl expression by siRNA attenuates both thrombin- and sphingosine 1-phosphate (S1P)-mediated structural changes in ECs, specifically spatially-defined changes in elastic modulus and distribution of critical proteins. These results indicate that c-Abl kinase is an important determinant of cortical actin-based cytoskeletal rearrangement. Our findings directly bridge the gap between kinase activity, structural complexity, and functional connectivity across varied length-scales, and suggest that manipulation of c-Abl kinase activity may be a potential target for the treatment of pulmonary barrier disorders.
• Whitaker, M. E., Nair, V., Sinari, S., Dherange, P. A., Natarajan, B., Trutter, L., Brittain, E. L., Hemnes, A. R., Austin, E. D., Patel, K., Black, S. M., Garcia, J. G., Yuan Md PhD, J. X., Vanderpool, R. R., Rischard, F., Makino, A., Bedrick, E. J., & Desai, A. A. (2018). Diabetes Mellitus Associates with Increased Right Ventricular Afterload and Remodeling in Pulmonary Arterial Hypertension. The American journal of medicine, 131(6), 702.e7-702.e13.
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  Diabetes mellitus is associated with left ventricular hypertrophy and dysfunction. Parallel studies have also reported associations between diabetes mellitus and right ventricular dysfunction and reduced survival in patients with pulmonary arterial hypertension. However, the impact of diabetes mellitus on the pulmonary vasculature has not been well characterized. We hypothesized that diabetes mellitus and hyperglycemia could specifically influence right ventricular afterload and remodeling in patients with Group I pulmonary arterial hypertension, providing a link to their known susceptibility to right ventricular dysfunction.
• Belvitch, P., Brown, M. E., Brinley, B. N., Letsiou, E., Rizzo, A. N., Garcia, J. G., & Dudek, S. M. (2017). The ARP 2/3 complex mediates endothelial barrier function and recovery. Pulmonary circulation, 7(1), 200-210.
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  Pulmonary endothelial cell (EC) barrier dysfunction and recovery is critical to the pathophysiology of acute respiratory distress syndrome. Cytoskeletal and subsequent cell membrane dynamics play a key mechanistic role in determination of EC barrier integrity. Here, we characterizAQe the actin related protein 2/3 (Arp 2/3) complex, a regulator of peripheral branched actin polymerization, in human pulmonary EC barrier function through studies of transendothelial electrical resistance (TER), intercellular gap formation, peripheral cytoskeletal structures and lamellipodia. Compared to control, Arp 2/3 inhibition with the small molecule inhibitor CK-666 results in a reduction of baseline barrier function (1,241 ± 53 vs 988 ± 64 ohm;  
• Bime, C., Fiero, M., Lu, Z., Oren, E., Berry, C. E., Parthasarathy, S., & Garcia, J. G. (2017). High Positive End-Expiratory Pressure Is Associated with Improved Survival in Obese Patients with Acute Respiratory Distress Syndrome. The American journal of medicine, 130(2), 207-213.
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  In acute respiratory distress syndrome, minimizing lung injury from repeated collapse and reopening of alveoli by applying a high positive end-expiratory pressure improves oxygenation without influencing mortality. Obesity causes alveolar atelectasis, thus suggesting that a higher positive end-expiratory pressure might be more protective among the obese. We hypothesized that the effect of applying a high positive end-expiratory pressure on mortality from acute respiratory distress syndrome would differ by obesity status.
• Black, S., Yuan, J., Garcia, A. N., Jacobson, J. R., Garcia, J. G., Wu, X., Zemskov, E., Desai, A., Gross, C., & Wang, T. (2017). Endothelial cell signaling and ventilator-induced lung injury: molecular mechanisms, genomic analyses, and therapeutic targets. American journal of physiology. Lung cellular and molecular physiology, 312(4), L452-L476.
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  Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS). Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The pathobiology of VILI and ARDS shares many inflammatory features including increases in lung vascular permeability due to loss of endothelial cell barrier integrity resulting in alveolar flooding. While there have been advances in the understanding of certain elements of VILI and ARDS pathobiology, such as defining the importance of lung inflammatory leukocyte infiltration and highly induced cytokine expression, a deep understanding of the initiating and regulatory pathways involved in these inflammatory responses remains poorly understood. Prevailing evidence indicates that loss of endothelial barrier function plays a primary role in the development of VILI and ARDS. Thus this review will focus on the latest knowledge related to 1) the key role of the endothelium in the pathogenesis of VILI; 2) the transcription factors that relay the effects of excessive mechanical stress in the endothelium; 3) the mechanical stress-induced posttranslational modifications that influence key signaling pathways involved in VILI responses in the endothelium; 4) the genetic and epigenetic regulation of key target genes in the endothelium that are involved in VILI responses; and 5) the need for novel therapeutic strategies for VILI that can preserve endothelial barrier function.
• Brown, M. F., Wang, T., Oita, R. C., Molugu, T. R., Huang, A., Garcia, J. G., Chawla, U., & Brown, M. F. (2017). Self-Association and Conformational Stability of NAMPT Protein. Biophysical Journal, 112(3), 56a. doi:10.1016/j.bpj.2016.11.337
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  Nicotinamide phosphoribosyltransferase (NAMPT), also known as Visfatin and Pre B-cell colony enhancing factor (PBEF), is a rate-limiting enzyme in the salvage pathway required for nicotinamide adenine dinucleotide (NAD) biosynthesis. It is a highly conserved 52-kDa protein, found in living species from bacteria to humans [1]. The protein serves as a cytokine and is involved in cellular regulation influencing cancer, ischemia, obesity, and type-II diabetes [2, 3]. Despite being involved in many regulatory pathways, there is a paucity of information concerning the function of NAMPT, due to the limitations of in vivo assays, and lack of expression systems for the protein. Here, we successfully expressed the NAMPT protein using the pET-SUMO expression vector in E.coli strain SHuffle containing a hexa-His tag for protein purification. Activity assays demonstrated functionality of the protein. Moreover, initial biophysical characterization of the protein using circular dichroism revealed secondary structural elements consistent with crystallographic data. Dynamic light scattering showed the protein exists as large oligomeric units potentially involved in the NAMPT signal amplification pathway. Hydropathy analysis indicated possible hydrophobic patches on the protein surface that explains the native oligomeric state. Most striking, we discovered that NAMPT can be solubilized in n-dodecyl-β-D-maltopyranoside detergent in monomeric form. These findings open opportunities for further structural and functional investigations. Presently we are optimizing conditions for NMR experiments on NAMPT protein. These methods [4] are complementary to X-ray crystallography, and provide valuable information on the structure and dynamics, offering an important tool for understanding biological functioning.[1] T. Wang et al. (2006) Nat. Struct. Mol. Biol 13, 661.[2] S. M. Camp et al. (2015) Sci. Rep. 5, 13135.[3] A. Fukuhara et al. (2005) Science 307, 426.[4] T. R. Molugu et al. (2016) Chem. Rev. (in press).
• Brown, R. V., Wang, T., Chappeta, V. R., Wu, G., Onel, B., Chawla, R., Quijada, H., Camp, S. M., Chiang, E. T., Lassiter, Q. R., Lee, C., Phanse, S., Turnidge, M. A., Zhao, P., Garcia, J. G., Gokhale, V., Yang, D., & Hurley, L. H. (2017). The Consequences of Overlapping G-Quadruplexes and i-Motifs in the Platelet-Derived Growth Factor Receptor β Core Promoter Nuclease Hypersensitive Element Can Explain the Unexpected Effects of Mutations and Provide Opportunities for Selective Targeting of Both Structures by Small Molecules To Downregulate Gene Expression. Journal of the American Chemical Society, 139(22), 7456-7475.
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  The platelet-derived growth factor receptor β (PDGFR-β) signaling pathway is a validated and important target for the treatment of certain malignant and nonmalignant pathologies. We previously identified a G-quadruplex-forming nuclease hypersensitive element (NHE) in the human PDGFR-β promoter that putatively forms four overlapping G-quadruplexes. Therefore, we further investigated the structures and biological roles of the G-quadruplexes and i-motifs in the PDGFR-β NHE with the ultimate goal of demonstrating an alternate and effective strategy for molecularly targeting the PDGFR-β pathway. Significantly, we show that the primary G-quadruplex receptor for repression of PDGFR-β is the 3'-end G-quadruplex, which has a GGA sequence at the 3'-end. Mutation studies using luciferase reporter plasmids highlight a novel set of G-quadruplex point mutations, some of which seem to provide conflicting results on effects on gene expression, prompting further investigation into the effect of these mutations on the i-motif-forming strand. Herein we characterize the formation of an equilibrium between at least two different i-motifs from the cytosine-rich (C-rich) sequence of the PDGFR-β NHE. The apparently conflicting mutation results can be rationalized if we take into account the single base point mutation made in a critical cytosine run in the PDGFR-β NHE that dramatically affects the equilibrium of i-motifs formed from this sequence. We identified a group of ellipticines that targets the G-quadruplexes in the PDGFR-β promoter, and from this series of compounds, we selected the ellipticine analog GSA1129, which selectively targets the 3'-end G-quadruplex, to shift the dynamic equilibrium in the full-length sequence to favor this structure. We also identified a benzothiophene-2-carboxamide (NSC309874) as a PDGFR-β i-motif-interactive compound. In vitro, GSA1129 and NSC309874 downregulate PDGFR-β promoter activity and transcript in the neuroblastoma cell line SK-N-SH at subcytotoxic cell concentrations. GSA1129 also inhibits PDGFR-β-driven cell proliferation and migration. With an established preclinical murine model of acute lung injury, we demonstrate that GSA1129 attenuates endotoxin-mediated acute lung inflammation. Our studies underscore the importance of considering the effects of point mutations on structure formation from the G- and C-rich sequences and provide further evidence for the involvement of both strands and associated structures in the control of gene expression.
• Cairns, C. B., Bollinger, K., & Garcia, J. G. (2017). A Transformative Approach to Academic Medicine: The Partnership Between the University of Arizona and Banner Health. Academic medicine : journal of the Association of American Medical Colleges, 92(1), 20-22.
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  The University of Arizona Health Network (UAHN) was a modestly successful health care delivery organization with a vibrant academic portfolio and stable finances. By 2013, however, market forces, health care financing changes, and the burden of technology and informatics upgrades led to a compromised financial position at UAHN, a situation experienced by many academic medical centers. Concurrently, Banner Health had been interested in forming an academic partnership to enhance innovation, including the incorporation of new approaches into health care delivery, and to recruit high-quality providers to the organization. In 2015, the University of Arizona (UA) and Banner Health entered into a unique partnership known as Banner - University Medicine. The objective was to create a statewide system that provides reliable, compassionate, high-quality health care across all of its providers and facilities and to make a 30-year commitment to UA's College of Medicine in Tucson and the College of Medicine in Phoenix to support the State of Arizona's position as a first-tier research and training destination with world-class physicians. The goal of the Banner - University Medicine partnership is to create a nationally leading organization that transforms health care by delivering better care, enhanced service, and lower costs through new approaches focused on wellness. Key elements of this partnership are highlighted in this Commentary, including the unique governance structure of the Academic Management Council, the creation of the Academic Enhancement Fund to support the UA Colleges of Medicine in Tucson and Phoenix, and novel approaches to medical education, research, innovation, and care.
• Celedón, J. C., Burchard, E. G., Schraufnagel, D., Castillo-Salgado, C., Schenker, M., Balmes, J., Neptune, E., Cummings, K. J., Holguin, F., Riekert, K. A., Wisnivesky, J. P., Garcia, J. G., Roman, J., Kittles, R., Ortega, V. E., Redline, S., Mathias, R., Thomas, A., Samet, J., , Ford, J. G., et al. (2017). An American Thoracic Society/National Heart, Lung, and Blood Institute Workshop Report: Addressing Respiratory Health Equality in the United States. Annals of the American Thoracic Society, 14(5), 814-826.
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  Health disparities related to race, ethnicity, and socioeconomic status persist and are commonly encountered by practitioners of pediatric and adult pulmonary, critical care, and sleep medicine in the United States. To address such disparities and thus progress toward equality in respiratory health, the American Thoracic Society and the National Heart, Lung, and Blood Institute convened a workshop in May of 2015. The workshop participants addressed health disparities by focusing on six topics, each of which concluded with a panel discussion that proposed recommendations for research on racial, ethnic, and socioeconomic disparities in pulmonary, critical care, and sleep medicine. Such recommendations address best practices to advance research on respiratory health disparities (e.g., characterize broad ethnic groups into subgroups known to differ with regard to a disease of interest), risk factors for respiratory health disparities (e.g., study the impact of new tobacco or nicotine products on respiratory diseases in minority populations), addressing equity in access to healthcare and quality of care (e.g., conduct longitudinal studies of the impact of the Affordable Care Act on respiratory and sleep disorders), the impact of personalized medicine on disparities research (e.g., implement large studies of pharmacogenetics in minority populations), improving design and methodology for research studies in respiratory health disparities (e.g., use study designs that reduce participants' burden and foster trust by engaging participants as decision-makers), and achieving equity in the pulmonary, critical care, and sleep medicine workforce (e.g., develop and maintain robust mentoring programs for junior faculty, including local and external mentors). Addressing these research needs should advance efforts to reduce, and potentially eliminate, respiratory, sleep, and critical care disparities in the United States.
• Chen, J., Sysol, J. R., Singla, S., Zhao, S., Yamamura, A., Valdez-Jasso, D., Abbasi, T., Shioura, K. M., Sahni, S., Reddy, V., Sridhar, A., Gao, H., Torres, J., Camp, S. M., Tang, H., Ye, S. Q., Comhair, S., Dweik, R., Hassoun, P., , Yuan, J. X., et al. (2017). Nicotinamide Phosphoribosyltransferase Promotes Pulmonary Vascular Remodeling and Is a Therapeutic Target in Pulmonary Arterial Hypertension. Circulation, 135(16), 1532-1546.
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  Pulmonary arterial hypertension is a severe and progressive disease, a hallmark of which is pulmonary vascular remodeling. Nicotinamide phosphoribosyltransferase (NAMPT) is a cytozyme that regulates intracellular nicotinamide adenine dinucleotide levels and cellular redox state, regulates histone deacetylases, promotes cell proliferation, and inhibits apoptosis. We hypothesized that NAMPT promotes pulmonary vascular remodeling and that inhibition of NAMPT could attenuate pulmonary hypertension.
• Desai, A. A., Lei, Z., Bahroos, N., Maienschein-Cline, M., Saraf, S. L., Zhang, X., Shah, B. N., Nouraie, S. M., Abbasi, T., Patel, A. R., Lang, R. M., Lussier, Y., Garcia, J. G., Gordeuk, V. R., & Machado, R. F. (2017). Association of circulating transcriptomic profiles with mortality in sickle cell disease. Blood, 129(22), 3009-3016.
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  Sickle cell disease (SCD) complications are associated with increased morbidity and risk of mortality. We sought to identify a circulating transcriptomic profile predictive of these poor outcomes in SCD. Training and testing cohorts consisting of adult patients with SCD were recruited and prospectively followed. A pathway-based signature derived from grouping peripheral blood mononuclear cell transcriptomes distinguished 2 patient clusters with differences in survival in the training cohort. These findings were validated in a testing cohort in which the association between cluster 1 molecular profiling and mortality remained significant in a fully adjusted model. In a third cohort of West African children with SCD, cluster 1 differentiated SCD severity using a published scoring index. Finally, a risk score composed of assigning weights to cluster 1 profiling, along with established clinical risk factors using tricuspid regurgitation velocity, white blood cell count, history of acute chest syndrome, and hemoglobin levels, demonstrated a higher hazard ratio for mortality in both the training and testing cohorts compared with clinical risk factors or cluster 1 data alone. Circulating transcriptomic profiles are a powerful method to risk-stratify severity of disease and poor outcomes in both children and adults, respectively, with SCD and highlight potential associated molecular pathways.
• Hecker, L., Garcia, J. G., Wang, T., Colson, B., Knox, A., Mohamed, M., Quijada, H., Desai, A., Ahmad, K., Shin, Y. J., & Palumbo, S. (2017). Dysregulated Nox4 ubiquitination contributes to redox imbalance and age-related severity of acute lung injury. American journal of physiology. Lung cellular and molecular physiology, 312(3), L297-L308.
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  Acute respiratory distress syndrome (ARDS) is a devastating critical illness disproportionately affecting the elderly population, with both higher incidence and mortality. The integrity of the lung endothelial cell (EC) monolayer is critical for preservation of lung function. However, mechanisms mediating EC barrier regulation in the context of aging remain unclear. We assessed the severity of acute lung injury (ALI) in young (2 mo) and aged (18 mo) mice using a two-hit preclinical model. Compared with young cohorts, aged mice exhibited increased ALI severity, with greater vascular permeability characterized by elevated albumin influx and levels of bronchoalveolar lavage (BAL) cells (neutrophils) and protein. Aged/injured mice also demonstrated elevated levels of reactive oxygen species (ROS) in the BAL, which was associated with upregulation of the ROS-generating enzyme, Nox4. We evaluated the role of aging in human lung EC barrier regulation utilizing a cellular model of replicative senescence. Senescent EC populations were defined by increases in β-galactosidase activity and p16 levels. In response to lipopolysaccharide (LPS) challenge, senescent ECs demonstrate exacerbated permeability responses compared with control "young" ECs. LPS challenge led to a rapid induction of Nox4 expression in both control and senescent ECs, which was posttranslationally mediated via the proteasome/ubiquitin system. However, senescent ECs demonstrated deficient Nox4 ubiquitination, resulting in sustained expression of Nox4 and alterations in cellular redox homeostasis. Pharmacological inhibition of Nox4 in senescent ECs reduced LPS-induced alterations in permeability. These studies provide insight into the roles of Nox4/senescence in EC barrier responses and offer a mechanistic link to the increased incidence and mortality of ARDS associated with aging.
• Herazo-Maya, J. D., Sun, J., Molyneaux, P. L., Li, Q., Villalba, J. A., Tzouvelekis, A., Lynn, H., Juan-Guardela, B. M., Risquez, C., Osorio, J. C., Yan, X., Michel, G., Aurelien, N., Lindell, K. O., Klesen, M. J., Moffatt, M. F., Cookson, W. O., Zhang, Y., Garcia, J. G., , Noth, I., et al. (2017). Validation of a 52-gene risk profile for outcome prediction in patients with idiopathic pulmonary fibrosis: an international, multicentre, cohort study. The Lancet. Respiratory medicine, 5(11), 857-868.
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  The clinical course of idiopathic pulmonary fibrosis (IPF) is unpredictable. Clinical prediction tools are not accurate enough to predict disease outcomes.
• Huang, L. S., Jiang, P., Feghali-Bostwick, C., Reddy, S. P., Garcia, J. G., & Natarajan, V. (2017). Lysocardiolipin acyltransferase regulates TGF-β mediated lung fibroblast differentiation. Free radical biology & medicine, 112, 162-173.
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  Lysocardiolipin acyltransferase (LYCAT), a cardiolipin remodeling enzyme, plays a key role in mitochondrial function and vascular development. We previously reported that reduced LYCAT mRNA levels in peripheral blood mononuclear cells correlated with poor pulmonary function outcomes and decreased survival in IPF patients. Further LYCAT overexpression reduced lung fibrosis, and LYCAT knockdown accentuated experimental pulmonary fibrosis. NADPH Oxidase 4 (NOX4) expression and oxidative stress are known to contribute to lung fibroblast differentiation and progression of fibrosis. In this study, we investigated the role of LYCAT in TGF-β mediated differentiation of human lung fibroblasts to myofibroblasts, and whether this occurred through mitochondrial superoxide and NOX4 mediated hydrogen peroxide (HO) generation. Our data indicated that LYCAT expression was up-regulated in primary lung fibroblasts isolated from IPF patients and bleomycin-challenged mice, compared to controls. In vitro, siRNA-mediated SMAD3 depletion inhibited TGF-β stimulated LYCAT expression in human lung fibroblasts. ChIP immunoprecipitation assay revealed TGF-β stimulated SMAD2/3 binding to the endogenous LYCAT promoter, and mutation of the SMAD2/3 binding sites (-179/-183 and -540/-544) reduced TGF-β-stimulated LYCAT promoter activity. Overexpression of LYCAT attenuated TGF-β-induced mitochondrial and intracellular oxidative stress, NOX4 expression and differentiation of human lung fibroblasts. Further, pretreatment with Mito-TEMPO, a mitochondrial superoxide scavenger, blocked TGF-β-induced mitochondrial superoxide NOX4 expression and differentiation of human lung fibroblasts. Treatment of human lung fibroblast with NOX1/NOX4 inhibitor, GKT137831, also attenuated TGF-β induced fibroblast differentiation and mitochondrial oxidative stress. Collectively, these results suggest that LYCAT is a negative regulator of TGF-β-induced lung fibroblast differentiation by modulation of mitochondrial superoxide and NOX4 dependent HO generation, and this may serve as a potential therapeutic target for human lung fibrosis.
• Huang, Y., Ma, S. F., Espindola, M. S., Vij, R., Oldham, J. M., Huffnagle, G. B., Erb-Downward, J. R., Flaherty, K. R., Moore, B. B., White, E. S., Zhou, T., Li, J., Lussier, Y. A., Han, M. K., Kaminski, N., Garcia, J. G., Hogaboam, C. M., Martinez, F. J., Noth, I., & , C. I. (2017). Microbes Are Associated with Host Innate Immune Response in Idiopathic Pulmonary Fibrosis. American journal of respiratory and critical care medicine, 196(2), 208-219.
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  Differences in the lung microbial community influence idiopathic pulmonary fibrosis (IPF) progression. Whether the lung microbiome influences IPF host defense remains unknown.
• Kumar, S., Sun, X., Noonepalle, S. K., Lu, Q., Zemskov, E., Wang, T., Aggarwal, S., Gross, C., Sharma, S., Desai, A. A., Hou, Y., Dasarathy, S., Qu, N., Reddy, V., Lee, S. G., Cherian-Shaw, M., Yuan, J. X., Catravas, J. D., Rafikov, R., , Garcia, J. G., et al. (2017). Hyper-activation of pp60 limits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury. Free radical biology & medicine, 102, 217-228.
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  The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60 activity and this correlated with an increase in nitric oxide (NO) production, but also an increase in NOS derived superoxide, peroxynitrite formation and 3-nitrotyrosine (3-NT) levels. These effects could be simulated by the over-expression of a constitutively active pp60 (Y527FSrc) mutant and attenuated by over-expression of dominant negative pp60 mutant or reducing pp60 expression. LPS induces both RhoA nitration and endothelial barrier disruption and these events were attenuated when pp60 expression was reduced. Endothelial NOS uncoupling correlated with an increase in the levels of asymmetric dimethylarginine (ADMA) in both LPS exposed and Y527FSrc over-expressing PAEC. The effects in PAEC were also recapitulated when we transiently over-expressed Y527FSrc in the mouse lung. Finally, we found that the pp60--mediated decrease in DDAH activity was mediated by the phosphorylation of DDAH II at Y207 and that a Y207F mutant DDAH II was resistant to pp60-mediated inhibition. We conclude that pp60 can directly inhibit DDAH II and this is involved in the increased ADMA levels that enhance eNOS uncoupling during the development of ALI.
• Mascarenhas, J. B., Tchourbanov, A. Y., Fan, H., Danilov, S. M., Wang, T., & Garcia, J. G. (2017). Mechanical Stress and Single Nucleotide Variants Regulate Alternative Splicing of the MYLK Gene. American journal of respiratory cell and molecular biology, 56(1), 29-37.
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  The nonmuscle (nm) myosin light-chain kinase isoform (MLCK), encoded by the MYLK gene, is a vital participant in regulating vascular barrier responses to mechanical and inflammatory stimuli. We determined that MYLK is alternatively spliced, yielding functionally distinct nmMLCK splice variants including nmMLCK2, a splice variant highly expressed in vascular endothelial cells (EC) and associated with reduced EC barrier integrity. We demonstrated previously that the nmMLCK2 variant lacks exon 11, which encodes a key regulatory region containing two differentially phosphorylated tyrosine residues (Y and Y) that influence vascular barrier function during inflammation. In this study, we used minigene constructs and RT-PCR to interrogate biophysical factors (mechanical stress) and genetic variants (MYLK single-nucleotide polymorphisms [SNPs]) that are potentially involved in regulating MYLK alternative splicing and nmMLCK2 generation. Human lung EC exposed to pathologic mechanical stress (18% cyclic stretch) produced increased nmMLCK2 expression relative to levels of nmMLCK1 with alternative splicing significantly influenced by MYLK SNPs rs77323602 and rs147245669. In silico analyses predicted that these variants would alter exon 11 donor and acceptor sites for alternative splicing, computational predictions that were confirmed by minigene studies. The introduction of rs77323602 favored wild-type nmMLCK expression, whereas rs147245669 favored alternative splicing and deletion of exon 11, yielding increased nmMLCK2 expression. Finally, lymphoblastoid cell lines selectively harboring these MYLK SNPs (rs77323602 and rs147245669) directly validated SNP-specific effects on MYLK alternative splicing and nmMLCK2 generation. Together, these studies demonstrate that mechanical stress and MYLK SNPs regulate MYLK alternative splicing and generation of a splice variant, nmMLCK2, that contributes to the severity of inflammatory injury.
• Slepian, M. J., Sheriff, J., Hutchinson, M., Tran, P., Bajaj, N., Garcia, J. G., Scott Saavedra, S., & Bluestein, D. (2017). Shear-mediated platelet activation in the free flow: Perspectives on the emerging spectrum of cell mechanobiological mechanisms mediating cardiovascular implant thrombosis. Journal of biomechanics, 50, 20-25.
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  Shear-mediated platelet activation (SMPA) is central in thrombosis of implantable cardiovascular therapeutic devices. Despite the morbidity and mortality associated with thrombosis of these devices, our understanding of mechanisms operative in SMPA, particularly in free flowing blood, remains limited. Herein we present and discuss a range of emerging mechanisms for consideration for "free flow" activation under supraphysiologic shear. Further definition and manipulation of these mechanisms will afford opportunities for novel pharmacologic and mechanical strategies to limit SMPA and enhance overall implant device safety.
• Sun, X., Mathew, B., Sammani, S., Jacobson, J. R., & Garcia, J. G. (2017). Simvastatin-induced sphingosine 1-phosphate receptor 1 expression is KLF2-dependent in human lung endothelial cells. Pulmonary circulation, 7(1), 117-125.
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  We have demonstrated that simvastatin and sphingosine 1-phosphate (S1P) both attenuate increased vascular permeability in preclinical models of acute respiratory distress syndrome. However, the underlying mechanisms remain unclear. As Krüppel-like factor 2 (KLF2) serves as a critical regulator for cellular stress response in endothelial cells (EC), we hypothesized that simvastatin enhances endothelial barrier function via increasing expression of the barrier-promoting S1P receptor, , via a KLF2-dependent mechanism. luciferase reporter promoter activity in human lung artery EC (HPAEC) was tested after simvastatin (5 μM), and S1PR1 and KLF2 protein expression detected by immunoblotting. , transcription and expression of S1PR1 and KLF2 in mice lungs were detected by microarray profiling and immunoblotting after exposure to simvastatin (10 mg/kg). Endothelial barrier function was measured by trans-endothelial electrical resistance with the S1PR1 agonist FTY720-(S)-phosphonate. Both and gene expression (mRNA, protein) were significantly increased by simvastatin and . promoter activity was significantly increased by simvastatin (
• Szilágyi, K. L., Liu, C., Zhang, X., Wang, T., Fortman, J. D., Zhang, W., & Garcia, J. G. (2017). Epigenetic contribution of the myosin light chain kinase gene to the risk for acute respiratory distress syndrome. Translational research : the journal of laboratory and clinical medicine, 180, 12-21.
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  Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome with a considerable case fatality rate (∼30%-40%). Health disparities exist with African descent (AD) subjects exhibiting greater mortality than European descent (ED) individuals. Myosin light chain kinase is encoded by MYLK, whose genetic variants are implicated in ARDS pathogenesis and may influence ARDS mortality. As baseline population-specific epigenetic changes, that is, cytosine modifications, have been observed between AD and ED individuals, epigenetic variations in MYLK may provide insights into ARDS disparities. We compared methylation levels of MYLK cytosine-guanine dinucleotides (CpGs) between ARDS patients and intensive care unit (ICU) controls overall and by ethnicity in a nested case-control study of 39 ARDS cases and 75 non-ARDS ICU controls. Two MYLK CpG sites (cg03892735 and cg23344121) were differentially modified between ARDS subjects and controls (P 
• Talwar, A., Garcia, J. G., Tsai, H., Moreno, M., Lahm, T., Zamanian, R. T., Machado, R., Kawut, S. M., Selej, M., Mathai, S., D'Anna, L. H., Sahni, S., Rodriquez, E. J., Channick, R., Fagan, K., Gray, M., Armstrong, J., Rodriguez Lopez, J., de Jesus Perez, V., & , P. C. (2017). Health Disparities in Patients with Pulmonary Arterial Hypertension: A Blueprint for Action. An Official American Thoracic Society Statement. American journal of respiratory and critical care medicine, 196(8), e32-e47.
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  Health disparities have a major impact in the quality of life and clinical care received by minorities in the United States. Pulmonary arterial hypertension (PAH) is a rare cardiopulmonary disorder that affects children and adults and that, if untreated, results in premature death. The impact of health disparities in the diagnosis, treatment, and clinical outcome of patients with PAH has not been systematically investigated.
• Tang, H., Sun, X., Desai, A. A., Yuan, J. X., Tang, H., Sun, X., Song, S., Mcdermott, K. M., Makino, A., Garcia, J. G., Desai, A. A., Cordery, A. G., Black, S. M., & Babicheva, A. (2017). Inhibition of PHD2 Induces Endothelial-to-Mesenchymal Transition in Pulmonary Vascular Endothelial Cells. The FASEB Journal, 31. doi:10.1096/fasebj.31.1_supplement.1016.9
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  RATIONALEPulmonary arterial hypertension (PAH) is a fatal disease that predominantly affect young women. Enhanced proliferation and/or reduced apoptosis of pulmonary vascular endothelial cells (PVE...
• Tikhomirova, V. E., Kost, O. A., Kryukova, O. V., Golukhova, E. Z., Bulaeva, N. I., Zholbaeva, A. Z., Bokeria, L. A., Garcia, J. G., & Danilov, S. M. (2017). ACE phenotyping in human heart. PloS one, 12(8), e0181976.
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  Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, is expressed as a type-1 membrane glycoprotein on the surface of different cells, including endothelial cells of the heart. We hypothesized that the local conformation and, therefore, the properties of heart ACE could differ from lung ACE due to different microenvironment in these organs.
• Vanderpool, R. R., Desai, A. A., Knapp, S. M., Simon, M. A., Abidov, A., Yuan, J. X., Garcia, J. G., Hansen, L. M., Knoper, S. R., Naeije, R., & Rischard, F. P. (2017). How prostacyclin therapy improves right ventricular function in pulmonary arterial hypertension. The European respiratory journal, 50(2).
• Wang, X., Bleher, R., Wang, L., Garcia, J. G., Dudek, S. M., Shekhawat, G. S., & Dravid, V. P. (2017). Imatinib Alters Agonists-mediated Cytoskeletal Biomechanics in Lung Endothelium. Scientific reports, 7(1), 14152.
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  The endothelium serves as a size-selective barrier and tightly controls the fluid exchange from the circulation to the surrounding tissues. In this study, a multiplexed microscopy characterization is developed to study the spatio-temporal effects of Abl kinases on endothelial cytoskeletal structure using AFM, SEM, and immunofluorescence. Sphingosine 1-phosphate (S1P) produces significant endothelial barrier enhancement by means of peripheral actin rearrangement. However, Abl kinase inhibition by imatinib reduces rapid redistribution of the important cytoskeletal proteins to the periphery and their association with the cortical actin ring. Herein, it moderates the thickness of the cortical actin ring, and diminishes the increase in elastic modulus at the periphery and cytoplasm. These findings demonstrate that imatinib attenuates multiple cytoskeletal changes associated with S1P-mediated endothelial barrier enhancement and suggest a novel role for Abl kinases in mediating these S1P effects. These observations bridge the gap between molecule dynamics, structure complexity and function connectivity across varied length-scales to improve our understanding on human pulmonary endothelial barrier regulation. Moreover, our study suggests a framework for understanding form-function relationships in other biomechanical subsystems, wherein complex hierarchical organization programmed from the molecular scale to the cellular and tissue levels has an intimate relationship to the overall physiological function.
• Wu, K., Zhang, Q., Wu, X., Lu, W., Tang, H., Liang, Z., Gu, Y., Song, S., Ayon, R. J., Wang, Z., McDermott, K. M., Balistrieri, A., Wang, C., Black, S. M., Garcia, J. G., Makino, A., Yuan, J. X., & Wang, J. (2017). Chloroquine is a potent pulmonary vasodilator that attenuates hypoxia-induced pulmonary hypertension. British journal of pharmacology, 174(22), 4155-4172.
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  Sustained pulmonary vasoconstriction and excessive pulmonary vascular remodelling are two major causes of elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension. The purpose of this study was to investigate whether chloroquine induced relaxation in the pulmonary artery (PA) and attenuates hypoxia-induced pulmonary hypertension (HPH).
• Yuan, J., Makino, A., Garcia, J. G., Mcdermott, K. M., Rischard, F., Desai, A., Black, S., Khalpey, Z. I., Cordery, A. G., Sun, X., Tang, H., Babicheva, A., Dash, S., Yamamura, H., Yamamura, A., Ayon, R. J., & Song, S. (2017). Capsaicin-induced Ca2+ signaling is enhanced via upregulated TRPV1 channels in pulmonary artery smooth muscle cells from patients with idiopathic PAH. American journal of physiology. Lung cellular and molecular physiology, 312(3), L309-L325.
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  Capsaicin is an active component of chili pepper and a pain relief drug. Capsaicin can activate transient receptor potential vanilloid 1 (TRPV1) channels to increase cytosolic Ca2+ concentration ([Ca2+]cyt). A rise in [Ca2+]cyt in pulmonary artery smooth muscle cells (PASMCs) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. In this study, we observed that a capsaicin-induced increase in [Ca2+]cyt was significantly enhanced in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with normal PASMCs from healthy donors. In addition, the protein expression level of TRPV1 in IPAH PASMCs was greater than in normal PASMCs. Increasing the temperature from 23 to 43°C, or decreasing the extracellular pH value from 7.4 to 5.9 enhanced capsaicin-induced increases in [Ca2+]cyt; the acidity (pH 5.9)- and heat (43°C)-mediated enhancement of capsaicin-induced [Ca2+]cyt increases were greater in IPAH PASMCs than in normal PASMCs. Decreasing the extracellular osmotic pressure from 310 to 200 mOsmol/l also increased [Ca2+]cyt, and the hypo-osmolarity-induced rise in [Ca2+]cyt was greater in IPAH PASMCs than in healthy PASMCs. Inhibition of TRPV1 (with 5'-IRTX or capsazepine) or knockdown of TRPV1 (with short hairpin RNA) attenuated capsaicin-, acidity-, and osmotic stretch-mediated [Ca2+]cyt increases in IPAH PASMCs. Capsaicin induced phosphorylation of CREB by raising [Ca2+]cyt, and capsaicin-induced CREB phosphorylation were significantly enhanced in IPAH PASMCs compared with normal PASMCs. Pharmacological inhibition and knockdown of TRPV1 attenuated IPAH PASMC proliferation. Taken together, the capsaicin-mediated [Ca2+]cyt increase due to upregulated TRPV1 may be a critical pathogenic mechanism that contributes to augmented Ca2+ influx and excessive PASMC proliferation in patients with IPAH.
• Zhou, T., Casanova, N., Pouladi, N., Wang, T., Lussier, Y., Knox, K. S., & Garcia, J. G. (2017). Identification of Jak-STAT signaling involvement in sarcoidosis severity via a novel microRNA-regulated peripheral blood mononuclear cell gene signature. Scientific reports, 7(1), 4237.
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  Sarcoidosis is a granulomatous lung disorder of unknown cause. The majority of individuals with sarcoidosis spontaneously achieve full remission (uncomplicated sarcoidosis), however, ~20% of sarcoidosis-affected individuals experience progressive lung disease or cardiac and nervous system involvement (complicated sarcoidosis). We investigated peripheral blood mononuclear cell (PBMC) microRNA and protein-coding gene expression data from healthy controls and patients with uncomplicated or complicated sarcoidosis. We identified 46 microRNAs and 1,559 genes that were differentially expressed across a continuum of sarcoidosis severity (healthy control → uncomplicated sarcoidosis → complicated sarcoidosis). A total of 19 microRNA-mRNA regulatory pairs were identified within these deregulated microRNAs and mRNAs, which consisted of 17 unique protein-coding genes yielding a 17-gene signature. Pathway analysis of the 17-gene signature revealed Jak-STAT signaling pathway as the most significantly represented pathway. A severity score was assigned to each patient based on the expression of the 17-gene signature and a significant increasing trend in the severity score was observed from healthy control, to uncomplicated sarcoidosis, and finally to complicated sarcoidosis. In addition, this microRNA-regulated gene signature differentiates sarcoidosis patients from healthy controls in independent validation cohorts. Our study suggests that PBMC gene expression is useful in diagnosis of sarcoidosis.
• Barreto-Luis, A., Pino-Yanes, M., Corrales, A., Campo, P., Callero, A., Acosta-Herrera, M., Cumplido, J., Ma, S. F., Martinez-Tadeo, J., Villar, J., Garcia, J. G., Carrillo, T., Carracedo, ., Blanca, M., & Flores, C. (2016). Genome-wide association study in Spanish identifies ADAM metallopeptidase with thrombospondin type 1 motif, 9 (ADAMTS9), as a novel asthma susceptibility gene. The Journal of allergy and clinical immunology, 137(3), 964-6.
• Bime, C., Poongkunran, C., Borgstrom, M., Natt, B., Desai, H., Parthasarathy, S., & Garcia, J. G. (2016). Racial Differences in Mortality from Severe Acute Respiratory Failure in the United States, 2008-2012. Annals of the American Thoracic Society, 13(12), 2184-2189.
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  Racial disparities in health and healthcare in the United States are well documented and are increasingly recognized in acute critical illnesses such as sepsis and acute respiratory failure.
• Bime, C., Zhou, T., Wang, T., Slepian, M. J., Garcia, J. G., & Hecker, L. (2016). Reactive oxygen species-associated molecular signature predicts survival in patients with sepsis. Pulmonary circulation, 6(2), 196-201.
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  Sepsis-related multiple organ dysfunction syndrome is a leading cause of death in intensive care units. There is overwhelming evidence that oxidative stress plays a significant role in the pathogenesis of sepsis-associated multiple organ failure; however, reactive oxygen species (ROS)-associated biomarkers and/or diagnostics that define mortality or predict survival in sepsis are lacking. Lung or peripheral blood gene expression analysis has gained increasing recognition as a potential prognostic and/or diagnostic tool. The objective of this study was to identify ROS-associated biomarkers predictive of survival in patients with sepsis. In-silico analyses of expression profiles allowed the identification of a 21-gene ROS-associated molecular signature that predicts survival in sepsis patients. Importantly, this signature performed well in a validation cohort consisting of sepsis patients aggregated from distinct patient populations recruited from different sites. Our signature outperforms randomly generated signatures of the same signature gene size. Our findings further validate the critical role of ROSs in the pathogenesis of sepsis and provide a novel gene signature that predicts survival in sepsis patients. These results also highlight the utility of peripheral blood molecular signatures as biomarkers for predicting mortality risk in patients with sepsis, which could facilitate the development of personalized therapies.
• Brown, M. F., Wang, T., Oita, R. C., Molugu, T. R., Garcia, J. G., Chawla, U., & Brown, M. F. (2016). Thermal Denaturation and Domain Stability of NAMPT Protein. Biophysical Journal, 110(3), 44a. doi:10.1016/j.bpj.2015.11.304
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  Nicotinamide phosphoribosyltransferase (NAMPT) is also known as Pre-B cell colony enhancing factor (PBEF) or visceral fat protein (Visfatin). It is a highly conserved 52-kDa protein, found in living species from bacteria to humans [1], and acts as a rate-limiting step in the salvage pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. NAMPT is involved in several inflammatory deceases notably, sepsis, acute lung injury, and rheumatoid arthritis [2]. It is also known to activate insulin receptor causing type II diabetes [3]. Because of such medicinal relevance, NAMPT has been extensively investigated by X-ray crystallography, aiming at developing inhibitors [1]. However, physiologically relevant biochemical characterization including nuclear magnetic resonance (NMR) spectroscopy for NAMPT is scarce. NMR methods are complementary to X-ray crystallography, and provide valuable information on the structure and functional dynamics simultaneously. They offer an important tool for understanding biological functioning [4]. Bacterial expression, biochemical characterization, and thermal stability studies on NAMPT have been undertaken with the objective of optimizing conditions for NMR experiments. Secondary structure estimates using circular dichroism (CD) spectroscopy are consistent with crystallographic data. Thermal unfolding studies indicated an unusually high degree of thermal stability of NAMPT showing a melting temperature of about 90 °C. Results for dynamic light scattering (DLS) experiments showed a salt-dependent multimeric state. Presently, we are optimizing solvent conditions for obtaining the stable oligomeric state relevant to NMR experiments. Calorimetric studies are also under progress. We successfully expressed NAMPT in E.coli and are developing strategies for better yields and isotope labelling. [1] T. Wang et al. (2006) Nat. Struct. Mol. Biol. 13, 661. [2] S. M. Camp et al. (2015) Sci. Rep. 5, 13135. [3] A. Fukuhara et al. (2005) Science 307, 426. [4] T. R. Molugu et al. (2015) Concepts Magn. Reson. (in press).
• Camp, S. M., Chiang, E. T., Sun, C., Usatyuk, P. V., Bittman, R., Natarajan, V., Garcia, J. G., & Dudek, S. M. (2016). "Pulmonary Endothelial Cell Barrier Enhancement by Novel FTY720 Analogs: Methoxy-FTY720, Fluoro-FTY720, and β-Glucuronide-FTY720". Chemistry and physics of lipids, 194, 85-93.
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  Effective therapeutic agents are lacking for the prevention and reversal of vascular leak, a frequent pathophysiologic result of inflammatory processes such as acute respiratory distress syndrome (ARDS) and sepsis. We previously demonstrated the potent barrier-enhancing effects of related compounds sphingosine 1-phosphate (S1P), the pharmaceutical agent FTY720, and its analog (S)-FTY720 phosphonate (Tys) in models of inflammatory lung injury. In this study, we characterize additional novel FTY720 analogs for their potential to reduce vascular leak as well as utilize them as tools to better understand the mechanisms by which this class of agents modulates permeability. Transendothelial resistance (TER) and labeled dextran studies demonstrate that (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F), and β-glucuronide-FTY720 (FTY-G) compounds display in vitro barrier-enhancing properties comparable or superior to FTY720 and S1P. In contrast, the (S)-methoxy-FTY720 ((S)-OMe-FTY) analog disrupts lung endothelial cell (EC) barrier integrity in TER studies in association with actin stress fiber formation and robust intracellular calcium release, but independent of myosin light chain or ERK phosphorylation. Additional mechanistic studies with (R)-OMe-FTY, FTY-F, and FTY-G suggest that lung EC barrier enhancement is mediated through lipid raft signaling, Gi-linked receptor coupling to downstream tyrosine phosphorylation events, and S1PR1-dependent receptor ligation. These results provide important mechanistic insights into modulation of pulmonary vascular barrier function by FTY720-related compounds and highlight common signaling events that may assist the development of novel therapeutic tools in the prevention or reversal of the pulmonary vascular leak that characterizes ARDS.
• Danilov, S. M., Lünsdorf, H., Akinbi, H. T., Nesterovitch, A. B., Epshtein, Y., Letsiou, E., Kryukova, O. V., Piegeler, T., Golukhova, E. Z., Schwartz, D. E., Dull, R. O., Minshall, R. D., Kost, O. A., & Garcia, J. G. (2016). Lysozyme and bilirubin bind to ACE and regulate its conformation and shedding. Scientific reports, 6, 34913.
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  Angiotensin I-converting enzyme (ACE) hydrolyzes numerous peptides and is a critical participant in blood pressure regulation and vascular remodeling. Elevated tissue ACE levels are associated with increased risk for cardiovascular and respiratory disorders. Blood ACE concentrations are determined by proteolytic cleavage of ACE from the endothelial cell surface, a process that remains incompletely understood. In this study, we identified a novel ACE gene mutation (Arg532Trp substitution in the N domain of somatic ACE) that increases blood ACE activity 7-fold and interrogated the mechanism by which this mutation significantly increases blood ACE levels. We hypothesized that this ACE mutation disrupts the binding site for blood components which may stabilize ACE conformation and diminish ACE shedding. We identified the ACE-binding protein in the blood as lysozyme and also a Low Molecular Weight (LMW) ACE effector, bilirubin, which act in concert to regulate ACE conformation and thereby influence ACE shedding. These results provide mechanistic insight into the elevated blood level of ACE observed in patients on ACE inhibitor therapy and elevated blood lysozyme and ACE levels in sarcoidosis patients.
• Desai, A. A., Knox, K. S., Yuan, J. X., Sprissler, R., Nair, V., Lynn, H., Larson, B. T., Knox, K. S., Gupta, A., Garcia, J. G., Desai, A. A., & Cordery, A. G. (2016). Abstract 17494: Exome Sequencing Reveals a Novel SNP in TRPC6 in Pulmonary Arterial Hypertension. Circulation.
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  Introduction: The Ca2+ sensing receptor TRPC6 has been reported to promote pulmonary vascular disease. A functional promoter SNP (-254(C/G) has also been previously reported to be over-represented in patients with PAH. We hypothesized that there may be other novel SNPs associated with PAH in TRPC6. Methods: Whole-exome sequencing (WES) was performed on lung tissue DNA collected at the time of transplant or autopsy from 12 patients with PAH (WHO Group I). Standard BWA, SAMTOOLS, and GATK protocols were used to analyze WES data. Filtered SNPs on were compared against 1000 genomes and dbSNP databases for case-control analysis. In a separate PAH cohort (n=47, WHO Group I) with available clinical data, genotyping of a top candidate SNP, rs191383391, was performed via PCR. Association between genotype [wild-type (GG), homozygous recessive (TT)] and clinical phenotypes including right heart catheterization-defined pulmonary vascular resistance (PVRi), cardiac index (CI), cardiac MRI-defined right ventricular eje...
• Desai, A., Black, S., Wang, T., Garcia, J. G., Sun, X., Adyshev, D., Kelly, G. T., Camp, S. M., & Elangovan, V. R. (2016). Endotoxin- and mechanical stress-induced epigenetic changes in the regulation of the nicotinamide phosphoribosyltransferase promoter. Pulmonary circulation, 6(4), 539-544.
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  Mechanical ventilation, a lifesaving intervention for patients with acute respiratory distress syndrome (ARDS), also unfortunately contributes to excessive mechanical stress and impaired lung physiological and structural integrity. We have elsewhere established the pivotal role of increased nicotinamide phosphoribosyltransferase (NAMPT) transcription and secretion as well as its direct binding to the toll-like receptor 4 (TLR4) in the progression of this devastating syndrome; however, regulation of this critical gene in ventilator-induced lung injury (VILI) is not well characterized. On the basis of an emerging role for epigenetics in enrichment of VILI and CpG sites within the NAMPT promoter and 5'UTR, we hypothesized that NAMPT expression and downstream transcriptional events are influenced by epigenetic mechanisms. Concomitantly, excessive mechanical stress of human pulmonary artery endothelial cells or lipopolysaccharide (LPS) treatment led to both reduced DNA methylation levels in the NAMPT promoter and increased gene transcription. Histone deacetylase inhibition by trichostatin A or Sirt-1-silencing RNA attenuates LPS-induced NAMPT expression. Furthermore, recombinant NAMPT administration induced TLR4-dependent global H3K9 hypoacetylation. These studies suggest a complex epigenetic regulatory network of NAMPT in VILI and ARDS and open novel strategies for combating VILI and ARDS.
• Duarte, J. D., Desai, A. A., Sysol, J. R., Abbasi, T., Patel, A. R., Lang, R. M., Gupta, A., Garcia, J. G., Gordeuk, V. R., & Machado, R. F. (2016). Genome-Wide Analysis Identifies IL-18 and FUCA2 as Novel Genes Associated with Diastolic Function in African Americans with Sickle Cell Disease. PloS one, 11(9), e0163013.
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  Diastolic dysfunction is common in sickle cell disease (SCD), and is associated with an increased risk of mortality. However, the molecular pathogenesis underlying this development is poorly understood. The aim of this study was to identify a gene expression profile that is associated with diastolic function in SCD, potentially elucidating molecular mechanisms behind diastolic dysfunction development.
• Indik, J. H., Nair, V., Rafikov, R., Nyotowidjojo, I. S., Bisla, J., Kansal, M., Parikh, D. S., Robinson, M., Desai, A., Oberoi, M., Gupta, A., Abbasi, T., Khalpey, Z., Patel, A. R., Lang, R. M., Dudley, S. C., Choi, B. R., Garcia, J. G., Machado, R. F., & Desai, A. A. (2016). Associations of Prolonged QTc in Sickle Cell Disease. PloS one, 11(10), e0164526.
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  Sudden death is a leading cause of mortality in sickle cell disease, implicating ventricular tachyarrhythmias. Prolonged QTc on an electrocardiogram (ECG), commonly seen with myocardial ischemia, is a known risk for polymorphic ventricular tachycardia (VT). We hypothesized that prolonged QTc is associated with mortality in sickle cell disease. ECG were analyzed from a cohort of 224 sickle patients (University of Illinois at Chicago, UIC) along with available laboratory, and echocardiographic findings, and from another cohort of 38 patients (University of Chicago, UC) for which cardiac MRI and free heme values were also measured. In the UIC cohort, QTc was potentially related to mortality with a hazard ratio (HR) of 1.22 per 10ms, (P = 0.015), and a HR = 3.19 (P = 0.045) for a QTc>480ms. In multivariate analyses, QTc remained significantly associated with survival after adjusting for inpatient ECG status (HR 1.26 per 10ms interval, P = 0.010) and genotype status [HR 1.21 per 10ms interval, P = 0.037). QTc trended toward association with mortality after adjusting for both LDH and hydroxyurea use (HR 1.21 per 10ms interval, P = 0.062) but was not significant after adjusting for TRV. In univariate analyses, QTc was related to markers of hemolysis including AST (P = 0.031), hemoglobin (P = 0.014), TR velocity (P = 0.036), higher in inpatients (P
• Knox, K. S., Suryanarayana, P., Desai, A. A., Yuan, J. X., Yarlagadda, V., Whitaker, M., Suryanarayana, P., Shewale, A., Rischard, F., Riaz, I. B., Patel, K., Natarajan, B., Nair, V., Knox, K. S., Khalpey, Z., Garcia, J. G., Dherange, P., Desai, A. A., & Black, S. M. (2016). Abstract 18384: Hispanic Disparities in PAH: Multi-Modality Validation of Increased Susceptibility to Right Ventricular Dysfunction. Circulation, 134.
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  Introduction: Hispanics are known to have increased baseline right ventricular (RV) wall thickness and cardiovascular risk factors compared to non-Hispanics. We, therefore, hypothesized that Hispanics have reduced RV function in response to increased pulmonary afterload compared to non-Hispanics. Methods: A total of 127 WHO Groups I and IV pulmonary arterial hypertension (PAH) patients were prospectively enrolled and underwent echocardiography and right heart catheterization (RHC) within 4 months, with a subset also performing six-minute walk test (6MWT, n=86). After comparing baseline characteristics between self-reported Hispanics (n=28) and non-Hispanics (n=99), ethnicity effects were assessed using hierarchical linear regression models adjusted for age, gender, indexed pulmonary vascular resistance (PVRi), PAH medication use, and days from echo to RHC. Results: Baseline characteristics between the two groups were not significantly different except for age, which was higher in non-Hispanics (60.9+13.7 yrs) compared to Hispanics (51.9+12.2 yrs, P Conclusions: Hispanics with PAH exhibit increased pathological RV remodeling and systolic dysfunction validated by both echo and RHC with decreased trends in functional capacity (6MWT) compared to non-Hispanics. This susceptibility may be influenced, in part, by increased pulmonary artery elastance and relatively higher but “normal” wedge pressures.
• Mirsaeidi, M., Banoei, M. M., Nienow, C. K., Abassi, T., Hakim, A., Schraufnagel, D., Winston, B. W., Sweiss, N., Baughman, R., Garcia, J. G., & Machado, R. (2016). Plasma metabolomic profile in fibrosing pulmonary sarcoidosis. Sarcoidosis, vasculitis, and diffuse lung diseases : official journal of WASOG, 33(1), 29-38.
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  There is no known marker to screen patients with sarcoidosis to determine the risk of progression to pulmonary fibrosis. We aimed to identify potential noninvasive biomarkers for early detection of pulmonary fibrosing sarcoidosis.
• Mirsaeidi, M., Vu, A., Zhang, W., Arbieva, Z., Zhang, C., Abbasi, T., Hakim, A., Schraufnagel, D., Sweiss, N., Baughman, R., Garcia, J. G., & Machado, R. F. (2016). Annexin A11 is associated with pulmonary fibrosis in African American patients with sarcoidosis. Sarcoidosis, vasculitis, and diffuse lung diseases : official journal of WASOG, 33(4), 418-422.
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  Not available.
• Pouladi, N., Bime, C., Garcia, J. G., & Lussier, Y. A. (2016). Complex genetics of pulmonary diseases: lessons from genome-wide association studies and next-generation sequencing. Translational research : the journal of laboratory and clinical medicine, 168, 22-39.
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  The advent of high-throughput technologies has provided exceptional assistance for lung scientists to discover novel genetic variants underlying the development and progression of complex lung diseases. However, the discovered variants thus far do not explain much of the estimated heritability of complex lung diseases. Here, we review the literature of successfully used genome-wide association studies (GWASs) and identified the polymorphisms that reproducibly underpin the susceptibility to various noncancerous complex lung diseases or affect therapeutic responses. We also discuss the inherent limitations of GWAS approaches and how the use of next-generation sequencing technologies has furthered our understanding about the genetic determinants of these diseases. Next, we describe the contribution of the metagenomics to understand the interactions of the airways microbiome with lung diseases. We then highlight the urgent need for new integrative genomics-phenomics methods to more effectively interrogate and understand multiple downstream "omics" (eg, chromatin modification patterns). Finally, we address the scarcity of genetic studies addressing under-represented populations such as African Americans and Hispanics.
• Rafikova, O., Meadows, M. L., Kinchen, J. M., Mohney, R. P., Maltepe, E., Desai, A. A., Yuan, J. X., Garcia, J. G., Fineman, J. R., Rafikov, R., & Black, S. M. (2016). Metabolic Changes Precede the Development of Pulmonary Hypertension in the Monocrotaline Exposed Rat Lung. PloS one, 11(3), e0150480.
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  There is increasing interest in the potential for metabolic profiling to evaluate the progression of pulmonary hypertension (PH). However, a detailed analysis of the metabolic changes in lungs at the early stage of PH, characterized by increased pulmonary artery pressure but prior to the development of right ventricle hypertrophy and failure, is lacking in a preclinical animal model of PH. Thus, we undertook a study using rats 14 days after exposure to monocrotaline (MCT), to determine whether we could identify early stage metabolic changes prior to the manifestation of developed PH. We observed changes in multiple pathways associated with the development of PH, including activated glycolysis, increased markers of proliferation, disruptions in carnitine homeostasis, increased inflammatory and fibrosis biomarkers, and a reduction in glutathione biosynthesis. Further, our global metabolic profile data compare favorably with prior work carried out in humans with PH. We conclude that despite the MCT-model not recapitulating all the structural changes associated with humans with advanced PH, including endothelial cell proliferation and the formation of plexiform lesions, it is very similar at a metabolic level. Thus, we suggest that despite its limitations it can still serve as a useful preclinical model for the study of PH.
• Rojo de la Vega, M., Dodson, M., Gross, C., Mansour, H. M., Lantz, R. C., Chapman, E., Wang, T., Black, S. M., Garcia, J. G., & Zhang, D. D. (2016). Role of Nrf2 and Autophagy in Acute Lung Injury. Current pharmacology reports, 2(2), 91-101.
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  Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are the clinical manifestations of severe lung damage and respiratory failure. Characterized by severe inflammation and compromised lung function, ALI/ARDS result in very high mortality of affected individuals. Currently, there are no effective treatments for ALI/ARDS, and ironically, therapies intended to aid patients (specifically mechanical ventilation, MV) may aggravate the symptoms. Key events contributing to the development of ALI/ARDS are: increased oxidative and proteotoxic stresses, unresolved inflammation, and compromised alveolar-capillary barrier function. Since the airways and lung tissues are constantly exposed to gaseous oxygen and airborne toxicants, the bronchial and alveolar epithelial cells are under higher oxidative stress than other tissues. Cellular protection against oxidative stress and xenobiotics is mainly conferred by Nrf2, a transcription factor that promotes the expression of genes that regulate oxidative stress, xenobiotic metabolism and excretion, inflammation, apoptosis, autophagy, and cellular bioenergetics. Numerous studies have demonstrated the importance of Nrf2 activation in the protection against ALI/ARDS, as pharmacological activation of Nrf2 prevents the occurrence or mitigates the severity of ALI/ARDS. Another promising new therapeutic strategy in the prevention and treatment of ALI/ARDS is the activation of autophagy, a bulk protein and organelle degradation pathway. In this review, we will discuss the strategy of concerted activation of Nrf2 and autophagy as a preventive and therapeutic intervention to ameliorate ALI/ARDS.
• Saraf, S. L., Oh, A. L., Patel, P. R., Jalundhwala, Y., Sweiss, K., Koshy, M., Campbell-Lee, S., Gowhari, M., Hassan, J., Peace, D., Quigley, J. G., Khan, I., Molokie, R. E., Hsu, L. L., Mahmud, N., Levinson, D. J., Pickard, A. S., Garcia, J. G., Gordeuk, V. R., & Rondelli, D. (2016). Nonmyeloablative Stem Cell Transplantation with Alemtuzumab/Low-Dose Irradiation to Cure and Improve the Quality of Life of Adults with Sickle Cell Disease. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 22(3), 441-8.
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  Allogeneic hematopoietic stem cell transplantation (HSCT) is rarely performed in adult patients with sickle cell disease (SCD). We utilized the chemotherapy-free, alemtuzumab/total body irradiation 300 cGy regimen with sirolimus as post-transplantation immunosuppression in 13 high-risk SCD adult patients between November 2011 and June 2014. Patients received matched related donor (MRD) granulocyte colony-stimulating factor-mobilized peripheral blood stem cells, including 2 cases that were ABO incompatible. Quality-of-life (QoL) measurements were performed at different time points after HSCT. All 13 patients initially engrafted. A stable mixed donor/recipient chimerism was maintained in 12 patients (92%), whereas 1 patient not compliant with sirolimus experienced secondary graft failure. With a median follow-up of 22 months (range, 12 to 44 months) there was no mortality, no acute or chronic graft-versus-host disease (GVHD), and no grades 3 or 4 extramedullary toxicities. At 1 year after transplantation, patients with stable donor chimerism have normalized hemoglobin concentrations and improved cardiopulmonary and QoL parameters including bodily pain, general health, and vitality. In 4 patients, sirolimus was stopped without rejection or SCD-related complications. These results underscore the successful use of a chemotherapy-free regimen in MRD HSCT for high-risk adult SCD patients and demonstrates a high cure rate, absence of GVHD or mortality, and improvement in QoL including the applicability of this regimen in ABO mismatched cases (NCT number 01499888).
• Singla, S., Zhou, T., Javaid, K., Abbasi, T., Casanova, N., Zhang, W., Ma, S. F., Wade, M. S., Noth, I., Sweiss, N. J., Garcia, J. G., & Machado, R. F. (2016). Expression profiling elucidates a molecular gene signature for pulmonary hypertension in sarcoidosis. Pulmonary circulation, 6(4), 465-471.
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  Pulmonary hypertension (PH), when it complicates sarcoidosis, carries a poor prognosis, in part because it is difficult to detect early in patients with worsening respiratory symptoms. Pathogenesis of sarcoidosis occurs via incompletely characterized mechanisms that are distinct from the mechanisms of pulmonary vascular remodeling well known to occur in conjunction with other chronic lung diseases. To address the need for a biomarker to aid in early detection as well as the gap in knowledge regarding the mechanisms of PH in sarcoidosis, we used genome-wide peripheral blood gene expression analysis and identified an 18-gene signature capable of distinguishing sarcoidosis patients with PH ( = 8), sarcoidosis patients without PH ( = 17), and healthy controls ( = 45). The discriminative accuracy of this 18-gene signature was 100% in separating sarcoidosis patients with PH from those without it. If validated in a large replicate cohort, this signature could potentially be used as a diagnostic molecular biomarker for sarcoidosis-associated PH.
• Song, S., Jacobson, K. N., McDermott, K. M., Reddy, S. P., Cress, A. E., Tang, H., Dudek, S. M., Black, S. M., Garcia, J. G., Makino, A., & Yuan, J. X. (2016). ATP promotes cell survival via regulation of cytosolic [Ca2+] and Bcl-2/Bax ratio in lung cancer cells. American journal of physiology. Cell physiology, 310(2), C99-114.
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  Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression.
• Sun, X., Elangovan, V. R., Shimizu, Y., Ma, S. F., Wang, T., & Garcia, J. G. (2016). Genetic and Epigenetic Regulation of Myosin Light Chain Kinase by Inflammatory Lung Disease Associated Polymorphisms. Journal of Investigative Medicine.
• Sun, X., Kellner, M., Desai, A. A., Wang, T., Lu, Q., Kangath, A., Qu, N., Klinger, C., Fratz, S., Yuan, J. X., Jacobson, J. R., Garcia, J. G., Rafikov, R., Fineman, J. R., & Black, S. M. (2016). Asymmetric Dimethylarginine Stimulates Akt1 Phosphorylation via Heat Shock Protein 70-Facilitated Carboxyl-Terminal Modulator Protein Degradation in Pulmonary Arterial Endothelial Cells. American journal of respiratory cell and molecular biology, 55(2), 275-87.
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  Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial nitric oxide synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at threonine (T) 308 and serine (S) 473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells to ADMA enhanced Akt1 phosphorylation at both threonine 308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog activity, or membrane Akt1 levels. Heat shock protein (Hsp) 90 plays a pivotal role in maintaining Akt1 activity, and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but, surprisingly, overexpression of a dominant-negative Hsp90 mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of dominant-negative Hsp90 increased Hsp70 levels, and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, carboxyl-terminal modulator protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMP protein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo, and that increasing CTMP levels may be an avenue to treat pulmonary hypertension.
• Sun, X., Rizzo, A. N., Letsiou, E., Garcia, J. G., & Dudek, S. M. (2016). ID: 124: ABL FAMILY KINASES MEDIATE LUNG VASCULAR PERMEABILITY AND INFLAMMATION IN ACUTE LUNG INJURY. Journal of Investigative Medicine, 64(4), 965-965. doi:10.1136/jim-2016-000120.110
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  Rationale Acute respiratory distress syndrome (ARDS) is a life-threatening disease process in which overwhelming inflammation causes disruption of the pulmonary endothelial cell (EC) barrier, leading to leakage of fluid and inflammatory cells from the blood stream into the airspaces. Current research aims to identify agents that could both decrease inflammation and increase pulmonary vascular barrier integrity. Recently published work suggests that imatinib, an FDA-approved Abl family kinase inhibitor, may attenuate vascular leak and inflammation; however the mechanism underlying these effects is not completely understood. In the present study we explored the effects of LPS on the expression of the Abl family kinases, c-Abl and Arg, as well as the effects of Abl family kinases on LPS-induced vascular permeability and inflammation. Methods In silico analyses of the promoter regions of Abl1 (encodes c-Abl) and Abl2 (encodes Arg) were analyzed for potential responsive elements using the online programs Genomatix, TFsearch, and Jaspar. Cultured human pulmonary artery ECs were challenged with LPS (100 ng/mL, 24 hrs), harvested using RNeasy kit and reverse transcribed to cDNA. RT-PCR was performed to assess alterations in the expression of Abl1 and Abl2 after LPS challenge. In separate studies, siRNA was used to selectively silence either c-Abl or Arg and inter-endothelial gap formation was assessed by measuring FITC-dextran binding to a biotinylated avidin substrate. Complementary immunofluorescence studies were carried out to assess effects on adherens junction distributions. Western blotting was used to assess the effects of c-Abl and Arg silencing on NFkB phosphorylation. Results In silico analyses revealed that c-Abl contains two antioxidant responsive elements, whereas Arg contains two mechanical stress responsive elements. LPS treatment caused an increase in the mRNA expression of c-Abl (1.5 fold, p Conclusions The Abl family kinases c-Abl and Arg play complementary but distinct roles in mediating vascular permeability and inflammation following LPS challenge. The promoter of Abl1 (c-Abl) contains antioxidant response elements and LPS causes an increase in c-Abl expression. Additionally, LPS increases the mRNA expression of c-Abl, but not Arg. C-Abl contributes to LPS-induced NFκB signaling; whereas Arg contributes to inter-endothelial gap formation and adherens junction stability. Inhibition of both of these kinases may be of benefit in patients with ARDS.
• Tang, H., Yamamura, A., Yamamura, H., Song, S., Fraidenburg, D. R., Chen, J., Gu, Y., Pohl, N. M., Zhou, T., Jiménez-Pérez, L., Ayon, R. J., Desai, A. A., Goltzman, D., Rischard, F., Khalpey, Z., Black, S. M., Garcia, J. G., Makino, A., & Yuan, J. X. (2016). Pathogenic role of calcium-sensing receptors in the development and progression of pulmonary hypertension. American journal of physiology. Lung cellular and molecular physiology, 310(9), L846-59.
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  An increase in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and a critical stimulation for PASMC proliferation and migration. Previously, we demonstrated that expression and function of calcium sensing receptors (CaSR) in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH) and animals with experimental pulmonary hypertension (PH) were greater than in PASMC from normal subjects and control animals. However, the mechanisms by which CaSR triggers Ca(2+) influx in PASMC and the implication of CaSR in the development of PH remain elusive. Here, we report that CaSR functionally interacts with TRPC6 to regulate [Ca(2+)]cyt in PASMC. Downregulation of CaSR or TRPC6 with siRNA inhibited Ca(2+)-induced [Ca(2+)]cyt increase in IPAH-PASMC (in which CaSR is upregulated), whereas overexpression of CaSR or TRPC6 enhanced Ca(2+)-induced [Ca(2+)]cyt increase in normal PASMC (in which CaSR expression level is low). The upregulated CaSR in IPAH-PASMC was also associated with enhanced Akt phosphorylation, whereas blockade of CaSR in IPAH-PASMC attenuated cell proliferation. In in vivo experiments, deletion of the CaSR gene in mice (casr(-/-)) significantly inhibited the development and progression of experimental PH and markedly attenuated acute hypoxia-induced pulmonary vasoconstriction. These data indicate that functional interaction of upregulated CaSR and upregulated TRPC6 in PASMC from IPAH patients and animals with experimental PH may play an important role in the development and progression of sustained pulmonary vasoconstriction and pulmonary vascular remodeling. Blockade or downregulation of CaSR and/or TRPC6 with siRNA or miRNA may be a novel therapeutic strategy to develop new drugs for patients with pulmonary arterial hypertension.
• Tang, H., Yuan, J. X., Yamamura, H., Yamamura, A., Tang, H., Song, S., Makino, A., Gu, Y., Garcia, J. G., Fraidenburg, D. R., Chen, J., Black, S. M., & Ayon, R. J. (2016). Calcium-sensing Receptor, Coupled with Transient Receptor Potential Canonical Channels 6, Regulates Cytosolic [Ca2+] in the Development and Progression of Pulmonary Hypertension. The FASEB Journal.
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  RationaleElevated pulmonary arterial pressure (PAP) in patients with pulmonary arterial hypertension (PAH) is mainly caused by increased pulmonary vascular resistance (PVR), due primarily to sustai...
• Tao, S., Rojo de la Vega, M., Quijada, H., Wondrak, G. T., Wang, T., Garcia, J. G., & Zhang, D. D. (2016). Bixin protects mice against ventilation-induced lung injury in an NRF2-dependent manner. Scientific reports, 6, 18760.
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  Mechanical ventilation (MV) is a therapeutic intervention widely used in the clinic to assist patients that have difficulty breathing due to lung edema, trauma, or general anesthesia. However, MV causes ventilator-induced lung injury (VILI), a condition characterized by increased permeability of the alveolar-capillary barrier that results in edema, hemorrhage, and neutrophil infiltration, leading to exacerbated lung inflammation and oxidative stress. This study explored the feasibility of using bixin, a canonical NRF2 inducer identified during the current study, to ameliorate lung damage in a murine VILI model. In vitro, bixin was found to activate the NRF2 signaling pathway through blockage of ubiquitylation and degradation of NRF2 in a KEAP1-C151 dependent manner; intraperitoneal (IP) injection of bixin led to pulmonary upregulation of the NRF2 response in vivo. Remarkably, IP administration of bixin restored normal lung morphology and attenuated inflammatory response and oxidative DNA damage following MV. This observed beneficial effect of bixin derived from induction of the NRF2 cytoprotective response since it was only observed in Nrf2(+/+) but not in Nrf2(-/-) mice. This is the first study providing proof-of-concept that NRF2 activators can be developed into pharmacological agents for clinical use to prevent patients from lung injury during MV treatment.
• Viswanathan, P., Ephstein, Y., Garcia, J. G., Cho, M., & Dudek, S. M. (2016). Differential elastic responses to barrier-altering agonists in two types of human lung endothelium. Biochemical and biophysical research communications, 478(2), 599-605.
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  Vascular integrity is primarily determined by endothelial cell (EC) cytoskeletal structure that is differentially regulated by various stimuli. In this study, atomic force microscopy (AFM) was used to characterize structural and mechanical properties in the cytoskeleton of cultured human pulmonary artery EC (HPAEC) and human lung microvascular EC (HLMVEC) by determining elastic properties (Young's modulus) in response to endogenous barrier protective agents sphingosine 1-phosphate (S1P) and hepatocyte growth factor (HGF), or the barrier disruptive molecule thrombin. Initial studies in unstimulated cells indicate higher baseline peripheral elastic modulus values in HPAEC (mean 2.9 KPa) than in HLMVEC (1.8 KPa). After 30 min of stimulation, S1P induced the highest Young's modulus increase (6.1 KPa) compared to the other barrier enhancing stimuli, HGF (5.8 KPa) and the pharmaceutical agent and S1P analog FTY720 (4.1 KPa). In contrast, the barrier disruptive agent thrombin decreased values from 2.5 KPa to 0.7 KPa depending on the cell type and treatment time. AFM topographical imaging supports these quantitative biophysical data regarding differential peripheral elastic properties in EC. Overall, these AFM studies provide novel insights into the biomechanical properties of human lung EC that regulate vascular barrier function and have potential applicability to pathophysiologic vascular leak syndromes such as acute lung injury.
• Wang, T., Mathew, B., Wu, X., Shimizu, Y., Rizzo, A. N., Dudek, S. M., Weichselbaum, R. R., Jacobson, J. R., Hecker, L., & Garcia, J. G. (2016). Nonmuscle myosin light chain kinase activity modulates radiation-induced lung injury. Pulmonary circulation, 6(2), 234-9.
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  Radiotherapy as a primary treatment for thoracic malignancies induces deleterious effects, such as acute or subacute radiation-induced lung injury (RILI). Although the molecular etiology of RILI is controversial and likely multifactorial, a potentially important cellular target is the lung endothelial cytoskeleton that regulates paracellular gap formation and the influx of macromolecules and fluid to the alveolar space. Here we investigate the central role of a key endothelial cytoskeletal regulatory protein, the nonmuscle isoform of myosin light chain kinase (nmMLCK), in an established murine RILI model. Our results indicate that thoracic irradiation significantly augmented nmMLCK protein expression and enzymatic activity in murine lungs. Furthermore, genetically engineered mice harboring a deletion of the nmMLCK gene (nmMLCK(-/-) mice) exhibited protection from RILI, as assessed by attenuated vascular leakage and leukocyte infiltration. In addition, irradiated wild-type mice treated with two distinct MLCK enzymatic inhibitors, ML-7 and PIK (peptide inhibitor of kinase), also demonstrated attenuated RILI. Taken together, these data suggests a key role for nmMLCK in vascular barrier regulation in RILI and warrants further examination of RILI strategies that target nmMLCK.
• Wang, T., Sun, X., Shimizu, Y., Ma, S., Garcia, J. G., & Elangovan, R. (2016). ID: 110: GENETIC AND EPIGENETIC REGULATION OF MYOSIN LIGHT CHAIN KINASE BY INFLAMMATORY LUNG DISEASE ASSOCIATED POLYMORPHISMS. Journal of Investigative Medicine, 64(4), 963-964. doi:10.1136/jim-2016-000120.107
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  Rationale Myosin light chain kinase (MLCK), a central cytoskeletal regulator encoded by MYLK gene, regulates muscle contraction, cell migration, endothelial cell–cell adhesion, and barrier function, thereby playing key pathophysiological roles in lung inflammatory diseases. We previously identified that MYLK single nucleotide polymorphisms (SNPs) as well as haplotypes are significantly associated with severe sepsis, acute lung injury and asthma in African Americans (AA) and European Americans (EA). Here we examined genetic and epigenetic regulation of the MYLK promoter as well as the effects of SNPs on MLCK expression and activity, thereby influencing cytoskeletal balance and cell integrity. Methods A series of nested deletions from the ∼2.5 kb putative promoter fragment were fused to luciferase reporter vectors, and transfected into human lung endothelium. We next evaluated the influence of ARDS and asthma associated SNPs on transcription factor (TF) binding and promoter activity. Exon SNP rs2700408 and intronic SNP rs11714297 were associated with ARDS in AA (GWAS) and EA (Gao et al, 2006), respectively. Rs57186134 has high LD with rs936170, one asthma associated SNP in AA (Gao et al, 2007). The DNA fragments containing SNPs were generated by site-directed mutagenesis. Transcription factor binding to the MYLK promoter was detected by protein-DNA electrophoretic mobility shift assay. Genetic regulation of MYLK and influences of disease-associated SNPs from previous studies were measured by luciferase promoter activity assays following challenge with inflammatory factors and mechanical stretch. Results Deletion construct luciferase reporter analysis revealed that the MYLK promoter for nmMLCK contains distal inhibitory and proximal enhancing regulatory regions. Human endothelial cell challenge with either 18% cyclic stretch, demethylation agents (5-Aza), or inflammatory factor TNFα and IL-4 significantly up-regulate MYLK promoter activities (p Conclusion These findings suggest that the MYLK gene is transcriptionally regulated by mechanical stress and inflammatory factors, and modulated by SNPs associated with lung inflammatory diseases. These functional insights further strengthen the concept that MYLKcontributes to inflammatory disease susceptibility and represents a molecular target in complex lung disorders.
• Xie, L., Chiang, E. T., Wu, X., Kelly, G. T., Kanteti, P., Singleton, P. A., Camp, S. M., Zhou, T., Dudek, S. M., Natarajan, V., Wang, T., Black, S. M., Garcia, J. G., & Jacobson, J. R. (2016). Regulation of Thrombin-Induced Lung Endothelial Cell Barrier Disruption by Protein Kinase C Delta. PloS one, 11(7), e0158865.
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  Protein Kinase C (PKC) plays a significant role in thrombin-induced loss of endothelial cell (EC) barrier integrity; however, the existence of more than 10 isozymes of PKC and tissue-specific isoform expression has limited our understanding of this important second messenger in vascular homeostasis. In this study, we show that PKCδ isoform promotes thrombin-induced loss of human pulmonary artery EC barrier integrity, findings substantiated by PKCδ inhibitory studies (rottlerin), dominant negative PKCδ construct and PKCδ silencing (siRNA). In addition, we identified PKCδ as a signaling mediator upstream of both thrombin-induced MLC phosphorylation and Rho GTPase activation affecting stress fiber formation, cell contraction and loss of EC barrier integrity. Our inhibitor-based studies indicate that thrombin-induced PKCδ activation exerts a positive feedback on Rho GTPase activation and contributes to Rac1 GTPase inhibition. Moreover, PKD (or PKCμ) and CPI-17, two known PKCδ targets, were found to be activated by PKCδ in EC and served as modulators of cytoskeleton rearrangement. These studies clarify the role of PKCδ in EC cytoskeleton regulation, and highlight PKCδ as a therapeutic target in inflammatory lung disorders, characterized by the loss of barrier integrity, such as acute lung injury and sepsis.
• Zhang, X., Shah, B. N., Zhang, W., Saraf, S. L., Miasnikova, G., Sergueeva, A., Ammosova, T., Niu, X., Nouraie, M., Nekhai, S., Castro, O., Gladwin, M. T., Prchal, J. T., Garcia, J. G., Machado, R. F., & Gordeuk, V. R. (2016). A genetic variation associated with plasma erythropoietin and a non-coding transcript of PRKAR1A in sickle cell disease. Human molecular genetics, 25(20), 4601-4609.
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  Blood erythropoietin (EPO) increases primarily to hypoxia. In sickle cell anaemia (homozygous HBBE6V; HbSS), plasma EPO is elevated due to hemolytic anaemia-related hypoxia. Hydroxyurea treatment reduces haemolysis and anaemia by increasing foetal haemoglobin, which leads to lower hypoxic transcriptional responses in blood mononuclear cells but paradoxically further increases EPO. To investigate this apparent hypoxia-independent EPO regulation, we assessed two sickle cell disease (SCD) cohorts for genetic associations with plasma EPO, by prioritizing 237,079 quantitative trait loci for expression level and/or transcript isoform variations of 12,727 genes derived from SCD blood mononuclear cells. We found an association between the T allele of SNP rs60684937 and increased plasma EPO (n = 567, combined P = 5.5 × 10 − 8 adjusted for haemoglobin and hydroxyurea) and validated it in independent SCD patients (n = 183, P = 0.018). The T allele of rs60684937 was associated with a relatively increased expression of a non-coding transcript of PRKAR1A (cAMP-dependent protein kinase type I-alpha regulatory subunit) in 58 SCD patients (P = 7.9 × 10 − 7) and 58 HapMap Yoruba samples (P = 0.0011). In conclusion, we demonstrate that plasma EPO elevation with hydroxyurea in SCD is independent of hypoxic responses and that genetic variation at SNP rs60684937 may contribute to EPO regulation through a cAMP-dependent protein kinase A pathway.
• Zhou, T., Zhou, T., White, E. S., Vij, R., Oldham, J. M., Noth, I., Moore, B., Martinez, F. J., Ma, S., Lussier, Y. A., Li, J., Huffnagle, G. B., Huang, Y., Hogaboam, C. M., Han, M. K., Garcia, J. G., Flaherty, K. R., Espindola, M. S., & Erb-downward, J. R. (2016). Microbes mediated host innate immune response in idiopathic pulmonary fibrosis. European Respiratory Journal, 48. doi:10.1183/13993003.congress-2016.pa881
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  Background: The host immune response to lung microbes in idiopathic pulmonary fibrosis (IPF) is unclear. Objective: Investigate immune pathways implicated by presence of operational taxonomic units (OTU) microbiota from bronchoscopy IPF lung samples, and if bacterial CpG-oligodeoxynucleotide (CpG-ODN) motifs mediated fibroblast TLR9 activation is involved. Methods: Paired gene expression arrays and 16S rDNA sequencing data of 68 IPF patients were retrieved from COMET study. R package “WGCNA” were used to construct gene co-expression modules. Each eigengene value was correlated with each trait. Results: Figure 1 showed 10 modules and their respective positive/negative correlation with each trait. Magenta module significantly correlated with the D L CO ( p =0.007), species richness ( p =5x10 -6 ), and OTU1302 ( Pseudomonas ) ( p =0.0001). Purple module correlated OTU1341 ( Prevotella ) ( p =0.003) and OTU1348 ( Staphylococcus ) ( p =0.03) and negatively correlated with CpG response ( p =0.03). D L CO and CpG response significantly correlated with blue module with opposite directions. Canonical pathways enriched in magenta, purple, and blue modules are integrin signaling, regulation of epithelial-mesenchymal transition, and Toll-like receptor signaling, respectively. CONCLUSIONS: The integrated analysis of host transcriptome with lung microbiota and CpG-ODN response unveiled underlying molecular mechanism of microbes mediated host innate immune response in IPF.
• van Es, N., van der Hulle, T., van Es, J., den Exter, P. L., Douma, R. A., Goekoop, R. J., Mos, I. C., Garcia, J. G., Kamphuisen, P. W., Huisman, M. V., Klok, F. A., Büller, H. R., & Bossuyt, P. M. (2016). PO-07 - Excluding pulmonary embolism in cancer patients using the Wells rule and age-adjusted D-dimer testing: an individual patient data meta-analysis. Thrombosis research, 140 Suppl 1, S179.
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  Among patients with clinically suspected pulmonary embolism (PE), imaging and anticoagulant treatment can be safely withheld in approximately one-third of patients based on the combination of a "PE unlikely" Wells score and a D-dimer below the age-adjusted threshold. The clinical utility of this diagnostic approach in cancer patients is less clear.
• Acosta-Herrera, M., Pino-Yanes, M., Ma, S. F., Barreto-Luis, A., Corrales, A., Cumplido, J., Pérez-Rodríguez, E., Campo, P., Eng, C., García-Robaina, J. C., Quintela, I., Villar, J., Blanca, M., Carracedo, ., Carrillo, T., Garcia, J. G., Torgerson, D. G., Burchard, E. G., & Flores, C. (2015). Fine mapping of the myosin light chain kinase (MYLK) gene replicates the association with asthma in populations of Spanish descent. The Journal of allergy and clinical immunology, 136(4), 1116-8.e9.
• Camp, S. M., Ceco, E., Evenoski, C. L., Danilov, S. M., Zhou, T., Chiang, E. T., Moreno-Vinasco, L., Mapes, B., Zhao, J., Gursoy, G., Brown, M. E., Adyshev, D. M., Siddiqui, S. S., Quijada, H., Sammani, S., Letsiou, E., Saadat, L., Yousef, M., Wang, T., , Liang, J., et al. (2015). Unique Toll-Like Receptor 4 Activation by NAMPT/PBEF Induces NFκB Signaling and Inflammatory Lung Injury. Scientific reports, 5, 13135.
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  Ventilator-induced inflammatory lung injury (VILI) is mechanistically linked to increased NAMPT transcription and circulating levels of nicotinamide phosphoribosyl-transferase (NAMPT/PBEF). Although VILI severity is attenuated by reduced NAMPT/PBEF bioavailability, the precise contribution of NAMPT/PBEF and excessive mechanical stress to VILI pathobiology is unknown. We now report that NAMPT/PBEF induces lung NFκB transcriptional activities and inflammatory injury via direct ligation of Toll-like receptor 4 (TLR4). Computational analysis demonstrated that NAMPT/PBEF and MD-2, a TLR4-binding protein essential for LPS-induced TLR4 activation, share ~30% sequence identity and exhibit striking structural similarity in loop regions critical for MD-2-TLR4 binding. Unlike MD-2, whose TLR4 binding alone is insufficient to initiate TLR4 signaling, NAMPT/PBEF alone produces robust TLR4 activation, likely via a protruding region of NAMPT/PBEF (S402-N412) with structural similarity to LPS. The identification of this unique mode of TLR4 activation by NAMPT/PBEF advances the understanding of innate immunity responses as well as the untoward events associated with mechanical stress-induced lung inflammation.
• Camp, S. M., Chiang, E. T., Sun, C., Usatyuk, P. V., Bittman, R., Natarajan, V., Garcia, J. G., & Dudek, S. M. (2015). Pulmonary endothelial cell barrier enhancement by novel FTY720 analogs: methoxy-FTY720, fluoro-FTY720, and β-glucuronide-FTY720. Chemistry and physics of lipids, 191, 16-24.
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  Effective therapeutic agents are lacking for the prevention and reversal of vascular leak, a frequent pathophysiologic result of inflammatory processes such as acute respiratory distress syndrome (ARDS) and sepsis. We previously demonstrated the potent barrier-enhancing effects of related compounds sphingosine 1-phosphate (S1P), the pharmaceutical agent FTY720, and its analog (S)-FTY720 phosphonate (Tys) in models of inflammatory lung injury. In this study, we characterize additional novel FTY720 analogs for their potential to reduce vascular leak as well as utilize them as tools to better understand the mechanisms by which this class of agents modulates permeability. Transendothelial resistance (TER) and labeled dextran studies demonstrate that (R)-methoxy-FTY720 ((R)-OMe-FTY), (R)/(S)-fluoro-FTY720 (FTY-F), and β-glucuronide-FTY720 (FTY-G) compounds display in vitro barrier-enhancing properties comparable or superior to FTY720 and S1P. In contrast, the (S)-methoxy-FTY720 ((S)-OMe-FTY) analog disrupts lung endothelial cell (EC) barrier integrity in TER studies in association with actin stress fiber formation and robust intracellular calcium release, but independent of myosin light chain or ERK phosphorylation. Additional mechanistic studies with (R)-OMe-FTY, FTY-F, and FTY-G suggest that lung EC barrier enhancement is mediated through lipid raft signaling, Gi-linked receptor coupling to downstream tyrosine phosphorylation events, and S1PR1-dependent receptor ligation. These results provide important mechanistic insights into modulation of pulmonary vascular barrier function by FTY720-related compounds and highlight common signaling events that may assist the development of novel therapeutic tools in the prevention or reversal of the pulmonary vascular leak that characterizes ARDS.
• Casanova, N., Zhou, T., Knox, K. S., & Garcia, J. G. (2015). Identifying Novel Biomarkers in Sarcoidosis Using Genome-Based Approaches. Clinics in chest medicine, 36(4), 621-630.
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  This article briefly reviews conventional biomarkers used clinically to (1) support a diagnosis and (2) monitor disease progression in patients with sarcoidosis. Potential new biomarkers identified by genome-wide screening and the approaches to discover these biomarkers are described.
• Chen, W., Epshtein, Y., Ni, X., Dull, R. O., Cress, A. E., Garcia, J. G., & Jacobson, J. R. (2015). Role of Integrin β4 in Lung Endothelial Cell Inflammatory Responses to Mechanical Stress. Scientific reports, 5, 16529.
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  Simvastatin, an HMG-CoA reductase inhibitor, has lung vascular-protective effects that are associated with decreased agonist-induced integrin β4 (ITGB4) tyrosine phosphorylation. Accordingly, we hypothesized that endothelial cell (EC) protection by simvastatin is dependent on these effects and sought to further characterize the functional role of ITGB4 as a mediator of EC protection in the setting of excessive mechanical stretch at levels relevant to ventilator-induced lung injury (VILI). Initially, early ITGB4 tyrosine phosphorylation was confirmed in human pulmonary artery EC subjected to excessive cyclic stretch (18% CS). EC overexpression of mutant ITGB4 with specific tyrosines mutated to phenylalanine (Y1440, Y1526 Y1640, or Y1422) resulted in significantly attenuated CS-induced cytokine expression (IL6, IL-8, MCP-1, and RANTES). In addition, EC overexpression of ITGB4 constructs with specific structural deletions also resulted in significantly attenuated CS-induced inflammatory cytokine expression compared to overexpression of wildtype ITGB4. Finally, mice expressing a mutant ITGB4 lacking a cytoplasmic signaling domain were found to have attenuated lung injury after VILI-challenge (VT = 40 ml/kg, 4 h). Our results provide mechanistic insights into the anti-inflammatory properties of statins and may ultimately lead to novel strategies targeted at ITGB4 signaling to treat VILI.
• Choi, S., Camp, S. M., Dan, A., Garcia, J. G., Dudek, S. M., & Leckband, D. E. (2015). A genetic variant of cortactin linked to acute lung injury impairs lamellipodia dynamics and endothelial wound healing. American journal of physiology. Lung cellular and molecular physiology, 309(9), L983-94.
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  Inflammatory mediators released in acute lung injury (ALI) trigger the disruption of interendothelial junctions, leading to loss of vascular barrier function, protein-rich pulmonary edema, and severe hypoxemia. Genetic signatures that predict patient recovery or disease progression are poorly defined, but recent genetic screening of ALI patients has identified an association between lung inflammatory disease and a single nucleotide polymorphism (SNP) in the gene for the actin-binding and barrier-regulatory protein cortactin. This study investigated the impact of this disease-linked cortactin variant on wound healing processes that may contribute to endothelial barrier restoration. A microfabricated platform was used to quantify wound healing in terms of gap closure speed, lamellipodia dynamics, and cell velocity. Overexpression of wild-type cortactin in endothelial cells (ECs) improved directional cell motility and enhanced lamellipodial protrusion length, resulting in enhanced gap closure rates. By contrast, the cortactin SNP impaired wound closure and cell locomotion, consistent with the observed reduction in lamellipodial protrusion length and persistence. Overexpression of the cortactin SNP in lung ECs mitigated the barrier-enhancing activity of sphingosine 1-phosphate. These findings suggest that this common cortactin variant may functionally contribute to ALI predisposition by impeding endothelial wound healing.
• Desai, A. A., Yuan, J. X., Rafikova, O., Rafikov, R., Meadows, M. L., Garcia, J. G., Desai, A. A., & Black, S. M. (2015). 314 - Metabolic Changes Precede the Development of Pulmonary Hypertension in Monocrotaline Model. Free Radical Biology and Medicine, 87, S140-S141. doi:10.1016/j.freeradbiomed.2015.10.364
• Desai, A., Choi, B., Dudley, S. C., Kittles, R., Machado, R. F., Garcia, J. G., Hillery, C., Indik, J. H., Goldman, S., Juneman, E. B., Groth, J., Nair, N., Rutledge, C., Kanady, J., Fleming, I., Batai, K., Weigand, K., Shi, G., Kim, T. Y., , Gupta, G., et al. (2018). IL-18 is a novel mediator of prolonged QTc and ventricular arrhythmias associated with Sickle Cell Disease. Proceedings of the National Academy of Sciences.
• Feldman, A. M., Runge, M. S., Garcia, J. G., & Rubenstein, A. H. (2015). American medical education at a crossroads. Science translational medicine, 7(285), 285fs17.
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  New medical-education models in which research plays a modest role could engender a two-tiered educational system, cause a reduction in the physician-scientist pipeline, and diminish the translation of biomedical advances.
• Fu, P., Usatyuk, P. V., Jacobson, J., Cress, A. E., Garcia, J. G., Salgia, R., & Natarajan, V. (2015). Role played by paxillin and paxillin tyrosine phosphorylation in hepatocyte growth factor/sphingosine-1-phosphate-mediated reactive oxygen species generation, lamellipodia formation, and endothelial barrier function. Pulmonary circulation, 5(4), 619-30.
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  Paxillin is a multifunctional and multidomain focal adhesion adaptor protein. It serves as an important scaffolding protein at focal adhesions by recruiting and binding to structural and signaling molecules. Paxillin tyrosine phosphorylation at Y31 and Y118 is important for paxillin redistribution to focal adhesions and angiogenesis. Hepatocyte growth factor (HGF) and sphingosine-1-phosphate (S1P) are potent stimulators of lamellipodia formation, a prerequisite for endothelial cell migration. The role played by paxillin and its tyrosine phosphorylated forms in HGF- or S1P-induced lamellipodia formation and barrier function is unclear. HGF or S1P stimulated lamellipodia formation, tyrosine phosphorylation of paxillin at Y31 and Y118, and c-Abl in human lung microvascular endothelial cells (HLMVECs). Knockdown of paxillin with small interfering RNA (siRNA) or transfection with paxillin mutants (Y31F or Y118F) mitigated HGF- or S1P-induced lamellipodia formation, translocation of p47 (phox) to lamellipodia, and reactive oxygen species (ROS) generation in HLMVECs. Furthermore, exposure of HLMVECs to HGF or S1P stimulated c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 in a time-dependent fashion, and down-regulation of c-Abl with siRNA attenuated HGF- or S1P-mediated lamellipodia formation, translocation of p47 (phox) to lamellipodia, and endothelial barrier enhancement. In vivo, knockdown of paxillin with siRNA in mouse lungs attenuated ventilator-induced lung injury. Together, these results suggest that c-Abl-mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates HGF- or S1P-mediated lamellipodia formation, ROS generation in lamellipodia, and endothelial permeability.
• Fu, P., Usatyuk, P. V., Lele, A., Harijith, A., Gregorio, C. C., Garcia, J. G., Salgia, R., & Natarajan, V. (2015). c-Abl mediated tyrosine phosphorylation of paxillin regulates LPS-induced endothelial dysfunction and lung injury. American journal of physiology. Lung cellular and molecular physiology, 308(10), L1025-38.
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  Paxillin is phosphorylated at multiple residues; however, the role of tyrosine phosphorylation of paxillin in endothelial barrier dysfunction and acute lung injury (ALI) remains unclear. We used siRNA and site-specific nonphosphorylable mutants of paxillin to abrogate the function of paxillin to determine its role in lung endothelial permeability and ALI. In vitro, lipopolysaccharide (LPS) challenge of human lung microvascular endothelial cells (HLMVECs) resulted in enhanced tyrosine phosphorylation of paxillin at Y31 and Y118 with no significant change in Y181 and significant barrier dysfunction. Knockdown of paxillin with siRNA attenuated LPS-induced endothelial barrier dysfunction and destabilization of VE-cadherin. LPS-induced paxillin phosphorylation at Y31 and Y118 was mediated by c-Abl tyrosine kinase, but not by Src and focal adhesion kinase. c-Abl siRNA significantly reduced LPS-induced endothelial barrier dysfunction. Transfection of HLMVECs with paxillin Y31F, Y118F, and Y31/118F double mutants mitigated LPS-induced barrier dysfunction and VE-cadherin destabilization. In vivo, the c-Abl inhibitor AG957 attenuated LPS-induced pulmonary permeability in mice. Together, these results suggest that c-Abl mediated tyrosine phosphorylation of paxillin at Y31 and Y118 regulates LPS-mediated pulmonary vascular permeability and injury.
• Huang, L. S., Berdyshev, E. V., Tran, J. T., Xie, L., Chen, J., Ebenezer, D. L., Mathew, B., Gorshkova, I., Zhang, W., Reddy, S. P., Harijith, A., Wang, G., Feghali-Bostwick, C., Noth, I., Ma, S. F., Zhou, T., Ma, W., Garcia, J. G., & Natarajan, V. (2015). Sphingosine-1-phosphate lyase is an endogenous suppressor of pulmonary fibrosis: role of S1P signalling and autophagy. Thorax, 70(12), 1138-48.
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  Idiopathic pulmonary fibrosis (IPF) is characterised by accumulation of fibroblasts and myofibroblasts and deposition of extracellular matrix proteins. Sphingosine-1-phosphate (S1P) signalling plays a critical role in pulmonary fibrosis.
• Huang, Y., Ma, S. F., Vij, R., Oldham, J. M., Herazo-Maya, J., Broderick, S. M., Strek, M. E., White, S. R., Hogarth, D. K., Sandbo, N. K., Lussier, Y. A., Gibson, K. F., Kaminski, N., Garcia, J. G., & Noth, I. (2015). A functional genomic model for predicting prognosis in idiopathic pulmonary fibrosis. BMC pulmonary medicine, 15, 147.
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  The course of disease for patients with idiopathic pulmonary fibrosis (IPF) is highly heterogeneous. Prognostic models rely on demographic and clinical characteristics and are not reproducible. Integrating data from genomic analyses may identify novel prognostic models and provide mechanistic insights into IPF.
• Letsiou, E., Rizzo, A. N., Sammani, S., Naureckas, P., Jacobson, J. R., Garcia, J. G., & Dudek, S. M. (2015). Differential and opposing effects of imatinib on LPS- and ventilator-induced lung injury. American journal of physiology. Lung cellular and molecular physiology, 308(3), L259-69.
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  Endothelial dysfunction underlies the pathophysiology of vascular disorders such as acute lung injury (ALI) syndromes. Recent work has identified the Abl family kinases (c-Abl and Arg) as important regulators of endothelial cell (EC) barrier function and suggests that their inhibition by currently available pharmaceutical agents such as imatinib may be EC protective. Here we describe novel and differential effects of imatinib in regulating lung pathophysiology in two clinically relevant experimental models of ALI. Imatinib attenuates endotoxin (LPS)-induced vascular leak and lung inflammation in mice but exacerbates these features in a mouse model of ventilator-induced lung injury (VILI). We next explored these discrepant observations in vitro through investigation of the roles for Abl kinases in cultured lung EC. Imatinib attenuates LPS-induced lung EC permeability, restores VE-cadherin junctions, and reduces inflammation by suppressing VCAM-1 expression and inflammatory cytokine (IL-8 and IL-6) secretion. Conversely, in EC exposed to pathological 18% cyclic stretch (CS) (in vitro model of VILI), imatinib decreases VE-cadherin expression, disrupts cell-cell junctions, and increases IL-8 levels. Downregulation of c-Abl expression with siRNA attenuates LPS-induced VCAM-1 expression, whereas specific reduction of Arg reduces VE-cadherin expression in 18% CS-challenged ECs to mimic the imatinib effects. In summary, imatinib exhibits pulmonary barrier-protective and anti-inflammatory effects in LPS-injured mice and lung EC; however, imatinib exacerbates VILI as well as dysfunction in 18% CS-EC. These findings identify the Abl family kinases as important modulators of EC function and potential therapeutic targets in lung injury syndromes.
• Letsiou, E., Sammani, S., Zhang, W., Zhou, T., Quijada, H., Moreno-Vinasco, L., Dudek, S. M., & Garcia, J. G. (2015). Pathologic mechanical stress and endotoxin exposure increases lung endothelial microparticle shedding. American journal of respiratory cell and molecular biology, 52(2), 193-204.
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  Acute lung injury (ALI) results from infectious challenges and from pathologic lung distention produced by excessive tidal volume delivered during mechanical ventilation (ventilator-induced lung injury [VILI]) and is characterized by extensive alveolar and vascular dysfunction. Identification of novel ALI therapies is hampered by the lack of effective ALI/VILI biomarkers. We explored endothelial cell (EC)-derived microparticles (EMPs) (0.1-1 μm) as potentially important markers and potential mediators of lung vascular injury in preclinical models of ALI and VILI. We characterized EMPs (annexin V and CD31 immunoreactivity) produced from human lung ECs exposed to physiologic or pathologic mechanical stress (5 or 18% cyclic stretch [CS]) or to endotoxin (LPS). EC exposure to 18% CS or to LPS resulted in increased EMP shedding compared with static cells (∼ 4-fold and ∼ 2.5-fold increases, respectively). Proteomic analysis revealed unique 18% CS-derived (n = 10) and LPS-derived EMP proteins (n = 43). VILI-challenged mice (40 ml/kg, 4 h) exhibited increased plasma and bronchoalveolar lavage CD62E (E-selectin)-positive MPs compared with control mice. Finally, mice receiving intratracheal instillation of 18% CS-derived EMPs displayed significant lung inflammation and injury. These findings indicate that ALI/VILI-producing stimuli induce significant shedding of distinct EMP populations that may serve as potential ALI biomarkers and contribute to the severity of lung injury.
• Mathew, B., Takekoshi, D., Sammani, S., Epshtein, Y., Sharma, R., Smith, B. D., Mitra, S., Desai, A. A., Weichselbaum, R. R., Garcia, J. G., & Jacobson, J. R. (2015). Role of GADD45a in murine models of radiation- and bleomycin-induced lung injury. American journal of physiology. Lung cellular and molecular physiology, 309(12), L1420-9.
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  We previously reported protective effects of GADD45a (growth arrest and DNA damage-inducible gene 45 alpha) in murine ventilator-induced lung injury (VILI) via effects on Akt-mediated endothelial cell signaling. In the present study we investigated the role of GADD45a in separate murine models of radiation- and bleomycin-induced lung injury. Initial studies of wild-type mice subjected to single-dose thoracic radiation (10 Gy) confirmed a significant increase in lung GADD45a expression within 24 h and persistent at 6 wk. Mice deficient in GADD45a (GADD45a(-/-)) demonstrated increased susceptibility to radiation-induced lung injury (RILI, 10 Gy) evidenced by increased bronchoalveolar lavage (BAL) fluid total cell counts, protein and albumin levels, and levels of inflammatory cytokines compared with RILI-challenged wild-type animals at 2 and 4 wk. Furthermore, GADD45a(-/-) mice had decreased total and phosphorylated lung Akt levels both at baseline and 6 wk after RILI challenge relative to wild-type mice while increased RILI susceptibility was observed in both Akt(+/-) mice and mice treated with an Akt inhibitor beginning 1 wk prior to irradiation. Additionally, overexpression of a constitutively active Akt1 transgene reversed RILI-susceptibility in GADD45a(-/-) mice. In separate studies, lung fibrotic changes 2 wk after treatment with bleomycin (0.25 U/kg IT) was significantly increased in GADD45a(-/-) mice compared with wild-type mice assessed by lung collagen content and histology. These data implicate GADD45a as an important modulator of lung inflammatory responses across different injury models and highlight GADD45a-mediated signaling as a novel target in inflammatory lung injury clinically.
• Qian, Z., Zhou, T., Gurguis, C. I., Xu, X., Wen, Q., Lv, J., Fang, F., Hecker, L., Cress, A. E., Natarajan, V., Jacobson, J. R., Zhang, D. D., Garcia, J. G., & Wang, T. (2015). Nuclear factor, erythroid 2-like 2-associated molecular signature predicts lung cancer survival. Scientific reports, 5, 16889.
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  Nuclear factor, erythroid 2-like 2 (NFE2L2), a transcription factor also known as NF-E2-related factor 2 (Nrf2), is a key cytoprotective gene that regulates critical antioxidant and stress-responsive genes. Nrf2 has been demonstrated to be a promising therapeutic target and useful biomarker in malignant disease. We hypothesized that NFE2L2-mediated gene expression would reflect cancer severity and progression. We conducted a meta-analysis of microarray data for 240 NFE2L2-mediated genes that were enriched in tumor tissues. We then developed a risk scoring system based on NFE2L2 gene expression profiling and designated 50 tumor-associated genes as the NFE2L2-associated molecular signature (NAMS). We tested the relationship between this gene expression signature and both recurrence-free survival and overall survival in lung cancer patients. We find that NAMS predicts clinical outcome in the training cohort and in 12 out of 20 validation cohorts. Cox proportional hazard regressions indicate that NAMS is a robust prognostic gene signature, independent of other clinical and pathological factors including patient age, gender, smoking, gene alteration, MYC level, and cancer stage. NAMS is an excellent predictor of recurrence-free survival and overall survival in human lung cancer. This gene signature represents a promising prognostic biomarker in human lung cancer.
• Rizzo, A. N., Sammani, S., Esquinca, A. E., Jacobson, J. R., Garcia, J. G., Letsiou, E., & Dudek, S. M. (2015). Imatinib attenuates inflammation and vascular leak in a clinically relevant two-hit model of acute lung injury. American journal of physiology. Lung cellular and molecular physiology, 309(11), L1294-304.
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  Acute lung injury/acute respiratory distress syndrome (ALI/ARDS), an illness characterized by life-threatening vascular leak, is a significant cause of morbidity and mortality in critically ill patients. Recent preclinical studies and clinical observations have suggested a potential role for the chemotherapeutic agent imatinib in restoring vascular integrity. Our prior work demonstrates differential effects of imatinib in mouse models of ALI, namely attenuation of LPS-induced lung injury but exacerbation of ventilator-induced lung injury (VILI). Because of the critical role of mechanical ventilation in the care of patients with ARDS, in the present study we pursued an assessment of the effectiveness of imatinib in a "two-hit" model of ALI caused by combined LPS and VILI. Imatinib significantly decreased bronchoalveolar lavage protein, total cells, neutrophils, and TNF-α levels in mice exposed to LPS plus VILI, indicating that it attenuates ALI in this clinically relevant model. In subsequent experiments focusing on its protective role in LPS-induced lung injury, imatinib attenuated ALI when given 4 h after LPS, suggesting potential therapeutic effectiveness when given after the onset of injury. Mechanistic studies in mouse lung tissue and human lung endothelial cells revealed that imatinib inhibits LPS-induced NF-κB expression and activation. Overall, these results further characterize the therapeutic potential of imatinib against inflammatory vascular leak.
• Shen, K., Ramirez, B., Mapes, B., Shen, G. R., Gokhale, V., Brown, M. E., Santarsiero, B., Ishii, Y., Dudek, S. M., Wang, T., & Garcia, J. G. (2015). Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants. PloS one, 10(6), e0130515.
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  The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.
• Shimizu, Y., Camp, S. M., Sun, X., Zhou, T., Wang, T., & Garcia, J. G. (2015). Sp1-mediated nonmuscle myosin light chain kinase expression and enhanced activity in vascular endothelial growth factor-induced vascular permeability. Pulmonary circulation, 5(4), 707-15.
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  Despite the important role played by the nonmuscle isoform of myosin light chain kinase (nmMLCK) in vascular barrier regulation and the implication of both nmMLCK and vascular endothelial growth factor (VEGF) in the pathogenesis of acute respiratory distress syndrome (ARDS), the role played by nmMLCK in VEGF-induced vascular permeability is poorly understood. In this study, the role played by nmMLCK in VEGF-induced vascular hyperpermeability was investigated. Human lung endothelial cell barrier integrity in response to VEGF is examined in both the absence and the presence of nmMLCK small interfering RNAs. Levels of nmMLCK messenger RNA (mRNA), protein, and promoter activity expression were monitored after VEGF stimulation in lung endothelial cells. nmMYLK promoter activity was assessed using nmMYLK promoter luciferase reporter constructs with a series of nested deletions. nmMYLK transcriptional regulation was further characterized by examination of a key transcriptional factor. nmMLCK plays an important role in VEGF-induced permeability. We found that activation of the VEGF signaling pathway in lung endothelial cells increases MYLK gene product at both mRNA and protein levels. Increased nmMLCK mRNA and protein expression is a result of increased nmMYLK promoter activity, regulated in part by binding of the Sp1 transcription factor on triggering by the VEGF signaling pathway. Taken together, these findings suggest that MYLK is an important ARDS candidate gene and a therapeutic target that is highly influenced by excessive VEGF concentrations in the inflamed lung.
• Tang, H., Chen, J., Fraidenburg, D. R., Song, S., Sysol, J. R., Drennan, A. R., Offermanns, S., Ye, R. D., Bonini, M. G., Minshall, R. D., Garcia, J. G., Machado, R. F., Makino, A., & Yuan, J. X. (2015). Deficiency of Akt1, but not Akt2, attenuates the development of pulmonary hypertension. American journal of physiology. Lung cellular and molecular physiology, 308(2), L208-20.
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  Pulmonary vascular remodeling, mainly attributable to enhanced pulmonary arterial smooth muscle cell proliferation and migration, is a major cause for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with pulmonary hypertension. The signaling cascade through Akt, comprised of three isoforms (Akt1-3) with distinct but overlapping functions, is involved in regulating cell proliferation and migration. This study aims to investigate whether the Akt/mammalian target of rapamycin (mTOR) pathway, and particularly which Akt isoform, contributes to the development and progression of pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Compared with the wild-type littermates, Akt1(-/-) mice were protected against the development and progression of chronic HPH, whereas Akt2(-/-) mice did not demonstrate any significant protection against the development of HPH. Furthermore, pulmonary vascular remodeling was significantly attenuated in the Akt1(-/-) mice, with no significant effect noted in the Akt2(-/-) mice after chronic exposure to normobaric hypoxia (10% O2). Overexpression of the upstream repressor of Akt signaling, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), and conditional and inducible knockout of mTOR in smooth muscle cells were also shown to attenuate the rise in right ventricular systolic pressure and the development of right ventricular hypertrophy. In conclusion, Akt isoforms appear to have a unique function within the pulmonary vasculature, with the Akt1 isoform having a dominant role in pulmonary vascular remodeling associated with HPH. The PTEN/Akt1/mTOR signaling pathway will continue to be a critical area of study in the pathogenesis of pulmonary hypertension, and specific Akt isoforms may help specify therapeutic targets for the treatment of pulmonary hypertension.
• Wang, L., Bittman, R., Garcia, J. G., & Dudek, S. M. (2015). Junctional complex and focal adhesion rearrangement mediates pulmonary endothelial barrier enhancement by FTY720 S-phosphonate. Microvascular research, 99, 102-9.
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  Modulation of pulmonary vascular barrier function is an important clinical goal given the devastating effects of vascular leak in acute lung injury (ALI). We previously demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine 1-phosphate (S1P) and FTY720, has more potent pulmonary barrier protective effects than these agents in vitro and in mouse models of ALI. Tys preserves expression of the barrier-promoting S1P1 receptor (S1PR1), whereas S1P and FTY720 induce its ubiquitination and degradation. Here we further characterize the novel barrier promoting effects of Tys in cultured human pulmonary endothelial cells (EC).
• Wang, T., Zhou, T., Saadat, L., & Garcia, J. G. (2015). A MYLK variant regulates asthmatic inflammation via alterations in mRNA secondary structure. European journal of human genetics : EJHG, 23(6), 874-6.
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  Myosin light-chain kinase (MYLK) is a gene known to be significantly associated with severe asthma in African Americans. Here we further examine the molecular function of a single-nucleotide polymorphism (SNP), located in the non-muscle myosin light-chain kinase isoform (nmMLCK), in asthma susceptibility and pathobiology. We identified nmMLCK variant (reference SNP: rs9840993, NM_053025: 721C>T, c.439C>T) with a distinct mRNA secondary structure from the other variants. The nmMLCK variant (721C) secondary structure exhibits increased stability with an elongated half-life in the human endothelial cell, and greater efficiency in protein translation initiation owing to an increased accessibility to translation start site. Finally, nmMLCK expression of 721C- and 721T-containing MYLK transgenes were compared in nmMLCK(-/-) mice and confirmed deleterious effects of nmMLCK expression on asthmatic indices and implicated the augmented influence of MYLK 721C>T (c.439C>T) SNP on asthma severity. The confirmation of the novel mechanism of the regulation of asthmatic inflammation by a MYLK advances knowledge of the genetic basis for asthma disparities, and further suggests the potential of nmMLCK as a therapeutic target. Our study suggests that in addition to altering protein structure and function, non-synonymous SNPs may also lead to phenotypic disparity by altering protein expression.
• Wang, X., Bleher, R., Brown, M. E., Garcia, J. G., Dudek, S. M., Shekhawat, G. S., & Dravid, V. P. (2015). Nano-Biomechanical Study of Spatio-Temporal Cytoskeleton Rearrangements that Determine Subcellular Mechanical Properties and Endothelial Permeability. Scientific reports, 5, 11097.
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  The endothelial cell (EC) lining of the pulmonary vascular system forms a semipermeable barrier between blood and the interstitium and regulates various critical biochemical functions. Collectively, it represents a prototypical biomechanical system, where the complex hierarchical architecture, from the molecular scale to the cellular and tissue level, has an intimate and intricate relationship with its biological functions. We investigated the mechanical properties of human pulmonary artery endothelial cells (ECs) using atomic force microscopy (AFM). Concurrently, the wider distribution and finer details of the cytoskeletal nano-structure were examined using fluorescence microscopy (FM) and scanning transmission electron microscopy (STEM), respectively. These correlative measurements were conducted in response to the EC barrier-disrupting agent, thrombin, and barrier-enhancing agent, sphingosine 1-phosphate (S1P). Our new findings and analysis directly link the spatio-temporal complexities of cell re-modeling and cytoskeletal mechanical properties alteration. This work provides novel insights into the biomechanical function of the endothelial barrier and suggests similar opportunities for understanding the form-function relationship in other biomechanical subsystems.
• Wolberger, C., Wang, T., Vaisitti, T., Serra, S., Rossi, D., Rizzi, M., Raffaelli, N., Mazzola, F., Maffei, R., Inghirami, G., Garcia, J. G., Gaidano, G., Deaglio, S., Coscia, M., Brusa, D., Audrito, V., & Arruga, F. (2015). Extracellular nicotinamide phosphoribosyltransferase (NAMPT) promotes M2 macrophage polarization in chronic lymphocytic leukemia.. Blood, 125(1), 111-23. doi:10.1182/blood-2014-07-589069
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  Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in nicotinamide adenine dinucleotide biosynthesis. In the extracellular compartment, it exhibits cytokine-/adipokinelike properties, suggesting that it stands at the crossroad between metabolism and inflammation. Here we show that both intracellular and extracellular NAMPT levels are increased in cells and plasma of chronic lymphocytic leukemia (CLL) patients. The extracellular form (eNAMPT) is produced by CLL lymphocytes upon B-cell receptor, Toll-like receptor, and nuclear factor κB (NF-κB) signaling pathway activation. eNAMPT is important for differentiation of resting monocytes, polarizing them toward tumor-supporting M2 macrophages. These cells express high levels of CD163, CD206, and indoleamine 2,3-dioxygenase and secrete immunosuppressive (interleukin [IL] 10, CC chemokine ligand 18) and tumor-promoting (IL-6, IL-8) cytokines. NAMPT-primed M2 macrophages activate extracellular-regulated kinase 1/2, signal transducer and activator of transcription 3, and NF-κB signaling; promote leukemic cell survival; and reduce T-cell responses. These effects are independent of the enzymatic activity of NAMPT, as inferred from the use of an enzymatically inactive mutant. Overall, these results reveal that eNAMPT is a critical element in the induction of an immunosuppressive and tumor-promoting microenvironment of CLL.
• Zhang, X., Zhang, W., Saraf, S. L., Nouraie, M., Han, J., Gowhari, M., Hassan, J., Miasnikova, G., Sergueeva, A., Nekhai, S., Kittles, R., Machado, R. F., Garcia, J. G., Gladwin, M. T., Steinberg, M. H., Sebastiani, P., McClain, D. A., & Gordeuk, V. R. (2015). Genetic polymorphism of APOB is associated with diabetes mellitus in sickle cell disease. Human genetics, 134(8), 895-904.
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  Environmental variations have strong influences in the etiology of type 2 diabetes mellitus. In this study, we investigated the genetic basis of diabetes in patients with sickle cell disease (SCD), a Mendelian disorder accompanied by distinct physiological conditions of hypoxia and hyperactive erythropoiesis. Compared to the general African American population, the prevalence of diabetes as assessed in two SCD cohorts of 856 adults was low, but it markedly increased with older age and overweight. Meta-analyses of over 5 million single-nucleotide polymorphisms (SNPs) in the two SCD cohorts identified a SNP, rs59014890, the C allele of which associated with diabetes risk at P = 3.2 × 10(-8) and, surprisingly, associated with decreased APOB expression in peripheral blood mononuclear cells (PBMCs). The risk allele of the APOB polymorphism was associated with overweight in 181 SCD adolescents, with diabetes risk in 592 overweight, non-SCD African Americans ≥ 45 years of age, and with elevated plasma lipid concentrations in general populations. In addition, lower expression level of APOB in PBMCs was associated with higher values for percent hemoglobin A1C and serum total cholesterol and triglyceride concentrations in patients with Chuvash polycythemia, a congenital disease with elevated hypoxic responses and increased erythropoiesis at normoxia. Our study reveals a novel, environment-specific genetic polymorphism that may affect key metabolic pathways contributing to diabetes in SCD.
• Zhang, X., Zhang, W., Shah, B. N., Sergueeva, A. I., Nouraie, M., Niu, X., Nekhai, S., Miasnikova, G. Y., Machado, R. F., Gordeuk, V. R., Gladwin, M. T., Garcia, J. G., Castro, O. L., & Ammosova, T. (2015). Hydroxyurea Treatment Is Associated with Elevated Serum Erythropoietin Concentration but Suppressed Global Hypoxic Transcriptional Responses in Sickle Cell Disease. Blood, 126(23), 3380-3380. doi:10.1182/blood.v126.23.3380.3380
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  Background The level of distorted erythrocytes due to polymerization of hemoglobin S in sickle cell disease (SCD) (Science 1949;110:543) is a major determinant of the severity of hemolysis and microvascular occlusion ( Lancet 2010;376:2018). Erythropoietin (EPO) is elevated in SCD due to hemolytic anemia and a related increase in hypoxia-inducible factors (HIFs) ( Eur J Haematol 2007;78:183). Hydroxyurea (HU) is widely used in the treatment of SCD. HU inhibits ribonucleotide reductase ( Semin Oncol 1992;19(3 Suppl 9):1-10) and promotes γ globin synthesis thereby increasing HbF-containing erythrocytes (F cells) while suppressing sickle β hemoglobin production ( J Clin Invest 1984;74:652 and 2003;111:231). Increased level of F cells reduces hemolysis and ameliorates clinical complications in SCD. We and others have observed an increase in serum EPO level with HU treatment in SCD despite an increase in the hemoglobin concentration, and we hypothesized that this may be due to the known increased affinity of hemoglobin F for oxygen and related tissue hypoxia ( Blood 2009;114:4639). Methods Messenger RNA from peripheral blood mononuclear cells (PBMCs) was profiled using Affymetrix Human Exon 1.0 ST Array. Hypoxic transcriptional alteration was defined in 15 Chuvash polycythemia (CP) patients vs. 17 control individuals. CP leads to constitutive up-regulation of HIFs in the absence of anemia or hypoxia. Transcriptional alteration in SCD was determined in 13 HbSS subjects without HU treatment vs. 16 control individuals, and that induced by HU treatment was determined in 19 HbSS subjects with vs. 13 without HU treatment. For meta-analysis on serum EPO concentration, genomic DNA isolated from PBMCs was hybridized to the Illumina Human 610-Quad SNP array. Genotypes were imputed to 1000 genomes project phase 1 data. A linear regression model was applied adjusting for age, gender, hemoglobin concentration, and HU treatment. Results Gene expression changes by HbSS highly correlated with those associated with homozygous VHLR200W (Pearson's r=0.79, Figure 1A). At 5% false discovery rate (FDR), expression levels of 377 genes were altered in both VHLR200W homozygotes and HbSS by >1.2 fold. For these hypoxic genes, the correlation of expression changes between HbSS and homozygous VHLR200W reached r=0.97 (Figure 1B). In contrast to our hypothesis, HU treatment in general suppressed expression changes induced by HbSS (r=-0.85, Figure 1C), especially for the hypoxic genes (r=-0.95, Figure 1D). In VHLR200W homozygotes, 62 of the hypoxic genes correlated with plasma EPO levels (adjusted P
• Adyshev, D. M., Elangovan, V. R., Moldobaeva, N., Mapes, B., Sun, X., & Garcia, J. G. (2014). Mechanical stress induces pre-B-cell colony-enhancing factor/NAMPT expression via epigenetic regulation by miR-374a and miR-568 in human lung endothelium. American journal of respiratory cell and molecular biology, 50(2), 409-18.
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  Increased lung vascular permeability and alveolar edema are cardinal features of inflammatory conditions such as acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). We previously demonstrated that pre-B-cell colony-enhancing factor (PBEF)/NAMPT, the proinflammatory cytokine encoded by NAMPT, participates in ARDS and VILI inflammatory syndromes. The present study evaluated posttranscriptional regulation of PBEF/NAMPT gene expression in human lung endothelium via 3'-untranslated region (UTR) microRNA (miRNA) binding. In silico analysis identified hsa-miR-374a and hsa-miR-568 as potential miRNA candidates. Increased PBEF/NAMPT transcription (by RT-PCR) and expression (by Western blotting) induced by 18% cyclic stretch (CS) (2 h: 3.4 ± 0.06 mRNA fold increase (FI); 10 h: 1.5 ± 0.06 protein FI) and by LPS (4 h: 3.8 ± 0.2 mRNA FI; 48 h: 2.6 ± 0.2 protein FI) were significantly attenuated by transfection with mimics of hsa-miR-374a or hsa-miR-568 (40-60% reductions each). LPS and 18% CS increased the activity of a PBEF/NAMPT 3'-UTR luciferase reporter (2.4-3.25 FI) with induction reduced by mimics of each miRNA (44-60% reduction). Specific miRNA inhibitors (antagomirs) for each PBEF/NAMPT miRNA significantly increased the endogenous PBEF/NAMPT mRNA (1.4-3.4 ± 0.1 FI) and protein levels (1.2-1.4 ± 0.1 FI) and 3'-UTR luciferase activity (1.4-1.7 ± 0.1 FI) compared with negative antagomir controls. Collectively, these data demonstrate that increased PBEF/NAMPT expression induced by bioactive agonists (i.e., excessive mechanical stress, LPS) involves epigenetic regulation with hsa-miR-374a and hsa-miR-568, representing novel therapeutic strategies to reduce inflammatory lung injury.
• Belvitch, P., Adyshev, D., Elangovan, V. R., Brown, M. E., Naureckas, C., Rizzo, A. N., Siegler, J. H., Garcia, J. G., & Dudek, S. M. (2014). Proline-rich region of non-muscle myosin light chain kinase modulates kinase activity and endothelial cytoskeletal dynamics. Microvascular research, 95, 94-102.
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  Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two-fold increase in cortactin binding (p
• Chen, J., Tang, H., Sysol, J. R., Moreno-Vinasco, L., Shioura, K. M., Chen, T., Gorshkova, I., Wang, L., Huang, L. S., Usatyuk, P. V., Sammani, S., Zhou, G., Raj, J. U., Garcia, J. G., Berdyshev, E., Yuan, J. X., Natarajan, V., & Machado, R. F. (2014). The sphingosine kinase 1/sphingosine-1-phosphate pathway in pulmonary arterial hypertension. American journal of respiratory and critical care medicine, 190(9), 1032-43.
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  Sphingosine kinases (SphKs) 1 and 2 regulate the synthesis of the bioactive sphingolipid sphingosine-1-phosphate (S1P), an important lipid mediator that promotes cell proliferation, migration, and angiogenesis.
• Chen, W., Sharma, R., Rizzo, A. N., Siegler, J. H., Garcia, J. G., & Jacobson, J. R. (2014). Role of claudin-5 in the attenuation of murine acute lung injury by simvastatin. American journal of respiratory cell and molecular biology, 50(2), 328-36.
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  The statins are now recognized to have pleiotropic properties, including augmentation of endothelial barrier function. To explore the mechanisms involved, we investigated the effect of simvastatin on endothelial cell (EC) tight junctions. Western blotting of human pulmonary artery ECs treated with simvastatin (5 μM) confirmed a significant time-dependent increase (16-48 h) in claudin-5 protein expression compared with controls, without detectable alterations in zonula occludens-1 or occludin. These effects were associated with membrane translocation of VE-cadherin, whereas translocation of vascular endothelial cadherin (VE-cadherin; silencing RNA) inhibited simvastatin-induced claudin-5 up-regulation. Moreover, simvastatin treatment of ECs induced increased phosphorylation of both FoxO1 and β-catenin, transcriptional regulators of claudin-5 expression mediated by VE-cadherin. Subsequently, we found no effect of claudin-5 silencing on EC barrier protection by simvastatin in response to thrombin stimulation, as measured by either transendothelial electrical resistance or by EC monolayer flux of FITC-dextran (2,000 kD). However, silencing of claudin-5 did significantly attenuate simvastatin-mediated EC barrier protection in response to thrombin, as measured by monolayer flux of sodium fluorescein (376 Da). Finally, employing a murine model of LPS-induced acute lung injury, there was no effect of claudin-5 silencing in vivo (intratracheal injection) on bronchoalveolar lavage fluid protein or cell counts, but LPS-induced lung tissue extravasation of the small molecular weight markers, sodium fluorescein and Hochst stain (562 Da), were significantly increased in claudin-5-silenced animals compared with simvastatin-treated control animals. These findings implicate a distinct mechanism underlying size-selective endothelial barrier-protective properties of statins, and may ultimately lead to new novel therapeutic targets for patients with acute lung injury.
• Danilov, S. M., Wade, M. S., Schwager, S. L., Douglas, R. G., Nesterovitch, A. B., Popova, I. A., Hogarth, K. D., Bhardwaj, N., Schwartz, D. E., Sturrock, E. D., & Garcia, J. G. (2014). A novel angiotensin I-converting enzyme mutation (S333W) impairs N-domain enzymatic cleavage of the anti-fibrotic peptide, AcSDKP. PloS one, 9(2), e88001.
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  Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis.
• Desai, A. A., Patel, A. R., Ahmad, H., Groth, J. V., Thiruvoipati, T., Turner, K., Yodwut, C., Czobor, P., Artz, N., Machado, R. F., Garcia, J. G., & Lang, R. M. (2014). Mechanistic insights and characterization of sickle cell disease-associated cardiomyopathy. Circulation. Cardiovascular imaging, 7(3), 430-437.
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  Cardiovascular disease is an important cause of morbidity and mortality in sickle cell disease (SCD). We sought to characterize sickle cell cardiomyopathy using multimodality noninvasive cardiovascular testing and identify potential causative mechanisms.
• Eadon, M. T., Jacob, A., Cunningham, P. N., Quigg, R. J., Garcia, J. G., & Alexander, J. J. (2014). Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells. Immunology, 143(3), 363-73.
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  Blood-brain barrier (BBB) disturbance is a crucial occurrence in many neurological diseases, including systemic lupus erythematosus (SLE). Our previous studies showed that experimental lupus serum altered the integrity of the mouse brain endothelial layer, an important constituent of the BBB. Complement activation occurs in lupus with increased circulating complement components. Using a genomics approach, we identified the microRNA (miRNA) altered in mouse brain endothelial cells (bEnd3) by lupus serum and the complement protein, C5a. Of the 318 miRNA evaluated, 23 miRNAs were altered by lupus serum and 32 were altered by C5a alone compared with controls. Seven miRNAs (P < 0 · 05) were differentially expressed by both treatments: mmu-miR-133a*, mmu-miR-193*, mmu-miR-26b, mmu-miR-28*, mmu-miR-320a, mmu-miR-423-3p and mmu-miR-509-5p. The microarray results were validated by quantitative RT-PCR. In line with the in vitro results, expression of miR-26b and miR-28* were also significantly up-regulated in lupus mouse brain which was reduced by C5a receptor inhibition. Target prediction analysis revealed miR gene targets encoding components involved in inflammation, matrix arrangement, and apoptosis, pathways known to play important roles in central nervous system lupus. Our findings suggest that the miRNAs reported in this study may represent novel therapeutic targets in central nervous system lupus and other similar neuroinflammatory settings.
• Goldman, J. L., Sammani, S., Kempf, C., Saadat, L., Letsiou, E., Wang, T., Moreno-Vinasco, L., Rizzo, A. N., Fortman, J. D., & Garcia, J. G. (2014). Pleiotropic effects of interleukin-6 in a "two-hit" murine model of acute respiratory distress syndrome. Pulmonary circulation, 4(2), 280-8.
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  Patients with acute respiratory distress syndrome (ARDS) exhibit elevated levels of interleukin-6 (IL-6), which correlate with increased morbidity and mortality. The exact role of IL-6 in ARDS has proven difficult to study because it exhibits either pro- or anti-inflammatory actions in mouse models of lung injury, depending on the model utilized. In order to improve understanding of the role of this complex cytokine in ARDS, we evaluated IL-6 using the clinically relevant combination of lipopolysaccharide (LPS) and ventilator-induced lung injury (VILI) in IL-6(-/-) mice. Bronchoalveolar lavage fluid (BAL), whole-lung tissue, and histology were evaluated for inflammatory markers of injury. Transendothelial electrical resistance was used to evaluate the action of IL-6 on endothelial cells in vitro. In wild-type mice, the combination model showed a significant increase in lung injury compared to either LPS or VILI alone. IL-6(-/-) mice exhibited a statistically significant decrease in BAL cellular inflammation as well as lower histologic scores for lung injury, changes observed only in the combination model. A paradoxical increase in BAL total protein was observed in IL-6(-/-) mice exposed to LPS, suggesting that IL-6 provides protection from vascular leakage. However, in vitro data showed that IL-6, when combined with its soluble receptor, actually caused a significant increase in endothelial cell permeability, suggesting that the protection seen in vivo was likely due to complex interactions of IL-6 and other inflammatory mediators rather than to direct effects of IL-6. These studies suggest that a dual-injury model exhibits utility in evaluating the pleiotropic effects of IL-6 in ARDS on inflammatory cells and lung endothelium.
• Han, M. K., Zhou, Y., Murray, S., Tayob, N., Noth, I., Lama, V. N., Moore, B. B., White, E. S., Flaherty, K. R., Huffnagle, G. B., Martinez, F. J., & , C. I. (2014). Lung microbiome and disease progression in idiopathic pulmonary fibrosis: an analysis of the COMET study. The Lancet. Respiratory medicine, 2(7), 548-56.
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  The role of the lung microbiome in the pathogenesis of idiopathic pulmonary fibrosis is unknown. We investigated whether unique microbial signatures were associated with progression of idiopathic pulmonary fibrosis.
• Hecker, L., Zhang, D. D., Wang, T., Jiang, T., Hecker, L., & Garcia, J. G. (2014). Abstract 20541: Nicotinamide Phosphoribosyltransferase (NAMPT) Modulates Oxidative Stress via Nrf2: Roles of Intracellular and Extracellular NAMPT. Circulation, 130.
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  We previously identified the nicotinamide phosphoribosyltransferase (NAMPT, also known as pre-B cell colony enhancing factor or PBEF), as a candidate gene promoting both acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI) with circulating extracellular NAMPT (eNAMPT) potently inducing NFkB signaling in lung endothelium. We also confirmed the intracellular NAMPT (iNAMPT) enzymatic activity is strongly associated with apoptosis inhibition in neutrophils, which prolongs the half-life of infiltrated neutrophil, to exacerbate acute lung inflammation. In this study, we further characterize the novel function of NAMPT via oxidative stress modulation through Nrf2, which is a key regulator in cellular adaptive response to pro-inflammatory oxidative stimulation. Direct exposure to eNAMPT activates Nrf2, and its down-stream signaling, by upregulation of cellular Nrf2 protein levels. In addition, eNAMPT promotes NAMPT expression, in a positive feedback loop to increase both iNAMPT and eNAMPT, presumably via Nrf2 dependent oxidative stress transcriptional pathways. However, NAMPT protein expression is increased by Nrf2 knockdown (by specific siRNA) in human pulmonary endothelial cells or in lung tissue of Nrf2 knockout mice, which suggests that NAMPT expression is regulated by Nrf2, via non-classic transcriptional pathways. Interestingly, over-expressed iNAMPT directly binds to Nrf2, thereby inhibiting its transcriptional activity; in addition, over-expressed iNAMPT binds and inhibits Keap1, which leads to accumulation of Nrf2 in the cytosol. These novel findings characterize the complex modification of intracellular adaptive response to oxidative stress via Nrf2, by extracellular NAMPT, as well as intracellular NAMPT. These studies provide new molecular mechanisms of NAMPT-mediated acute lung inflammation and oxidative stress modulation.
• Huang, L. S., Mathew, B., Li, H., Zhao, Y., Ma, S. F., Noth, I., Reddy, S. P., Harijith, A., Usatyuk, P. V., Berdyshev, E. V., Kaminski, N., Zhou, T., Zhang, W., Zhang, Y., Rehman, J., Kotha, S. R., Gurney, T. O., Parinandi, N. L., Lussier, Y. A., , Garcia, J. G., et al. (2014). The mitochondrial cardiolipin remodeling enzyme lysocardiolipin acyltransferase is a novel target in pulmonary fibrosis. American journal of respiratory and critical care medicine, 189(11), 1402-15.
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  Lysocardiolipin acyltransferase (LYCAT), a cardiolipin-remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, is necessary for maintaining normal mitochondrial function and vascular development. We hypothesized that modulation of LYCAT expression in lung epithelium regulates development of pulmonary fibrosis.
• Kittles, R. A., Zhang, Y., Zhang, X., Zhang, W., Parker, M., Machado, R. F., Kittles, R. A., Gordeuk, V. R., Gladwin, M. T., Garcia, J. G., Batai, K., & Abbasi, T. (2014). European Ancestry Is Associated with Acute Chest Syndrome in Sickle Cell Disease. Blood, 124(21), 4051-4051. doi:10.1182/blood.v124.21.4051.4051
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  Background : Acute chest syndrome (ACS) is a major cause of morbidity and mortality in patients with sickle cell disease (SCD). The elucidation of genetic modifiers and mechanisms responsible for the phenotypic variability in ACS and SCD would have important clinical implications such as the identification of individuals at high risk for ACS complications and the potential for use of individualized preventive and therapeutic strategies that could ultimately improve outcomes for patients with SCD. We hypothesize that the development of ACS is significantly influenced by ancestry. Methods : Autosomal SNPs were genotyped with the Affymetrix PanAFR array in a University of Illinois cohort and with the Illumina 6.0 platform in the NIH Walk-PHaSST cohort. We merged the two GWAS datasets and there were 100,079 SNPs available after removing SNPs with > 2 alleles and Hardy-Weinberg Equilibrium (HWE) P 0.2, the total number of SNPs available for ancestry estimates was 354. Genetic ancestry was estimated using HapMap YRI and CEU genotypes as ancestral population ( K =2) using STRUCTUR 2.1. We ran STRUCTURE under the admixture model using prior population information and assuming allele frequencies among populations, with a burn-in length of 30,000 for 70,000 repetitions. Logistic regression was used to examine the association between European ancestry (EA) and Acute Chest Syndrome. Age, gender, hemoglobin genotype (HbSS/HbSβ0 vs. others), and use of hydroxyurea were additionally considered during modeling. Results: Seven hundred and thirty patients (UIC= 233, Walk-PHaSST = 497) were included in the analysis (Table 1). In binary logistic regression analysis adjusting for age, gender, Hb genotypes (severe vs. mild) and hydroxyurea use, EA was associated with and increase in ACS risk in both cohorts and when the cohorts where combined (UIC P=0.02, Walk-PHaSST P=0.001, combined P
• Mascarenhas, J. B., Brown, M., Wang, T., Mouneimne, G., Mascarenhas, J. B., Gregorio, C. C., Garcia, J. G., Dudek, S. M., & Brown, M. E. (2014). Abstract 20484: Enah/Vasp-Like Protein is a Critical Component in S1P-Mediated Endothelial Lamellipodia Formation. Circulation, 130.
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  Ena/VASP like protein, or EVL, is an actin-binding protein that regulates cancer cell lamellipodia protrusive activity and cell motility via an actomyosin contractility-dependent mechanism. The function of EVL in human lung endothelial cell (EC) barrier regulation, especially by the endogenous bioactive lipid mediator sphingosine-1-phosphate (S1P), is largely unknown. In this current study, we demonstrated that EVL is an active component in S1P-mediated EC barrier enhancement and lamellipodia formation. Compared to other focal adhesion (FA) proteins such as paxillin, EVL protein expression is very low in human pulmonary endothelial cells (ECs). S1P (1 μM) challenge stimulates translocation of cytosolic EVL to FAs in ECs, which was attenuated by EVL knockdown (KD) by its selective siRNA. S1P also promoted significant EVL translocation to lamellipodia, further confirmed by tracking translocation of EVL-GFP fusion protein upon S1P stimulation in a time-dependent manner. In addition, S1P-mediated cortical act...
• Mascarenhas, J. B., Brown, M., Wang, T., Steven, D. M., Mascarenhas, J. B., Garcia, J. G., & Brown, M. E. (2014). Abstract 20512: Cortactin Binds to a Novel Focal Adhesion Protein, EVL, During Endothelial Cytoskeleton Rearrangement. Circulation, 130.
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  The endothelial cell (EC) cytoskeleton is a complex array of proteins intimately involved in the diverse cell shape changes critical to dynamic regulation of lung vascular barrier integrity, particularly in the phases of onset and recovery during ARDS and VILI. Cortactin is structurally designed to be a central actin filament regulatory protein with an SH3 domain that directly binds non-muscle myosin light chain kinase (nmMLCK) and other effectors, and an acidic N-terminus that directly binds to Arp2/3, the protein complex regulating actin branching. In this study, we demonstrate cortactin binding to a focal adhesion protein, EVL, an actin polymerization-enhancing effector involved in linear actin filament formation and cortactin-mediated EC cytoskeleton regulation. To confirm the binding of cortactin to other cytoskeleton effector proteins, including nmMLCK, we generated a unique GFP-tagged proline-deficient nmMLCK mutant predicted to alter cortactin SH3 binding to MLCK. EC transfected with GFP-tagged proline-deficient nmMLCK mutant construct exhibit significantly higher stress fiber density and reduced localization in the cell periphery, highlighting the regulatory influence of cortactin on nmmLCK spatial localization. Cortactin-nmMLCK interaction is increased by tyrosine phosphorylation of either nmMLCK or cortactin by c-Abl, during S1P mediated cytoskeleton rearrangement. Importantly, c-Abl-mediated nmMLCK phosphorylation reverses nmMLCK’s inhibitory effects on cortactin/Arp2/3-induced pyrene-labeled actin polymerization. Cortactin binds to EVL, at basal level, with binding promoted during cytoskeleton rearrangement stimulated by S1P. The binding with EVL is critical for the cortactin translocation and interaction with nmMLCK in lamellipodia, as the silencing of EVL by siRNA significantly attenuated these essential cellular events (lamellipodia formation, cortactin translocation to lamellipodia, and binding of cortactin with nmMLCK) during cytoskeleton rearrangement. These findings reveal novel cellular mechanisms of endothelial cytoskeleton rearrangement and barrier function in ARDS and VILI.
• Mascarenhas, J. B., Hecker, L., Zhang, D. D., Yuan, J. X., Wang, T., Quijada, H., Mascarenhas, J. B., Lussier, Y. A., Jiang, T., Hecker, L., Garcia, J. G., & Elangovan, V. R. (2014). Abstract 20538: Nrf2 Signaling Ameliorates Ventilator-Induced Lung Inflammation. Circulation, 130.
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  Ventilator-induced lung injury (VILI) is an acute lung injury that develops during mechanical ventilation with high tidal volume in patients in the intensive care unit (ICU). VILI is a common entity with an unacceptable mortality rate (~20%). As no current therapies exist for VILI, novel therapeutic targets for VILI and mechanical stress-induced tissue injury are needed. In this study, we defined Nrf2 signaling in VILI and mechanical stress-induced endothelial cell pathobiology. Nrf2 is a transcription factor that alters ROS balance and is a novel therapeutic target for VILI. First, we defined Nrf2 as a sensory marker for mechanical stress-induced endothelial cell injury. Antioxidant response element (ARE) luciferase plasmids were transfected to both endothelial cells and murine lung tissues. EC exposed to 18% cyclic stretch (18% CS) or mice exposed to excessive tidal volume ventilation (40 ml/kg) resulted in ARE activation, site of Nrf2 binding. We next confirmed Nrf2 signaling is essential for gene expression dysregulation by 18% CS via Nrf2 knockdown that significantly ameliorates 18% CS-induced gene expression pattern, including signaling pathways regulating endothelial barrier function including MYLK. Finally, we validated that Nrf2 is a novel and effective therapeutic target for VILI in pre-clinical models of VILI. Nrf2 activator sulforaphane (SF, 11.5 mg/kg) significantly attenuated VILI (40 ml/kg, 4 hrs)-induced lung inflammation, including BAL protein leakage and white blood cell infiltration. These novel findings characterized Nrf2 as a sensor for mechanical stress, a central mediator of mechanical stress induced gene expression dysregulation, and a novel therapeutic target for VILI.
• Mascarenhas, J. B., Wang, T., Tchourbanov, A., Mascarenhas, J. B., Lau, S. S., Granzier, H., Garcia, J. G., Elangovan, V. R., & Danilov, S. M. (2014). Abstract 20441: Mechanism of Mechanical Stress-Regulated Non-Muscle Myosin Light Chain Kinase Alternative Splicing, Implication for Acute Lung Inflammation. Circulation, 130.
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  MYLK, the gene encoding the critical cytoskeletal effector, myosin light chain kinase (MLCK), is a compelling candidate gene in the inflammatory lung injuries observed in acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). We demonstrated that nmMLCK exists as two highly expressed proteins in lung endothelium: wild type nmMLCK1 (1914 aa) and the spliced variant, nmMLCK2 (1845 aa) that shares a primary structure that is identical to nmMLCK1 except for the absence of 69 amino acids encoded by the spliced out exon 11. Both edemagenic and barrier-enhancing agonists stimulate nmMLCK1 phosphorylation at Y464 & Y471, to promote nmMLCK1 enzymatic activity and cellular localization. The regulatory mechanisms governing MYLK expression and nmMLCK1 and nmMLCK2 EC barrier response activities are poorly understood. We studied nmMLCK1/nmMLCK2 mRNA ratios from human lung EC exposed to pathological 18% cyclic stretch (18% CS, 4 hr) by RNA sequencing (RNA Seq, BGI America) and identified increased nmMLCK transcript levels elicited by 18% CS. Specifically, nmMLCK2, but not nmMLCK1 was significantly elevated by 18% CS, results confirmed by qPCR. In silico evaluation identified an intronic splicing inhibitory element (TAGGGAG) in the boundary region in intron 10 as a potential binding site for heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), a nuclear protein known to regulate alternate splicing (Splice Scan II analysis). Consistent with hnRNPA1 modulation of splicing, we found hnRNPA1 upregulation in EC exposed to 18% CS, and in lung tissues from VILI- or LPS-challenged mice, highly suggesting a regulatory role of hnRNPA1 in nmMLCK splicing during inflammatory lung injury. Our study further confirmed a dysregulated alternative splicing pattern of non-muscle variant of MYLK by splicing factor hnRNPA1 upon inflammation stimuli. We believe these studies contribute to the understanding of the complex and functional role of nmMLCK in endothelial barrier regulation during ARDS or VILI.
• Mascarenhas, J. B., Zhang, D. D., Wang, T., Mascarenhas, J. B., Jiang, T., Garcia, J. G., & Elangovan, V. R. (2014). Abstract 20527: Nrf2 Signaling Modulates Human Endothelial Cell Barrier Regulation by S1P. Circulation, 130.
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  Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet-derived phospholipid, sphingosine-1-phosphate (S1P), decreases EC permeability in vitro and in vivo, features of obvious therapeutic potential. Oxidative stress is a common primary or secondary cause of vascular hyperpermeability in multiple pathological conditions, including inflammation, and exposure to excessive mechanical stress. Thus it is important to understand the relationship between S1P-mediated vascular protection and oxidative stress-induced barrier disruption. We examined the interaction of S1P and human pulmonary artery EC signaling and barrier regulation by the oxidative stress signaling mediator, Nrf2. Human pulmonary ECs pre-treated with either tBHQ (Nrf2 inducer) or Brusatol (Nrf2 inhibitor), were evaluated by transendothelial electric resistance (TER) in response to S1P or thrombin. tBHQ clearly augmented S1P-mediated endothelial barrier enhancement. In parallel, brusatol reduced the S1P-mediated endothelial barrier enhancement. In addition, neither tBHQ or brusatol modified the peak effects of thrombin-induced endothelial barrier disruption. However, tBHQ accelerated the recovery of endothelial barrier disruption induced by thrombin, whereas brusatol retarded EC recovery process. Furthermore, we demonstrated that tBHQ enhanced S1P mediated cortical actin polymerization and barrier enhancement, whereas Brusotal enhanced stress fiber formation in endothelial cells and inhibited S1P-mediated cortical actin formation confirming the active role of Nrf2 signaling in S1P-mediated endothelial barrier regulation. The effect of Brusatol on endothelial barrier function was confirmed by Nrf2 siRNA. These functional analyses confirm the importance of Nrf2 signaling in EC barrier restoration, a highly relevant response in acute lung inflammation resolution and lung fluid balance and further define a novel role of Nrf2-dependent signaling in vascular barrier regulation and cytoskeleton rearrangements
• Mascarenhas, J. B., Zhou, T., Zhou, T., Wang, T., Mascarenhas, J. B., Kittles, R. A., Jacobson, J. R., Garcia, J. G., Epstein, Y., Cress, A. E., & Chen, W. (2014). Abstract 20295: Key Role of Integrin Alpha6/Beta4 in Endothelial Cell Barrier Regulation. Circulation, 130.
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  The dynamic regulation of lung endothelial cell (EC) barrier integrity is essential in the initiation and resolution of inflammation during acute lung injury (ALI) and ventilator-induced lung injury (VILI). In previous studies, we identified integrin beta β4 (ITGβ4) as a key focal adhesion (FA) component, involved in human lung EC barrier enhancement by simvastatin. The clear functional role and key contribution of ITGβ4 in endothelial barrier regulation vascular permeability is largely unknown. We demonstrated that reduced ITGβ4 expression (siRNA attenuated sphingosine-1-phosphate (S1P, 1 μM) or hepatocyte growth factor (HGF, 25 ng/ml) mediated endothelial barrier enhancement. At the same time, S1P receptor 1 (S1PR1) or HGF receptor cMet knockdown also attenuated S1P- or HGF-mediated endothelial barrier enhancement. Consistent with these findings, in situ proximity ligation assays revealed that ITGβ4, S1PR1, and cMet, translocated together to the cell periphery as co-localized proteins after HGF treatment. We further confirmed that the interaction of c-Met and S1PR1 with ITGβ4 was dependent upon ITGβ4 phosphorylation, which is essential for HGF-mediated barrier enhancement. The selective antibody targeting integrin α6 (ITGα6, 10 μg/ml), the only binding partner of ITGβ4, attenuates S1P-mediated endothelial barrier enhancement. Furthermore, this antibody retarded barrier recovery after thrombin (0.5 U/ml), an EC barrier disruptor. These findings highly suggest ITGβ4 is essential participant in EC barrier regulation and paracellular gap closure. We next exposed genetically-engineered mice expressing a mutant ITGβ4 that lacks the cytoplasmic tail to ventilator-induced lung injury (VILI). The absence of the cytoplasmic tail conferred significant attenuation of inflammatory lung injury compared to wild-type controls with reduced BAL protein and cell counts and lung inflammation. These data suggest that the ITGβ4 cytoplasmic domain is an important participant in both pro- and anti-inflammatory responses to the excessive mechanical stress produced by VILI. In summary, ITGβ4 is a central regulator of endothelial barrier function, and a novel therapeutic target for acute lung inflammation.
• Mirsaeidi, M., Machado, R. F., Garcia, J. G., & Schraufnagel, D. E. (2014). Nontuberculous mycobacterial disease mortality in the United States, 1999-2010: a population-based comparative study. PloS one, 9(3), e91879.
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  Environmental nontuberculous mycobacteria (NTM) are ubiquitous organisms with which humans commonly interact. The epidemiologic characteristics of NTM diseases including mortality rate and its associated factors remain largely unknown. In this study, we explored the geographical area of exposure and mortality and comorbid conditions of affected persons to determine environment, host, and host-pathogen interactive factors.
• Mitra, S., Wade, M. S., Sun, X., Moldobaeva, N., Flores, C., Ma, S. F., Zhang, W., Garcia, J. G., & Jacobson, J. R. (2014). GADD45a promoter regulation by a functional genetic variant associated with acute lung injury. PloS one, 9(6), e100169.
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  Growth arrest DNA damage inducible alpha (GADD45a) is a stress-induced gene we have shown to participate in the pathophysiology of ventilator-induced lung injury (VILI) via regulation of mechanical stress-induced Akt ubiquitination and phosphorylation. The regulation of GADD45a expression by mechanical stress and its relationship with acute lung injury (ALI) susceptibility and severity, however, remains unknown.
• Moreno-Vinasco, L., Quijada, H., Sammani, S., Siegler, J., Letsiou, E., Deaton, R., Saadat, L., Zaidi, R. S., Messana, J., Gann, P. H., Machado, R. F., Ma, W., Camp, S. M., Wang, T., & Garcia, J. G. (2014). Nicotinamide phosphoribosyltransferase inhibitor is a novel therapeutic candidate in murine models of inflammatory lung injury. American journal of respiratory cell and molecular biology, 51(2), 223-8.
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  We previously identified the intracellular nicotinamide phosphoribosyltransferase (iNAMPT, aka pre-B-cell colony enhancing factor) as a candidate gene promoting acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI) with circulating nicotinamide phosphoribosyltransferase potently inducing NF-κB signaling in lung endothelium. iNAMPT also synthesizes intracellular nicotinamide adenine dinucleotide (iNAD) in response to extracellular oxidative stress, contributing to the inhibition of apoptosis via ill-defined mechanisms. We now further define the role of iNAMPT activity in the pathogenesis of ARDS/VILI using the selective iNAMPT inhibitor FK-866. C57/B6 mice were exposed to VILI (40 ml/kg, 4 h) or LPS (1.5 mg/kg, 18 h) after osmotic pump delivery of FK-866 (100 mg/kg/d, intraperitoneally). Assessment of total bronchoalveolar lavage (BAL) protein, polymorphonuclear neutrophil (PMN) levels, cytokine levels (TNF-α, IL-6, IL-1α), lung iNAD levels, and injury scores revealed that FK-866-mediated iNAMPT inhibition successfully reduced lung tissue iNAD levels, BAL injury indices, inflammatory cell infiltration, and lung injury scores in LPS- and VILI-exposed mice. FK-866 further increased lung PMN apoptosis, as reflected by caspase-3 activation in BAL PMNs. These findings support iNAMPT inhibition via FK-866 as a novel therapeutic agent for ARDS via enhanced apoptosis in inflammatory PMNs.
• Ni, X., Epshtein, Y., Chen, W., Zhou, T., Xie, L., Garcia, J. G., & Jacobson, J. R. (2014). Interaction of integrin β4 with S1P receptors in S1P- and HGF-induced endothelial barrier enhancement. Journal of cellular biochemistry, 115(6), 1187-95.
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  We previously reported sphingosine 1-phosphate (S1P) and hepatocyte growth factor (HGF) augment endothelial cell (EC) barrier function and attenuate murine acute lung inury (ALI). While the mechanisms underlying these effects are not fully understood, S1P and HGF both transactivate the S1P receptor, S1PR1 and integrin β4 (ITGB4) at membrane caveolin-enriched microdomains (CEMs). In the current study, we investigated the roles of S1PR2 and S1PR3 in S1P/HGF-mediated EC signaling and their associations with ITGB4. Our studies confirmed ITGB4 and S1PR2/3 are recruited to CEMs in human lung EC in response to either S1P (1 µM, 5 min) or HGF (25 ng/ml, 5 min). Co-immunoprecipitation experiments identified an S1P/HGF-mediated interaction of ITGB4 with both S1PR2 and S1PR3. We then employed an in situ proximity ligation assay (PLA) to confirm a direct ITGB4-S1PR3 association induced by S1P/HGF although a direct association was not detectable between S1PR2 and ITGB4. S1PR1 knockdown (siRNA), however, abrogated S1P/HGF-induced ITGB4-S1PR2 associations while there was no effect on ITGB4-S1PR3 associations. Moreover, PLA confirmed a direct association between S1PR1 and S1PR2 induced by S1P and HGF. Finally, silencing of S1PR2 significantly attenuated S1P/HGF-induced EC barrier enhancement as measured by transendothelial resistance while silencing of S1PR3 significantly augmented S1P/HGF-induced barrier enhancement. These results confirm an important role for S1PR2 and S1PR3 in S1P/HGF-mediated EC barrier responses that are associated with their complex formation with ITGB4. Our findings elucidate novel mechanisms of EC barrier regulation that may ultimately lead to new therapeutic targets for disorders characterized by increased vascular permeability including ALI.
• Sammani, S., Sammani, S., Letsiou, E., Garcia, J. G., & Dudek, S. M. (2014). Increased endothelial-derived microparticles in ventilator-induced lung injury. European Respiratory Journal, 44.
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  Ventilator-induced lung injury (VILI) is a consequence of mechanical ventilation and contributes to the pathophysiology of lung injury syndromes. The role of the vascular endothelium in VILI remains largely unknown. Endothelial microparticles (EMPs) are membrane vesicles shed by activated or apoptotic cells. We have previously demonstrated that human lung endothelial cells (EC) exposed to pathologic cyclic stretch (in vitro model of VILI) shed increased numbers of EMPs that mediate lung injury. In this study, we characterize EMPs in a murine model of VILI. C57BL/6J mice were subjected to high tidal volume (40mL/kg) mechanical ventilation (VILI). Spontaneously breathing (SB) mice served as control. After 4h, blood/BAL were collected and microparticles (MPs) were isolated by differential centrifugation. MPs were quantified after staining with a marker of endothelial activation, CD62E (Flow cytometry). Plasma MPs were also analyzed by immunoblotting for VE-cadherin. The purity and characteristics (size 0.1 to 1μm) of the isolated MP populations were confirmed by electron microscopy and flow cytometry. VILI-mice exhibited significant lung injury and increased circulating CD62E positive MPs (1.38-fold) compared to SB mice. In addition, MP lysates contained increased VE-cadherin levels in VILI-challenged mice compared to SB. We next determined the levels of EMPs in the alveolar space. CD62E(+) BAL MPs were increased by 1.87-fold in VILI-challenged mice compared to SB mice. Our results suggest that high levels of EMPs in blood and BAL during mechanical ventilation may represent VILI-induced endothelial dysfunction. EMPs are promising novel biomarkers and mediators of lung injury. Funding: AHA.
• Sammani, S., Sammani, S., Rizzo, A. N., Letsiou, E., Garcia, J. G., Esquinca, A., & Dudek, S. M. (2014). Abstract 13333: Imatinib Attenuates LPS-induced Inflammation and Vascular Leakage in Clinically Relevant Murine Models of Acute Lung Injury. Circulation, 130.
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  Introduction: Acute lung injury (ALI) is characterized by inflammation-induced disruption of the endothelial cell (EC) barrier lining the pulmonary vasculature, which causes leakage of fluid and inflammatory cells into the airspaces. The mechanisms underlying ALI are critically dependent on EC cytoskeletal regulation of vascular permeability. The Abl family kinases Abl and Abl Related Gene (Arg) are key mediators of the endothelial cytoskeleton. The pharmaceutical agent imatinib inhibits these kinases and is used clinically for the treatment of multiple different malignancies. Hypothesis: Imatinib attenuates LPS-induced inflammation and vascular leakage in murine ALI and retains its protective properties when combined with high tidal volume (Tv) mechanical ventilation (MV). Methods: As a clinically relevant combination model of ALI stimuli, animals were given intratracheal (i.t) LPS (0.5mg/kg) (vs. PBS), allowed to recover for 20 hours, and then intubated for MV (Tv 30ml/kg, RR 75/min, PEEP 0 cm H2O, 4hrs). Imatinib (75mg/kg) (vs. vehicle) was given i.p. 30 min before LPS and 30 min before MV. For post-injury experiments, imatinib (75mg/kg) (vs. vehicle) was given 4 hours after LPS (1mg/kg) and animals were harvested after 18 hrs. Bronchoalveolar lavage fluid (BAL) was analyzed for total cell counts, neutrophil number, total protein, albumin content, and cytokine levels. Lung tissues were used for MPO activity, albumin content, histology, and Evans Blue dye extravasation. Results: Imatinib attenuated inflammation in both combination model studies and post-LPS treatment studies as measured by BAL cell count, PMN number, MPO activity, TNFα levels, and H&E staining. Additionally, in both models, imatinib decreased vascular leakage as measured by total protein, BAL and lung tissue albumin, and Evans Blue dye extravasation. Conclusions: Imatinib attenuates LPS-induced inflammation and vascular leak in mice. Additionally, imatinib is protective in a clinically relevant murine model of combined LPS and mechanical ventilation injury. Imatinib may have potential for translational use in ALI patients.
• Sun, X., Elangovan, V. R., Mapes, B., Camp, S. M., Sammani, S., Saadat, L., Ceco, E., Ma, S. F., Flores, C., MacDougall, M. S., Quijada, H., Liu, B., Kempf, C. L., Wang, T., Chiang, E. T., & Garcia, J. G. (2014). The NAMPT promoter is regulated by mechanical stress, signal transducer and activator of transcription 5, and acute respiratory distress syndrome-associated genetic variants. American journal of respiratory cell and molecular biology, 51(5), 660-7.
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  Increased nicotinamide phosphoribosyltransferase (NAMPT) transcription is mechanistically linked to ventilator-induced inflammatory lung injury (VILI), with VILI severity attenuated by reduced NAMPT bioavailability. The molecular mechanisms of NAMPT promoter regulation in response to excessive mechanical stress remain poorly understood. The objective of this study was to define the contribution of specific transcription factors, acute respiratory distress syndrome (ARDS)-associated single nucleotide polymorphisms (SNPs), and promoter demethylation to NAMPT transcriptional regulation in response to mechanical stress. In vivo NAMPT protein expression levels were examined in mice exposed to high tidal volume mechanical ventilation. In vitro NAMPT expression levels were examined in human pulmonary artery endothelial cells exposed to 5 or 18% cyclic stretch (CS), with NAMPT promoter activity assessed using NAMPT promoter luciferase reporter constructs with a series of nested deletions. In vitro NAMPT transcriptional regulation was further characterized by measuring luciferase activity, DNA demethylation, and chromatin immunoprecipitation. VILI-challenged mice exhibited significantly increased NAMPT expression in bronchoalveolar lavage leukocytes and in lung endothelium. A mechanical stress-inducible region (MSIR) was identified in the NAMPT promoter from -2,428 to -2,128 bp. This MSIR regulates NAMPT promoter activity, mRNA expression, and signal transducer and activator of transcription 5 (STAT5) binding, which is significantly increased by 18% CS. In addition, NAMPT promoter activity was increased by pharmacologic promoter demethylation and inhibited by STAT5 silencing. ARDS-associated NAMPT promoter SNPs rs59744560 (-948G/T) and rs7789066 (-2,422A/G) each significantly elevated NAMPT promoter activity in response to 18% CS in a STAT5-dependent manner. Our results show that NAMPT is a key novel ARDS therapeutic target and candidate gene with genetic/epigenetic transcriptional regulation in response to excessive mechanical stress.
• Sweiss, N. J., Lower, E. E., Mirsaeidi, M., Dudek, S., Garcia, J. G., Perkins, D., Finn, P. W., & Baughman, R. P. (2014). Rituximab in the treatment of refractory pulmonary sarcoidosis. The European respiratory journal, 43(5), 1525-8.
• Sweiss, N. J., Noth, I., Mirsaeidi, M., Zhang, W., Naureckas, E. T., Hogarth, D. K., Strek, M., Caligiuri, P., Machado, R. F., Niewold, T. B., Garcia, J. G., Pangan, A. L., & Baughman, R. P. (2014). Efficacy Results of a 52-week Trial of Adalimumab in the Treatment of Refractory Sarcoidosis. Sarcoidosis, vasculitis, and diffuse lung diseases : official journal of WASOG, 31(1), 46-54.
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  Infliximab, a chimeric, monoclonal, anti-TNF antibody has been shown to be safe and efficacious for refractory sarcoidosis, we investigated whether adalimumab, a fully human, anti-TNF monoclonal antibody, is similarly safe and efficacious in refractory pulmonary sarcoidosis.
• Usatyuk, P. V., Fu, P., Mohan, V., Epshtein, Y., Jacobson, J. R., Gomez-Cambronero, J., Wary, K. K., Bindokas, V., Dudek, S. M., Salgia, R., Garcia, J. G., & Natarajan, V. (2014). Role of c-Met/phosphatidylinositol 3-kinase (PI3k)/Akt signaling in hepatocyte growth factor (HGF)-mediated lamellipodia formation, reactive oxygen species (ROS) generation, and motility of lung endothelial cells. The Journal of biological chemistry, 289(19), 13476-91.
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  Hepatocyte growth factor (HGF) mediated signaling promotes cell proliferation and migration in a variety of cell types and plays a key role in tumorigenesis. As cell migration is important to angiogenesis, we characterized HGF-mediated effects on the formation of lamellipodia, a pre-requisite for migration using human lung microvascular endothelial cells (HLMVECs). HGF, in a dose-dependent manner, induced c-Met phosphorylation (Tyr-1234/1235, Tyr-1349, Ser-985, Tyr-1003, and Tyr-1313), activation of PI3k (phospho-Yp85) and Akt (phospho-Thr-308 and phospho-Ser-473) and potentiated lamellipodia formation and HLMVEC migration. Inhibition of c-Met kinase by SU11274 significantly attenuated c-Met, PI3k, and Akt phosphorylation, suppressed lamellipodia formation and endothelial cell migration. LY294002, an inhibitor of PI3k, abolished HGF-induced PI3k (Tyr-458), and Akt (Thr-308 and Ser-473) phosphorylation and suppressed lamellipodia formation. Furthermore, HGF stimulated p47(phox)/Cortactin/Rac1 translocation to lamellipodia and ROS generation. Moreover, inhibition of c-Met/PI3k/Akt signaling axis and NADPH oxidase attenuated HGF- induced lamellipodia formation, ROS generation and cell migration. Ex vivo experiments with mouse aortic rings revealed a role for c-Met signaling in HGF-induced sprouting and lamellipodia formation. Taken together, these data provide evidence in support of a significant role for HGF-induced c-Met/PI3k/Akt signaling and NADPH oxidase activation in lamellipodia formation and motility of lung endothelial cells.
• Wang, L., Sammani, S., Moreno-Vinasco, L., Letsiou, E., Wang, T., Camp, S. M., Bittman, R., Garcia, J. G., & Dudek, S. M. (2014). FTY720 (s)-phosphonate preserves sphingosine 1-phosphate receptor 1 expression and exhibits superior barrier protection to FTY720 in acute lung injury. Critical care medicine, 42(3), e189-99.
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  Effective therapies are needed to reverse the increased vascular permeability that characterizes acute inflammatory diseases such as acute lung injury. FTY720 is a pharmaceutical analog of the potent barrier-enhancing phospholipid, sphingosine 1-phosphate. Because both FTY720 and sphingosine 1-phosphate have properties that may limit their usefulness in patients with acute lung injury, alternative compounds are needed for therapeutic use. The objective of this study is to characterize the effects of FTY720 (S)-phosphonate, a novel analog of FTY720-phosphate, on variables of pulmonary vascular permeability in vitro and alveolar-capillary permeability in vivo.
• Wang, T., Moreno-Vinasco, L., Ma, S. F., Zhou, T., Shimizu, Y., Sammani, S., Epshtein, Y., Watterson, D. M., Dudek, S. M., & Garcia, J. G. (2014). Nonmuscle myosin light chain kinase regulates murine asthmatic inflammation. American journal of respiratory cell and molecular biology, 50(6), 1129-35.
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  Myosin light chain kinase (MLCK; gene code, MYLK) is a multifunctional enzyme involved in isoform-specific nonmuscle (nm) and smooth muscle contraction, inflammation, and vascular permeability, processes directly relevant to asthma pathobiology. In this report, we highlight the contribution of the nm isoform (nmMLCK) to asthma susceptibility and severity, supported by studies in two lines of transgenic mice with knocking out nmMLCK or selectively overexpressing nmMLCK in endothelium. These mice were sensitized to exhibit ovalbumin-mediated allergic inflammation. Genetically engineered mice with targeted nmMLCK deletion (nmMLCK(-/-)) exhibited significant reductions in lung inflammation and airway hyperresponsiveness. Conversely, mice with overexpressed nmMLCK in endothelium (nmMLCK(ec/ec)) exhibited elevated susceptibility and severity in asthmatic inflammation. In addition, reduction of nmMLCK expression in pulmonary endothelium by small interfering RNA results in reduced asthmatic inflammation in wild-type mice. These pathophysiological assessments demonstrate the positive contribution of nmMLCK to asthmatic inflammation, and a clear correlation of the level of nmMLCK with the degree of experimental allergic inflammation. This study confirms MYLK as an asthma candidate gene, and verifies nmMLCK as a novel molecular target in asthmatic pathobiology.
• Wolfson, R. K., Mapes, B., & Garcia, J. G. (2014). Excessive mechanical stress increases HMGB1 expression in human lung microvascular endothelial cells via STAT3. Microvascular research, 92, 50-55.
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  Ventilator-induced lung injury (VILI) occurs when the lung parenchyma and vasculature are exposed to repetitive and excessive mechanical stress via mechanical ventilation utilized as supportive care for the adult respiratory distress syndrome (ARDS). VILI induces gene expression and systemic release of inflammatory mediators that contribute to the multi-organ dysfunction and morbidity and mortality of ARDS. HMGB1, an intracellular transcription factor with cytokine properties, is a late mediator in sepsis and ARDS pathobiology, however, the role of HMGB1 in VILI remains poorly described. We now report HMGB1 expression in human lung microvessel endothelial cells (ECs) exposed to excessive, equibiaxial mechanical stress, an in vitro correlate of VILI. We determined that high amplitude cyclic stretch (18% CS) increased HMGB1 expression (2-4-fold) via a signaling pathway with critical involvement of the transcription factor, STAT3. Concomitant exposure to 18% CS and oxidative stress (H₂O₂) augmented HMGB1 expression (~13 fold increase) whereas lipopolysaccharide (LPS) challenge increased HMGB1 expression in static EC, but not in 18% CS-challenged EC. In contrast, physiologic, low amplitude cyclic stretch (5% CS) attenuated both oxidative H₂O₂- and LPS-induced increases in HMGB1 expression, suggesting that physiologic mechanical stress is protective. These results indicate that HMGB1 gene expression is markedly responsive to VILI-mediated mechanical stress, an effect that is augmented by oxidative stress. We speculate that VILI-induced HMGB1 expression acts locally to increase vascular permeability and alveolar flooding, thereby exacerbating systemic inflammatory responses and increasing the likelihood of multi-organ dysfunction.
• Yu, G., Yang, X., Tzouvelekis, A., Sakamoto, K., Noth, I., Meyers, M., Ma, S., Lynn, H., Lindell, K. O., Klesen, M., Kaminski, N., Juan-guardela, B., Herazo-maya, J. D., Gibson, K. F., Garcia, J. G., & Deiullis, J. (2014). Discovery and validation of peripheral blood gene expression profiles predictive of poor outcome and disease progression in idiopathic pulmonary fibrosis. European Respiratory Journal, 44.
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  Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease associated with increase mortality. In this study we aim to identify and validate peripheral blood mononuclear cell (PBMC) gene expression profiles predictive of poor outcomes and IPF progression Methods: We collected transplant free survival (TFS) data, extracted RNA from PBMCs and performed Microarrays in IPF patients from a discovery (N=45) and replication cohort (N=75) at the University of Chicago and Pittsburgh, respectively. Significance analysis of Microarrays was used to identify genes associated with TFS. Hierarchical clustering and qRT-PCR were used to validate Microarray findings. Transcript counts were measured by the nCounter system (NanoString) in a larger (N=120), prospectively followed IPF cohort (University of Pittsburgh), at multiple time points per patient (N=425). A linear mixed model was used to identify significant associations between FVC and FVC% with gene expressions over time after adjusting for differences in age, gender and immunosuppression use. Results: Hierarchical clustering based on 52 outcome predictive genes distinguished two clusters of IPF patients with significant differences in TFS (HR:2.08,CI:1.29-3.32,P=0.017). No differences in age, gender or immunosuppression use were identified between patients in these clusters. 43 and 40 genes of the 52-gene profile were significantly associated with changes over time in FVC and FVC%, respectively, by the linear mixed model (P
• Zhang, X., Zhang, W., Ma, S. F., Desai, A. A., Saraf, S., Miasniakova, G., Sergueeva, A., Ammosova, T., Xu, M., Nekhai, S., Abbasi, T., Casanova, N. G., Steinberg, M. H., Baldwin, C. T., Sebastiani, P., Prchal, J. T., Kittles, R., Garcia, J. G., Machado, R. F., & Gordeuk, V. R. (2014). Hypoxic response contributes to altered gene expression and precapillary pulmonary hypertension in patients with sickle cell disease. Circulation, 129(16), 1650-8.
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  We postulated that the hypoxic response in sickle cell disease (SCD) contributes to altered gene expression and pulmonary hypertension, a complication associated with early mortality.
• Zhang, X., Zhang, W., Ma, S. F., Miasniakova, G., Sergueeva, A., Ammosova, T., Xu, M., Nekhai, S., Nourai, M., Wade, M. S., Prchal, J. T., Garcia, J. G., Machado, R. F., & Gordeuk, V. R. (2014). Iron deficiency modifies gene expression variation induced by augmented hypoxia sensing. Blood cells, molecules & diseases, 52(1), 35-45.
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  In congenital Chuvash polycythemia (CP), VHL(R200W) homozygosity leads to elevated hypoxia inducible factor (HIF) levels at normoxia. CP is often treated by phlebotomy resulting in iron deficiency, permitting us to examine the separate and synergistic effects of iron deficiency and HIF signaling on gene expression. We compared peripheral blood mononuclear cell gene expression profiles of eight VHL(R200W) homozygotes with 17 wildtype individuals with normal iron status and found 812 up-regulated and 2120 down-regulated genes at false discovery rate of 0.05. Among differential genes we identified three major gene regulation modules involving induction of innate immune responses, alteration of carbohydrate and lipid metabolism, and down-regulation of cell proliferation, stress-induced apoptosis and T-cell activation. These observations suggest molecular mechanisms for previous observations in CP of lower blood sugar without increased insulin and low oncogenic potential. Studies including 16 additional VHL(R200W) homozygotes with low ferritin indicated that iron deficiency enhanced the induction effect of VHL(R200W) for 50 genes including hemoglobin synthesis loci but suppressed the effect for 107 genes enriched for HIF-2 targets. This pattern is consistent with potentiation of HIF-1α protein stability by iron deficiency but a trend for down-regulation of HIF-2α translation by iron deficiency overriding an increase in HIF-2α protein stability.
• Zhou, T., Wang, T., & Garcia, J. G. (2014). Expression of nicotinamide phosphoribosyltransferase-influenced genes predicts recurrence-free survival in lung and breast cancers. Scientific reports, 4, 6107.
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  Nicotinamide phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the salvage pathway of nicotinamide adenine dinucleotide biosynthesis. NAMPT protein is a secreted plasma biomarker in inflammation and in cancer. The NAMPT enzymatic inhibitor, FK866, acts as an inducer of apoptosis and is a cancer therapeutic candidate, however, little is known regarding the influence of NAMPT on cancer biological mechanisms or on the prognosis of human cancers. We interrogated known microarray data sets to define NAMPT knockdown-influenced gene expression to demonstrate that reduced NAMPT expression strongly dysregulates cancer biology signaling pathways. Comparisons of gene expression datasets of four cancer types generated a N39 molecular signature exhibiting consistent dysregulated expression in multiple cancer tissues. The N39 signature provides a significant and independent prognostic tool of human recurrence-free survival in lung and breast cancers. Despite the absence of clear elucidation of molecular mechanisms, this study validates NAMPT as a novel "oncogene" with a central role in carcinogenesis. Furthermore, the N39 signature provides a potentially useful tool for prediction of recurrence-free survival in lung and breast cancer and validates NAMPT as a novel and effective therapeutic target in cancer.
• Zhou, T., Wang, T., & Garcia, J. G. (2014). Genes influenced by the non-muscle isoform of Myosin light chain kinase impact human cancer prognosis. PloS one, 9(4), e94325.
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  The multifunctional non-muscle isoform of myosin light chain kinase (nmMLCK) is critical to the rapid dynamic coordination of the cytoskeleton involved in cancer cell proliferation and migration. We identified 45 nmMLCK-influenced genes by bioinformatic filtering of genome-wide expression in wild type and nmMLCK knockout (KO) mice exposed to preclinical models of murine acute inflammatory lung injury, pathologies that are well established to include nmMLCK as an essential participant. To determine whether these nmMLCK-influenced genes were relevant to human cancers, the 45 mouse genes were matched to 38 distinct human orthologs (M38 signature) (GeneCards definition) and underwent Kaplan-Meier survival analysis in training and validation cohorts. These studies revealed that in training cohorts, the M38 signature successfully identified cancer patients with poor overall survival in breast cancer (P
• Zhu, X., Wang, H., Qian, Y., Machado, R., Li, J., Lee, C., Hoek, T. L., Garcia, J. G., Chen, J., & Bunney, R. (2014). Abstract 223: PBEF/Nampt Protects Against Mouse Sudden Cardiac Arrest Injury. Circulation, 130.
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  Introduction: Pre-B-cell colony-enhancing factor or nicotinamide phosphoribosyltransferase (PBEF/Nampt) is critical for NAD+ biosynthesis and metabolism. Given the important role of early metabolic recovery to sudden cardiac arrest (SCA) survival, we hypothesized that PBEF/Nampt deficient mice would demonstrate worse SCA outcomes after cardiopulmonary resuscitation (CPR). Methods: C57BL6 wild type and PBEF/Nampt+/- mice underwent 8 min of KCl-induced SCA and were monitored for 4 h survival after CPR. Heart, brain and blood PBEF/Nampt concentrations were measured by Western blot and ELISA. Tissue NAD+ concentrations and blood markers of metabolic recovery including plasma lactate, glucose and insulin were measured. Results: At baseline, heart and brain tissue levels of PBEF/Nampt were significantly decreased by 50% in PBEF/Nampt+/- mice with no change of NAD+ concentrations. By 4 h after CPR PBEF/Nampt+/- mice had worse survival, with 83% dead of cardiovascular collapse (n=18) compared to 45% of WT mice (n...
• Abbasi, T., & Garcia, J. G. (2013). Sphingolipids in lung endothelial biology and regulation of vascular integrity. Handbook of experimental pharmacology, 201-26.
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  Of the multiple and diverse homeostatic events that involve the lung vascular endothelium, participation in preserving vascular integrity and therefore organ function is paramount. We were the first to show that the lipid growth factor and angiogenic factor, sphingosine-1-phosphate, is a critical agonist involved in regulation of human lung vascular barrier function (Garcia et al. J Clin Invest, 2011). Utilizing both in vitro models and preclinical murine, rat, and canine models of acute and chronic inflammatory lung injury, we have shown that S1Ps, as well as multiple S1P analogues such as FTY720 and ftysiponate, serve as protective agents limiting the disruption of the vascular EC monolayer in the pulmonary microcirculation and attenuate parenchymal accumulation of inflammatory cells and high protein containing extravasated fluid, thereby reducing interstitial and alveolar edema. The vasculo-protective mechanism of these therapeutic effects occurs via ligation of specific G-protein-coupled receptors and an intricate interplay of S1P with other factors (such as MAPKS, ROCKs, Rho, Rac1) with rearrangement of the endothelial cytoskeleton to form strong cortical actin rings in the cell periphery and enhanced cell-to-cell and cell-to-matrix tethering dynamics. This cascade leads to reinforcement of focal adhesions and paracellular junctional complexes via cadherin, paxillin, catenins, and zona occludens. S1P through its interaction with Rac and Rho influences the cytoskeletal rearrangement indicated in the later stages of angiogenesis as a stabilizing force, preventing excessive vascular permeability. These properties translate into a therapeutic potential for acute and chronic inflammatory lung injuries. S1P has potential for providing a paradigm shift in the approach to disruption of critical endothelial gatekeeper function, loss of lung vascular integrity, and increased vascular permeability, defining features of acute lung injury (ALI), and may prove to exhibit an intrinsically protective role in the pulmonary vasculature ameliorating agonist- or sepsis-induced pulmonary injury and vascular leakage.
• Adyshev, D. M., Dudek, S. M., Moldobaeva, N., Kim, K. M., Ma, S. F., Kasa, A., Garcia, J. G., & Verin, A. D. (2013). Ezrin/radixin/moesin proteins differentially regulate endothelial hyperpermeability after thrombin. American journal of physiology. Lung cellular and molecular physiology, 305(3), L240-55.
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  Endothelial cell (EC) barrier disruption induced by inflammatory agonists such as thrombin leads to potentially lethal physiological dysfunction such as alveolar flooding, hypoxemia, and pulmonary edema. Thrombin stimulates paracellular gap and F-actin stress fiber formation, triggers actomyosin contraction, and alters EC permeability through multiple mechanisms that include protein kinase C (PKC) activation. We previously have shown that the ezrin, radixin, and moesin (ERM) actin-binding proteins differentially participate in sphingosine-1 phosphate-induced EC barrier enhancement. Phosphorylation of a conserved threonine residue in the COOH-terminus of ERM proteins causes conformational changes in ERM to unmask binding sites and is considered a hallmark of ERM activation. In the present study we test the hypothesis that ERM proteins are phosphorylated on this critical threonine residue by thrombin-induced signaling events and explore the role of the ERM family in modulating thrombin-induced cytoskeletal rearrangement and EC barrier function. Thrombin promotes ERM phosphorylation at this threonine residue (ezrin Thr567, radixin Thr564, moesin Thr558) in a PKC-dependent fashion and induces translocation of phosphorylated ERM to the EC periphery. Thrombin-induced ERM threonine phosphorylation is likely synergistically mediated by protease-activated receptors PAR1 and PAR2. Using the siRNA approach, depletion of either moesin alone or of all three ERM proteins significantly attenuates thrombin-induced increase in EC barrier permeability (transendothelial electrical resistance), cytoskeletal rearrangements, paracellular gap formation, and accumulation of phospho-myosin light chain. In contrast, radixin depletion exerts opposing effects on these indexes. These data suggest that ERM proteins play important differential roles in the thrombin-induced modulation of EC permeability, with moesin promoting barrier dysfunction and radixin opposing it.
• Adyshev, D. M., Moldobaeva, N., Mapes, B., Elangovan, V., & Garcia, J. G. (2013). MicroRNA regulation of nonmuscle myosin light chain kinase expression in human lung endothelium. American journal of respiratory cell and molecular biology, 49(1), 58-66.
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  Increased lung vascular permeability, the consequence of endothelial cell (EC) barrier dysfunction, is a cardinal feature of inflammatory conditions such as acute lung injury and sepsis and leads to lethal physiological dysfunction characterized by alveolar flooding, hypoxemia, and pulmonary edema. We previously demonstrated that the nonmuscle myosin light chain kinase isoform (nmMLCK) plays a key role in agonist-induced pulmonary EC barrier regulation. The present study evaluated posttranscriptional regulation of MYLK expression, the gene encoding nmMLCK, via 3' untranslated region (UTR) binding by microRNAs (miRNAs) with in silico analysis identifying hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, and hsa-miR-1290 as miRNA candidates. We identified increased MYLK gene transcription induced by TNF-α (24 h; 4.7 ± 0.45 fold increase [FI]), LPS (4 h; 2.85 ± 0.15 [FI]), and 18% cyclic stretch (24 h; 4.6 ± 0.24 FI) that was attenuated by transfection of human lung ECs with mimics of hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, or hsa-miR-1290 (20-80% reductions by each miRNA). TNF-α, LPS, and 18% cyclic stretch each increased the activity of a MYLK 3'UTR luciferase reporter (2.5-7.0 FI) with induction reduced by mimics of each miRNA (30-60% reduction). MiRNA inhibitors (antagomirs) for each MYLK miRNA significantly increased 3'UTR luciferase activity (1.2-2.3 FI) and rescued the decreased MLCK-3'UTR reporter activity produced by miRNA mimics (70-110% increases for each miRNA; P < 0.05). These data demonstrate that increased human lung EC expression of MYLK by bioactive agonists (excessive mechanical stress, LPS, TNF-α) is regulated in part by specific miRNAs (hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, and hsa-miR-1290), representing a novel therapeutic strategy for reducing inflammatory lung injury.
• Arce, F. T., Meckes, B., Camp, S. M., Garcia, J. G., Dudek, S. M., & Lal, R. (2013). Heterogeneous elastic response of human lung microvascular endothelial cells to barrier modulating stimuli. Nanomedicine : nanotechnology, biology, and medicine, 9(7), 875-84.
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  In this study we employ atomic force microscopy, supported by finite element analysis and fluorescence microscopy, to characterize the elastic properties accompanying cytoskeletal structural rearrangements of lung microvascular endothelial cells in response to barrier altering stimuli. Statistical analysis of elasticity data obtained from multiple cells demonstrates a heterogeneous cellular elastic response to barrier-enhancing and barrier-disrupting agents; sphingosine 1-phosphate (S1P) and thrombin, respectively. A small but detectable (10%) increase in the average elastic modulus of all cells is observed for S1P, which is accompanied by a corresponding significant decrease in cell thickness. Variable effects of thrombin on these parameters were observed. To account for possible substrate effects in our elasticity analysis, we analyzed only the low-force sections of the force-displacement curves and utilized a finite-thickness correction to the Hertzian model. Our finite element analysis results substantiate this approach. The heterogeneous elastic behavior correlates with differential cytoskeletal rearrangements observed with fluorescence microscopy.
• Bress, A., Han, J., Patel, S. R., Desai, A. A., Mansour, I., Groo, V., Progar, K., Shah, E., Stamos, T. D., Wing, C., Garcia, J. G., Kittles, R., & Cavallari, L. H. (2013). Association of aldosterone synthase polymorphism (CYP11B2 -344T>C) and genetic ancestry with atrial fibrillation and serum aldosterone in African Americans with heart failure. PloS one, 8(7), e71268.
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  The objective of this study was to examine the extent to which aldosterone synthase genotype (CYP11B2) and genetic ancestry correlate with atrial fibrillation (AF) and serum aldosterone in African Americans with heart failure. Clinical data, echocardiographic measurements, and a genetic sample for determination of CYP11B2 -344T>C (rs1799998) genotype and genetic ancestry were collected from 194 self-reported African Americans with chronic, ambulatory heart failure. Genetic ancestry was determined using 105 autosomal ancestry informative markers. In a sub-set of patients (n = 126), serum was also collected for determination of circulating aldosterone. The CYP11B2 -344C allele frequency was 18% among the study population, and 19% of patients had AF. Multiple logistic regression revealed that the CYP11B2 -344CC genotype was a significant independent predictor of AF (OR 12.7, 95% CI 1.60-98.4, p = 0.0150, empirical p = 0.011) while holding multiple clinical factors, left atrial size, and percent European ancestry constant. Serum aldosterone was significantly higher among patients with AF (p = 0.036), whereas increased West African ancestry was inversely correlated with serum aldosterone (r = -0.19, p = 0.037). The CYP11B2 -344CC genotype was also overrepresented among patients with extreme aldosterone elevation (≥90th percentile, p = 0.0145). In this cohort of African Americans with chronic ambulatory heart failure, the CYP11B2 -344T>C genotype was a significant independent predictor of AF while holding clinical, echocardiographic predictors, and genetic ancestry constant. In addition, increased West African ancestry was associated with decreased serum aldosterone levels, potentially providing an explanation for the lower risk for AF observed among African Americans.
• Chen, J., Feng, G., Guo, Q., Wardenburg, J. B., Lin, S., Inoshima, I., Deaton, R., Yuan, J. X., Garcia, J. G., Machado, R. F., Otto, M., & Wunderink, R. G. (2013). Transcriptional events during the recovery from MRSA lung infection: a mouse pneumonia model. PloS one, 8(8), e70176.
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  Community associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an emerging threat to human health throughout the world. Rodent MRSA pneumonia models mainly focus on the early innate immune responses to MRSA lung infection. However, the molecular pattern and mechanisms of recovery from MRSA lung infection are largely unknown. In this study, a sublethal mouse MRSA pneumonia model was employed to investigate late events during the recovery from MRSA lung infection. We compared lung bacterial clearance, bronchoalveolar lavage fluid (BALF) characterization, lung histology, lung cell proliferation, lung vascular permeability and lung gene expression profiling between days 1 and 3 post MRSA lung infection. Compared to day 1 post infection, bacterial colony counts, BALF total cell number and BALF protein concentration significantly decreased at day 3 post infection. Lung cDNA microarray analysis identified 47 significantly up-regulated and 35 down-regulated genes (p
• Desai, A. A., Casanova, N. G., Zhang, X., Zhang, W., Xu, M., Steinberg, M. H., Sergueeva, A. I., Sebastiani, P., Prchal, J. T., Nekhai, S. A., Miasnikova, G. Y., Machado, R. F., Ma, S., Kittles, R. A., Gordeuk, V. R., Garcia, J. G., Desai, A. A., Casanova, N. G., Baldwin, C. T., , Ammosova, T., et al. (2013). Genetic Association Of a MAPK8 Expression Quantitative Trait Locus With Pre-Capillary Pulmonary Hypertension In Sickle Cell Disease. Blood, 122(21), 991-991. doi:10.1182/blood.v122.21.991.991
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  Sickle cell disease (SCD) is associated with pleiotropic clinical outcomes, the severity of which exhibits remarkable inter-individual variability. Investigations of the pathophysiology of SCD have focused on the adverse effects of vaso-occlusion, chronic inflammation, and hemolysis. SCD is also characterized by up-regulation of the hypoxic response to chronic anemia. We postulated that the up-regulated hypoxic response in SCD contributes to altered gene expression that might impact pulmonary hypertension, a complication associated with early mortality. To identify genes regulated by the hypoxic response and not other effects of chronic anemia, we compared expression variation in peripheral blood mononuclear cells from 13 sickle cell anemia untreated with hydroxyurea and 15 Chuvash polycythemia (CP) patients characterized by homozygous VHL R200W -induced constitutive up-regulation of hypoxia inducible factors in the absence of anemia or hypoxia. Gene expression of both cohorts was profiled on identical Affymetrix exon arrays. The degree and direction of differential gene expression were highly correlated between sickle cell anemia and CP (Spearman’s ρ = 0.73 between regression coefficients of differential gene expression), suggesting that 53% of expression variation in sickle cell anemia is related to hypoxic transcriptional responses. At 5% false discovery rate (FDR), 1040 genes exhibited a >1.15 fold change in both sickle cell anemia and VHL R200W homozygotes, among which 297 were up-regulated and 743 down-regulated. Hypoxia strongly induced inflammatory response pathways but suppressed T-cell activation in sickle cell anemia. MAPK8 , encoding a mitogen-activated protein kinase important for stress-induced apoptosis, T-cell differentiation and inflammatory responses, was a hypoxia down-regulated gene and played a central role in hypoxic gene regulation in sickle cell anemia according to gene network analysis. To assess the genetic contribution to hypoxic transcriptional variation among SCD patients, we mapped expression quantitative trait loci (eQTL) for the 1,040 hypoxia response genes. Association mapping with a focus on local regulatory polymorphisms in 61 SCD patients identified eQTL for 103 of the hypoxia response genes at 5% FDR. We further tested the hypothesis that these hypoxic eQTL potentially underlie heterogeneity in risk of pulmonary hypertension in an additional SCD cohort (University of Illinois cohort). In this cohort, the A allele of an eQTL of MAPK8 , rs10857560, was associated with pre-capillary pulmonary hypertension defined as mean pulmonary artery pressure ≥25 and pulmonary capillary wedge pressure ≤15 mm Hg at right heart catheterization (allele frequency=0.66; OR=4.4, P=0.00037, n=238). This association was confirmed in another independent cohort (Walk-PHaSST cohort) (allele frequency=0.65; OR=7.2, P=0.0025, n=519). The homozygous AA genotype of rs10857560, which was associated with decreased MAPK8 expression, was present in all 14 identified pre-capillary pulmonary hypertension cases among the combined 757 SCD patients (P = 6 x 10-6 by the Fisher exact test). Our study demonstrates a prominent hypoxic transcription component in SCD that in part contributes to pre-capillary pulmonary hypertension. Disclosures: No relevant conflicts of interest to declare.
• Ephstein, Y., Singleton, P. A., Chen, W., Wang, L., Salgia, R., Kanteti, P., Dudek, S. M., Garcia, J. G., & Jacobson, J. R. (2013). Critical role of S1PR1 and integrin β4 in HGF/c-Met-mediated increases in vascular integrity. The Journal of biological chemistry, 288(4), 2191-200.
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  Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met. We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin β4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.
• Garcia, J. G. (2013). Erratum: Simvastatin attenuates radiation-induced murine lung injury and dysregulated lung gene expression (American Journal of Respiratory Cell and Molecular Biology (2011) 44 (415-422)). American Journal of Respiratory Cell and Molecular Biology, 49(4), 688-688. doi:10.1165/rcmb.2013.494688
• Garcia, J. G. (2013). Erratum: Type 2 Deiodinase and Host Responses of Sepsis and Acute Lung Injury. American Journal of Respiratory Cell and Molecular Biology, 49(4), 689-689. doi:10.1165/rcmb.2013.494689
• Garcia, J. G., & Sznajder, J. I. (2013). Healthcare disparities in patients with acute respiratory distress syndrome. Toward equity. American journal of respiratory and critical care medicine, 188(6), 631-2.
• Herazo-Maya, J. D., Noth, I., Duncan, S. R., Kim, S., Ma, S. F., Tseng, G. C., Feingold, E., Juan-Guardela, B. M., Richards, T. J., Lussier, Y., Huang, Y., Vij, R., Lindell, K. O., Xue, J., Gibson, K. F., Shapiro, S. D., Garcia, J. G., & Kaminski, N. (2013). Peripheral blood mononuclear cell gene expression profiles predict poor outcome in idiopathic pulmonary fibrosis. Science translational medicine, 5(205), 205ra136.
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  We aimed to identify peripheral blood mononuclear cell (PBMC) gene expression profiles predictive of poor outcomes in idiopathic pulmonary fibrosis (IPF) by performing microarray experiments of PBMCs in discovery and replication cohorts of IPF patients. Microarray analyses identified 52 genes associated with transplant-free survival (TFS) in the discovery cohort. Clustering the microarray samples of the replication cohort using the 52-gene outcome-predictive signature distinguished two patient groups with significant differences in TFS. We studied the pathways associated with TFS in each independent microarray cohort and identified decreased expression of "The costimulatory signal during T cell activation" Biocarta pathway and, in particular, the genes CD28, ICOS, LCK, and ITK, results confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A proportional hazards model, including the qRT-PCR expression of CD28, ICOS, LCK, and ITK along with patient's age, gender, and percent predicted forced vital capacity (FVC%), demonstrated an area under the receiver operating characteristic curve of 78.5% at 2.4 months for death and lung transplant prediction in the replication cohort. To evaluate the potential cellular source of CD28, ICOS, LCK, and ITK expression, we analyzed and found significant correlation of these genes with the PBMC percentage of CD4(+)CD28(+) T cells in the replication cohort. Our results suggest that CD28, ICOS, LCK, and ITK are potential outcome biomarkers in IPF and should be further evaluated for patient prioritization for lung transplantation and stratification in drug studies.
• Huang, L. S., Berdyshev, E., Mathew, B., Fu, P., Gorshkova, I. A., He, D., Ma, W., Noth, I., Ma, S. F., Pendyala, S., Reddy, S. P., Zhou, T., Zhang, W., Garzon, S. A., Garcia, J. G., & Natarajan, V. (2013). Targeting sphingosine kinase 1 attenuates bleomycin-induced pulmonary fibrosis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 27(4), 1749-60.
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  Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease, wherein transforming growth factor β (TGF-β) and sphingosine-1-phosphate (S1P) contribute to the pathogenesis of fibrosis. However, the in vivo contribution of sphingosine kinase (SphK) in fibrotic processes has not been documented. Microarray analysis of blood mononuclear cells from patients with IPF and SphK1- or SphK2-knockdown mice and SphK inhibitor were used to assess the role of SphKs in fibrogenesis. The expression of SphK1/2 negatively correlated with lung function and survival in patients with IPF. Also, the expression of SphK1 was increased in lung tissues from patients with IPF and bleomycin-challenged mice. Knockdown of SphK1, but not SphK2, increased survival and resistance to pulmonary fibrosis in bleomycin-challenged mice. Administration of SphK inhibitor reduced bleomycin-induced mortality and pulmonary fibrosis in mice. Knockdown of SphK1 or treatment with SphK inhibitor attenuated S1P generation and TGF-β secretion in a bleomycin-induced lung fibrosis mouse model that was accompanied by reduced phosphorylation of Smad2 and MAPKs in lung tissue. In vitro, bleomycin-induced expression of SphK1 in lung fibroblast was found to be TGF-β dependent. Taken together, these data indicate that SphK1 plays a critical role in the pathology of lung fibrosis and is a novel therapeutic target.
• Huang, L. S., Fu, P., Patel, P., Harijith, A., Sun, T., Zhao, Y., Garcia, J. G., Chun, J., & Natarajan, V. (2013). Lysophosphatidic acid receptor-2 deficiency confers protection against bleomycin-induced lung injury and fibrosis in mice. American journal of respiratory cell and molecular biology, 49(6), 912-22.
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  Idiopathic pulmonary fibrosis is a devastating disease characterized by alveolar epithelial cell injury, the accumulation of fibroblasts/myofibroblasts, and the deposition of extracellular matrix proteins. Lysophosphatidic acid (LPA) signaling through its G protein-coupled receptors is critical for its various biological functions. Recently, LPA and LPA receptor 1 were implicated in lung fibrogenesis. However, the role of other LPA receptors in fibrosis remains unclear. Here, we use a bleomycin-induced pulmonary fibrosis model to investigate the roles of LPA2 in pulmonary fibrogenesis. In the present study, we found that LPA2 knockout (Lpar2(-/-)) mice were protected against bleomycin-induced lung injury, fibrosis, and mortality, compared with wild-type control mice. Furthermore, LPA2 deficiency attenuated the bleomycin-induced expression of fibronectin (FN), α-smooth muscle actin (α-SMA), and collagen in lung tissue, as well as levels of IL-6, transforming growth factor-β (TGF-β), and total protein in bronchoalveolar lavage fluid. In human lung fibroblasts, the knockdown of LPA2 attenuated the LPA-induced expression of TGF-β1 and the differentiation of lung fibroblasts to myofibroblasts, resulting in the decreased expression of FN, α-SMA, and collagen, as well as decreased activation of extracellular regulated kinase 1/2, Akt, Smad3, and p38 mitogen-activated protein kinase. Moreover, the knockdown of LPA2 with small interfering RNA also mitigated the TGF-β1-induced differentiation of lung fibroblasts. In addition, LPA2 deficiency significantly attenuated the bleomycin-induced apoptosis of alveolar and bronchial epithelial cells in the mouse lung. Together, our data indicate that the knockdown of LPA2 attenuated bleomycin-induced lung injury and pulmonary fibrosis, and this may be related to an inhibition of the LPA-induced expression of TGF-β and the activation and differentiation of fibroblasts.
• Liu, B., Sun, X., Suyeoka, G., Garcia, J. G., & Leiderman, Y. I. (2013). TGFβ signaling induces expression of Gadd45b in retinal ganglion cells. Investigative ophthalmology & visual science, 54(2), 1061-9.
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  Growth arrest and DNA damage protein 45b (Gadd45b) functions as an intrinsic neuroprotective molecule protecting retinal ganglion cells (RGCs) from injury. This study was performed to elucidate further the induction pathway of Gadd45b expression in RGCs.
• Man, M., Close, S. L., Shaw, A. D., Bernard, G. R., Douglas, I. S., Kaner, R. J., Payen, D., Vincent, J. L., Fossceco, S., Janes, J. M., Leishman, A. G., O'Brien, L., Williams, M. D., & Garcia, J. G. (2013). Beyond single-marker analyses: mining whole genome scans for insights into treatment responses in severe sepsis. The pharmacogenomics journal, 13(3), 218-26.
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  Management of severe sepsis, an acute illness with high morbidity and mortality, suffers from the lack of effective biomarkers and largely empirical predictions of disease progression and therapeutic responses. We conducted a genome-wide association study using a large randomized clinical trial cohort to discover genetic biomarkers of response to therapy and prognosis utilizing novel approaches, including combination markers, to overcome limitations of single-marker analyses. Sepsis prognostic models were dominated by clinical variables with genetic markers less informative. In contrast, evidence for gene-gene interactions were identified for sepsis treatment responses with genetic biomarkers dominating models for predicting therapeutic responses, yielding candidates for replication in other cohorts.
• Mathew, B., Jacobson, J. R., Siegler, J. H., Moitra, J., Blasco, M., Xie, L., Unzueta, C., Zhou, T., Evenoski, C., Al-Sakka, M., Sharma, R., Huey, B., Bulent, A., Smith, B., Jayaraman, S., Reddy, N. M., Reddy, S. P., Fingerle-Rowson, G., Bucala, R., , Dudek, S. M., et al. (2013). Role of migratory inhibition factor in age-related susceptibility to radiation lung injury via NF-E2-related factor-2 and antioxidant regulation. American journal of respiratory cell and molecular biology, 49(2), 269-78.
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  Microvascular injury and increased vascular leakage are prominent features of radiation-induced lung injury (RILI), and often follow cancer-associated thoracic irradiation. Our previous studies demonstrated that polymorphisms in the gene (MIF) encoding macrophage migratory inhibition factor (MIF), a multifunctional pleiotropic cytokine, confer susceptibility to acute inflammatory lung injury and increased vascular permeability, particularly in senescent mice. In this study, we exposed wild-type and genetically engineered mif(-/-) mice to 20 Gy single-fraction thoracic radiation to investigate the age-related role of MIF in murine RILI (mice were aged 8 wk, 8 mo, or 16 mo). Relative to 8-week-old mice, decreased MIF was observed in bronchoalveolar lavage fluid and lung tissue of 8- to 16-month-old wild-type mice. In addition, radiated 8- to 16-month-old mif(-/-) mice exhibited significantly decreased bronchoalveolar lavage fluid total antioxidant concentrations with progressive age-related decreases in the nuclear expression of NF-E2-related factor-2 (Nrf2), a transcription factor involved in antioxidant gene up-regulation in response to reactive oxygen species. This was accompanied by decreases in both protein concentrations (NQO1, GCLC, and heme oxygenase-1) and mRNA concentrations (Gpx1, Prdx1, and Txn1) of Nrf2-influenced antioxidant gene targets. In addition, MIF-silenced (short, interfering RNA) human lung endothelial cells failed to express Nrf2 after oxidative (H2O2) challenge, an effect reversed by recombinant MIF administration. However, treatment with an antioxidant (glutathione reduced ester), but not an Nrf2 substrate (N-acetyl cysteine), protected aged mif(-/-) mice from RILI. These findings implicate an important role for MIF in radiation-induced changes in lung-cell antioxidant concentrations via Nrf2, and suggest that MIF may contribute to age-related susceptibility to thoracic radiation.
• Natarajan, V., Dudek, S. M., Jacobson, J. R., Moreno-Vinasco, L., Huang, L. S., Abassi, T., Mathew, B., Zhao, Y., Wang, L., Bittman, R., Weichselbaum, R., Berdyshev, E., & Garcia, J. G. (2013). Sphingosine-1-phosphate, FTY720, and sphingosine-1-phosphate receptors in the pathobiology of acute lung injury. American journal of respiratory cell and molecular biology, 49(1), 6-17.
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  Acute lung injury (ALI) attributable to sepsis or mechanical ventilation and subacute lung injury because of ionizing radiation (RILI) share profound increases in vascular permeability as a key element and a common pathway driving increased morbidity and mortality. Unfortunately, despite advances in the understanding of lung pathophysiology, specific therapies do not yet exist for the treatment of ALI or RILI, or for the alleviation of unremitting pulmonary leakage, which serves as a defining feature of the illness. A critical need exists for new mechanistic insights that can lead to novel strategies, biomarkers, and therapies to reduce lung injury. Sphingosine 1-phosphate (S1P) is a naturally occurring bioactive sphingolipid that acts extracellularly via its G protein-coupled S1P1-5 as well as intracellularly on various targets. S1P-mediated cellular responses are regulated by the synthesis of S1P, catalyzed by sphingosine kinases 1 and 2, and by the degradation of S1P mediated by lipid phosphate phosphatases, S1P phosphatases, and S1P lyase. We and others have demonstrated that S1P is a potent angiogenic factor that enhances lung endothelial cell integrity and an inhibitor of vascular permeability and alveolar flooding in preclinical animal models of ALI. In addition to S1P, S1P analogues such as 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720), FTY720 phosphate, and FTY720 phosphonates offer therapeutic potential in murine models of lung injury. This translational review summarizes the roles of S1P, S1P analogues, S1P-metabolizing enzymes, and S1P receptors in the pathophysiology of lung injury, with particular emphasis on the development of potential novel biomarkers and S1P-based therapies for ALI and RILI.
• Noth, I., Zhang, Y., Ma, S. F., Flores, C., Barber, M., Huang, Y., Broderick, S. M., Wade, M. S., Hysi, P., Scuirba, J., Richards, T. J., Juan-Guardela, B. M., Vij, R., Han, M. K., Martinez, F. J., Kossen, K., Seiwert, S. D., Christie, J. D., Nicolae, D., , Kaminski, N., et al. (2013). Genetic variants associated with idiopathic pulmonary fibrosis susceptibility and mortality: a genome-wide association study. The Lancet. Respiratory medicine, 1(4), 309-317.
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  Idiopathic pulmonary fibrosis (IPF) is a devastating disease that probably involves several genetic loci. Several rare genetic variants and one common single nucleotide polymorphism (SNP) of MUC5B have been associated with the disease. Our aim was to identify additional common variants associated with susceptibility and ultimately mortality in IPF.
• Nutescu, E. A., Drozda, K., Bress, A. P., Galanter, W. L., Stevenson, J., Stamos, T. D., Desai, A. A., Duarte, J. D., Gordeuk, V., Peace, D., Kadkol, S. S., Dodge, C., Saraf, S., Garofalo, J., Krishnan, J. A., Garcia, J. G., & Cavallari, L. H. (2013). Feasibility of implementing a comprehensive warfarin pharmacogenetics service. Pharmacotherapy, 33(11), 1156-64.
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  To determine the procedural feasibility of a pharmacist-led interdisciplinary service for providing genotype-guided warfarin dosing for hospitalized patients newly starting warfarin.
• Ortiz, H., Codina, A., & , e. r. (2013). [The Spanish Association of Surgeon's audited teaching programme for rectal cancer. Results after six years]. Cirugia espanola, 91(8), 496-503.
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  The Spanish Rectal Cancer Project of the Spanish Association of Surgeons was established in 2006. The main objective of this observational study was to assess the results obtained by the hospitals trained in the period 2006-2011, in order to evaluate whether this initiative has allowed acheivement of the observed quality standards in the Norwegian Rectal Cancer Project.
• Peljto, A. L., Zhang, Y., Fingerlin, T. E., Ma, S. F., Garcia, J. G., Richards, T. J., Silveira, L. J., Lindell, K. O., Steele, M. P., Loyd, J. E., Gibson, K. F., Seibold, M. A., Brown, K. K., Talbert, J. L., Markin, C., Kossen, K., Seiwert, S. D., Murphy, E., Noth, I., , Schwarz, M. I., et al. (2013). Association between the MUC5B promoter polymorphism and survival in patients with idiopathic pulmonary fibrosis. JAMA, 309(21), 2232-9.
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  Current prediction models of mortality in idiopathic pulmonary fibrosis (IPF), which are based on clinical and physiological parameters, have modest value in predicting which patients will progress. In addition to the potential for improving prognostic models, identifying genetic and molecular features that are associated with IPF mortality may provide insight into the underlying mechanisms of disease and inform clinical trials.
• Pillai, V. B., Sundaresan, N. R., Kim, G., Samant, S., Moreno-Vinasco, L., Garcia, J. G., & Gupta, M. P. (2013). Nampt secreted from cardiomyocytes promotes development of cardiac hypertrophy and adverse ventricular remodeling. American journal of physiology. Heart and circulatory physiology, 304(3), H415-26.
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  Nicotinamide phosphoribosyltransferase (Nampt) is an important coenzyme involved in cellular redox reactions. Inside the cell, Nampt (iNampt) functions as a rate-limiting enzyme in the NAD salvage pathway, and outside the cell (eNampt), it acts as a proinflammatory cytokine. High-circulating levels of Nampt are reported in different pathological conditions. This study was designed to examine the role of Nampt in the development of cardiac hypertrophy and ventricular remodeling. We studied the hypertrophic response in Nampt heterozygous (+/-) knockout and cardiac-specific overexpressing Nampt transgenic mice. Whereas Nampt(+/-) mice were protected against agonist (isoproterenol and angiotensin II)-induced hypertrophy, Nampt transgenic mice spontaneously developed cardiac hypertrophy at 6 mo of age. Experiments conducted to gain insight into the mechanism revealed that treatment of cardiomyocytes with recombinant (eNampt) or overexpression with Nampt-synthesizing adenovirus vector (Ad.Nampt) induced cardiomyocyte hypertrophy. The prohypertrophic effects of eNampt and Ad.Nampt were blocked by the addition of a Nampt-blocking antibody into cultures, thus suggesting that Nampt was in fact invoking hypertrophic response of cardiomyocytes by acting on the cell surface receptors. We also found increased Nampt levels in the supernatant of cardiomyocyte cultures subjected to stress by either serum starvation or H(2)O(2) treatment. Exploration of signaling pathways in Nampt-induced cardiac hypertrophy and fibrosis revealed increased activation of mitogen-activated protein kinases, namely, JNK1, p38, and ERK. This was also associated with increased calcineurin levels and nuclear factor of activated T-cell localization into the nucleus. From these studies we conclude that cardiomyocytes are capable of secreting Nampt during stress, and exogenous Nampt is a positive regulator of cardiac hypertrophy and adverse ventricular remodeling.
• Sun, X., Ma, S. F., Wade, M. S., Acosta-Herrera, M., Villar, J., Pino-Yanes, M., Zhou, T., Liu, B., Belvitch, P., Moitra, J., Han, Y. J., Machado, R., Noth, I., Natarajan, V., Dudek, S. M., Jacobson, J. R., Flores, C., & Garcia, J. G. (2013). Functional promoter variants in sphingosine 1-phosphate receptor 3 associate with susceptibility to sepsis-associated acute respiratory distress syndrome. American journal of physiology. Lung cellular and molecular physiology, 305(7), L467-77.
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  The genetic mechanisms underlying the susceptibility to acute respiratory distress syndrome (ARDS) are poorly understood. We previously demonstrated that sphingosine 1-phosphate (S1P) and the S1P receptor S1PR3 are intimately involved in lung inflammatory responses and vascular barrier regulation. Furthermore, plasma S1PR3 protein levels were shown to serve as a biomarker of severity in critically ill ARDS patients. This study explores the contribution of single nucleotide polymorphisms (SNPs) of the S1PR3 gene to sepsis-associated ARDS. S1PR3 SNPs were identified by sequencing the entire gene and tagging SNPs selected for case-control association analysis in African- and ED samples from Chicago, with independent replication in a European case-control study of Spanish individuals. Electrophoretic mobility shift assays, luciferase activity assays, and protein immunoassays were utilized to assess the functionality of associated SNPs. A total of 80 variants, including 29 novel SNPs, were identified. Because of limited sample size, conclusive findings could not be drawn in African-descent ARDS subjects; however, significant associations were found for two promoter SNPs (rs7022797 -1899T/G; rs11137480 -1785G/C), across two ED samples supporting the association of alleles -1899G and -1785C with decreased risk for sepsis-associated ARDS. In addition, these alleles significantly reduced transcription factor binding to the S1PR3 promoter; reduced S1PR3 promoter activity, a response particularly striking after TNF-α challenge; and were associated with lower plasma S1PR3 protein levels in ARDS patients. These highly functional studies support S1PR3 as a novel ARDS candidate gene and a potential target for individualized therapy.
• Sysol, J. R., Kempf, C., Helton, M. N., Dong, Y., Zhu, D., Sun, H., Garcia, J. G., Machado, R. F., & Chen, J. (2013). Evaluation of a reliable and cost-effective method of DNA isolation for mouse genotyping. Biotechnology letters, 35(4), 509-14.
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  Genotyping is commonly used to define specific gene alterations or the presence of transgenes in mice. This procedure is typically done using DNA isolated from mouse tail tissue. Although there are commercially available kits for tail DNA isolation, they can be time consuming and costly for routine genotyping. In this study, we describe a rapid, "crude" DNA isolation method using mouse tail tissue and compare it to a frequently used, commercially available kit in the genotyping of over 1,000 total mice from 8 genetic lines. Our genotyping results were obtained faster and less expensively but with the same success rate (Crude method: 97.7 %, Kit method: 98.4 %). To our knowledge, this is the first systematic study to compare the reliability of this crude DNA isolation method for mouse genotyping compared to a commercially available kit.
• Tauler, J., Rizzo, A. N., Garcia, J. G., & Borowicz, S. J. (2013). A potential role for myosin light chain kinase in lung adenocarcinoma tumorigenesis.. Journal of Clinical Oncology, 31(15_suppl), e22051-e22051. doi:10.1200/jco.2013.31.15_suppl.e22051
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  e22051 Background: Treatment of non-small cell lung cancer remains a clinical challenge despite the migration from cytotoxic chemotherapies to targeted agents. Myosin light chain kinase (MLCK) is a...
• Usatyuk, P. V., Burns, M., Mohan, V., Pendyala, S., He, D., Ebenezer, D. L., Harijith, A., Fu, P., Huang, L. S., Bear, J. E., Garcia, J. G., & Natarajan, V. (2013). Coronin 1B regulates S1P-induced human lung endothelial cell chemotaxis: role of PLD2, protein kinase C and Rac1 signal transduction. PloS one, 8(5), e63007.
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  Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs) with the bioactive lipid, sphingosine-1-phosphate (S1P) rapidly stimulates coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA) targeting coronin 1B (∼36%), PLD2 (∼45%) or Rac1 (∼50%) compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, ε and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis.
• Zhang, W., Rondelli, D., Patel, P. R., Mahmud, D., & Garcia, J. G. (2013). Genomic Signature Predicts Resistance To Busulfan In AML Cell Lines. Blood, 122(21), 3850-3850. doi:10.1182/blood.v122.21.3850.3850
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  The alkylating agent Busulfan (Bu) is commonly used in the pre-transplant conditioning regimen for patients with acute myeloid leukemia (AML). Relapse after transplant is in part due to resistance to Bu. Our study was aimed at identifying a genomic signature that could predict resistance to Bu in AML cells. In order to obtain a list of candidate genes which may correlate with increasing resistance to Bu we performed a linear regression analysis controlled for cancer type on NCI60 Stanford cDNA data and GI50 values. This produced a list of 111 genes correlating with increasing Busulfan GI50 (p
• Zhang, W., Szilagyi, K. L., & Garcia, J. G. (2013). Exploring DNA Methylation of MYLK as a Contributor to Acute Respiratory Distress Syndrome Disparities. Journal of Pulmonary and Respiratory Medicine, 03(04). doi:10.4172/2161-105x.1000e127
• Zhang, W., Zhou, T., Ma, S. F., Machado, R. F., Bhorade, S. M., & Garcia, J. G. (2013). MicroRNAs Implicated in Dysregulation of Gene Expression Following Human Lung Transplantation. Translational respiratory medicine, 1(1).
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  Lung transplantation remains the only viable treatment option for the majority of patients with advanced lung diseases. However, 5-year post-transplant survival rates remain low primarily secondary to chronic rejection. Novel insights from global gene expression profiles may provide molecular phenotypes and therapeutic targets to improve outcomes after lung transplantation.
• Zhou, T., Zhou, T., Wang, T., Sun, X., Shimizu, Y., & Garcia, J. G. (2013). Abstract 18761: Non-Muscle Myosin Light Chain Kinase Mediates Vascular Endothelial Growth Factor Induced Vascular Hyperpermeability via Specificity Protein 1 During Asthmatic Inflammation. Circulation, 128.
• Ahmad, H., Gayat, E., Yodwut, C., Abduch, M. C., Patel, A. R., Weinert, L., Desai, A., Tsang, W., Garcia, J. G., Lang, R. M., & Mor-Avi, V. (2012). Evaluation of myocardial deformation in patients with sickle cell disease and preserved ejection fraction using three-dimensional speckle tracking echocardiography. Echocardiography (Mount Kisco, N.Y.), 29(8), 962-9.
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  Sickle cell disease (SCD) is a hemoglobinopathy that affects one in 500 African Americans. Although it is well established that patients with SCD have left ventricular (LV) diastolic dysfunction, it is not clear whether they have subtle LV systolic dysfunction despite preserved ejection fraction (EF). We used three-dimensional speckle tracking echocardiography (3DSTE) to assess changes in both systolic and diastolic LV function in SCD.
• Ashley, E. A., Hershberger, R. E., Caleshu, C., Ellinor, P. T., Garcia, J. G., Herrington, D. M., Ho, C. Y., Johnson, J. A., Kittner, S. J., Macrae, C. A., Mudd-Martin, G., Rader, D. J., Roden, D. M., Scholes, D., Sellke, F. W., Towbin, J. A., Van Eyk, J., Worrall, B. B., & , A. H. (2012). Genetics and cardiovascular disease: a policy statement from the American Heart Association. Circulation, 126(1), 142-57.
• Brown, M. E., Shekhawat, G. S., Mustata, G. M., Ishikawa, Y., Garcia, J. G., Dudek, S. M., Dravid, V. P., Brown, M. E., & Avasthy, S. (2012). Sub-cellular Dynamic Mechanical Response Measurements of Live Human Pulmonary Artery Endothelial Cells with Atomic Force Microscopy. Microscopy and Microanalysis, 18(S2), 1622-1623. doi:10.1017/s1431927612009968
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  Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.
• Calfee, C. S., Gallagher, D., Abbott, J., Thompson, B. T., Matthay, M. A., & , N. A. (2012). Plasma angiopoietin-2 in clinical acute lung injury: prognostic and pathogenetic significance. Critical care medicine, 40(6), 1731-7.
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  Angiopoietin-2 is a proinflammatory mediator of endothelial injury in animal models, and increased plasma angiopoietin-2 levels are associated with poor outcomes in patients with sepsis-associated acute lung injury. Whether angiopoietin-2 levels are modified by treatment strategies in patients with acute lung injury is unknown.
• Chen, W., Sammani, S., Mitra, S., Ma, S. F., Garcia, J. G., & Jacobson, J. R. (2012). Critical role for integrin-β4 in the attenuation of murine acute lung injury by simvastatin. American journal of physiology. Lung cellular and molecular physiology, 303(4), L279-85.
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  The statins are a class of 3-hydroxy-3-methylglutaryl-coenzyme A-reductase inhibitors that are recognized to have pleiotropic properties. We previously reported the attenuation of LPS-induced murine acute lung injury (ALI) by simvastatin in vivo and identified relevant effects of simvastatin on endothelial cell (EC) signaling, activation, and barrier function in vitro. In particular, simvastatin induces the upregulation of integrin-β4, which in turn inhibits EC inflammatory responses via attenuation of MAPK signaling. The role of integrin-β4 in murine ALI protection by simvastatin, however, is unknown. We initially confirmed a time- and dose-dependent effect of simvastatin on increased integrin-β4 mRNA expression in human lung EC with peak protein expression evident at 16 h. Subsequently, reciprocal immunoprecipitation demonstrated an attenuation of LPS-induced integrin-β4 tyrosine phosphorylation by simvastatin (5 μM, 16 h). Increased expression of EC inflammatory cytokines [IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, regulated on activation normal T cell expressed and secreted (RANTES)] by LPS (500 ng/ml, 4 h) was also significantly attenuated by simvastatin pretreatment (5 μM, 16 h), but this effect was reversed by cotreatment with an integrin-β4-blocking antibody. Finally, although simvastatin (20 mg/kg) conferred significant protection in murine ALI as evidenced by decreased bronchoalveolar lavage fluid cell counts, protein, inflammatory cytokines (IL-6, IL-1β, MCP-1, RANTES), decreased Evans blue dye albumin extravasation in lung tissue, and changes on lung histology, these effects were reversed by the integrin-β4-blocking antibody (IV, 1 mg/kg, 2 h before LPS). These findings support integrin-β4 as an important mediator of ALI protection by simvastatin and implicate signaling by integrin-β4 as a novel therapeutic target in patients with ALI.
• Desai, A. A., Yodwut, C., Weinert, L., Turner, K. M., Trevino, S., Thiruvoipati, T., Patel, A. R., Mor-avi, V., Machado, R., Lang, R. M., Garcia, J. G., Desai, A. A., Czobor, P., Artz, N., & Ahmad, H. (2012). Mechanistic insights and characterization of cardiomyopathy due to Sickle Cell Disease. Journal of Cardiovascular Magnetic Resonance, 14(1), 1-2. doi:10.1186/1532-429x-14-s1-o78
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  Summary We sought to characterize the features of sickle cell cardiomyopathy and to identify causative mechanisms using comprehensive cardiac magnetic resonance, echocardiography, and arterial tonometry. We found that sickle cell cardiomyopathy is characterized by 4-chamber dilation, myocardial fibrosis, abnormal myocardial perfusion reserve, diastolic dysfunction, and only rarely myocardial iron overload. Left ventricular dilation and myocardial fibrosis are associated with increased blood transfusion requirements; where as, diastolic dysfunction is due to increased aortic stiffness. Background Cardiovascular disease is an important cause of morbidity and mortality in adults with sickle cell disease (SCD). We sought to characterize the features of SCD cardiomyopathy and to identify causative mechanisms. Methods Stable adults with SCD (n=38) and matched controls (n=13) were prospectively recruited to undergo 1) multiparametric cardiovascular magnetic resonance (CMR) (i. e. steady state free precession cine, regadenoson first pass myocardial perfusion, phase sensitive inversion recovery late gadolinium enhancement (LGE), and myocardial and hepatic multi-echo time (TE) single breathhold T2* imaging), 2) echocardiography (TTE), and 3) applanation tonometry (pulse wave analysis). Chamber size and function were measured from CMR using method of disks. Myocardial perfusion reserve index (MPRi) was calculated from time intensity curves generated from first pass perfusion images as the stress to rest ratio of mid-ventricular myocardium upslope (normalized by the left ventricular cavity upslope). LGE was considered present if the signal intensity (SI) was >5 standard deviations above normal remote myocardium and if seen in 2 consecutive slices or 2 imaging planes. Myocardial and hepatic T2* times were calculated using the formula ΔTE/ ln(SI TE2/SI TE1). Presence of diastolic dysfunction (DD) was determined using American Society of Echocardiography criteria. Aortic augmentation index was determined using standard tonometry methods.
• Desai, A. A., Zhou, T., Ahmad, H., Zhang, W., Mu, W., Trevino, S., Wade, M. S., Raghavachari, N., Kato, G. J., Peters-Lawrence, M. H., Thiruvoipati, T., Turner, K., Artz, N., Huang, Y., Patel, A. R., Yuan, J. X., Gordeuk, V. R., Lang, R. M., Garcia, J. G., & Machado, R. F. (2012). A novel molecular signature for elevated tricuspid regurgitation velocity in sickle cell disease. American journal of respiratory and critical care medicine, 186(4), 359-68.
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  An increased tricuspid regurgitation jet velocity (TRV > 2.5 m/s) and pulmonary hypertension defined by right heart catheterization both independently confer increased mortality in sickle cell disease (SCD).
• Flores, C., Ma, S. F., Pino-Yanes, M., Wade, M. S., Pérez-Méndez, L., Kittles, R. A., Wang, D., Papaiahgari, S., Ford, J. G., Kumar, R., & Garcia, J. G. (2012). African ancestry is associated with asthma risk in African Americans. PloS one, 7(1), e26807.
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  Asthma is a common complex condition with clear racial and ethnic differences in both prevalence and severity. Asthma consultation rates, mortality, and severe symptoms are greatly increased in African descent populations of developed countries. African ancestry has been associated with asthma, total serum IgE and lower pulmonary function in African-admixed populations. To replicate previous findings, here we aimed to examine whether African ancestry was associated with asthma susceptibility in African Americans. In addition, we examined for the first time whether African ancestry was associated with asthma exacerbations.
• Frank, K. M., Zhou, T., Moreno-Vinasco, L., Hollett, B., Garcia, J. G., & Bubeck Wardenburg, J. (2012). Host response signature to Staphylococcus aureus alpha-hemolysin implicates pulmonary Th17 response. Infection and immunity, 80(9), 3161-9.
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  Staphylococcus aureus pneumonia causes significant morbidity and mortality. Alpha-hemolysin (Hla), a pore-forming cytotoxin of S. aureus, has been identified through animal models of pneumonia as a critical virulence factor that induces lung injury. In spite of considerable molecular knowledge of how this cytotoxin injures the host, the precise host response to Hla in the context of infection remains poorly understood. We employed whole-genome expression profiling of infected lungs to define the host response to wild-type S. aureus compared with the response to an Hla-deficient isogenic mutant in experimental pneumonia. These data provide a complete expression profile at 4 and at 24 h postinfection, revealing a unique response to the toxin-expressing strain. Gene ontogeny analysis revealed significant differences in the extracellular matrix and cardiomyopathy pathways, both of which govern cellular interactions in the tissue microenvironment. Evaluation of individual transcript responses to Hla-secreting staphylococci was notable for upregulation of host cytokine and chemokine genes, including the p19 subunit of interleukin-23. Consistent with this observation, the cellular immune response to infection was characterized by a prominent Th17 response to the wild-type pathogen. These findings define specific host mRNA responses to Hla-producing S. aureus, coupling the pulmonary Th17 response to the secretion of this cytotoxin. Expression profiling to define the host response to a single virulence factor proved to be a valuable tool in identifying pathways for further investigation in S. aureus pneumonia. This approach may be broadly applicable to the study of bacterial toxins, defining host pathways that can be targeted to mitigate toxin-induced disease.
• Gorshkova, I., Zhou, T., Mathew, B., Jacobson, J. R., Takekoshi, D., Bhattacharya, P., Smith, B., Aydogan, B., Weichselbaum, R. R., Natarajan, V., Garcia, J. G., & Berdyshev, E. V. (2012). Inhibition of serine palmitoyltransferase delays the onset of radiation-induced pulmonary fibrosis through the negative regulation of sphingosine kinase-1 expression. Journal of lipid research, 53(8), 1553-68.
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  The enforcement of sphingosine-1-phosphate (S1P) signaling network protects from radiation-induced pneumonitis. We now demonstrate that, in contrast to early postirradiation period, late postirradiation sphingosine kinase-1 (SphK1) and sphingoid base-1-phosphates are associated with radiation-induced pulmonary fibrosis (RIF). Using the mouse model, we demonstrate that RIF is characterized by a marked upregulation of S1P and dihydrosphingosine-1-phosphate (DHS1P) levels in the lung tissue and in circulation accompanied by increased lung SphK1 expression and activity. Inhibition of sphingolipid de novo biosynthesis by targeting serine palmitoyltransferase (SPT) with myriocin reduced radiation-induced pulmonary inflammation and delayed the onset of RIF as evidenced by increased animal lifespan and decreased expression of markers of fibrogenesis, such as collagen and α-smooth muscle actin (α-SMA), in the lung. Long-term inhibition of SPT also decreased radiation-induced SphK activity in the lung and the levels of S1P-DHS1P in the lung tissue and in circulation. In vitro, inhibition or silencing of serine palmitoyltransferase attenuated transforming growth factor-β1 (TGF-β)-induced upregulation of α-SMA through the negative regulation of SphK1 expression in normal human lung fibroblasts. These data demonstrate a novel role for SPT in regulating TGF-β signaling and fibrogenesis that is linked to the regulation of SphK1 expression and S1P-DHS1P formation.
• Han, Y. J., Ma, S. F., Wade, M. S., Flores, C., & Garcia, J. G. (2012). An intronic MYLK variant associated with inflammatory lung disease regulates promoter activity of the smooth muscle myosin light chain kinase isoform. Journal of molecular medicine (Berlin, Germany), 90(3), 299-308.
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  Intronic single-nucleotide polymorphisms (SNPs) are commonly associated with complex diseases but exhibit unknown biologic functionality. Myosin light-chain kinase (MLCK), a central cytoskeletal regulator encoded by MYLK, plays a key pathophysiological role in complex diseases including acute lung injury (ALI) and asthma. We studied the potential regulatory roles of two intronic MYLK SNPs (rs936170 and rs820336) previously associated with ALI and asthma. Due to their genomic location at the junction encoding the non-muscle and smooth muscle MLCK (smMLCK) isoforms, we first identified the transcription start site (TSS) of the smMLCK isoform, and isolated a 2,954-bp DNA fragment upstream of the smMLCK TSS. Serial 5' deletion of the fragment revealed a proximal promoter region exhibiting strong promoter activity with potential inhibitory elements in the distal region. Site-directed mutageneses and luciferase reporter assays showed no effect of the distal promoter SNP rs936170 on smMLCK promoter activity. In contrast, SNP rs820336, located in an enhancer/repressor region downstream of TSS, was identified to regulate smMLCK promoter activity in an allelic-dependent manner. The A allele interrupted the binding site for Forkhead box protein N1 (FOXN1), a transcription factor governing expression of immune response genes. Silencing of FOXN1 expression (siRNA) reduced FOXN1 interaction with cis-regulatory elements in proximity to rs820336 and significantly decreased smMLCK expression. These functional insights into the involvement of intronic MYLK SNPs further strengthen the concept that MYLK contributes to inflammatory disease susceptibility and represents an attractive molecular target in complex inflammatory disorders.
• Kazee, N., & Garcia, J. G. (2012). Urban Health: Combating Disparities With Local Data Edited by Steven Whitman, Ami M. Shah, and Maureen R. Benjamins. American Journal of Epidemiology, 175(7), 725-726. doi:10.1093/aje/kws124
• Mekontso Dessap, A., Voiriot, G., Zhou, T., Marcos, E., Dudek, S. M., Jacobson, J. R., Machado, R., Adnot, S., Brochard, L., Maitre, B., & Garcia, J. G. (2012). Conflicting physiological and genomic cardiopulmonary effects of recruitment maneuvers in murine acute lung injury. American journal of respiratory cell and molecular biology, 46(4), 541-50.
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  Low tidal volume ventilation, although promoting atelectasis, is a protective strategy against ventilator-induced lung injury. Deep inflation (DI) recruitment maneuvers restore lung volumes, but potentially compromise lung parenchymal and vascular function via repetitive overdistention. Our objective was to examine cardiopulmonary physiological and transcriptional consequences of recruitment maneuvers. C57/BL6 mice challenged with either PBS or LPS via aspiration were placed on mechanical ventilation (5 h) using low tidal volume inflation (TI; 8 μl/g) alone or in combination with intermittent DIs (0.75 ml twice/min). Lung mechanics during TI ventilation significantly deteriorated, as assessed by forced oscillation technique and pressure-volume curves. DI mitigated the TI-induced alterations in lung mechanics, but induced a significant rise in right ventricle systolic pressures and pulmonary vascular resistances, especially in LPS-challenged animals. In addition, DI exacerbated the LPS-induced genome-wide lung inflammatory transcriptome, with prominent dysregulation of a gene cluster involving vascular processes, as well as increases in cytokine concentrations in bronchoalveolar lavage fluid and plasma. Gene ontology analyses of right ventricular tissue expression profiles also identified inflammatory signatures, as well as apoptosis and membrane organization ontologies, as potential elements in the response to acute pressure overload. Our results, although confirming the improvement in lung mechanics offered by DI, highlight a detrimental impact in sustaining inflammatory response and exacerbating lung vascular dysfunction, events contributing to increases in right ventricle afterload. These novel insights should be integrated into the clinical assessment of the risk/benefit of recruitment maneuver strategies.
• Petrov, M. N., Shilo, V. Y., Tarasov, A. V., Schwartz, D. E., Garcia, J. G., Kost, O. A., & Danilov, S. M. (2012). Conformational changes of blood ACE in chronic uremia. PloS one, 7(11), e49290.
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  The pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes.
• Pitroda, S. P., Zhou, T., Sweis, R. F., Filippo, M., Labay, E., Beckett, M. A., Mauceri, H. J., Liang, H., Darga, T. E., Perakis, S., Khan, S. A., Sutton, H. G., Zhang, W., Khodarev, N. N., Garcia, J. G., & Weichselbaum, R. R. (2012). Tumor endothelial inflammation predicts clinical outcome in diverse human cancers. PloS one, 7(10), e46104.
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  Vascular endothelial cells contribute to the pathogenesis of numerous human diseases by actively regulating the stromal inflammatory response; however, little is known regarding the role of endothelial inflammation in the growth of human tumors and its influence on the prognosis of human cancers.
• Sammani, S., Moreno, L., Letsiou, E., Garcia, J. G., Dudek, S. M., Chiang, E. T., & Camp, S. M. (2012). C-Abl Inhibition Exacerbates Mechanical Ventilation-Induced Lung Injury In A Murine Model. The FASEB Journal, 26. doi:10.1096/fasebj.26.1_supplement.lb552
• Sun, X., Singleton, P. A., Letsiou, E., Zhao, J., Belvitch, P., Sammani, S., Chiang, E. T., Moreno-Vinasco, L., Wade, M. S., Zhou, T., Liu, B., Parastatidis, I., Thomson, L., Ischiropoulos, H., Natarajan, V., Jacobson, J. R., Machado, R. F., Dudek, S. M., & Garcia, J. G. (2012). Sphingosine-1-phosphate receptor-3 is a novel biomarker in acute lung injury. American journal of respiratory cell and molecular biology, 47(5), 628-36.
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  The inflamed lung exhibits oxidative and nitrative modifications of multiple target proteins, potentially reflecting disease severity and progression. We identified sphingosine-1-phosphate receptor-3 (S1PR3), a critical signaling molecule mediating cell proliferation and vascular permeability, as a nitrated plasma protein in mice with acute lung injury (ALI). We explored S1PR3 as a potential biomarker in murine and human ALI. In vivo nitrated and total S1PR3 concentrations were determined by immunoprecipitation and microarray studies in mice, and by ELISA in human plasma. In vitro nitrated S1PR3 concentrations were evaluated in human lung vascular endothelial cells (ECs) or within microparticles shed from ECs after exposure to barrier-disrupting agonists (LPS, low-molecular-weight hyaluronan, and thrombin). The effects of S1PR3-containing microparticles on EC barrier function were assessed by transendothelial electrical resistance (TER). Nitrated S1PR3 was identified in the plasma of murine ALI and in humans with severe sepsis-induced ALI. Elevated total S1PR3 plasma concentrations (> 251 pg/ml) were linked to sepsis and ALI mortality. In vitro EC exposure to barrier-disrupting agents induced S1PR3 nitration and the shedding of S1PR3-containing microparticles, which significantly reduced TER, consistent with increased permeability. These changes were attenuated by reduced S1PR3 expression (small interfering RNAs). These results suggest that microparticles containing nitrated S1PR3 shed into the circulation during inflammatory lung states, and represent a novel ALI biomarker linked to disease severity and outcome.
• Tauler, J., Sun, X., Siegler, J., Moreno-vinasco, L., & Garcia, J. G. (2012). Abstract 352: hnRNP A2/B1 regulates S1PR3 expression in lung cancer. Cancer Research, 72, 352-352. doi:10.1158/1538-7445.am2012-352
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  Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Several clinical trials have shown that elevated expression of heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) in exfoliated bronchial epithelial cells correlates with eventual development of lung cancer. Cells with high levels of hnRNP A2/B1 expression have also been reported to manifest multiple molecular features of genetic injury. The hnRNPs are a complex group of RNA-binding proteins which play a key role in mRNA processing: stability, splicing, export and subcellular localization and participate also in telomere biogenesis. To elucidate the mechanism that links hnRNP A2/B1 overexpression with lung cancer progression we have knocked down hnRNPA2/B1 mRNA in non-small cell lung cancer (NSCLC) cell line A549 with a stable siRNA vector-based system. Silencing hnRNP A2/B1 increased proliferation and decreased migration in A549 and H1703 cell lines. These changes in migration and proliferation in A549 and H1703 hnRNPA2/B1-silenced cell lines correlated with an increase of E-Cadherin expression in both cell lines at mRNA and protein level. This increase in E-Cadherin expression was accompanied with a down-regulation of E-Cadherin inhibitors Twist1 and Snai1. We have proposed that hnRNP A2/B1 modulates epithelial-mesenchymal transition (EMT) in lung cancer cell lines that present a mesenchymal or intermediate phenotype such as H1703 and A549. It has been proposed that EMT may induce metastasis of cells with a mesenchymal phenotype. After metastatic seeding, EMT would be reversed trough mesenchymal-epithelial transition (MET) allowing tumor growth. However, mechanisms that regulate reversion of EMT are still poorly characterized. Interestingly, sphingosine 1-phospate (S1P) is an important angiogenic factor and regulator of vascular permeability. Microarray expression analysis revealed that silencing hnRNP A2/B1 decreases expression of the sphingosine 1-phosphate receptor 3 (S1PR3), a pro-migration RhoGTPAse activating S1P receptor, in A549 cells. Therefore, Moreover, S1P also stimulates motility of cancer cells through S1P receptors. It has been described that S1PR3 regulates migration and invasion in breast, prostate and gastric cancer. In A549 cells, S1PR3 is the most abundant receptor for S1P. Here we report that down regulation of hnRNP A2/B1, in lung cancer cells, decreases S1PR3 expression at the mRNA and protein level. We propose that further study of the mechanistic link between hnRNP A2/B1, S1P function and S1P receptors may allow a better understanding of the molecular mechanisms involved in invasion, metastasis. It may also provide new insights regarding the interaction between endothelial and epithelial cells and the regulation of EMT during tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 352. doi:1538-7445.AM2012-352
• Usatyuk, P. V., Singleton, P. A., Pendyala, S., Kalari, S. K., He, D., Gorshkova, I. A., Camp, S. M., Moitra, J., Dudek, S. M., Garcia, J. G., & Natarajan, V. (2012). Novel role for non-muscle myosin light chain kinase (MLCK) in hyperoxia-induced recruitment of cytoskeletal proteins, NADPH oxidase activation, and reactive oxygen species generation in lung endothelium. The Journal of biological chemistry, 287(12), 9360-75.
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  We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.
• Valentine, S. L., Sapru, A., Higgerson, R. A., Spinella, P. C., Flori, H. R., Graham, D. A., Brett, M., Convery, M., Christie, L. M., Karamessinis, L., Randolph, A. G., , P. A., & , A. R. (2012). Fluid balance in critically ill children with acute lung injury. Critical care medicine, 40(10), 2883-9.
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  In the Fluid and Catheter Treatment Trial (NCT00281268), adults with acute lung injury randomized to a conservative vs. liberal fluid management protocol had increased days alive and free of mechanical ventilator support (ventilator-free days). Recruiting sufficient children with acute lung injury into a pediatric trial is challenging. A Bayesian statistical approach relies on the adult trial for the a priori effect estimate, requiring fewer patients. Preparing for a Bayesian pediatric trial mirroring the Fluid and Catheter Treatment Trial, we aimed to: 1) identify an inverse association between fluid balance and ventilator-free days; and 2) determine if fluid balance over time is more similar to adults in the Fluid and Catheter Treatment Trial liberal or conservative arms.
• Wang, T., Garcia, J. G., & Zhang, W. (2012). Epigenetic Regulation in Particulate Matter-Mediated Cardiopulmonary Toxicities: A Systems Biology Perspective. Current pharmacogenomics and personalized medicine, 10(4), 314-321.
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  Particulate matter (PM) air pollution exerts significant adverse health effects in global populations, particularly in developing countries with extensive air pollution. Understanding of the mechanisms of PM-induced health effects including the risk for cardiovascular diseases remains limited. In addition to the direct cellular physiological responses such as mitochondrial dysfunction and oxidative stress, PM mediates remarkable dysregulation of gene expression, especially in cardiovascular tissues. The PM-mediated gene dysregulation is likely to be a complex mechanism affected by various genetic and non-genetic factors. Notably, PM is known to alter epigenetic markers (e.g., DNA methylation and histone modifications), which may contribute to air pollution-mediated health consequences including the risk for cardiovascular diseases. Notably, epigenetic changes induced by ambient PM exposure have emerged to play a critical role in gene regulation. Though the underlying mechanism(s) are not completely clear, the available evidence suggests that the modulated activities of DNA methyltransferase (DNMT), histone acetylase (HAT) and histone deacetylase (HDAC) may contribute to the epigenetic changes induced by PM or PM-related chemicals. By employing genome-wide epigenomic and systems biology approaches, PM toxicogenomics could conceivably progress greatly with the potential identification of individual epigenetic loci associated with dysregulated gene expression after PM exposure, as well the interactions between epigenetic pathways and PM. Furthermore, novel therapeutic targets based on epigenetic markers could be identified through future epigenomic studies on PM-mediated cardiopulmonary toxicities. These considerations collectively inform the future population health applications of genomics in developing countries while benefiting global personalized medicine at the same time.
• Wang, T., Lang, G. D., Moreno-Vinasco, L., Huang, Y., Goonewardena, S. N., Peng, Y. J., Svensson, E. C., Natarajan, V., Lang, R. M., Linares, J. D., Breysse, P. N., Geyh, A. S., Samet, J. M., Lussier, Y. A., Dudley, S., Prabhakar, N. R., & Garcia, J. G. (2012). Particulate matter induces cardiac arrhythmias via dysregulation of carotid body sensitivity and cardiac sodium channels. American journal of respiratory cell and molecular biology, 46(4), 524-31.
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  The mechanistic links between exposure to airborne particulate matter (PM) pollution and the associated increases in cardiovascular morbidity and mortality, particularly in people with congestive heart failure (CHF), have not been identified. To advance understanding of this issue, genetically engineered mice (CREB(A133)) exhibiting severe dilated cardiomyopathic changes were exposed to ambient PM collected in Baltimore. CREB(A133) mice, which display aberrant cardiac physiology and anatomy reminiscent of human CHF, displayed evidence of basal autonomic aberrancies (compared with wild-type mice) with PM exposure via aspiration, producing significantly reduced heart rate variability, respiratory dysynchrony, and increased ventricular arrhythmias. Carotid body afferent nerve responses to hypoxia and hyperoxia-induced respiratory depression were pronounced in PM-challenged CREB(A133) mice, and denervation of the carotid bodies significantly reduced PM-mediated cardiac arrhythmias. Genome-wide expression analyses of CREB(A133) left ventricular tissues demonstrated prominent Na(+) and K(+) channel pathway gene dysregulation. Subsequent PM challenge increased tyrosine phosphorylation and nitration of the voltage-gated type V cardiac muscle α-subunit of the Na(+) channel encoded by SCN5A. Ranolazine, a Na(+) channel modulator that reduces late cardiac Na(+) channel currents, attenuated PM-mediated cardiac arrhythmias and shortened PM-elongated QT intervals in vivo. These observations provide mechanistic insights into the epidemiologic findings in susceptibility of human CHF populations to PM exposure. Our results suggest a multiorgan pathobiology inherent to the CHF phenotype that is exaggerated by PM exposure via heightened carotid body sensitivity and cardiac Na(+) channel dysfunction.
• Wang, T., Shen, K., & Garcia, J. G. (2012). MYLK (myosin light chain kinase). Atlas of genetics and cytogenetics in oncology and haematology. doi:10.4267/2042/48362
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  Review on MYLK (myosin light chain kinase), with data on DNA, on the protein encoded, and where the gene is implicated.
• Wang, T., Wang, L., Moreno-Vinasco, L., Lang, G. D., Siegler, J. H., Mathew, B., Usatyuk, P. V., Samet, J. M., Geyh, A. S., Breysse, P. N., Natarajan, V., & Garcia, J. G. (2012). Particulate matter air pollution disrupts endothelial cell barrier via calpain-mediated tight junction protein degradation. Particle and fibre toxicology, 9, 35.
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  Exposure to particulate matter (PM) is a significant risk factor for increased cardiopulmonary morbidity and mortality. The mechanism of PM-mediated pathophysiology remains unknown. However, PM is proinflammatory to the endothelium and increases vascular permeability in vitro and in vivo via ROS generation.
• Wang, T., Wang, L., Zaidi, S. R., Sammani, S., Siegler, J., Moreno-Vinasco, L., Mathew, B., Natarajan, V., & Garcia, J. G. (2012). Hydrogen sulfide attenuates particulate matter-induced human lung endothelial barrier disruption via combined reactive oxygen species scavenging and Akt activation. American journal of respiratory cell and molecular biology, 47(4), 491-6.
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  Exposure to particulate air pollution is associated with increased cardiopulmonary morbidity and mortality, although the pathogenic mechanisms are poorly understood. We previously demonstrated that particulate matter (PM) exposure triggers massive oxidative stress in vascular endothelial cells (ECs), resulting in the loss of EC integrity and lung vascular hyperpermeability. We investigated the protective role of hydrogen sulfide (H(2)S), an endogenous gaseous molecule present in the circulation, on PM-induced human lung EC barrier disruption and pulmonary inflammation. Alterations in EC monolayer permeability, as reflected by transendothelial electrical resistance (TER), the generation of reactive oxygen species (ROS), and murine pulmonary inflammatory responses, were studied after exposures to PM and NaSH, an H(2)S donor. Similar to N-acetyl cysteine (5 mM), NaSH (10 μM) significantly scavenged PM-induced EC ROS and inhibited the oxidative activation of p38 mitogen-activated protein kinase. Concurrent with these events, NaSH (10 μM) activated Akt, which helps maintain endothelial integrity. Both of these pathways contribute to the protective effect of H(2)S against PM-induced endothelial barrier dysfunction. Furthermore, NaSH (20 mg/kg) reduced vascular protein leakage, leukocyte infiltration, and proinflammatory cytokine release in bronchoalveolar lavage fluids in a murine model of PM-induced lung inflammation. These data suggest a potentially protective role for H(2)S in PM-induced inflammatory lung injury and vascular hyperpermeability.
• Zhang, X., Zhang, W., Xu, M., Wade, M. S., Sergueeva, A. I., Prchal, J. T., Nekhai, S., Miasniakova, G., Machado, R. F., Ma, S., Gordeuk, V. R., Garcia, J. G., & Ammosova, T. (2012). Iron Deficiency Modifies Gene Expression Variation Induced by Augmented Hypoxia Sensing. Blood, 120(21), 1765-1765. doi:10.1182/blood.v120.21.1765.1765
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  Abstract 1765 Hypoxia may cause pulmonary and brain edema, pulmonary hypertension, aberrant metabolism and early mortality. To better understand pathological processes associated with hypoxia, we examined gene expression in Chuvash polycythemia blood mononuclear cells. Chuvash polycythemia is a congenital disorder of up-regulated hypoxic response at normoxia wherein VHL R200W homozygosity leads to elevated hypoxia inducible factor (HIF)-1 and HIF-2 levels, thromboses, pulmonary hypertension, lower systemic blood pressure (SBP) and increased mortality. VHL R200W homozygotes are often treated by phlebotomy resulting in iron deficiency, allowing us to evaluate an interaction of augmented hypoxia sensing with iron deficiency. Expression profiling of 8 VHL R200W homozygotes and 17 VHL wildtype individuals, matched for normal iron status as reflected in serum ferritin concentration, revealed altered regulation of 3069 genes at false discovery rate VHL R200W homozygotes. Genes induced by homozygous VHL R200W were enriched in immune response pathways; those repressed in RNA transcription and protein synthesis pathways. Forty-two genes showed a >1.5-fold change in expression level, mostly (74%) an increase. Seven showed a >2-fold increase: CA1 (carbonic anhydrase), SELENBP1 (selenium binding protein 1), IL1B (interleukin 1 beta), SLC4A1 (solute carrier family 4 member 1), HBB (hemoglobin beta), and AHSP (alpha hemoglobin stabilizing protein). Additional studies including 16 VHL R200W homozygotes with low ferritin indicated that iron deficiency enhanced the induction effect of VHL R200W for 51 of the 847 upregulated genes and suppressed the induction effect for 108 of the upregulated genes. Genes further upregulated by iron deficiency included CA1 , CSDA (cold shock domain protein A), BCL2L1 (BCL-2 like 1), BPGM (2,3-bisphosphoglycerate mutase), DCAF12 (DDB1 and CUL4 associated factor 12), FECH (ferrochelatase), SELENBP1 and SLC4A1. Genes for which iron deficiency suppressed the induction included inflammatory and immune pathway genes such as CASP5, CXCL16, IFI30, IFI35, IRF5, LILRB1, NOD2, RELT, TCIRG1 and TNFAIP2 . A number of the genes with altered regulation in VHL R200W homozygotes might modify risk of thrombosis (upregulated: F3, SERPINE1, SERPINB2, SERPING1 , PLAUR, THBD; down regulated: SERBP1 ), elevated systolic pulmonary artery pressure (upregulated: HTR1B, THBS1 ; downregulated: S1PR1, STIM2), or benign hemangioma (downregulated: CCM2). However, expression of these genes tended not to be influenced by iron status. VEGF was induced in VHL R200W homozygotes and surprisingly this induction was suppressed by iron deficiency. Expression relationships suggested a broad effect of VHL R200W in reducing systolic blood pressure through inducing VEGF. We demonstrate that many genes have commensurate changes of their expression by both iron deficiency and VHL R200W associated normoxic up-regulation of HIFs, as expected. However, there are genes that are regulated asynchronously. Further research is needed to define the molecular bases of separate regulation of genes by HIFs and iron status and to define relative risks and benefits of therapeutic phlebotomy for polycythemia. The resulting elucidation of the genomic pathways affecting predisposition to thromboses, pulmonary hypertension, lower systolic blood pressure and the interaction of augmented hypoxia sensing with iron deficiency should have broad implications leading to a better understanding of the pathophysiology of many diseases and the development of targeted therapies. Disclosures: No relevant conflicts of interest to declare.
• Zhang, X., Zhang, W., Xu, M., Wade, M. S., Sergueeva, A. I., Prchal, J. T., Nekhai, S., Miasniakova, G., Machado, R., Ma, S., Gordeuk, V. R., Garcia, J. G., & Ammosova, T. (2012). The Hypoxic Response and Altered Gene Expression in Patients with Sickle Cell Disease. Blood, 120(21), 3245-3245. doi:10.1182/blood.v120.21.3245.3245
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  Abstract 3245 Sickle cell disease (SCD) and Chuvash polycythemia (CP) are both monogenic hematologic disorders, the first resulting in hemolytic anemia and the second in polycythemia related to an upregulated hypoxic response at normoxia. Specifically, homozygosity for the VHL R200W mutation leads to increased levels of the transcription factors hypoxia inducible factor (HIF)-1 and HIF-2 at normoxia and altered transcription of many genes. Much attention in the pathophysiology of SCD has focused on the adverse effects of chronic inflammation, enhanced cellular adhesion pathways and hemolytic rate. However, the chronic anemia of SCD is associated with an upregulation of the hypoxic response as evidenced by high erythropoietin concentrations. In this prospective study, we compared gene expression alterations in peripheral blood mononuclear cells in SCD and CP. Our pre-specified hypothesis was that we could identify hypoxia-mediated gene expression alterations in SCD through a comparison with altered gene expression in CP and reveal molecular pathways that are potentially shared by these two conditions. We prospectively compared gene expression profiles in two independent cohorts, an SCD cohort comprised of 22 SCD patients and 19 African American controls and a CP cohort comprised of 8 VHL R200W homozygotes and 17 Chuvash controls with wildtype VHL . Because iron deficiency can influence the hypoxic response, we excluded iron-deficient subjects from each cohort. We used the identical Affymetrix exon array platform for both cohorts. Differential gene expression was highly correlated between the two conditions, with Spearman correlation ρ = 0.75 between their expression profiles across 16,642 genes, suggesting that 56% of expression variation triggered by beta hemoglobin mutation in SCD may be explained by hypoxic transcriptional responses. A small portion of differential genes were highly induced in both conditions. Among 54 genes up-regulated >1.5-fold in SCD patients, 24 (44%) overlapped with the 31 genes up-regulated >1.5-fold in VHL R200W homozygotes. The genes highly induced in both conditions included FAM46C (family with sequence similarity 46 member C), SELENBP1 (selenium binding protein 1), IL1B (interleukin 1, beta), MOP-1, SNCA (synuclein, alpha), GMPR (guanosine monophosphate reductase), BPGM (2,3-bisphosphoglycerate mutase), SLC25A37 (solute carrier family 25 member 37), CA1 (carbonic anhydrase I), DCAF12 (DDB1 and CUL4 associated factor 12), EPB42 (erythrocyte membrane protein band 4.2), AHSP (alpha hemoglobin stabilizing protein), SLC4A1 (solute carrier family 4 member 1), HBB (hemoglobin, beta), HBD (hemoglobin, delta), CSDA (cold shock domain protein A), FECH (ferrochelatase), BCL2L1 (BCL2-like 1), OSBP2 (oxysterol binding protein 2), APOBEC3A (apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3A), IFIT1 (interferon-induced protein with tetratricopeptide repeats 1), IFIT3 (interferon-induced protein with tetratricopeptide repeats 3), IFI44L (interferon-induced protein 44-like), and IFI27 (interferon, alpha-inducible protein 27). Three genes were down-regulated >1.5-fold in SCD patients. One of these, GIMAP7 (GTPase, IMAP family member 7), was among the 11 genes down-regulated >1.5-fold in VHL R200W homozygotes. These results suggest that there is a broad upregulation of the hypoxic response in SCD and that the hypoxic response may underlie or interact with an important proportion of the clinical and pathophysiologic manifestations of SCD. Disclosures: No relevant conflicts of interest to declare.
• Zhang, X., Zhang, W., Xu, M., Wade, M. S., Sergueeva, A. I., Sable, C., Prchal, J. T., Nekhai, S., Miasniakova, G., Machado, R., Ma, S., Gordeuk, V. R., Garcia, J. G., & Ammosova, T. (2012). Novel Putative Polymorphism in SERPINC1 Encoding Antithrombin III Is Implicated in Elevated Estimated Systolic Pulmonary Pressure in Patients with Chuvash Polycythemia.. Blood, 120(21), 2869-2869. doi:10.1182/blood.v120.21.2869.2869
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  Abstract 2869 Chuvash polycythemia (CP) is characterized by homozygosity for the R200W mutation in the von Hippel Lindau gene ( VHL ). This rare genetic disorder causes elevated levels of hypoxia inducible factor (HIF)-1 and HIF-2 that trigger constitutive hypoxia responses at normoxia. Hypoxia is a recognized cause of pulmonary hypertension. We recently reported that systolic pulmonary artery pressure (SPAP) estimated by echocardiography-determined tricuspid regurgitation velocity (TRV) was elevated in 120 CP patients compared to 31 Chuvash controls (P = 0.005), and that increasing age (P = 0.001), increasing systemic pulse pressure (P = 0.003) and lower serum ferritin concentration (P = 0.009) were independent predictors of higher estimated SPAP in CP patients [Sable et al., 2012]. In this study, we profiled gene expression for 16,642 genes in peripheral blood mononuclear cells (PBMCs) derived from a cohort of 43 CP patients, and measured the TRV for these individuals. Based on a prospectively chosen criterion of TRV ≥2.5 m/sec, 20 patients were classified as having elevated level of estimated SPAP and 23 were normal. Gene expression level between these two SPAP groups appeared to be homogenous. However, we identified 4777 genes at false discovery rate (FDR) Further analysis of probe level data revealed a potential genetic polymorphism located within exon 7 of SERPINC1 , encoding antithrombin III, at a site where a disease-associated SNP has not been reported. The minor allele of this polymorphism, which we designated “B”, was highly enriched in the elevated estimated SPAP group (P=0.0009), and classification based on the putative SERPINC1 genotypes strengthens the interaction effect with pulse pressure. Analysis of the gene expression data for the 43 CP patients with an additive genetic model of SERPINC1 identified 1902 differential genes at FDR P adjusted −10 ). Several genes involved in smooth muscle contraction, regulation of blood pressure, and angiogenesis were up-regulated by the putative B allele, for example, HTR5A (serotonin receptor 5A), TAC3 (tachykinin 3), ADRA1D (adrenergic alpha-1D- receptor), BDKRB1 (bradykinin receptor B1), BDKRB2 (bradykinin receptor B1), EDN2 (endothelin 2), PTGER1 (prostaglandin E receptor 1), PTGIR (prostaglandin I2 receptor), APLNR (apelin receptor), RAMP2 (receptor activity modifying protein 2), MYH6 (myosin heavy chain 6), MYH7 (myosin heavy chain 7), MYL2 (myosin light chain 2), MYLK2 (myosin light chain kinase 2), MYLK3 (myosin light chain kinase 3), ACE (angiotensin I converting enzyme 1), ATP2A1 (ATPase cardiac muscle fast twitch 1), ADCY4 (adenylate cyclase 4), and PRKAA2 (protein kinase AMP-activated alpha 2 catalytic subunit). On the other hand, genes whose malfunction has been associated with familial pulmonary hypertension, including BMPR2 (bone morphogenetic protein receptor, type II), SMAD5 (SMAD family member 5), and ACVR2A (activin A receptor, type IIA), were among the 782 genes down regulated by the B allele. These results suggest that alterations in SERPINC1 may be implicated in the development of elevated SPAP in patients with CP. Antithrombin III deficiency is a potential factor in chronic thromboembolic pulmonary hypertension, but our results suggest that the putative B polymorphism may have effects beyond the coagulation cascade. Whether this is due to the SERPINC1 polymorphism itself or to another locus in linkage disequilibrium will require our future planned studies, beginning with sequencing of SERPINC1 exon 7 and identification of the polymorphism. Disclosures: No relevant conflicts of interest to declare.
• Zhou, T., Zhan, W., Weichselbaum, R. R., Rondelli, D., Patel, P. R., Mahmud, D., & Garcia, J. G. (2012). Preclinical Model to Predict Anti-Leukemic Activity of Busulfan and Irradiation. Biology of Blood and Marrow Transplantation, 18(2), S291. doi:10.1016/j.bbmt.2011.12.509
• Zhou, T., Zhang, W., Sweiss, N. J., Chen, E. S., Moller, D. R., Knox, K. S., Ma, S. F., Wade, M. S., Noth, I., Machado, R. F., & Garcia, J. G. (2012). Peripheral blood gene expression as a novel genomic biomarker in complicated sarcoidosis. PloS one, 7(9), e44818.
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  Sarcoidosis, a systemic granulomatous syndrome invariably affecting the lung, typically spontaneously remits but in ~20% of cases progresses with severe lung dysfunction or cardiac and neurologic involvement (complicated sarcoidosis). Unfortunately, current biomarkers fail to distinguish patients with remitting (uncomplicated) sarcoidosis from other fibrotic lung disorders, and fail to identify individuals at risk for complicated sarcoidosis. We utilized genome-wide peripheral blood gene expression analysis to identify a 20-gene sarcoidosis biomarker signature distinguishing sarcoidosis (n = 39) from healthy controls (n = 35, 86% classification accuracy) and which served as a molecular signature for complicated sarcoidosis (n = 17). As aberrancies in T cell receptor (TCR) signaling, JAK-STAT (JS) signaling, and cytokine-cytokine receptor (CCR) signaling are implicated in sarcoidosis pathogenesis, a 31-gene signature comprised of T cell signaling pathway genes associated with sarcoidosis (TCR/JS/CCR) was compared to the unbiased 20-gene biomarker signature but proved inferior in prediction accuracy in distinguishing complicated from uncomplicated sarcoidosis. Additional validation strategies included significant association of single nucleotide polymorphisms (SNPs) in signature genes with sarcoidosis susceptibility and severity (unbiased signature genes - CX3CR1, FKBP1A, NOG, RBM12B, SENS3, TSHZ2; T cell/JAK-STAT pathway genes such as AKT3, CBLB, DLG1, IFNG, IL2RA, IL7R, ITK, JUN, MALT1, NFATC2, PLCG1, SPRED1). In summary, this validated peripheral blood molecular gene signature appears to be a valuable biomarker in identifying cases with sarcoidoisis and predicting risk for complicated sarcoidosis.
• Adyshev, D. M., Moldobaeva, N. K., Elangovan, V. R., Garcia, J. G., & Dudek, S. M. (2011). Differential involvement of ezrin/radixin/moesin proteins in sphingosine 1-phosphate-induced human pulmonary endothelial cell barrier enhancement. Cellular signalling, 23(12), 2086-96.
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  Endothelial cell (EC) barrier dysfunction induced by inflammatory agonists is a frequent pathophysiologic event in multiple diseases. The platelet-derived phospholipid sphingosine-1 phosphate (S1P) reverses this dysfunction by potently enhancing the EC barrier through a process involving Rac GTPase-dependent cortical actin rearrangement as an integral step. In this study we explored the role of the ezrin, radixin, and moesin (ERM) family of actin-binding linker protein in modulating S1P-induced human pulmonary EC barrier enhancement. S1P induces ERM translocation to the EC periphery and promotes ERM phosphorylation on a critical threonine residue (Ezrin-567, Radixin-564, Moesin-558). This phosphorylation is dependent on activation of PKC isoforms and Rac1. The majority of ERM phosphorylation on these critical threonine residues after S1P occurs in moesin and ezrin. Baseline radixin phosphorylation is higher than in the other two ERM proteins but does not increase after S1P. S1P-induced moesin and ezrin threonine phosphorylation is not mediated by the barrier enhancing receptor S1PR1 because siRNA downregulation of S1PR1 fails to inhibit these phosphorylation events, while stimulation of EC with the S1PR1-specific agonist SEW2871 fails to induce these phosphorylation events. Silencing of either all ERM proteins or radixin alone (but not moesin alone) reduced S1P-induced Rac1 activation and phosphorylation of the downstream Rac1 effector PAK1. Radixin siRNA alone, or combined siRNA for all three ERM proteins, dramatically attenuates S1P-induced EC barrier enhancement (measured by transendothelial electrical resistance (TER), peripheral accumulation of di-phospho-MLC, and cortical cytoskeletal rearrangement. In contrast, moesin depletion has the opposite effects on these parameters. Ezrin silencing partially attenuates S1P-induced EC barrier enhancement and cytoskeletal changes. Thus, despite structural similarities and reported functional redundancy, the ERM proteins differentially modulate S1P-induced alterations in lung EC cytoskeleton and permeability. These results suggest that ERM activation is an important regulatory event in EC barrier responses to S1P.
• Barca-Mayo, O., Liao, X. H., DiCosmo, C., Dumitrescu, A., Moreno-Vinasco, L., Wade, M. S., Sammani, S., Mirzapoiazova, T., Garcia, J. G., Refetoff, S., & Weiss, R. E. (2011). Role of type 2 deiodinase in response to acute lung injury (ALI) in mice. Proceedings of the National Academy of Sciences of the United States of America, 108(49), E1321-9.
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  Thyroid hormone (TH) metabolism, mediated by deiodinase types 1, 2, and 3 (D1, D2, and D3) is profoundly affected by acute illness. We examined the role of TH metabolism during ventilator-induced lung injury (VILI) in mice. Mice exposed to VILI recapitulated the serum TH findings of acute illness, namely a decrease in 3,5,3'-triiodothyronine (T(3)) and thyroid-stimulating hormone and an increase in reverse T(3). Both D2 immunoreactivity and D2 enzymatic activity were increased significantly. D1 and D3 activity did not change. Using D2 knockout (D2KO) mice, we determined whether the increase in D2 was an adaptive response. Although similar changes in serum TH levels were observed in D2KO and WT mice, D2KO mice exhibited greater susceptibility to VILI than WT mice, as evidenced by poorer alveoli integrity and quantified by lung chemokine and cytokine mRNA induction. These data suggest that an increase in lung D2 is protective against VILI. Similar findings of increased inflammatory markers were found in hypothyroid WT mice exposed to VILI compared with euthyroid mice, indicating that the lungs were functionally hypothyroid. Treatment of D2KO mice with T(3) reversed many of the lung chemokine and cytokine profiles seen in response to VILI, demonstrating a role for T(3) in the treatment of lung injury. We conclude that TH metabolism in the lung is linked to the response to inflammatory injury and speculate that D2 exerts its protective effect by making more TH available to the injured lung tissue.
• Berdyshev, E. V., Gorshkova, I., Usatyuk, P., Kalari, S., Zhao, Y., Pyne, N. J., Pyne, S., Sabbadini, R. A., Garcia, J. G., & Natarajan, V. (2011). Intracellular S1P generation is essential for S1P-induced motility of human lung endothelial cells: role of sphingosine kinase 1 and S1P lyase. PloS one, 6(1), e16571.
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  Earlier we have shown that extracellular sphingosine-1-phosphate (S1P) induces migration of human pulmonary artery endothelial cells (HPAECs) through the activation of S1P(1) receptor, PKCε, and PLD2-PKCζ-Rac1 signaling cascade. As endothelial cells generate intracellular S1P, here we have investigated the role of sphingosine kinases (SphKs) and S1P lyase (S1PL), that regulate intracellular S1P accumulation, in HPAEC motility.
• Calfee, C. S., Ware, L. B., Glidden, D. V., Eisner, M. D., Parsons, P. E., Thompson, B. T., Matthay, M. A., & , N. H. (2011). Use of risk reclassification with multiple biomarkers improves mortality prediction in acute lung injury. Critical care medicine, 39(4), 711-7.
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  Multiple single biomarkers have been associated with poor outcomes in acute lung injury; however, no single biomarker has sufficient discriminating power to clearly indicate prognosis. Using both derivation and replication cohorts, we tested novel risk reclassification methods to determine whether measurement of multiple plasma biomarkers at the time of acute lung injury diagnosis would improve mortality prediction in acute lung injury.
• Desai, A. A., Weinert, L., Turner, K. M., Trevino, S., Thiruvoipati, T., Patel, A. R., Machado, R., Lang, R. M., Jamison, E. B., Homaa, A., Garcia, J. G., Feehan, S., Desai, A. A., & Artz, N. (2011). Left ventricular function, morphology, and myocardial tissue characterization in Sickle Cell Disease: a multi-modality imaging study. Journal of Cardiovascular Magnetic Resonance, 13(1). doi:10.1186/1532-429x-13-s1-p334
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  Methods Thirty-one stable, African-American outpatients with SCD (mean age 32±8 yrs) prospectively underwent CMR (Philips 1.5 Tesla) and TTE (Philips iE33). Retrospectivelygated cines of left ventricular (LV) 2-, 3-, and 4-chamber, and short axis cine stack were obtained using SSFP (temporal resolution 25-40ms). Late gadolinium enhancement (LGE) images of the same views were obtained 10-20 minutes after infusion of Gd-DTPA (0.15 mmol/kg) using phase sensitive inversion recovery (TR 4.5 ms, TE 2.2ms, TI 250-300 ms, flip angle 30°, PSIR flip angle 5°). Single short-axis, mid-ventricular myocardial T2* slice and coronal, hepatic T2* slice were acquired with a single breath-hold, at six echo-times (2.3 to 14 msec) using a gradient echo sequence. Tissue T2* signal intensity was measured in LV septum and liver at two separate echo times and T2*= ΔTE/ln (SITE2/ SITE1) where ΔTE represents time difference between the two echo times and ITE1 and ITE2 represent signal intensity at echo time one and two. Myocardial and hepatic T2* were abnormal if
• Desai, A. A., Zhou, T., Zhou, T., Nouraie, M., Machado, R. F., Gordeuk, V. R., Garcia, J. G., & Desai, A. A. (2011). Genomic Assessment of Mortality in Sickle Cell Disease- A Novel Association with Cytokine Imbalance and T Cell Dysregulation. Blood, 118(21), 1081-1081. doi:10.1182/blood.v118.21.1081.1081
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  Abstract 1081 Rationale: Sickle cell disease (SCD) is characterized by systemic complications and significant clinical variability. While some patients never experience severe clinical events, others suffer lifelong morbidity and accelerated mortality. To address this variability, we prospectively followed patients with SCD at steady-state to assess outcomes and leveraged microarray analysis of RNA isolated from peripheral blood mononuclear cells (PBMCs) to identify novel gene expression patterns and signaling pathways that could be associated with survival in SCD. Methods: Forty-two adult patients with SCD at steady-state (no VOC or ACS episodes for >3 weeks) were followed for 5 years (median follow-up 1154 ± 317 days). At baseline enrollment, patients underwent phlebotomy for plasma and serum collection. The plasma was utilized to extract mRNA from PBMCs. The serum was used to assess targeted cytokine levels using high-sensitive cytokine kits. Microarray expression analysis of PBMC mRNA utilized an FDR 1.2. Student t-tests were performed for statistical significance. Results: Five out of forty-two patients (∼12%) died over the course of the study (median follow-up 1093 ± 320 days for surviving patients vs 1246 ± 325 days for patients who died, p=0.42). Although there were trends towards older age, higher tricuspid regurgitation jet velocity, NT-pro-BNP levels, AST levels, and lifetime number of transfusions in the patients that died, there were no statistically significant differences between groups (Table 1). Patients who died (n=5) exhibited 278 differentially regulated genes in comparison to those who survived (n=37). Gene ontology analysis revealed significantly represented T-cell receptor and immune mediated pathways, comprising seven out of the top ten pathways (p Conclusion: These preliminary data suggest that a PBMC genomic signature is a potential prognostic biomarker in SCD. Patients who subsequently died demonstrated a unique PBMC-gene expression profile with significant representation of specific T-cell and immune-mediated pathways. Concurrently, these at-risk patients also demonstrated higher serum IL-6 and IL-8 levels. Given that high circulating levels of these type 2 cytokines suppress both humoral and cell-mediated immune functions, we speculate that T-cells may play novel role in the variable morbidity and mortality observed in SCD. Disclosures: No relevant conflicts of interest to declare.
• Desai, A. A., Zhou, T., Zhou, T., Wade, M. S., Turner, K., Trevino, S., Thiruvoipati, T., Patel, A. R., Machado, R. F., Lang, R. M., Garcia, J. G., Desai, A. A., Artz, N., & Ahmad, H. (2011). Abstract 10132: Integration of microRNA Expression Profiling and a Validated Gene Signature from Peripheral Blood Mononuclear Cells in Pulmonary Hypertension Associated with Sickle Cell Disease. Circulation, 124.
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  Background: Pulmonary hypertension (PH) is a serious complication of sickle cell disease (SCD) associated with increased mortality. Gene expression profiles of peripheral blood mononuclear cells (PBMC) have been reported in pulmonary arterial hypertension and in SCD. We hypothesized that a gene expression signature from PBMC in patients with SCD may be utilized as a PH biomarker. Methods & Results: 30 patients with homozygous SCD prospectively underwent transthoracic echocardiography and phlebotomy for PBMC isolation. PH was defined as estimated right ventricular systolic pressure (RVSP)>30 mmHg with a peak tricuspid regurgitation velocity (TRV)>2.5m/s. Genome-wide gene expression profiles were compared between SCD cases with PH (n=18) and without PH (n=9) and correlated against PH severity using RVSP (correlation coefficient ρR) and TRV (ρT) as surrogates. Only genes with ρR^2 and ρT^2 >0.15 were considered differentially expressed for PH yielding 631 transcripts. A support vector machine analysis was then applied identifying a 10 gene signature which discriminated patients with and without echo-defined PH in the discovery cohort with 100% accuracy. The 10 gene signature was validated in an independent cohort of SCD patients with PH confirmed by right heart catheterization (n=13) and without PH (n=16) with 90% accuracy. To further identify significant pathways involved in PH in SCD, we correlated miRNA expression profiling in the discovery cohort against PH severity (ρR^2 or ρT^2 >0.15 considered differentially expressed) yielding 12 unique miRNAs. In silico analyses of the top PH miRNAs revealed strong binding predictions for miR-301a to polypeptide N-acetylgalactosaminyltransferase 13 (GALNT13), a PH signature gene, which was further validated by microarray data confirming correlation between miR-301a expression and GALNT13 in SCD-related PH. Conclusion: These expression data may have implications as a biomarker to screen an at-risk population in SCD for early PH diagnosis. Given the robust integration of miRNA and gene expression data yielding GALNT13, a glycosyltransferase enzyme involved in O-glycan synthesis, as a top candidate gene, we speculate that GALNT13 and glyocosylation patterns may play a novel role in SCD-related PH.
• Desai, A. A., Zhou, T., Zhou, T., Zhang, W., Yuan, J. X., Wade, M. S., Turner, K., Trevino, S., Thiruvoipati, T., Raghavachari, N., Patel, A. R., Machado, R. F., Lang, R. M., Kato, G. J., Gordeuk, V. R., Garcia, J. G., Desai, A. A., Artz, N., & Ahmad, H. (2011). Integration of Genomic and Genetic Approaches Highlight a Novel Validated Gene Signature for Pulmonary Hypertension Associated with Sickle Cell Disease. Blood, 118(21), 511-511. doi:10.1182/blood.v118.21.511.511
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  Abstract 511 Background: Pulmonary hypertension (PH) is a serious complication of sickle cell disease (SCD) associated with increased mortality. Gene expression profiles of peripheral blood mononuclear cells (PBMC) and genetic strategies have been studied in pulmonary arterial hypertension and in SCD. We hypothesized that a PBMC-derived gene signature in SCD patients may be utilized as a PH biomarker which may be further validated using integrated genomic and genetic strategies. Methods & Results: Twenty-seven patients with homozygous SCD underwent transthoracic echocardiography and PBMC isolation for genome—wide expression profiling. An independent SCD cohort (n=132) was genotyped using Affymetrix 6.0 SNP array and also underwent TTE. PH was defined as estimated right ventricular systolic pressure (RVSP)>30 mmHg with a peak tricuspid regurgitation velocity (TRV)>2.5m/s. Genome-wide mRNA and miRNA expression profiles were correlated against PH severity using RVSP (correlation coefficient ρ R ) and TRV (ρ T ) as surrogates. Utilizing a correlation threshold (ρ R 2 >0.15, ρ T 2 >0.15) for prioritization yielded 631 transcripts and 12 miRNAs. A support vector machine analysis on the transcripts identified a 10 gene signature which discriminated patients with and without echo-defined PH with 100% accuracy. This gene signature was then validated in an independent cohort of SCD patients with PH confirmed by right heart catheterization ( n =10) and without PH ( n =10) with 90% accuracy. In silico analyses of the top PH-related miRNAs revealed strong binding predictions of miR-301a to polypeptide N-acetylgalactosaminyltransferase 13 (GALNT13), a PH signature gene, which was further validated by microarray data confirming correlation between miR-301a and GALNT13 expression (p=0.024). Genome-wide association study in 132 adult SCD patients comparing echo-defined PH (n=51) versus no PH (n=81) revealed 12 significant SNPs, which were within or upstream to the PH signature genes (Table 1, P Disclosures: No relevant conflicts of interest to declare.
• Garcia, J. G. (2011). Focusing on the flood: targeting functional polymorphisms in ALI permeability pathways. American journal of respiratory and critical care medicine, 183(10), 1287-9.
• Garcia, J. G. (2011). Genomic investigations into acute inflammatory lung injury. Proceedings of the American Thoracic Society, 8(2), 167-72.
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  Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome, are complex illnesses involving the interplay of both environmental (such as mechanical ventilation) and genetic factors. To understand better the underlying mechanisms of pathogenesis associated with ALI, we recently identified several candidate genes by global expression profiling in preclinical models of ALI and relevant single-nucleotide polymorphisms. We summarize here several strategies successfully used to identify novel ALI candidate genes and detail the validation of variants in these genes as contributing factors to ALI pathobiology, conclusions based on functional analyses, and specific genetic association studies conducted in ALI cohorts. Continued insights into ALI pathogenesis and identification of genetic variants, which confer ALI risk and severity, promise to reveal novel molecular therapeutic targets that can be translated into personalized treatments to reduce the very high, unacceptable mortality of this disorder.
• Han, Y. J., Ma, S. F., Yourek, G., Park, Y. D., & Garcia, J. G. (2011). A transcribed pseudogene of MYLK promotes cell proliferation. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 25(7), 2305-12.
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  Pseudogenes are considered nonfunctional genomic artifacts of catastrophic pathways. Recent evidence, however, indicates novel roles for pseudogenes as regulators of gene expression. We tested the functionality of myosin light chain kinase pseudogene (MYLKP1) in human cells and tissues by RT-PCR, promoter activity, and cell proliferation assays. MYLKP1 is partially duplicated from the original MYLK gene that encodes nonmuscle and smooth muscle myosin light chain kinase (smMLCK) isoforms and regulates cell contractility and cytokinesis. Despite strong homology with the smMLCK promoter (∼ 89.9%), the MYLKP1 promoter is minimally active in normal bronchial epithelial cells but highly active in lung adenocarcinoma cells. Moreover, MYLKP1 and smMLCK exhibit negatively correlated transcriptional patterns in normal and cancer cells with MYLKP1 strongly expressed in cancer cells and smMLCK highly expressed in non-neoplastic cells. For instance, expression of smMLCK decreased (19.5 ± 4.7 fold) in colon carcinoma tissues compared to normal colon tissues. Mechanistically, MYLKP1 overexpression inhibits smMLCK expression in cancer cells by decreasing RNA stability, leading to increased cell proliferation. These studies provide strong evidence for the functional involvement of pseudogenes in carcinogenesis and suggest MYLKP1 as a potential novel diagnostic or therapeutic target in human cancers.
• Liu, K. D., Thompson, B. T., Ancukiewicz, M., Steingrub, J. S., Douglas, I. S., Matthay, M. A., Wright, P., Peterson, M. W., Rock, P., Hyzy, R. C., Anzueto, A., Truwit, J. D., & , N. I. (2011). Acute kidney injury in patients with acute lung injury: impact of fluid accumulation on classification of acute kidney injury and associated outcomes. Critical care medicine, 39(12), 2665-71.
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  It has been suggested that fluid accumulation may delay recognition of acute kidney injury. We sought to determine the impact of fluid balance on the incidence of nondialysis requiring acute kidney injury in patients with acute lung injury and to describe associated outcomes, including mortality.
• Ma, S. F., Xie, L., Pino-Yanes, M., Sammani, S., Wade, M. S., Letsiou, E., Siegler, J., Wang, T., Infusino, G., Kittles, R. A., Flores, C., Zhou, T., Prabhakar, B. S., Moreno-Vinasco, L., Villar, J., Jacobson, J. R., Dudek, S. M., & Garcia, J. G. (2011). Type 2 deiodinase and host responses of sepsis and acute lung injury. American journal of respiratory cell and molecular biology, 45(6), 1203-11.
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  The role of thyroid hormone metabolism in clinical outcomes of the critically ill remains unclear. Using preclinical models of acute lung injury (ALI), we assessed the gene and protein expression of type 2 deiodinase (DIO2), a key driver for synthesis of biologically active triiodothyronine, and addressed potential association of DIO2 genetic variants with ALI in a multiethnic cohort. DIO2 gene and protein expression levels in murine lung were validated by microarrays and immunoblotting. Lung injury was assessed by levels of bronchoalveolar lavage protein and leukocytes. Single-nucleotide polymorphisms were genotyped and ALI susceptibility association assessed. Significant increases in both DIO2 gene and D2 protein expression were observed in lung tissues from murine ALI models (LPS- and ventilator-induced lung injury), with expression directly increasing with the extent of lung injury. Mice with reduced levels of DIO2 expression (by silencing RNA) demonstrated reduced thyroxine levels in plasma and increased lung injury (increased bronchoalveolar lavage protein and leukocytes), suggesting a protective role for DIO2 in ALI. The G (Ala) allele of the Thr92Ala coding single-nucleotide polymorphism (rs225014) was protective in severe sepsis and severe sepsis-associated ALI after adjustments for age, sex, and genetic ancestry in a logistic regression model in European Americans. Our studies indicate that DIO2 is a novel ALI candidate gene, the nonsynonymous Thr92Ala coding variant of which confers ALI protection. Increased DIO2 expression may dampen the ALI inflammatory response, thereby strengthening the premise that thyroid hormone metabolism is intimately linked to the integrated response to inflammatory injury in critically ill patients.
• Mathew, B., Huang, Y., Jacobson, J. R., Berdyshev, E., Gerhold, L. M., Wang, T., Moreno-Vinasco, L., Lang, G., Zhao, Y., Chen, C. T., LaRiviere, P. J., Mauceri, H., Sammani, S., Husain, A. N., Dudek, S. M., Natarajan, V., Lussier, Y. A., Weichselbaum, R. R., & Garcia, J. G. (2011). Simvastatin attenuates radiation-induced murine lung injury and dysregulated lung gene expression. American journal of respiratory cell and molecular biology, 44(3), 415-22.
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  Novel therapies are desperately needed for radiation-induced lung injury (RILI), which, despite aggressive corticosteroid therapy, remains a potentially fatal and dose-limiting complication of thoracic radiotherapy. We assessed the utility of simvastatin, an anti-inflammatory and lung barrier-protective agent, in a dose- and time-dependent murine model of RILI (18-(25 Gy). Simvastatin reduced multiple RILI indices, including vascular leak, leukocyte infiltration, and histological evidence of oxidative stress, while reversing RILI-associated dysregulated gene expression, including p53, nuclear factor-erythroid-2-related factor, and sphingolipid metabolic pathway genes. To identify key regulators of simvastatin-mediated RILI protection, we integrated whole-lung gene expression data obtained from radiated and simvastatin-treated mice with protein-protein interaction network analysis (single-network analysis of proteins). Topological analysis of the gene product interaction network identified eight top-prioritized genes (Ccna2a, Cdc2, fcer1 g, Syk, Vav3, Mmp9, Itgam, Cd44) as regulatory nodes within an activated RILI network. These studies identify the involvement of specific genes and gene networks in RILI pathobiology, and confirm that statins represent a novel strategy to limit RILI.
• Mathew, B., Jacobson, J. R., Berdyshev, E., Huang, Y., Sun, X., Zhao, Y., Gerhold, L. M., Siegler, J., Evenoski, C., Wang, T., Zhou, T., Zaidi, R., Moreno-Vinasco, L., Bittman, R., Chen, C. T., LaRiviere, P. J., Sammani, S., Lussier, Y. A., Dudek, S. M., , Natarajan, V., et al. (2011). Role of sphingolipids in murine radiation-induced lung injury: protection by sphingosine 1-phosphate analogs. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 25(10), 3388-400.
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  Clinically significant radiation-induced lung injury (RILI) is a common toxicity in patients administered thoracic radiotherapy. Although the molecular etiology is poorly understood, we previously characterized a murine model of RILI in which alterations in lung barrier integrity surfaced as a potentially important pathobiological event and genome-wide lung gene mRNA levels identified dysregulation of sphingolipid metabolic pathway genes. We hypothesized that sphingolipid signaling components serve as modulators and novel therapeutic targets of RILI. Sphingolipid involvement in murine RILI was confirmed by radiation-induced increases in lung expression of sphingosine kinase (SphK) isoforms 1 and 2 and increases in the ratio of ceramide to sphingosine 1-phosphate (S1P) and dihydro-S1P (DHS1P) levels in plasma, bronchoalveolar lavage fluid, and lung tissue. Mice with a targeted deletion of SphK1 (SphK1(-/-)) or with reduced expression of S1P receptors (S1PR1(+/-), S1PR2(-/-), and S1PR3(-/-)) exhibited marked RILI susceptibility. Finally, studies of 3 potent vascular barrier-protective S1P analogs, FTY720, (S)-FTY720-phosphonate (fTyS), and SEW-2871, identified significant RILI attenuation and radiation-induced gene dysregulation by the phosphonate analog, fTyS (0.1 and 1 mg/kg i.p., 2×/wk) and to a lesser degree by SEW-2871 (1 mg/kg i.p., 2×/wk), compared with those in controls. These results support the targeting of S1P signaling as a novel therapeutic strategy in RILI.
• Mathew, B., Lennon, F. E., Siegler, J., Mirzapoiazova, T., Mambetsariev, N., Sammani, S., Gerhold, L. M., LaRiviere, P. J., Chen, C. T., Garcia, J. G., Salgia, R., Moss, J., & Singleton, P. A. (2011). The novel role of the mu opioid receptor in lung cancer progression: a laboratory investigation. Anesthesia and analgesia, 112(3), 558-67.
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  The possibility that μ opioid agonists can influence cancer recurrence is a subject of recent interest. Epidemiologic studies suggested that there were differences in cancer recurrence in breast and prostate cancer contingent on anesthetic regimens. In this study, we identify a possible mechanism for these epidemiologic findings on the basis of μ opioid receptor (MOR) regulation of Lewis lung carcinoma (LLC) tumorigenicity in cell and animal models.
• Mirzapoiazova, T., Moitra, J., Moreno-Vinasco, L., Sammani, S., Turner, J. R., Chiang, E. T., Evenoski, C., Wang, T., Singleton, P. A., Huang, Y., Lussier, Y. A., Watterson, D. M., Dudek, S. M., & Garcia, J. G. (2011). Non-muscle myosin light chain kinase isoform is a viable molecular target in acute inflammatory lung injury. American journal of respiratory cell and molecular biology, 44(1), 40-52.
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  Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non-muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (~50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody-conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6-signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation.
• Mitra, S., Sammani, S., Wang, T., Boone, D. L., Meyer, N. J., Dudek, S. M., Moreno-Vinasco, L., Garcia, J. G., & Jacobson, J. R. (2011). Role of growth arrest and DNA damage-inducible α in Akt phosphorylation and ubiquitination after mechanical stress-induced vascular injury. American journal of respiratory and critical care medicine, 184(9), 1030-40.
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  The stress-induced growth arrest and DNA damage-inducible a (GADD45a) gene is up-regulated by mechanical stress with GADD45a knockout (GADD45a(-/-)) mice demonstrating both increased susceptibility to ventilator-induced lung injury (VILI) and reduced levels of the cell survival and vascular permeability signaling effector (Akt). However, the functional role of GADD45a in the pathogenesis of VILI is unknown.
• Pendyala, S., Moitra, J., Kalari, S., Kleeberger, S. R., Zhao, Y., Reddy, S. P., Garcia, J. G., & Natarajan, V. (2011). Nrf2 regulates hyperoxia-induced Nox4 expression in human lung endothelium: identification of functional antioxidant response elements on the Nox4 promoter. Free radical biology & medicine, 50(12), 1749-59.
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  Reactive oxygen species (ROS) generated by vascular endothelial and smooth muscle cells contribute to the development and progression of vascular diseases. We have recently shown that hyperoxia enhances NADPH oxidase 4 (Nox4) expression, which regulates lung endothelial cell migration and angiogenesis. Regulation of Nox4 in the vasculature is poorly understood. The objective of this study was to identify the transcriptional factor(s) involved in regulation of endothelial Nox4. We found that hyperoxia-induced Nox4 expression was markedly reduced in Nrf2(-/-) mice, compared to Nrf2(+/+) mice. Exposure of human lung microvascular endothelial cells (HLMVECs) to hyperoxia stimulated Nrf2 translocation from the cytoplasm to the nucleus and increased Nox4 expression. Knockdown of Nrf2 expression using an siRNA approach attenuated basal Nox4 expression; however, it enhanced superoxide/ROS generation under both normoxia and hyperoxia. In silico analysis revealed the presence of at least three consensus sequences for the antioxidant response element (ARE) in the promoter region of Nox4. In transient transfections, hyperoxia stimulated Nox4 promoter activity in HLMVECs, and deletion of the -438 to -458 and -619 to -636 sequences markedly reduced hyperoxia-stimulated Nox4 promoter activation. ChIP analysis revealed an enhanced recruitment of Nrf2 to the endogenous Nox4 promoter spanning these two AREs after hyperoxic insult. Collectively, these results demonstrate, for the first time, a novel role for Nrf2 in regulating hyperoxia-induced Nox4 transcription via AREs in lung endothelium.
• Sammani, S., Park, K. S., Zaidi, S. R., Mathew, B., Wang, T., Huang, Y., Zhou, T., Lussier, Y. A., Husain, A. N., Moreno-Vinasco, L., Vigneswaran, W. T., & Garcia, J. G. (2011). A sphingosine 1-phosphate 1 receptor agonist modulates brain death-induced neurogenic pulmonary injury. American journal of respiratory cell and molecular biology, 45(5), 1022-7.
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  Lung transplantation remains the only viable therapy for patients with end-stage lung disease. However, the full utilization of this strategy is severely compromised by a lack of donor lung availability. The vast majority of donor lungs available for transplantation are from individuals after brain death (BD). Unfortunately, the early autonomic storm that accompanies BD often results in neurogenic pulmonary edema (NPE), producing varying degrees of lung injury or leading to primary graft dysfunction after transplantation. We demonstrated that sphingosine 1-phosphate (S1P)/analogues, which are major barrier-enhancing agents, reduce vascular permeability via the S1P1 receptor, S1PR1. Because primary lung graft dysfunction is induced by lung vascular endothelial cell barrier dysfunction, we hypothesized that the S1PR1 agonist, SEW-2871, may attenuate NPE when administered to the donor shortly after BD. Significant lung injury was observed after BD, with increases of approximately 60% in bronchoalveolar lavage (BAL) total protein, cell counts, and lung tissue wet/dry (W/D) weight ratios. In contrast, rats receiving SEW-2871 (0.1 mg/kg) 15 minutes after BD and assessed after 4 hours exhibited significant lung protection (∼ 50% reduction, P = 0.01), as reflected by reduced BAL protein/albumin, cytokines, cellularity, and lung tissue wet/dry weight ratio. Microarray analysis at 4 hours revealed a global impact of both BD and SEW on lung gene expression, with a differential gene expression of enriched immune-response/inflammation pathways across all groups. Overall, SEW served to attenuate the BD-mediated up-regulation of gene expression. Two potential biomarkers, TNF and chemokine CC motif receptor-like 2, exhibited gene array dysregulation. We conclude that SEW-2871 significantly attenuates BD-induced lung injury, and may serve as a potential candidate to improve human donor availability.
• Sweiss, N. J., Zhang, W., Franek, B. S., Kariuki, S. N., Moller, D. R., Patterson, K. C., Bennett, P., Girijala, L. R., Nair, V., Baughman, R. P., Garcia, J. G., & Niewold, T. B. (2011). Linkage of type I interferon activity and TNF-alpha levels in serum with sarcoidosis manifestations and ancestry. PloS one, 6(12), e29126.
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  Both type I interferon (IFN), also known as IFN-α and tumor necrosis factor alpha (TNF-α) have been implicated in the pathogenesis of sarcoidosis. We investigated serum levels of these cytokines in a large multi-ancestral sarcoidosis population to determine correlations between cytokine levels and disease phenotypes.
• Usatyuk, P. V., He, D., Bindokas, V., Gorshkova, I. A., Berdyshev, E. V., Garcia, J. G., & Natarajan, V. (2011). Photolysis of caged sphingosine-1-phosphate induces barrier enhancement and intracellular activation of lung endothelial cell signaling pathways. American journal of physiology. Lung cellular and molecular physiology, 300(6), L840-50.
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  Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates cellular functions by ligation via G protein-coupled S1P receptors. In addition to its extracellular action, S1P also has intracellular effects; however, the signaling pathways modulated by intracellular S1P remain poorly defined. We have previously demonstrated a novel pathway of intracellular S1P generation in human lung endothelial cells (ECs). In the present study, we examined the role of intracellular S1P generated by photolysis of caged S1P on EC barrier regulation and signal transduction. Intracellular S1P released from caged S1P caused mobilization of intracellular calcium, induced activation of MAPKs, redistributed cortactin, vascular endothelial cadherin, and β-catenin to cell periphery, and tightened endothelial barrier in human pulmonary artery ECs. Treatment of cells with pertussis toxin (PTx) had no effect on caged S1P-mediated effects on Ca(2+) mobilization, reorganization of cytoskeleton, cell adherens junction proteins, and barrier enhancement; however, extracellular S1P effects were significantly attenuated by PTx. Additionally, intracellular S1P also activated small GTPase Rac1 and its effector Ras GTPase-activating-like protein IQGAP1, suggesting involvement of these proteins in the S1P-mediated changes in cell-to-cell adhesion contacts. Downregulation of sphingosine kinase 1 (SphK1), but not SphK2, with siRNA or inhibition of SphK activity with an inhibitor 2-(p-hydroxyanilino)-4-(p-chlorophenyl) thiazole (CII) attenuated exogenously administrated S1P-induced EC permeability. Furthermore, S1P1 receptor inhibitor SB649164 abolished exogenous S1P-induced transendothelial resistance changes but had no effect on intracellular S1P generated by photolysis of caged S1P. These results provide evidence that intracellular S1P modulates signal transduction in lung ECs via signaling pathway(s) independent of S1P receptors.
• Wang, L., Chiang, E. T., Simmons, J. T., Garcia, J. G., & Dudek, S. M. (2011). FTY720-induced human pulmonary endothelial barrier enhancement is mediated by c-Abl. The European respiratory journal, 38(1), 78-88.
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  Strategies to improve pulmonary endothelial barrier function are needed to reverse the devastating effects of vascular leak in acute respiratory distress syndrome. FTY720 is a pharmaceutical analogue of the potent barrier-enhancing phospholipid sphingosine 1-phosphate (S1P). FTY720 decreases vascular permeability by an incompletely characterised mechanism that differs from S1P. Here, we describe its barrier-promoting effects on intracellular signalling and junctional assembly formation in human pulmonary endothelium. Permeability of cultured human pulmonary endothelial cells was assessed using transendothelial electrical resistance and dextran transwell assays. Junctional complex formation was assessed using membrane fractionation and immunofluorescence. Pharmacological inhibitors and small interfering (si)RNA were utilised to determine the effects of individual components on permeability. Unlike S1P, FTY720 failed to induce membrane translocation of adherens junction or tight junction proteins. β-catenin, occludin, claudin-5 or zona occludens protein (ZO)-1/ZO-2 siRNAs did not alter FTY720-induced barrier enhancement. FTY720 induced focal adhesion kinase (FAK) phosphorylation and focal adhesion formation, with FAK siRNA partially attenuating the prolonged phase of barrier enhancement. Inhibition of Src, protein kinase (PK)A, PKG, PKC or protein phosphatase 2A failed to alter FTY720-induced barrier enhancement. FTY720 increased c-Abl tyrosine kinase activity and c-Abl siRNA attenuated peak barrier enhancement after FTY720. FTY720 enhances endothelial barrier function by a novel pathway involving c-Abl signalling.
• Wolfson, R. K., Chiang, E. T., & Garcia, J. G. (2011). HMGB1 induces human lung endothelial cell cytoskeletal rearrangement and barrier disruption. Microvascular research, 81(2), 189-97.
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  Acute lung injury (ALI) results from loss of alveolar-capillary barrier integrity and the evolution of high-permeability pulmonary edema resulting in alveolar flooding and significant morbidity and mortality. HMGB1 is a late mediator of sepsis which uniquely participates in the evolution of sepsis and sepsis-induced ALI. The molecular events by which HMGB1 contributes to ALI remain poorly characterized. We characterized the role of HMGB1 in endothelial cell (EC) cytoskeletal rearrangement and vascular permeability, events essential to paracellular gap formation and barrier dysfunction characteristic of ALI. Initial experiments demonstrated HMGB1-mediated dose-dependent (5-20 μg/ml) decreases in transendothelial cell electrical resistance (TER) in the human pulmonary artery EC, a reflection of loss of barrier integrity. Furthermore, HMGB1 produced dose-dependent increases in paracellular gap formation in concert with loss of peripheral organized actin fibers, dissociation of cell-cell junctional cadherins, and the development of central stress fibers, a phenotypic change associated with increased contractile activity and increased EC permeability. Using siRNA strategies directed against known HMGB1 receptors (RAGE, TLR2, TLR4), we systematically determined that the receptor for advanced glycation end products (RAGE) is the primary receptor signaling HMGB1-induced TER decreases and paracellular gap formation via p38 MAP kinase activation and phosphorylation of the actin-binding protein, Hsp27. These studies add to the understanding of HMGB1-induced inflammatory events and vascular barrier disruption and offer the potential for clinical intervention in sepsis-induced ALI.
• Zhang, Y., Noth, I., Garcia, J. G., & Kaminski, N. (2011). A variant in the promoter of MUC5B and idiopathic pulmonary fibrosis. The New England journal of medicine, 364(16), 1576-7.
• Zhao, Y., Gorshkova, I. A., Berdyshev, E., He, D., Fu, P., Ma, W., Su, Y., Usatyuk, P. V., Pendyala, S., Oskouian, B., Saba, J. D., Garcia, J. G., & Natarajan, V. (2011). Protection of LPS-induced murine acute lung injury by sphingosine-1-phosphate lyase suppression. American journal of respiratory cell and molecular biology, 45(2), 426-35.
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  A defining feature of acute lung injury (ALI) is the increased lung vascular permeability and alveolar flooding, which leads to associated morbidity and mortality. Specific therapies to alleviate the unremitting vascular leak in ALI are not currently clinically available; however, our prior studies indicate a protective role for sphingosine-1-phosphate (S1P) in animal models of ALI with reductions in lung edema. As S1P levels are tightly regulated by synthesis and degradation, we tested the hypothesis that inhibition of S1P lyase (S1PL), the enzyme that irreversibly degrades S1P via cleavage, could ameliorate ALI. Intratracheal instillation of LPS to mice enhanced S1PL expression, decreased S1P levels in lung tissue, and induced lung inflammation and injury. LPS challenge of wild-type mice receiving 2-acetyl-4(5)-[1(R),2(S),3(R),4-tetrahydroxybutyl]-imidazole to inhibit S1PL or S1PL(+/-) mice resulted in increased S1P levels in lung tissue and bronchoalveolar lavage fluids and reduced lung injury and inflammation. Moreover, down-regulation of S1PL expression by short interfering RNA (siRNA) in primary human lung microvascular endothelial cells increased S1P levels, and attenuated LPS-mediated phosphorylation of p38 mitogen-activated protein kinase and I-κB, IL-6 secretion, and endothelial barrier disruption via Rac1 activation. These results identify a novel role for intracellularly generated S1P in protection against ALI and suggest S1PL as a potential therapeutic target.
• Zhou, T., Garcia, J. G., & Zhang, W. (2011). Integrating microRNAs into a system biology approach to acute lung injury. Translational research : the journal of laboratory and clinical medicine, 157(4), 180-90.
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  Acute lung injury (ALI), including the ventilator-induced lung injury (VILI) and the more severe acute respiratory distress syndrome (ARDS), are common and complex inflammatory lung diseases potentially affected by various genetic and nongenetic factors. Using the candidate gene approach, genetic variants associated with immune response and inflammatory pathways have been identified and implicated in ALI. Because gene expression is an intermediate phenotype that resides between the DNA sequence variation and the higher level cellular or whole-body phenotypes, the illustration of gene expression regulatory networks potentially could enhance understanding of disease susceptibility and the development of inflammatory lung syndromes. MicroRNAs (miRNAs) have emerged as a novel class of gene regulators that play critical roles in complex diseases including ALI. Comparisons of global miRNA profiles in animal models of ALI and VILI identified several miRNAs (eg, miR-146a and miR-155) previously implicated in immune response and inflammatory pathways. Therefore, via regulation of target genes in these biological processes and pathways, miRNAs potentially contribute to the development of ALI. Although this line of inquiry exists at a nascent stage, miRNAs have the potential to be critical components of a comprehensive model for inflammatory lung disease built by a systems biology approach that integrates genetic, genomic, proteomic, epigenetic as well as environmental stimuli information. Given their particularly recognized role in regulation of immune and inflammatory responses, miRNAs also serve as novel therapeutic targets and biomarkers for ALI/ARDS or VILI, thus facilitating the realization of personalized medicine for individuals with acute inflammatory lung disease.
• Zhou, T., Zhou, T., Zhang, W., Weichselbaum, R. R., Rondelli, D., Patel, P. R., Mahmud, N., Mahmud, D., & Garcia, J. G. (2011). Anti-Leukemic Activity Is Increased with Low Dose Busulfan and Irradiation: A Preclinical Model. Blood, 118(21), 4696-4696. doi:10.1182/blood.v118.21.4696.4696
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  Abstract 4696 Combinations of busulfan (Bu) and low dose total body irradiation are commonly used in conditioning regimens for allogeneic stem cell transplant in acute myeloid leukemia (AML). In this study we tested the in vitro effect of physiologic doses of Bu and ionizing radiation (IR) to address the question whether additive or synergistic antileukemic activity can be demonstrated in two leukemic cell lines, one sensitive (HL60) and one resistant (K562) to radiation. Cells were treated for 24 hours with Bu at 0, 12.5, 25, 50, 100 or 200 μ g/ml, or IR at 1.5, 3 or 6 Gy, or a combination of both with 12.5 or 25 μ g/ml for 24 hours, followed by IR at 1.5 or 3Gy. Cells were then tested for proliferation by thymidine uptake, apoptosis by surface expression of annexin V and caspase 3, and in-vitro colony formation assays. Exposure to Bu or IR for 24, 48 and 72 hours induced a dose dependent inhibition of proliferation and colony formation in both cell lines. However, 3 or 6 Gy of IR were both very effective against HL60 cells whereas a significant increase in cytotoxicity in K562 cells was observed only by using 6Gy (p=0.008). In-vitro treatment with either Bu or IR caused apoptotic cell death mainly in HL60 cells, but in a very limited amount in K562 cells. To test the effect of the combination of Bu and IR we utilized a low dose of Bu for 24 hours with low doses of radiation. HL60 cells were strongly inhibited by both agents when separate or combined. In contrast, treatment of the relatively radio-resistant K562 cells with low dose IR (3Gy) alone inhibited colony formation by 28%. The inhibitory effect increased to 84% when combined with low dose Bu. In order to identify genes associated with a response to Bu, we formed a linear regression model controlled for cancer type and also leukemic cell lines using GI50 and Stanford cDNA array data from the NCI-60. Among 7 genes (p
• Beiser, D. G., Wang, H., Li, J., Wang, X., Yordanova, V., Das, A., Mirzapoiazova, T., Garcia, J. G., Stern, S. A., & Vanden Hoek, T. L. (2010). Plasma and myocardial visfatin expression changes are associated with therapeutic hypothermia protection during murine hemorrhagic shock/resuscitation. Resuscitation, 81(6), 742-8.
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  Cytokine production during hemorrhagic shock (HS) could affect cardiac function during the hours after resuscitation. Visfatin is a recently described protein that functions both as a proinflammatory plasma cytokine and an intracellular enzyme within the nicotinamide adenine dinucleotide (NAD(+)) salvage pathway. We developed a mouse model of HS to study the effect of therapeutic hypothermia (TH) on hemodynamic outcomes and associated plasma and tissue visfatin content.
• Brown, M., Adyshev, D., Bindokas, V., Moitra, J., Garcia, J. G., & Dudek, S. M. (2010). Quantitative distribution and colocalization of non-muscle myosin light chain kinase isoforms and cortactin in human lung endothelium. Microvascular research, 80(1), 75-88.
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  Vascular barrier regulation is intimately linked to alterations in the distribution and configuration of the endothelial cell (EC) cytoskeleton in response to angiogenic and edemagenic agonists. Critical actin cytoskeletal rearrangement includes spatially directed increases in myosin light chain (MLC) phosphorylation, catalyzed by Ca(2+)/calmodulin-dependent non-muscle myosin light chain kinase variants (nmMLCK1- and -2), as well as association of nmMLCK with the actin-binding protein, cortactin. As these associations have proven difficult to quantify in a spatially specific manner, we now describe the utility of intensity correlation image analysis and the intensity correlation quotient (ICQ) to quantify colocalization in fixed and live cell imaging assays in human pulmonary artery EC. From baseline ICQ values averaging 0.216 reflecting colocalization of cortactin-DsRed with EGFP-nmMLCK fusion proteins in resting EC, thrombin-induced EC contraction significantly reduced cortactin-DsRed-EGFP-nmMLCK colocalization (nmMLCK1: ICQ=0.118; nmMLCK2: ICQ=0.091) whereas the potent EC barrier-protective agonist, sphingosine 1-phosphate (S1P), significantly increased nmMLCK-cortactin colocalization within lamellipodia (nmMLCK1: ICQ=0.275; nmMLCK2: ICQ=0.334). Over-expression of a cortactin-DsRed mutant fusion protein lacking the SH3 domain, known to be essential for cortactin-nmMLCK association, reduced baseline and S1P-mediated live cell colocalization with each nmMLCK variant (nmMLCK1: ICQ=0.160; nmMLCK2: ICQ=0.157). Similarly, expression of a truncated EGFP-nmMLCK2 mutant lacking cortactin- and actin-binding domains, markedly reduced basal localization in lamellipodia and abolished colocalization with cortactin-DsRed in lamellipodia after S1P (ICQ=-0.148). These data provide insights into the molecular basis for vascular barrier-regulatory cytoskeletal responses and support the utility of sophisticated imaging analyses and methodological assessment to quantify the critical nmMLCK and cortactin interaction during vascular barrier regulation.
• Chen, W., Garcia, J. G., & Jacobson, J. R. (2010). Integrin beta4 attenuates SHP-2 and MAPK signaling and reduces human lung endothelial inflammatory responses. Journal of cellular biochemistry, 110(3), 718-24.
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  We previously identified the marked upregulation of integrin beta4 in human lung endothelial cells (EC) treated with simvastatin, an HMG coA-reductase inhibitor with vascular-protective and anti-inflammatory properties in murine models of acute lung injury (ALI). We now investigate the role of integrin beta4 as a novel mediator of vascular inflammatory responses with a focus on mitogen-activated protein kinases (MAPK) signaling and the downstream expression of the inflammatory cytokines (IL-6 and IL-8) essential for the full elaboration of inflammatory lung injury. Silencing of integrin beta4 (siITGB4) in human lung EC resulted in significant increases in both basal and LPS-induced phosphorylation of ERK 1/2, JNK, and p38 MAPK, consistent with robust integrin beta4 regulation of MAPK activation. In addition, siITB4 increased both basal and LPS-induced expression of IL-6 and IL-8 mRNA and protein secretion into the media. We next observed that integrin beta4 silencing increased basal and LPS-induced phosphorylation of SHP-2, a protein tyrosine phosphatase known to modulate MAPK signaling. In contrast, inhibition of SHP-2 enzymatic activity (sodium stibogluconate) abrogated the increased ERK phosphorylation associated with integrin beta4 silencing in LPS-treated EC and attenuated the increases in levels of IL-6 and IL-8 in integrin-beta4-silenced EC. These findings highlight a novel negative regulatory role for integrin beta4 in EC inflammatory responses involving SHP-2-mediated MAPK signaling. Upregulation of integrin beta4 may represent an important element of the anti-inflammatory and vascular-protective properties of statins and provides a novel strategy to limit inflammatory vascular syndromes.
• Danilov, S. M., Balyasnikova, I. V., Danilova, A. S., Naperova, I. A., Arablinskaya, N. E., Borisov, S. E., Metzger, R., Franke, F. E., Schwartz, D. E., Gachok, I. V., Trakht, I. N., Kost, O. A., & Garcia, J. G. (2010). Conformational fingerprinting of the angiotensin I-converting enzyme (ACE). 1. Application in sarcoidosis. Journal of proteome research, 9(11), 5782-93.
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  Fine epitope mapping of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) revealed that the epitopes of all mAbs contained putative glycosylation sites. ACE glycosylation is both cell- and tissue-specific and, therefore, the local conformation of ACE produced by different cells could be also unique. The pattern of ACE binding by a set of mAbs to 16 epitopes of human ACE - "conformational fingerprint of ACE" - is the most sensitive marker of ACE conformation and could be cell- and tissue-specific. The recognition of ACEs by mAbs to ACE was estimated using an immune-capture enzymatic plate precipitation assay. Precipitation patterns of soluble recombinant ACE released from Chinese hamster ovary (CHO)-ACE cells was influenced by conditions that alter ACE glycosylation. This pattern was also strongly cell type specific. Patients with sarcoidosis exhibited conformational fingerprints of tissue ACE (lungs and lymph nodes), as well as blood ACE, which were distinct from controls. Conformational fingerprinting of ACE may detect ACE originated from the cells other than endothelial cells in the blood and when combined with elevated blood ACE levels in patients with sarcoidosis may potentially reflect extrapulmonary sarcoidosis involvement (bone marrow, spleen, liver). If proven true, this would serve as a biomarker of enormous potential clinical significance.
• Desai, A. A., Parikh, D. S., Moreno-vinasco, L., Gera, A., Garcia, J. G., & Desai, A. A. (2010). Role of Caldesmon in hypoxia-mediated endothelial cell response. The FASEB Journal, 24.
• Dudek, S. M., Chiang, E. T., Camp, S. M., Guo, Y., Zhao, J., Brown, M. E., Singleton, P. A., Wang, L., Desai, A., Arce, F. T., Lal, R., Van Eyk, J. E., Imam, S. Z., & Garcia, J. G. (2010). Abl tyrosine kinase phosphorylates nonmuscle Myosin light chain kinase to regulate endothelial barrier function. Molecular biology of the cell, 21(22), 4042-56.
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  Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl-mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.
• Jacob, A., Hack, B., Chiang, E., Garcia, J. G., Quigg, R. J., & Alexander, J. J. (2010). C5a alters blood-brain barrier integrity in experimental lupus. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 24(6), 1682-8.
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  The blood-brain barrier (BBB) is a crucial anatomic location in the brain. Its dysfunction complicates many neurodegenerative diseases, from acute conditions, such as sepsis, to chronic diseases, such as systemic lupus erythematosus (SLE). Several studies suggest an altered BBB in lupus, but the underlying mechanism remains unknown. In the current study, we observed a definite loss of BBB integrity in MRL/MpJ-Tnfrsf6(lpr) (MRL/lpr) lupus mice by IgG infiltration into brain parenchyma. In line with this result, we examined the role of complement activation, a key event in this setting, in maintenance of BBB integrity. Complement activation generates C5a, a molecule with multiple functions. Because the expression of the C5a receptor (C5aR) is significantly increased in brain endothelial cells treated with lupus serum, the study focused on the role of C5a signaling through its G-protein-coupled receptor C5aR in brain endothelial cells, in a lupus setting. Reactive oxygen species production increased significantly in endothelial cells, in both primary cells and the bEnd3 cell line treated with lupus serum from MRL/lpr mice, compared with those treated with control serum from MRL(+/+) mice. In addition, increased permeability monitored by changes in transendothelial electrical resistance, cytoskeletal remodeling caused by actin fiber rearrangement, and increased iNOS mRNA expression were observed in bEnd3 cells. These disruptive effects were alleviated by pretreating cells with a C5a receptor antagonist (C5aRant) or a C5a antibody. Furthermore, the structural integrity of the vasculature in MRL/lpr brain was maintained by C5aR inhibition. These results demonstrate the regulation of BBB integrity by the complement system in a neuroinflammatory setting. For the first time, a novel role of C5a in the maintenance of BBB integrity is identified and the potential of C5a/C5aR blockade highlighted as a promising therapeutic strategy in SLE and other neurodegenerative diseases.
• Lennon, F. E., Singleton, P. A., Siegler, J., Salgia, R., Moss, J., Moreno-vinasco, L., Mathew, B., Mambetsariev, N., Lennon, F. E., Gerhold, L., & Garcia, J. G. (2010). Effect of methylnaltrexone inhibition of the mu opioid receptor on Lewis lung carcinoma proliferation, invasion, and angiogenesis.. Journal of Clinical Oncology, 28(15_suppl), e13597-e13597. doi:10.1200/jco.2010.28.15_suppl.e13597
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  e13597 Background: Expression of the mu opioid receptor (MOR) is increased in patients with non-small cell lung cancer (NSCLC), a disease with poor prognosis and limited therapies. In this study, w...
• Mambetsariev, N., Mirzapoiazova, T., Mambetsariev, B., Sammani, S., Lennon, F. E., Garcia, J. G., & Singleton, P. A. (2010). Hyaluronic Acid binding protein 2 is a novel regulator of vascular integrity. Arteriosclerosis, thrombosis, and vascular biology, 30(3), 483-90.
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  The disruption of the endothelial cell barrier is a critical feature of inflammation and an important contributing factor to acute lung injury (ALI), an inflammatory condition that is a major cause of morbidity and mortality in critically ill patients. We evaluated the role of the extracellular serine protease, hyaluronic acid binding protein 2 (HABP2), in vascular barrier regulation.
• Moreno-Vinasco, L., & Garcia, J. G. (2010). Receptor tyrosine kinase inhibitors in rodent pulmonary hypertension. Advances in experimental medicine and biology, 661, 419-34.
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  Pulmonary hypertension (PH) is a disorder characterized by vascular remodeling and proliferation, a phenotype dependent upon unimpeded growth factor and kinase pathway activation with strong similarities to malignant tumors. This chapter details our novel application of the multikinase inhibitor, sorafenib, in rodent models of PH to improved hemodynamic parameters and attenuates PH structural changes1. Sorafenib is a Raf kinase inhibitor and our biochemical and genomic evidence supported the potential involvement of the MAPK cascade system and TGFB3 in PH development and the response to therapy. Integration of expression genomic analyses coupled with intense bioinformatics identified gene expression and ontology signatures in the development of PH and implicated the role of cytoskeletal protein such as caldesmon or nmMLCK as potentially key participants in PH-induced vascular remodeling and proliferation. Our studies suggest the PKI sorafenib as a potentially novel treatment for severe PH with the MAPK cascade a potential canonical target profoundly effecting vascular cytoskeletal -rearrangements and remodeling1.
• Peng, X. Q., Damarla, M., Skirball, J., Nonas, S., Wang, X. Y., Han, E. J., Hasan, E. J., Cao, X., Boueiz, A., Damico, R., Tuder, R. M., Sciuto, A. M., Anderson, D. R., Garcia, J. G., Kass, D. A., Hassoun, P. M., & Zhang, J. T. (2010). Protective role of PI3-kinase/Akt/eNOS signaling in mechanical stress through inhibition of p38 mitogen-activated protein kinase in mouse lung. Acta pharmacologica Sinica, 31(2), 175-83.
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  To test the hypothesis that PI3K/Akt/eNOS signaling has a protective role in a murine model of ventilation associated lung injury (VALI) through down-regulation of p38 MAPK signaling.
• Sammani, S., Moreno-Vinasco, L., Mirzapoiazova, T., Singleton, P. A., Chiang, E. T., Evenoski, C. L., Wang, T., Mathew, B., Husain, A., Moitra, J., Sun, X., Nunez, L., Jacobson, J. R., Dudek, S. M., Natarajan, V., & Garcia, J. G. (2010). Differential effects of sphingosine 1-phosphate receptors on airway and vascular barrier function in the murine lung. American journal of respiratory cell and molecular biology, 43(4), 394-402.
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  The therapeutic options for ameliorating the profound vascular permeability, alveolar flooding, and organ dysfunction that accompanies acute inflammatory lung injury (ALI) remain limited. Extending our previous finding that the intravenous administration of the sphingolipid angiogenic factor, sphingosine 1-phosphate (S1P), attenuates inflammatory lung injury and vascular permeability via ligation of S1PR(1), we determine that a direct intratracheal or intravenous administration of S1P, or a selective S1P receptor (S1PR(1)) agonist (SEW-2871), produces highly concentration-dependent barrier-regulatory responses in the murine lung. The intratracheal or intravenous administration of S1P or SEW-2871 at < 0.3 mg/kg was protective against LPS-induced murine lung inflammation and permeability. However, intratracheal delivery of S1P at 0.5 mg/kg (for 2 h) resulted in significant alveolar-capillary barrier disruption (with a 42% increase in bronchoalveolar lavage protein), and produced rapid lethality when delivered at 2 mg/kg. Despite the greater selectivity for S1PR(1), intratracheally delivered SEW-2871 at 0.5 mg/kg also resulted in significant alveolar-capillary barrier disruption, but was not lethal at 2 mg/kg. Consistent with the S1PR(1) regulation of alveolar/vascular barrier function, wild-type mice pretreated with the S1PR(1) inverse agonist, SB-649146, or S1PR(1)(+/-) mice exhibited reduced S1P/SEW-2871-mediated barrier protection after challenge with LPS. In contrast, S1PR(2)(-/-) knockout mice as well as mice with reduced S1PR(3) expression (via silencing S1PR3-containing nanocarriers) were protected against LPS-induced barrier disruption compared with control mice. These studies underscore the potential therapeutic effects of highly selective S1PR(1) receptor agonists in reducing inflammatory lung injury, and highlight the critical role of the S1P delivery route, S1PR(1) agonist concentration, and S1PR(1) expression in target tissues.
• Sammani, S., Zaidi, S. R., Toth, P. T., Sammani, S., Moreno-vinasco, L., Mirzapoiazova, T., Machado, R. F., Garcia, J. G., Dweik, R. A., Chiang, E. T., Ayetkin, M., & Archer, S. L. (2010). Novel role of Pre-B-Cell Colony Enhancing Factor (PBEF) in pulmonary arterial hypertension (PAH). The FASEB Journal, 24.
• Shah, C. V., Lanken, P. N., Localio, A. R., Gallop, R., Bellamy, S., Ma, S. F., Flores, C., Kahn, J. M., Finkel, B., Fuchs, B. D., Garcia, J. G., & Christie, J. D. (2010). An alternative method of acute lung injury classification for use in observational studies. Chest, 138(5), 1054-61.
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  In observational studies using acute lung injury (ALI) as an outcome, a spectrum of lung injury and difficult-to-interpret chest radiographs (CXRs) may hamper efforts to uncover risk factor associations. We assessed the impact of excluding patients with difficult-to-classify or equivocal ALI diagnosis on clinical and genetic risk factor associations for ALI after trauma.
• Singleton, P. A., Mambetsariev, N., Lennon, F. E., Mathew, B., Siegler, J. H., Moreno-Vinasco, L., Salgia, R., Moss, J., & Garcia, J. G. (2010). Methylnaltrexone potentiates the anti-angiogenic effects of mTOR inhibitors. Journal of angiogenesis research, 2(1), 5.
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  Recent cancer therapies include drugs that target both tumor growth and angiogenesis including mammalian target of rapamycin (mTOR) inhibitors. Since mTOR inhibitor therapy is associated with significant side effects, we examined potential agents that can reduce the therapeutic dose.
• Singleton, P. A., Mirzapoiazova, T., Guo, Y., Sammani, S., Mambetsariev, N., Lennon, F. E., Moreno-Vinasco, L., & Garcia, J. G. (2010). High-molecular-weight hyaluronan is a novel inhibitor of pulmonary vascular leakiness. American journal of physiology. Lung cellular and molecular physiology, 299(5), L639-51.
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  Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, a perturbation observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that enhance EC barrier integrity have important therapeutic implications. We observed that binding of high-molecular-weight hyaluronan (HMW-HA) to its cognate receptor CD44 within caveolin-enriched microdomains (CEM) enhances human pulmonary EC barrier function. Immunocytochemical analysis indicated that HMW-HA promotes redistribution of a significant population of CEM to areas of cell-cell contact. Quantitative proteomic analysis of CEM isolated from human EC demonstrated HMW-HA-mediated recruitment of cytoskeletal regulatory proteins (annexin A2, protein S100-A10, and filamin A/B). Inhibition of CEM formation [caveolin-1 small interfering RNA (siRNA) and cholesterol depletion] or silencing (siRNA) of CD44, annexin A2, protein S100-A10, or filamin A/B expression abolished HMW-HA-induced actin cytoskeletal reorganization and EC barrier enhancement. To confirm our in vitro results in an in vivo model of inflammatory lung injury with vascular hyperpermeability, we observed that the protective effects of HMW-HA on LPS-induced pulmonary vascular leakiness were blocked in caveolin-1 knockout mice. Furthermore, targeted inhibition of CD44 expression in the mouse pulmonary vasculature significantly reduced HMW-HA-mediated protection from LPS-induced hyperpermeability. These data suggest that HMW-HA, via CD44-mediated CEM signaling events, represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.
• Sun, X., Ma, S. F., Wade, M. S., Flores, C., Pino-Yanes, M., Moitra, J., Ober, C., Kittles, R., Husain, A. N., Ford, J. G., & Garcia, J. G. (2010). Functional variants of the sphingosine-1-phosphate receptor 1 gene associate with asthma susceptibility. The Journal of allergy and clinical immunology, 126(2), 241-9, 249.e1-3.
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  The genetic mechanisms underlying asthma remain unclear. Increased permeability of the microvasculature is a feature of asthma, and the sphingosine-1-phosphate receptor (S1PR1) is an essential participant regulating lung vascular integrity and responses to lung inflammation.
• Sweiss, N. J., Patterson, K., Sawaqed, R., Jabbar, U., Korsten, P., Hogarth, K., Wollman, R., Garcia, J. G., Niewold, T. B., & Baughman, R. P. (2010). Rheumatologic manifestations of sarcoidosis. Seminars in respiratory and critical care medicine, 31(4), 463-73.
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  Sarcoidosis is a systemic, clinically heterogeneous disease characterized by the development of granulomas. Any organ system can be involved, and patients may present with any number of rheumatologic symptoms. There are no U.S. Food and Drug Administration-approved therapies for the treatment of sarcoidosis. Diagnosing sarcoidosis becomes challenging, particularly when its complications cause patients' symptoms to mimic other conditions, including polymyositis, Sjögren syndrome, or vasculitis. This review presents an overview of the etiology of and biomarkers associated with sarcoidosis. We then provide a detailed description of the rheumatologic manifestations of sarcoidosis and present a treatment algorithm based on current clinical evidence for patients with sarcoid arthritis. The discussion will focus on characteristic findings in patients with sarcoid arthritis, osseous involvement in sarcoidosis, and sarcoid myopathy. Arthritic conditions that sometimes coexist with sarcoidosis are described as well. We present two cases of sarcoidosis with rheumatologic manifestations. Our intent is to encourage a multidisciplinary, translational approach to meet the challenges and difficulties in understanding and treating sarcoidosis.
• Sweiss, N. J., Salloum, R., Gandhi, S., Ghandi, S., Alegre, M. L., Sawaqed, R., Badaracco, M., Pursell, K., Pitrak, D., Baughman, R. P., Moller, D. R., Garcia, J. G., & Niewold, T. B. (2010). Significant CD4, CD8, and CD19 lymphopenia in peripheral blood of sarcoidosis patients correlates with severe disease manifestations. PloS one, 5(2), e9088.
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  Sarcoidosis is a poorly understood chronic inflammatory condition. Infiltration of affected organs by lymphocytes is characteristic of sarcoidosis, however previous reports suggest that circulating lymphocyte counts are low in some patients with the disease. The goal of this study was to evaluate lymphocyte subsets in peripheral blood in a cohort of sarcoidosis patients to determine the prevalence, severity, and clinical features associated with lymphopenia in major lymphocyte subsets.
• Wang, T., Chiang, E. T., Moreno-Vinasco, L., Lang, G. D., Pendyala, S., Samet, J. M., Geyh, A. S., Breysse, P. N., Chillrud, S. N., Natarajan, V., & Garcia, J. G. (2010). Particulate matter disrupts human lung endothelial barrier integrity via ROS- and p38 MAPK-dependent pathways. American journal of respiratory cell and molecular biology, 42(4), 442-9.
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  Epidemiologic studies have linked exposure to airborne pollutant particulate matter (PM) with increased cardiopulmonary mortality and morbidity. The mechanisms of PM-mediated lung pathophysiology, however, remain unknown. We tested the hypothesis that PM, via enhanced oxidative stress, disrupts lung endothelial cell (EC) barrier integrity, thereby enhancing organ dysfunction. Using PM collected from Ft. McHenry Tunnel (Baltimore, MD), we assessed PM-mediated changes in transendothelial electrical resistance (TER) (a highly sensitive measure of barrier function), reactive oxygen species (ROS) generation, and p38 mitogen-activated protein kinase (MAPK) activation in human pulmonary artery EC. PM induced significant dose (10-100 microg/ml)- and time (0-10 h)-dependent EC barrier disruption reflected by reduced TER values. Exposure of human lung EC to PM resulted in significant ROS generation, which was directly involved in PM-mediated EC barrier dysfunction, as N-acetyl-cysteine (NAC, 5 mM) pretreatment abolished both ROS production and barrier disruption induced by PM. Furthermore, PM induced p38 MAPK activation and HSP27 phosphorylation, events that were both attenuated by NAC. In addition, PM-induced EC barrier disruption was partially prevented by the p38 MAP kinase inhibitor SB203580 (10 microM) as well as by reduced expression of either p38 MAPK beta or HSP27 (siRNA). These results demonstrate that PM induces ROS generation in human lung endothelium, resulting in oxidative stress-mediated EC barrier disruption via p38 MAPK- and HSP27-dependent pathways. These findings support a novel mechanism for PM-induced lung dysfunction and adverse cardiopulmonary outcomes.
• Wang, T., Svensson, E. C., Samet, J. M., Prabhakar, N. R., Peng, Y., Natarajan, V., Moreno-vinasco, L., Lussier, Y. A., Linares, J. D., Lang, R. M., Lang, G. D., Huang, Y., Goonewardena, S., Geyh, A. S., Garcia, J. G., Breysse, P. N., & Beshai, J. F. (2010). Particulate Matter Exacerbates Respiratory and Cardiac Arrhythmias in Cardiomyopathic Mice via Heightened Carotid Body Sensitivity. Air Pollution and Health.
• Ware, L. B., Koyama, T., Billheimer, D. D., Wu, W., Bernard, G. R., Thompson, B. T., Brower, R. G., Standiford, T. J., Martin, T. R., Matthay, M. A., & , N. A. (2010). Prognostic and pathogenetic value of combining clinical and biochemical indices in patients with acute lung injury. Chest, 137(2), 288-96.
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  No single clinical or biologic marker reliably predicts clinical outcomes in acute lung injury (ALI)/ARDS. We hypothesized that a combination of biologic and clinical markers would be superior to either biomarkers or clinical factors alone in predicting ALI/ARDS mortality and would provide insight into the pathogenesis of clinical ALI/ARDS.
• Zaidi, R., Sammani, S., Moreno-vinasco, L., Machado, R. F., & Garcia, J. G. (2010). 242: Pre-B-Cell Colony Enhancing Factor (PBEF) Is a Novel target in Hypoxia-Induced Murine Pulmonary Hypertension. Journal of Heart and Lung Transplantation, 29(2), S83-S83. doi:10.1016/j.healun.2009.11.253
• Berdyshev, E. V., Gorshkova, I., Skobeleva, A., Bittman, R., Lu, X., Dudek, S. M., Mirzapoiazova, T., Garcia, J. G., & Natarajan, V. (2009). FTY720 inhibits ceramide synthases and up-regulates dihydrosphingosine 1-phosphate formation in human lung endothelial cells. The Journal of biological chemistry, 284(9), 5467-77.
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  Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent sphingosine 1-phosphate (S1P)-like action through S1P(1,3-5) receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with l-[U-(13)C, (15)N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, sphingosine, and S1P but increased levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent K(i) of 2.15 microm. FTY720-induced up-regulation of DHS1P level was mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells.
• Birukova, A. A., Arce, F. T., Moldobaeva, N., Dudek, S. M., Garcia, J. G., Lal, R., & Birukov, K. G. (2009). Endothelial permeability is controlled by spatially defined cytoskeletal mechanics: atomic force microscopy force mapping of pulmonary endothelial monolayer. Nanomedicine : nanotechnology, biology, and medicine, 5(1), 30-41.
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  Actomyosin contraction directly regulates endothelial cell (EC) permeability, but intracellular redistribution of cytoskeletal tension associated with EC permeability is poorly understood. We used atomic force microscopy (AFM), EC permeability assays, and fluorescence microscopy to link barrier regulation, cell remodeling, and cytoskeletal mechanical properties in EC treated with barrier-protective as well as barrier-disruptive agonists. Thrombin, vascular endothelial growth factor, and hydrogen peroxide increased EC permeability, disrupted cell junctions, and induced stress fiber formation. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, hepatocyte growth factor, and iloprost tightened EC barriers, enhanced peripheral actin cytoskeleton and adherens junctions, and abolished thrombin-induced permeability and EC remodeling. AFM force mapping and imaging showed differential distribution of cell stiffness: barrier-disruptive agonists increased stiffness in the central region, and barrier-protective agents decreased stiffness in the center and increased it at the periphery. Attenuation of thrombin-induced permeability correlates well with stiffness changes from the cell center to periphery. These results directly link for the first time the patterns of cell stiffness with specific EC permeability responses.
• Calfee, C. S., Eisner, M. D., Parsons, P. E., Thompson, B. T., Conner, E. R., Matthay, M. A., Ware, L. B., & , N. A. (2009). Soluble intercellular adhesion molecule-1 and clinical outcomes in patients with acute lung injury. Intensive care medicine, 35(2), 248-57.
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  To determine if levels of soluble intercellular adhesion molecule-1 (sICAM-1), a marker of alveolar epithelial and endothelial injury, differ in patients with hydrostatic pulmonary edema and acute lung injury (ALI) and are associated with clinical outcomes in patients with ALI.
• Camp, S. M., Bittman, R., Chiang, E. T., Moreno-Vinasco, L., Mirzapoiazova, T., Sammani, S., Lu, X., Sun, C., Harbeck, M., Roe, M., Natarajan, V., Garcia, J. G., & Dudek, S. M. (2009). Synthetic analogs of FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] differentially regulate pulmonary vascular permeability in vivo and in vitro. The Journal of pharmacology and experimental therapeutics, 331(1), 54-64.
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  Novel therapies are needed to address the vascular endothelial cell (EC) barrier disruption that occurs in inflammatory diseases such as acute lung injury (ALI). We previously demonstrated the potent barrier-enhancing effects of both sphingosine 1-phosphate (S1P) and the structurally similar compound FTY720 [2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol] in inflammatory lung injury. In this study, we examined the therapeutic potential of several novel FTY720 analogs to reduce vascular leak. Similar to S1P and FTY720, the (R)- and (S)-enantiomers of FTY720 phosphonate and enephosphonate analogs produce sustained EC barrier enhancement in vitro, as seen by increases in transendothelial electrical resistance (TER). In contrast, the (R)- and (S)-enantiomers of FTY720-regioisomeric analogs disrupt EC barrier integrity in a dose-dependent manner. Barrier-enhancing FTY720 analogs demonstrate a wider protective concentration range in vitro (1-50 microM) and greater potency than either S1P or FTY720. In contrast to FTY720-induced EC barrier enhancement, S1P and the FTY720 analogs dramatically increase TER within minutes in association with cortical actin ring formation. Unlike S1P, these FTY720 analogs exhibit differential phosphorylation effects without altering the intracellular calcium level. Inhibitor studies indicate that barrier enhancement by these analogs involves signaling via G(i)-coupled receptors, tyrosine kinases, and lipid rafts. Consistent with these in vitro responses, the (S)-phosphonate analog of FTY720 significantly reduces multiple indices of alveolar and vascular permeability in a lipopolysaccharide-mediated murine model of ALI (without significant alterations in leukocyte counts). These results demonstrate the capacity for FTY720 analogs to significantly decrease pulmonary vascular leakage and inflammation in vitro and in vivo.
• Chen, B., Yates, E., Huang, Y., Kogut, P., Ma, L., Turner, J. R., Tao, Y., Camoretti-Mercado, B., Lang, D., Svensson, E. C., Garcia, J. G., Gruber, P. J., Morrisey, E. E., & Solway, J. (2009). Alternative promoter and GATA5 transcripts in mouse. American journal of physiology. Gastrointestinal and liver physiology, 297(6), G1214-22.
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  GATA5 is a member of the GATA zinc finger transcription factor family involved in tissue-specific transcriptional regulation during cell differentiation and embryogenesis. Previous reports indicate that null mutation of the zebrafish GATA5 gene results in embryonic lethality, whereas deletion of exon 1 from the mouse GATA5 gene causes only derangement of female urogenital development. Here, we have identified an alternate promoter within intron 1 of the mouse GATA5 gene that transcribes a 2.5-kb mRNA that lacks exon 1 entirely but includes 82 bp from intron 1 and all of exons 2-6. The alternative promoter was active during transient transfection in cultured airway myocytes and bronchial epithelial cells, and it drove reporter gene expression in gastric epithelial cells in transgenic mice. The 2.5-kb alternative transcript encodes an NH(2)-terminally truncated "short GATA5" comprising aa 226-404 with a single zinc finger, which retains ability to transactivate the atrial natriuretic factor promoter (albeit less efficiently than full-length GATA5). Another new GATA5 transcript contains all of exons 1-5 and the 5' portion of exon 6 but lacks the terminal 1143 bp of the 3'-untranslated region from exon 6. These findings extend current understanding of the tissue distribution of GATA5 expression and suggests that GATA5 expression and function are more complex than previously appreciated.
• Chiang, E. T., Camp, S. M., Dudek, S. M., Brown, M. E., Usatyuk, P. V., Zaborina, O., Alverdy, J. C., & Garcia, J. G. (2009). Protective effects of high-molecular weight polyethylene glycol (PEG) in human lung endothelial cell barrier regulation: role of actin cytoskeletal rearrangement. Microvascular research, 77(2), 174-86.
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  Acute lung injury represents the result of multiple pathways initiated by local or systemic insults and is characterized by profound vascular permeability, pulmonary edema, and life-threatening respiratory failure. Permeability-reducing therapies are of potential clinical utility but are currently unavailable. We hypothesized that polyethylene glycol (PEG) compounds, inert and non-toxic polymers that serve as a surrogate mucin lining in intestinal epithelium, may attenuate agonist-mediated lung endothelial cell (EC) barrier dysfunction. High molecular weight PEG (PEG15-20) produced rapid, dose-dependent increases in transendothelial electrical resistance (TER) in human lung endothelium cultured on gold microelectrodes, reflecting increased paracellular integrity. The maximal effective concentration of 8% PEG induced a sustained 125% increase in TER (40 h), results similar to barrier-enhancing agonists such as sphingosine 1-phosphate (40% increase in TER). Maximal PEG barrier enhancement was achieved at 45-60 min and PEG effectively reversed both thrombin- and LPS-induced EC barrier dysfunction. Consistent with the increase in TER, immunofluorescent studies demonstrated that PEG produced significant cytoskeletal rearrangement with formation of well-defined cortical actin rings and lamellipodia containing the actin-binding proteins, cortactin and MLCK, known participants in cell-matrix and cell-cell junctional adhesion. Finally, PEG challenge induced rapid alterations in levels of MAP kinase and MLC phosphorylation. In summary, PEG joins a number of EC barrier-regulatory agents which rapidly activate barrier-enhancing signal transduction pathways which target the cytoskeleton and provides a potential therapeutic strategy in inflammatory lung injury.
• Erickson, S. E., Shlipak, M. G., Martin, G. S., Wheeler, A. P., Ancukiewicz, M., Matthay, M. A., Eisner, M. D., & , N. I. (2009). Racial and ethnic disparities in mortality from acute lung injury. Critical care medicine, 37(1), 1-6.
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  Little is known about the influence of race and ethnicity on mortality from acute lung injury (ALI). We sought to determine whether black race or Hispanic ethnicity is independently associated with mortality among patients with ALI.
• Finigan, J. H., Boueiz, A., Wilkinson, E., Damico, R., Skirball, J., Pae, H. H., Damarla, M., Hasan, E., Pearse, D. B., Reddy, S. P., Grigoryev, D. N., Cheadle, C., Esmon, C. T., Garcia, J. G., & Hassoun, P. M. (2009). Activated protein C protects against ventilator-induced pulmonary capillary leak. American journal of physiology. Lung cellular and molecular physiology, 296(6), L1002-11.
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  The coagulation system is central to the pathophysiology of acute lung injury. We have previously demonstrated that the anticoagulant activated protein C (APC) prevents increased endothelial permeability in response to edemagenic agonists in endothelial cells and that this protection is dependent on the endothelial protein C receptor (EPCR). We currently investigate the effect of APC in a mouse model of ventilator-induced lung injury (VILI). C57BL/6J mice received spontaneous ventilation (control) or mechanical ventilation (MV) with high (HV(T); 20 ml/kg) or low (LV(T); 7 ml/kg) tidal volumes for 2 h and were pretreated with APC or vehicle via jugular vein 1 h before MV. In separate experiments, mice were ventilated for 4 h and received APC 30 and 150 min after starting MV. Indices of capillary leakage included bronchoalveolar lavage (BAL) total protein and Evans blue dye (EBD) assay. Changes in pulmonary EPCR protein and Rho-associated kinase (ROCK) were assessed using SDS-PAGE. Thrombin generation was measured via plasma thrombin-antithrombin complexes. HV(T) induced pulmonary capillary leakage, as evidenced by significant increases in BAL protein and EBD extravasation, without significantly increasing thrombin production. HV(T) also caused significant decreases in pulmonary, membrane-bound EPCR protein levels and increases in pulmonary ROCK-1. APC treatment significantly decreased pulmonary leakage induced by MV when given either before or after initiation of MV. Protection from capillary leakage was associated with restoration of EPCR protein expression and attenuation of ROCK-1 expression. In addition, mice overexpressing EPCR on the pulmonary endothelium were protected from HV(T)-mediated injury. Finally, gene microarray analysis demonstrated that APC significantly altered the expression of genes relevant to vascular permeability at the ontology (e.g., blood vessel development) and specific gene (e.g., MAPK-associated kinase 2 and integrin-beta(6)) levels. These findings indicate that APC is barrier-protective in VILI and that EPCR is a critical participant in APC-mediated protection.
• Flores, C., Pérez-Méndez, L., Maca-Meyer, N., Muriel, A., Espinosa, E., Blanco, J., Sangüesa, R., Muros, M., Garcia, J. G., Villar, J., & , G. a. (2009). A common haplotype of the LBP gene predisposes to severe sepsis. Critical care medicine, 37(10), 2759-66.
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  To investigate whether common variants across the LBP gene contribute to the development of severe sepsis. Sepsis is the leading cause of multiple system organ dysfunction and death in critically ill patients. The lipopolysaccharide-binding protein is an acute-phase protein that plays a dominant role in the genesis of sepsis by initiating signal transduction pathways leading to the activation of the inflammatory host response.
• Fu, P., Birukova, A. A., Xing, J., Sammani, S., Murley, J. S., Garcia, J. G., Grdina, D. J., & Birukov, K. G. (2009). Amifostine reduces lung vascular permeability via suppression of inflammatory signalling. The European respiratory journal, 33(3), 612-24.
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  Despite an encouraging outcome of antioxidant therapy in animal models of acute lung injury, effective antioxidant agents for clinical application remain to be developed. The present study investigated the effect of pre-treatment with amifostine, a thiol antioxidant compound, on lung endothelial barrier dysfunction induced by Gram-negative bacteria wall-lipopolysaccharide (LPS). Endothelial permeability was monitored by changes in transendothelial electrical resistance. Cytoskeletal remodelling and reactive oxygen species (ROS) production was examined by immunofluorescence. Cell signalling was assessed by Western blot. Measurements of Evans blue extravasation, cell count and protein content in bronchoalveolar lavage fluid were used as in vivo parameters of lung vascular permeability. Hydrogen peroxide, LPS and interleukin-6 caused cytoskeletal reorganisation and increased permeability in the pulmonary endothelial cells, reflecting endothelial barrier dysfunction. These disruptive effects were inhibited by pre-treatment with amifostine and linked to the amifostine-mediated abrogation of ROS production and redox-sensitive signalling cascades, including p38, extracellular signal regulated kinase 1/2, mitogen-activated protein kinases and the nuclear factor-kappaB pathway. In vivo, concurrent amifostine administration inhibited LPS-induced oxidative stress and p38 mitogen-activated protein kinase activation, which was associated with reduced vascular leak and neutrophil recruitment to the lungs. The present study demonstrates, for the first time, protective effects of amifostine against lipopolysaccharide-induced lung vascular leak in vitro and in animal models of lipopolysaccharide-induced acute lung injury.
• Garcia, J. G. (2009). Concepts in microvascular endothelial barrier regulation in health and disease. Microvascular research, 77(1), 1-3.
• Kolozsvári, B., Szíjgyártó, Z., Bai, P., Gergely, P., Verin, A., Garcia, J. G., & Bakó, E. (2009). Role of calcineurin in thrombin-mediated endothelial cell contraction. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 75(5), 405-11.
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  Barrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. EC dysfunction elicited by thrombin was found to correlate with actin microfilament redistribution. It is known that calcineurin (Cn) is involved in thrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activity and the phosphorylation state of EC myosin light chain is also affected by Cn activity. Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed with GFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endothelial cells (BPAEC). Actin microfilaments were stained with Texas Red-phalloidin. Cytotoxic effects of transfections or treatments and the efficiency of transfections were assessed by flow cytometry. Treatment of BPAEC with Cn inhibitors (cyclosporin A and FK506) hindered recovery of the cells from thrombin-induced EC dysfunction. Inhibition of Cn in the absence of thrombin had no effect on cytoskeletal actin filaments. We detected attenuated thrombin-induced stress fiber formation and changes in cell shape only when cells were transfected with constitutively active CnA and not with various CnA isoforms. Flow cytometry (FCM) analysis has proved that cytotoxic effect of treatments is negligible. We observed that Cn is involved in the recovery from thrombin-induced EC dysfunction. Inhibition of Cn caused prolonged contractile effect, while overexpression of constitutively active CnA resulted in reduced thrombin-induced stress fiber formation.
• Krishnaswamy, S., Kanteti, R., Duke-Cohan, J. S., Loganathan, S., Liu, W., Ma, P. C., Sattler, M., Singleton, P. A., Ramnath, N., Innocenti, F., Nicolae, D. L., Ouyang, Z., Liang, J., Minna, J., Kozloff, M. F., Ferguson, M. K., Natarajan, V., Wang, Y. C., Garcia, J. G., , Vokes, E. E., et al. (2009). Ethnic differences and functional analysis of MET mutations in lung cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 15(18), 5714-23.
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  African Americans have higher incidence and poorer response to lung cancer treatment compared with Caucasians. However, the underlying molecular mechanisms for the significant ethnic difference are not known. The present study examines the ethnic differences in the type and frequency of MET proto-oncogene (MET) mutation in lung cancer and correlated them with other frequently mutated genes such as epidermal growth factor receptor (EGFR), KRAS2, and TP53.
• Lal, R., Lal, R., Garcia, J. G., Dudek, S. M., & Arce, F. M. (2009). Micromechanical Properties Of Fixed And Living Vascular Pulmonary Endothelial Cells Following Exposure To Barrier Enhancing And Barrier Disrupting Agents. Biophysical Journal, 96(3), 6-9. doi:10.1016/j.bpj.2008.12.3328
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  Disruption of pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event that occurs in multiple inflammatory disease processes. The actin cytoskeleton, an essential regulator of endothelial permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. We used atomic force microscopy (AFM) to characterize structural and mechanical changes in the F-actin cytoskeleton of cultured human pulmonary artery EC in response to both barrier-enhancing and barrier-disrupting conditions. The mechanical properties of both fixed and live cells were evaluated. Elastic modulus values in the range of 50-1000 kPa were typically measured for fixed cells, while much lower values of 1-40 kPa were characteristic of live cells. In fixed cells, a differential distribution of elasticity was observed after exposure to the barrier-enhancing compound S1P (sphingosine 1-phosphate) compared to that produced by the barrier-disrupting agonist, thrombin. After S1P, the elastic modulus was elevated primarily at the periphery, while thrombin treatment increased elasticity in the central region of the cell. These observations correspond with the distribution of F-actin in parallel-treated EC as detected by immunofluorescence. In living cells, thrombin generally increased the average elastic modulus over 60 minutes; however, the S1P response was more varied and subtle. Experiments are under way to confirm these preliminary observations. These results provide novel insights into the structural and mechanical properties that dynamically regulate pulmonary EC barrier function.
• Liu, P., Li, H., Cepeda, J., Zhang, L. Q., Cui, X., Garcia, J. G., & Ye, S. Q. (2009). Critical role of PBEF expression in pulmonary cell inflammation and permeability. Cell biology international, 33(1), 19-30.
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  Previous studies in our lab have identified pre-B-cell colony enhancing factor (PBEF) as a novel biomarker in acute lung injury. This study continues to elucidate the underlying molecular mechanism of PBEF in the pathogenesis of acute lung injury in pulmonary cell culture models. Our results revealed that IL-1beta induced PBEF expression in pulmonary vascular endothelial cells at the transcriptional level and a -1535 T-variant in the human PBEF gene promoter significantly attenuated its binding to an IL-1beta-induced unknown transcription factor. This may underlie the reduced expression of PBEF and thus the lower susceptibility to acute lung injury in -1535T carriers. Furthermore, overexpression of PBEF significantly augmented IL-8 secretion and mRNA expression by more than 6-fold and 2-fold in A549 cells and HPAEC, respectively. It also significantly augmented IL-1beta-mediated cell permeability by 44% in A549 cells and 65% in endothelial cells. The knockdown of PBEF expression significantly inhibited IL-1beta-stimulated IL-8 secretion and mRNA level by 60% and 70%, respectively, and the knockdown of PBEF expression also significantly attenuated IL-1beta-induced cell permeability by 29% in epithelial cells and 24% in endothelial cells. PBEF expression also affected the expression of two other inflammatory cytokines (IL-16 and CCR3 genes). These results suggest that PBEF is critically involved in pulmonary vascular and epithelial inflammation and permeability, which are hallmark features in the pathogenesis of acute lung injury. This study lends further support to our finding that PBEF is a potential new target in acute lung injury.
• Meyer, N. J., Huang, Y., Singleton, P. A., Sammani, S., Moitra, J., Evenoski, C. L., Husain, A. N., Mitra, S., Moreno-Vinasco, L., Jacobson, J. R., Lussier, Y. A., & Garcia, J. G. (2009). GADD45a is a novel candidate gene in inflammatory lung injury via influences on Akt signaling. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 23(5), 1325-37.
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  We explored the mechanistic involvement of the growth arrest and DNA damage-inducible gene GADD45a in lipopolysaccharide (LPS)- and ventilator-induced inflammatory lung injury (VILI). Multiple biochemical and genomic parameters of inflammatory lung injury indicated that GADD45a(-/-) mice are modestly susceptible to intratracheal LPS-induced lung injury and profoundly susceptible to high tidal volume VILI, with increases in microvascular permeability and bronchoalveolar lavage levels of inflammatory cytokines. Expression profiling of lung tissues from VILI-challenged GADD45a(-/-) mice revealed strong dysregulation in the B-cell receptor signaling pathway compared with wild-type mice and suggested the involvement of PI3 kinase/Akt signaling components. Western blot analyses of lung homogenates confirmed approximately 50% reduction in Akt protein levels in GADD45a(-/-) mice accompanied by marked increases in Akt ubiquitination. Electrical resistance measurements across human lung endothelial cell monolayers with either reduced GADD45a or Akt expression (siRNAs) revealed significant potentiation of LPS-induced human lung endothelial barrier dysfunction, which was attenuated by overexpression of a constitutively active Akt1 transgene. These studies validate GADD45a as a novel candidate gene in inflammatory lung injury and a significant participant in vascular barrier regulation via effects on Akt-mediated endothelial signaling.
• Pendyala, S., Usatyuk, P. V., Gorshkova, I. A., Garcia, J. G., & Natarajan, V. (2009). Regulation of NADPH oxidase in vascular endothelium: the role of phospholipases, protein kinases, and cytoskeletal proteins. Antioxidants & redox signaling, 11(4), 841-60.
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  The generation of reactive oxygen species (ROS) in the vasculature plays a major role in the genesis of endothelial cell (EC) activation and barrier function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase family of proteins is a major contributor of ROS associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. The NADPH oxidase in lung ECs has most of the components found in phagocytic oxidase, and recent studies show the expression of several homologues of Nox proteins in vascular cells. Activation of NADPH oxidase of nonphagocytic vascular cells is complex and involves assembly of the cytosolic (p47(phox), p67(phox), and Rac1) and membrane-associated components (Noxes and p22(phox)). Signaling pathways leading to NADPH oxidase activation are not completely defined; however, they do appear to involve the cytoskeleton and posttranslation modification of the components regulated by protein kinases, protein phosphatases, and phospholipases. Furthermore, several key components regulating NADPH oxidase recruitment, assembly, and activation are enriched in lipid microdomains to form a functional signaling platform. Future studies on temporal and spatial localization of Nox isoforms will provide new insights into the role of NADPH oxidase-derived ROS in the pathobiology of lung diseases.
• Sammani, S., Zaidi, R., Sammani, S., Moreno-vinasco, L., Mirzapoiazova, T., Machado, R., & Garcia, J. G. (2009). Abstract 5660: Essential Role of Non-muscle Myosin Light Chain Kinase in Hypoxia-induced Murine Pulmonary Hypertension. Circulation, 120.
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  Introduction: The pulmonary arterial hypertension (PAH) phenotype shares several pathways involved in cancer pathobiology. We previously demonstrated that the multi-kinase inhibitor, sorafenib, ameliorates rodent PAH (Moreno-Vinasco et al 2008) with genomic signatures which strongly implicated the endothelial cell cytoskeleton in the development of PAH vascular remodeling including non-muscle caldesmon, Rho-kinase and both smooth muscle- and non-muscle myosin light chain kinase (nmMLCK) isoforms. We hypothesized that the acto-myosin machinery present within lung endothelium are critical to the development of PAH in a murine model of hypoxia-induced pulmonary hypertension. Methods: Wild type (WT) and genetically engineered mice with targeted deletion of the nmMLCK allele were exposed to normoxia, or to hypoxia and SU5416, a VEGFR-1,2 inhibitor that induces severe PH (3.5 weeks). WT and nmMLCK KO mice were assessed for hemodynamics and histopathology. Results: Hypoxia-SU5416 WT mice developed pulmonary arte...
• Singleton, P. A., Chatchavalvanich, S., Fu, P., Xing, J., Birukova, A. A., Fortune, J. A., Klibanov, A. M., Garcia, J. G., & Birukov, K. G. (2009). Akt-mediated transactivation of the S1P1 receptor in caveolin-enriched microdomains regulates endothelial barrier enhancement by oxidized phospholipids. Circulation research, 104(8), 978-86.
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  Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, leading to increased mass transport across the vessel wall and leukocyte extravasation, the key mechanisms in pathogenesis of tissue inflammation and edema. We have previously demonstrated that OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) significantly enhances vascular endothelial barrier properties in vitro and in vivo and attenuates endothelial hyperpermeability induced by inflammatory and edemagenic agents via Rac and Cdc42 GTPase dependent mechanisms. These findings suggested potential important therapeutic value of barrier-protective oxidized phospholipids. In this study, we examined involvement of signaling complexes associated with caveolin-enriched microdomains (CEMs) in barrier-protective responses of human pulmonary ECs to OxPAPC. Immunoblotting from OxPAPC-treated ECs revealed OxPAPC-mediated rapid recruitment (5 minutes) to CEMs of the sphingosine 1-phosphate receptor (S1P(1)), the serine/threonine kinase Akt, and the Rac1 guanine nucleotide exchange factor Tiam1 and phosphorylation of caveolin-1, indicative of signaling activation in CEMs. Abolishing CEM formation (methyl-beta-cyclodextrin) blocked OxPAPC-mediated Rac1 activation, cytoskeletal reorganization, and EC barrier enhancement. Silencing (small interfering RNA) Akt expression blocked OxPAPC-mediated S1P(1) activation (threonine phosphorylation), whereas silencing S1P(1) receptor expression blocked OxPAPC-mediated Tiam1 recruitment to CEMs, Rac1 activation, and EC barrier enhancement. To confirm our in vitro results in an in vivo murine model of acute lung injury with pulmonary vascular hyperpermeability, we observed that selective lung silencing of caveolin-1 or S1P(1) receptor expression blocked OxPAPC-mediated protection from ventilator-induced lung injury. Taken together, these results suggest Akt-dependent transactivation of S1P(1) within CEMs is important for OxPAPC-mediated cortical actin rearrangement and EC barrier protection.
• Singleton, P. A., Moss, J., & Garcia, J. G. (2009). Methylnaltrexone potentiates the antiangiogenic effects of the mTOR inhibitor, temsirolimus. Journal of Clinical Oncology, 27. doi:10.1200/jco.2009.27.15_suppl.e14636
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  e14636 Background: Angiogenesis is important in cancer growth and metastasis. Recent therapeutic interventions cancer, include drugs such as temsirolimus that target both tumor growth and angiogene...
• Singleton, P. A., Pendyala, S., Gorshkova, I. A., Mambetsariev, N., Moitra, J., Garcia, J. G., & Natarajan, V. (2009). Dynamin 2 and c-Abl are novel regulators of hyperoxia-mediated NADPH oxidase activation and reactive oxygen species production in caveolin-enriched microdomains of the endothelium. The Journal of biological chemistry, 284(50), 34964-75.
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  Reactive oxygen species (ROS) generation, particularly by the endothelial NADPH oxidase family of proteins, plays a major role in the pathophysiology associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. We examined potential regulators of ROS production and discovered that hyperoxia treatment of human pulmonary artery endothelial cells induced recruitment of the vesicular regulator, dynamin 2, the non-receptor tyrosine kinase, c-Abl, and the NADPH oxidase subunit, p47(phox), to caveolin-enriched microdomains (CEMs). Silencing caveolin-1 (which blocks CEM formation) and/or c-Abl expression with small interference RNA inhibited hyperoxia-mediated tyrosine phosphorylation and association of dynamin 2 with p47(phox) and ROS production. In addition, treatment of human pulmonary artery endothelial cells with dynamin 2 small interfering RNA or the dynamin GTPase inhibitor, Dynasore, attenuated hyperoxia-mediated ROS production and p47(phox) recruitment to CEMs. Using purified recombinant proteins, we observed that c-Abl tyrosine-phosphorylated dynamin 2, and this phosphorylation increased p47(phox)/dynamin 2 association (change in the dissociation constant (K(d)) from 85.8 to 6.9 nm). Furthermore, exposure of mice to hyperoxia increased ROS production, c-Abl activation, dynamin 2 association with p47(phox), and pulmonary leak, events that were attenuated in the caveolin-1 knock-out mouse confirming a role for CEMs in ROS generation. These results suggest that hyperoxia induces c-Abl-mediated dynamin 2 phosphorylation required for recruitment of p47(phox) to CEMs and subsequent ROS production in lung endothelium.
• Singleton, P. A., Salgia, R., Moss, J., Mambetsariev, N., Lennon, F. E., & Garcia, J. G. (2009). Abstract C79: Methylnaltrexone inhibits EGF‐ and IGF‐induced human bronchioloalveolar carcinoma proliferation and migration. Molecular Cancer Therapeutics, 8. doi:10.1158/1535-7163.targ-09-c79
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  Introduction: Non‐small cell lung cancer (NSCLC) has a very poor prognosis and improved therapies are needed. Expression of the mu opioid receptor (MOR) is increased in metastatic sites of patients with NSCLC. Previous work by our laboratory and others suggested a role of opioids in several aspects of tumor growth (Wang et al., Anticancer Res, 2009; Mol Cancer Ther, 2008; Gupta et al., Cancer Res, 2002). In this study, we investigated whether inhibition of MOR by the peripheral MOR antagonist, methylnaltrexone (MNTX), attenuates EGF‐ and IGF‐mediated human bronchioloalveolar carcinoma (BAC) molecular pathways involved in NSCLC oncogenic potential. Methods: Human H358 BAC cells were pretreated with various siRNAs targeting MOR, Akt, cortactin, Gab1, PLC 1 or STAT3 or pretreated with MNTX (1.0–250 nM), 10‐(4 ‐(N‐diethylamino)butyl)‐2‐chlorophenoxazine (Akt inhibitor, 5 µM), U‐73122 (PLC inhibitor, 1 µM) or 6‐Nitrobenzo[b]thiophene‐1,1‐dioxide (STAT3 inhibitor, 1 µM) prior to the addition of EGF or IGF (10 ng/ml). Functional (cell proliferation and migration) and biochemical studies (immunoprecipitation, immunoblotting) were then conducted. Results: 14 NSCLC cell lines (7 adenocarcinomas, 3 SCC, 2 bronchoioloalveolar carcinoma, 1 large cell carcinoma, 1 adenosquamous carcinoma) were examined for MOR expression using immunoblotting techniques. MOR expression was increased in most NSCLC cell lines with BAC having the highest levels of MOR expression (7–9 fold higher that control primary lung epithelial or BEAS‐2B cells). EGF and IGF treatment of BAC (10 ng/ml, 5–30 minutes) stimulated MOR association with the EGF and IGF receptor, respectively. MNTX inhibited EGF‐ and IGF‐mediated BAC, but not control BEAS‐2B, proliferation and migration in a dose‐dependent manner (IC50 = ∼10 and 100 nM). On a mechanistic level, we observed that MNTX inhibited both EGF‐ and IGF‐induced recruitment of the adaper protein, Gab1 (GRB2‐associated binding protein 1), to the plasma membrane and complex formation with the EGF/IGF receptor in BAC. Silencing (siRNA) MOR, Gab1 or MNTX treatment inhibited EGF/IGF‐mediated Akt (serine/threonine kinase), cortactin (actin regulatory protein), PLC 1 (phospholipase) and STAT3 (Signal Transducer and Activator of Transcription protein) phosphorylation/activation, events which are involved in BAC oncogenic signaling. In addition, inhibiting (siRNA and/or chemical inhibitors) Akt, cortactin, PLC 1 and/or STAT3 attenuated both EGF‐ and IGF‐induced BAC growth and migration (50–90%). Conclusion: We have shown that MNTX inhibits EGF/IGF‐mediated oncogenic signaling through inhibition of MOR‐mediated Gab1 recruitment to the plasma membrane and consequent attenuation of Akt, cortactin, PLC 1 and STAT3 activation leading to reduced BAC proliferation and migration. Further study of this molecule as a potential therapeutic agent is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C79.
• Singleton, P. A., Siegler, J., Salgia, R., Moss, J., Moreno-vinasco, L., Mathew, B., Mambetsariev, N., Lennon, F. E., Gerhold, L. M., & Garcia, J. G. (2009). Abstract C78: The mu opioid receptor regulates Lewis lung carcinoma tumor growth and metastasis. Molecular Cancer Therapeutics, 8. doi:10.1158/1535-7163.targ-09-c78
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  Introduction: Expression of the mu opioid receptor (MOR) is increased in patients with non‐small cell lung cancer (NSCLC), a disease with poor prognosis and limited therapies. Previous work by our laboratory and others suggested a role of opioids in several aspects of tumor growth (Anticancer Res 2009;29:3195–3205, Mol Cancer Ther 2008;7:1669–1679, Cancer Res 2002;62:4491–4498). In this study, we investigated MOR as a potential therapeutic target for lung carcinoma. Methods: Lewis lung carcinoma (LLC) cells were treated with MOR siRNA or the peripheral MOR antagonist, methylnaltrexone (MNTX, 250 nM), prior to the addition of EGF (10 ng/ml), IGF (10 ng/ml), DAMGO (1 nM), morphine (1 nM) or serum (1, 5 or 10%). In vitro functional (cell proliferation and invasion) and biochemical studies (immunoblotting) were then conducted. For in vivo assays, C57BL/6J mice were injected intravenously with dual color (GFP‐RFP labeled) LLC cells, with or without MOR siRNA treatment. In another experiment, MOR knockout as well as wildtype control mice were injected with LLC cells (subcutaneously into the flank) and followed for 12 weeks. In a parallel experiment, LLC tumor bearing C57BL/6J mice received continuous infusion of MNTX (10 mg/kg/day) for 2 weeks. Tumor growth and lung metastasis were evaluated using tumor volume measurements and/or in vivo fluorescent microscopy (Olympus OV‐100) with or without intravenous injection of ProSense and/or MMP Sense probes. Results: Our data indicate that LLC cells have ∼5 fold increased expression of MOR (compared to primary lung epithelial or BEAS‐2B cells). Inhibition of MOR with siRNA or MNTX treatment reduced in vitro LLC proliferation (90%) and invasion (50–75%). In vivo lung metastasis was reduced by ∼75% in wildtype C57BL/6J mice with LLC lacking MOR (siRNA) versus control siRNA‐treated LLC (3 weeks post‐i.v. injection, quantitated with OV‐100). In addition, primary LLC tumor volume was substantially reduced (>90%) in MOR knockout mice compared to wildtype mice (flank injection). Finally, we observed that continuous infusion of MNTX (10 mg/kg/day) in mice with pre‐existing LLC primary tumors attenuates further tumor growth. Conclusion: We have shown that MOR is a potential therapeutic target for inhibition of LLC proliferation, invasion and metastasis. In addition, MNTX can attenuate LLC primary tumor growth. Further study of MNTX as a potential therapeutic agent is warranted. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C78.
• Sun, X., Shikata, Y., Wang, L., Ohmori, K., Watanabe, N., Wada, J., Shikata, K., Birukov, K. G., Makino, H., Jacobson, J. R., Dudek, S. M., & Garcia, J. G. (2009). Enhanced interaction between focal adhesion and adherens junction proteins: involvement in sphingosine 1-phosphate-induced endothelial barrier enhancement. Microvascular research, 77(3), 304-13.
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  Sphingosine 1-phosphate (S1P) is an important vascular barrier regulatory agonist which enhances the junctional integrity of human lung endothelial cell monolayers. We have now demonstrated that S1P induced cortical actin ring formation and redistribution of focal adhesion kinase (FAK) and paxillin to the cell periphery suggesting the critical role of cell-cell adhesion in endothelial barrier enhancement. Co-immunoprecipitation studies revealed increased association of VE-cadherin with FAK and paxillin in S1P-challenged human pulmonary artery endothelial cell (HPAEC) monolayers. Furthermore, S1P-induced enhancement of VE-cadherin interaction with alpha-catenin and beta-catenin was associated with the increased formation of FAK-beta-catenin protein complexes. Depletion of beta-catenin (siRNA) resulted in loss of S1P-mediated VE-cadherin association with FAK and paxillin rearrangement. Furthermore, transendothelial electrical resistance (an index of barrier function) demonstrated that beta-catenin siRNA significantly attenuated S1P-induced barrier enhancement. These results demonstrate a mechanism of S1P-induced endothelial barrier enhancement via beta-catenin-linked adherens junction and focal adhesion interaction.
• Usatyuk, P. V., Gorshkova, I. A., He, D., Zhao, Y., Kalari, S. K., Garcia, J. G., & Natarajan, V. (2009). Phospholipase D-mediated activation of IQGAP1 through Rac1 regulates hyperoxia-induced p47phox translocation and reactive oxygen species generation in lung endothelial cells. The Journal of biological chemistry, 284(22), 15339-52.
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  Phosphatidic acid generated by the activation of phospholipase D (PLD) functions as a second messenger and plays a vital role in cell signaling. Here we demonstrate that PLD-dependent generation of phosphatidic acid is critical for Rac1/IQGAP1 signal transduction, translocation of p47(phox) to cell periphery, and ROS production. Exposure of [(32)P]orthophosphate-labeled human pulmonary artery endothelial cells (HPAECs) to hyperoxia (95% O(2) and 5% CO(2)) in the presence of 0.05% 1-butanol, but not tertiary-butanol, stimulated PLD as evidenced by accumulation of [(32)P]phosphatidylbutanol. Infection of HPAECs with adenoviral constructs of PLD1 and PLD2 wild-type potentiated hyperoxia-induced PLD activation and accumulation of O(2)(.)/reactive oxygen species (ROS). Conversely, overexpression of catalytically inactive mutants of PLD (hPLD1-K898R or mPLD2-K758R) or down-regulation of expression of PLD with PLD1 or PLD2 siRNA did not augment hyperoxia-induced [(32)P]phosphatidylbutanol accumulation and ROS generation. Hyperoxia caused rapid activation and redistribution of Rac1, and IQGAP1 to cell periphery, and down-regulation of Rac1, and IQGAP1 attenuated hyperoxia-induced tyrosine phosphorylation of Src and cortactin and ROS generation. Further, hyperoxia-mediated redistribution of Rac1, and IQGAP1 to membrane ruffles, was attenuated by PLD1 or PLD2 small interference RNA, suggesting that PLD is upstream of the Rac1/IQGAP1 signaling cascade. Finally, small interference RNA for PLD1 or PLD2 attenuated hyperoxia-induced cortactin tyrosine phosphorylation and abolished Src, cortactin, and p47(phox) redistribution to cell periphery. These results demonstrate a role of PLD in hyperoxia-mediated IQGAP1 activation through Rac1 in tyrosine phosphorylation of Src and cortactin, as well as in p47(phox) translocation and ROS formation in human lung endothelial cells.
• Wadgaonkar, R., Patel, V., Grinkina, N., Romano, C., Liu, J., Zhao, Y., Sammani, S., Garcia, J. G., & Natarajan, V. (2009). Differential regulation of sphingosine kinases 1 and 2 in lung injury. American journal of physiology. Lung cellular and molecular physiology, 296(4), L603-13.
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  Two mammalian sphingosine kinase (SphK) isoforms, SphK1 and SphK2, possess identical kinase domains but have distinct kinetic properties and subcellular localizations, suggesting each has one or more specific roles in sphingosine-1-phosphate (S1P) generation. Although both kinases use sphingosine as a substrate to generate S1P, the mechanisms controlling SphK activation and subsequent S1P generation during lung injury are not fully understood. In this study, we established a murine lung injury model to investigate LPS-induced lung injury in SphK1 knockout (SphK1(-/-)) and wild-type (WT) mice. We found that SphK1(-/-) mice were much more susceptible to LPS-induced lung injury compared with their WT counterparts, quantified by multiple parameters including cytokine induction. Intriguingly, overexpression of WT SphK1 delivered by adenoviral vector to the lungs protected SphK1(-/-) mice from lung injury and attenuated the severity of the response to LPS. However, adenoviral overexpression of a SphK1 kinase-dead mutant (SphKKD) in SphK1(-/-) mouse lungs further exacerbated the response to LPS as well as the extent of lung injury. WT SphK2 adenoviral overexpression also failed to provide protection and, in fact, augmented the degree of LPS-induced lung injury. This suggested that, in vascular injury, S1P generated by SphK2 activation plays a distinctly separate role compared with SphK1-dependent S1P generation and survival signaling. Microarray and real-time RT-PCR analysis of SphK1 and SphK2 expression levels during lung injury revealed that, in WT mice, LPS treatment caused significantly enhanced SphK1 expression ( approximately 5x) levels within 6 h, which declined back to baseline levels by 24 h posttreatment. In contrast, expression of SphK2 was gradually induced following LPS treatment and was elevated within 24 h. Collectively, our results for the first time demonstrate distinct functional roles of the two SphK isoforms in the regulation of LPS-induced lung injury.
• Zhao, J., Singleton, P. A., Brown, M. E., Dudek, S. M., & Garcia, J. G. (2009). Phosphotyrosine protein dynamics in cell membrane rafts of sphingosine-1-phosphate-stimulated human endothelium: role in barrier enhancement. Cellular signalling, 21(12), 1945-60.
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  Sphingosine-1-phosphate (S1P), a lipid growth factor, is critical to the maintenance and enhancement of vascular barrier function via processes highly dependent upon cell membrane raft-mediated signaling events. Anti-phosphotyrosine 2 dimensional gel electrophoresis (2-DE) immunoblots confirmed that disruption of membrane raft formation (via methyl-beta-cyclodextrin) inhibits S1P-induced protein tyrosine phosphorylation. To explore S1P-induced dynamic changes in membrane rafts, we used 2-D techniques to define proteins within detergent-resistant cell membrane rafts which are differentially expressed in S1P-challenged (1microM, 5min) human pulmonary artery endothelial cells (EC), with 57 protein spots exhibiting >3-fold change. S1P induced the recruitment of over 20 cell membrane raft proteins exhibiting increasing levels of tyrosine phosphorylation including known barrier-regulatory proteins such as focal adhesion kinase (FAK), cortactin, p85alpha phosphatidylinositol 3-kinase (p85alphaPI3K), myosin light chain kinase (nmMLCK), filamin A/C, and the non-receptor tyrosine kinase, c-Abl. Reduced expression of either FAK, MLCK, cortactin, filamin A or filamin C by siRNA transfection significantly attenuated S1P-induced EC barrier enhancement. Furthermore, S1P induced cell membrane raft components, p-caveolin-1 and glycosphingolipid (GM1), to the plasma membrane and enhanced co-localization of membrane rafts with p-caveolin-1 and p-nmMLCK. These results suggest that S1P induces both the tyrosine phosphorylation and recruitment of key actin cytoskeletal proteins to membrane rafts, resulting in enhanced human EC barrier function.
• Zhao, Y., Usatyuk, P. V., Gorshkova, I. A., He, D., Wang, T., Moreno-Vinasco, L., Geyh, A. S., Breysse, P. N., Samet, J. M., Spannhake, E. W., Garcia, J. G., & Natarajan, V. (2009). Regulation of COX-2 expression and IL-6 release by particulate matter in airway epithelial cells. American journal of respiratory cell and molecular biology, 40(1), 19-30.
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  Particulate matter (PM) in ambient air is a risk factor for human respiratory and cardiovascular diseases. The delivery of PM to airway epithelial cells has been linked to release of proinflammatory cytokines; however, the mechanisms of PM-induced inflammatory responses are not well-characterized. This study demonstrates that PM induces cyclooxygenase (COX)-2 expression and IL-6 release through both a reactive oxygen species (ROS)-dependent NF-kappaB pathway and an ROS-independent C/EBPbeta pathway in human bronchial epithelial cells (HBEpCs) in culture. Treatment of HBEpCs with Baltimore PM induced ROS production, COX-2 expression, and IL-6 release. Pretreatment with N-acetylcysteine (NAC) or EUK-134, in a dose-dependent manner, attenuated PM-induced ROS production, COX-2 expression, and IL-6 release. The PM-induced ROS was significantly of mitochondrial origin, as evidenced by increased oxidation of the mitochondrially targeted hydroethidine to hydroxyethidium by reaction with superoxide. Exposure of HBEpCs to PM stimulated phosphorylation of NF-kappaB and C/EBPbeta, while the NF-kappaB inhibitor, Bay11-7082, or C/EBPbeta siRNA attenuated PM-induced COX-2 expression and IL-6 release. Furthermore, NAC or EUK-134 attenuated PM-induced activation of NF-kappaB; however, NAC or EUK-134 had no effect on phosphorylation of C/EBPbeta. In addition, inhibition of COX-2 partly attenuated PM-induced Prostaglandin E2 and IL-6 release.
• Arce, F. T., Whitlock, J. L., Birukova, A. A., Birukov, K. G., Arnsdorf, M. F., Lal, R., Garcia, J. G., & Dudek, S. M. (2008). Regulation of the micromechanical properties of pulmonary endothelium by S1P and thrombin: role of cortactin. Biophysical journal, 95(2), 886-94.
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  Disruption of pulmonary endothelial cell (EC) barrier function is a critical pathophysiologic event in highly morbid inflammatory conditions such as sepsis and acute respiratory disease stress syndrome. Actin cytoskeleton, an essential regulator of endothelial permeability, is a dynamic structure whose stimuli-induced rearrangement is linked to barrier modulation. Here, we used atomic force microscopy to characterize structural and mechanical changes in the F-actin cytoskeleton of cultured human pulmonary artery EC in response to both barrier-enhancing (induced by sphingosine 1-phosphate (S1P)) and barrier-disrupting (induced by thrombin) conditions. Atomic force microscopy elasticity measurements show differential effects: for the barrier protecting molecule S1P, the elastic modulus was elevated significantly on the periphery; for the barrier-disrupting molecule thrombin, on the other hand, it was elevated significantly in the central region of the cell. The force and elasticity maps correlate with F-actin rearrangements as identified by immunofluorescence analysis. Significantly, reduced expression (via siRNA) of cortactin, an actin-binding protein essential to EC barrier regulation, resulted in a shift in the S1P-mediated elasticity pattern to more closely resemble control, unstimulated endothelium.
• Archer, S. L., Gomberg-Maitland, M., Maitland, M. L., Rich, S., Garcia, J. G., & Weir, E. K. (2008). Mitochondrial metabolism, redox signaling, and fusion: a mitochondria-ROS-HIF-1alpha-Kv1.5 O2-sensing pathway at the intersection of pulmonary hypertension and cancer. American journal of physiology. Heart and circulatory physiology, 294(2), H570-8.
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  Pulmonary arterial hypertension (PAH) is a lethal syndrome characterized by vascular obstruction and right ventricular failure. Although the fundamental cause remains elusive, many predisposing and disease-modifying abnormalities occur, including endothelial injury/dysfunction, bone morphogenetic protein receptor-2 gene mutations, decreased expression of the O(2)-sensitive K(+) channel (Kv1.5), transcription factor activation [hypoxia-inducible factor-1alpha (HIF-1alpha) and nuclear factor-activating T cells], de novo expression of survivin, and increased expression/activity of both serotonin transporters and platelet-derived growth factor receptors. Together, these abnormalities create a cancerlike, proliferative, apoptosis-resistant phenotype in pulmonary artery smooth muscle cells (PASMCs). A possible unifying mechanism for PAH comes from studies of fawn-hooded rats, which manifest spontaneous PAH and impaired O(2) sensing. PASMC mitochondria normally produce reactive O(2) species (ROS) in proportion to P(O2). Superoxide dismutase 2 (SOD2) converts intramitochondrial superoxide to diffusible H(2)O(2), which serves as a redox-signaling molecule, regulating pulmonary vascular tone and structure through effects on Kv1.5 and transcription factors. O(2) sensing is mediated by this mitochondria-ROS-HIF-1alpha-Kv1.5 pathway. In PAH and cancer, mitochondrial metabolism and redox signaling are reversibly disordered, creating a pseudohypoxic redox state characterized by normoxic decreases in ROS, a shift from oxidative to glycolytic metabolism and HIF-1alpha activation. Three newly recognized mitochondrial abnormalities disrupt the mitochondria-ROS-HIF-1alpha-Kv1.5 pathway: 1) mitochondrial pyruvate dehydrogenase kinase activation, 2) SOD2 deficiency, and 3) fragmentation and/or hyperpolarization of the mitochondrial reticulum. The pyruvate dehydrogenase kinase inhibitor, dichloroacetate, corrects the mitochondrial abnormalities in experimental models of PAH and human cancer, causing a regression of both diseases. Mitochondrial abnormalities that disturb the ROS-HIF-1alpha-Kv1.5 O(2)-sensing pathway contribute to the pathogenesis of PAH and cancer and constitute promising therapeutic targets.
• Cai, H., Liu, D., & Garcia, J. G. (2008). CaM Kinase II-dependent pathophysiological signalling in endothelial cells. Cardiovascular research, 77(1), 30-4.
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  Calcium/calmodulin-dependent protein kinase II (CaM Kinase II) is a known modulator of cardiac pathophysiology. The present review uniquely focuses on novel CaM Kinase II-mediated endothelial cell signalling which, under pathophysiological conditions, may indirectly modulate cardiac functions via alterations in endothelial or endocardial responses. CaM Kinase II has four different isoforms and various splicing variants for each isoform. The endothelial cell CaM Kinase II isoforms are sensitive to KN93 and a threonine 286-mutated inhibitory peptide. In macrovascular endothelial cells derived from aortas, CaM Kinase II mediates redox-sensitive upregulation of endothelial nitric oxide synthase (eNOS) gene expression by hydrogen peroxide (H2O2) and oscillatory shear stress, and a rapid activation of eNOS in response to bradykinin. In endothelial cells derived from lung microvessels, CaM Kinase II mediates barrier dysfunction, particularly when activated by thrombin. In brain capillary endothelial cells, CaM Kinase II lies upstream of voltage-gated potassium channels and hypoxia-induced cell swelling. In both macrovascular and microvascular endothelial cells, CaM Kinase II mediates actin cytoskeleton reorganization via distinct p38 MAPK/HSP27 and ERK1/2/MLCK signalling pathways, respectively. Although understanding of endothelium-specific CaM Kinase II signalling is nascent, data accumulated so far have demonstrated a potentially significant role of CaM Kinase II in endothelial cell pathophysiology.
• Chen, W., Pendyala, S., Natarajan, V., Garcia, J. G., & Jacobson, J. R. (2008). Endothelial cell barrier protection by simvastatin: GTPase regulation and NADPH oxidase inhibition. American journal of physiology. Lung cellular and molecular physiology, 295(4), L575-83.
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  The statins, hydroxy-3-methylglutaryl-CoA reductase inhibitors that lower serum cholesterol, exhibit myriad clinical benefits, including enhanced vascular integrity. One potential mechanism underlying increased endothelial cell (EC) barrier function is inhibition of geranylgeranylation, a covalent modification enabling translocation of the small GTPases Rho and Rac to the cell membrane. While RhoA inhibition attenuates actin stress fiber formation and promotes EC barrier function, Rac1 inhibition at the cell membrane potentially prevents activation of NADPH oxidase and subsequent generation of superoxides known to induce barrier disruption. We examined the relative regulatory effects of simvastatin on RhoA, Rac1, and NADPH oxidase activities in the context of human pulmonary artery EC barrier protection. Confluent EC treated with simvastatin demonstrated significantly decreased thrombin-induced FITC-dextran permeability, a reflection of vascular integrity, which was linked temporally to simvastatin-mediated actin cytoskeletal rearrangement. Compared with Rho inhibition alone (Y-27632), simvastatin afforded additional protection against thrombin-mediated barrier dysfunction and attenuated LPS-induced EC permeability and superoxide generation. Statin-mediated inhibition of both Rac translocation to the cell membrane and superoxide production were attenuated by geranylgeranyl pyrophosphate (GGPP), indicating that these effects are due to geranylgeranylation inhibition. Finally, thrombin-induced EC permeability was modestly attenuated by reduced Rac1 expression (small interfering RNA), whereas these effects were made more pronounced by simvastatin pretreatment. Together, these data suggest EC barrier protection by simvastatin is due to dual inhibitory effects on RhoA and Rac1 as well as the attenuation of superoxide generation by EC NADPH oxidase and contribute to the molecular mechanistic understanding of the modulation of EC barrier properties by simvastatin.
• Christie, J. D., Ma, S. F., Aplenc, R., Li, M., Lanken, P. N., Shah, C. V., Fuchs, B., Albelda, S. M., Flores, C., & Garcia, J. G. (2008). Variation in the myosin light chain kinase gene is associated with development of acute lung injury after major trauma. Critical care medicine, 36(10), 2794-800.
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  Single nucleotide polymorphisms in the myosin light chain kinase (MYLK) gene have been implicated in the risk of sepsis-related acute lung injury and asthma. MYLK encodes protein isoforms involved in multiple components of the inflammatory response, including apoptosis, vascular permeability, and leukocyte diapedesis. We tested the association of MYLK gene variation in the development of acute lung injury in major trauma patients.
• Damico, R. L., Chesley, A., Johnston, L., Bind, E. P., Amaro, E., Nijmeh, J., Karakas, B., Welsh, L., Pearse, D. B., Garcia, J. G., & Crow, M. T. (2008). Macrophage migration inhibitory factor governs endothelial cell sensitivity to LPS-induced apoptosis. American journal of respiratory cell and molecular biology, 39(1), 77-85.
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  Human endothelial cells (EC) are typically resistant to the apoptotic effects of stimuli associated with lung disease. The determinants of this resistance remain incompletely understood. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine produced by human pulmonary artery EC (HPAEC). Its expression increases in response to various death-inducing stimuli, including lipopolysaccharide (LPS). We show here that silencing MIF expression by RNA interference (MIF siRNA) dramatically reduces MIF mRNA expression and the LPS-induced increase in MIF protein levels, thereby sensitizing HPAECs to LPS-induced cell death. Addition of recombinant human MIF (rhMIF) protein prevents the death-sensitizing effect of MIF siRNA. A common mediator of apoptosis resistance in ECs is the death effector domain (DED)-containing protein, FLIP (FLICE-like inhibitory protein). We show that LPS induces a transcription-independent increase in the short isoform of FLIP (FLIP(s)). This increase is blocked by MIF siRNA but restored with the addition of recombinant MIF protein (rHMIF). While FLIP(s) siRNA also sensitizes HPAECs to LPS-induced death, the addition of rhMIF does not affect this sensitization, placing MIF upstream of FLIP(s) in preventing HPAEC death. These studies demonstrate that MIF is an endogenous pro-survival factor in HPAECs and identify a novel mechanism for its role in apoptosis resistance through the regulation of FLIP(s). These results show that MIF can protect vascular endothelial cells from inflammation-associated cell damage.
• Desai, A. A., Hysi, P., & Garcia, J. G. (2008). Integrating genomic and clinical medicine: searching for susceptibility genes in complex lung diseases. Translational research : the journal of laboratory and clinical medicine, 151(4), 181-93.
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  The integration of molecular, genomic, and clinical medicine in the post-genome era provides the promise of novel information on genetic variation and pathophysiologic cascades. The current challenge is to translate these discoveries rapidly into viable biomarkers that identify susceptible populations and into the development of precisely targeted therapies. In this article, we describe the application of comparative genomics, microarray platforms, genetic epidemiology, statistical genetics, and bioinformatic approaches within examples of complex pulmonary pathobiology. Our search for candidate genes, which are gene variations that drive susceptibility to and severity of enigmatic acute and chronic lung disorders, provides a logical framework to understand better the evolution of genomic medicine. The dissection of the genetic basis of complex diseases and the development of highly individualized therapies remain lofty but achievable goals.
• Flores, C., Ma, S. F., Maresso, K., Wade, M. S., Villar, J., & Garcia, J. G. (2008). IL6 gene-wide haplotype is associated with susceptibility to acute lung injury. Translational research : the journal of laboratory and clinical medicine, 152(1), 11-7.
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  Experimental and clinical studies support the key role of interleukin 6 (IL-6), a potent proinflammatory cytokine, in the development of acute lung injury (ALI). Plasma IL-6 levels are influenced mainly by genetic determinants, and a -174G/C polymorphism of the gene has been recently associated with susceptibility to ALI. Here we aimed to validate the association of the IL6 gene with ALI in a case-control sample from Spain. DNA was isolated from 67 consecutive patients who fulfilled international criteria for severe sepsis and for ALI and 96 population-based controls drawn from the general population. Genotypes of the -174G/C polymorphism along with other 14 tagging variants of the IL6 gene were evaluated. Twenty polymorphisms unlinked to IL6 gene were additionally compared between cases and controls to rule out population stratification. None of the individual single-nucleotide polymorphisms was significantly associated with susceptibility to ALI. However, we found that a common haplotype from -1363 to +4835 from the transcription start site, and spanning the gene, conferred risk for susceptibility to ALI (odds ratio, 2.73; 95% confidence interval, 1.39-5.37; P = 0.003). Adjustment for relevant covariates did not modify this result. These data support the association of the IL6 gene with ALI susceptibility and illustrate the value of haplotype analysis as a robust approach for evaluating IL6 gene effects in association studies.
• Gorshkova, I., He, D., Berdyshev, E., Usatuyk, P., Burns, M., Kalari, S., Zhao, Y., Pendyala, S., Garcia, J. G., Pyne, N. J., Brindley, D. N., & Natarajan, V. (2008). Protein kinase C-epsilon regulates sphingosine 1-phosphate-mediated migration of human lung endothelial cells through activation of phospholipase D2, protein kinase C-zeta, and Rac1. The Journal of biological chemistry, 283(17), 11794-806.
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  The signaling pathways by which sphingosine 1-phosphate (S1P) potently stimulates endothelial cell migration and angiogenesis are not yet fully defined. We, therefore, investigated the role of protein kinase C (PKC) isoforms, phospholipase D (PLD), and Rac in S1P-induced migration of human pulmonary artery endothelial cells (HPAECs). S1P-induced migration was sensitive to S1P(1) small interfering RNA (siRNA) and pertussis toxin, demonstrating coupling of S1P(1) to G(i). Overexpression of dominant negative (dn) PKC-epsilon or -zeta, but not PKC-alpha or -delta, blocked S1P-induced migration. Although S1P activated both PLD1 and PLD2, S1P-induced migration was attenuated by knocking down PLD2 or expressing dnPLD2 but not PLD1. Blocking PKC-epsilon, but not PKC-zeta, activity attenuated S1P-mediated PLD stimulation, demonstrating that PKC-epsilon, but not PKC-zeta, was upstream of PLD. Transfection of HPAECs with dnRac1 or Rac1 siRNA attenuated S1P-induced migration. Furthermore, transfection with PLD2 siRNA, infection of HPAECs with dnPKC-zeta, or treatment with myristoylated PKC-zeta peptide inhibitor abrogated S1P-induced Rac1 activation. These results establish that S1P signals through S1P(1) and G(i) to activate PKC-epsilon and, subsequently, a PLD2-PKC-zeta-Rac1 cascade. Activation of this pathway is necessary to stimulate the migration of lung endothelial cells, a key component of the angiogenic process.
• Grigoryev, D. N., Mathai, S. C., Fisher, M. R., Girgis, R. E., Zaiman, A. L., Housten-Harris, T., Cheadle, C., Gao, L., Hummers, L. K., Champion, H. C., Garcia, J. G., Wigley, F. M., Tuder, R. M., Barnes, K. C., & Hassoun, P. M. (2008). Identification of candidate genes in scleroderma-related pulmonary arterial hypertension. Translational research : the journal of laboratory and clinical medicine, 151(4), 197-207.
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  We hypothesize that pulmonary arterial hypertension (PAH)-associated genes identified by expression profiling of peripheral blood mononuclear cells (PBMCs) from patients with idiopathic pulmonary arterial hypertension (IPAH) can also be identified in PBMCs from scleroderma patients with PAH (PAH-SSc). Gene expression profiles of PBMCs collected from IPAH (n = 9), PAH-SSc (n = 10) patients, and healthy controls (n = 5) were generated using HG_U133A_2.0 GeneChips and were processed by the RMA/GCOS_1.4/SAM_1.21 data analysis pipeline. Disease severity in consecutive patients was assessed by functional status and hemodynamic measurements. The expression profiles were analyzed using PAH severity-stratification, and identified candidate genes were validated with real-time polymerase chain reaction (PCR). Transcriptomics of PBMCs from IPAH patients was highly comparable with that of PMBCs from PAH-SSc patients. The PBMC gene expression patterns significantly correlate with right atrium pressure (RA) and cardiac index (CI), which are known predictors of survival in PAH. Array stratification by RA and CI identified 364 PAH-associated candidate genes. Gene ontology (GO) analysis revealed significant (Z(score) > 1.96) alterations in angiogenesis genes according to PAH severity: matrix metalloproteinase 9 (MMP9) and vascular endothelial growth factor (VEGF) were significantly upregulated in mild as compared with severe PAH and healthy controls, as confirmed by real-time PCR. These data demonstrate that PBMCs from patients with PAH-SSc carry distinct transcriptional expression. Furthermore, our findings suggest an association between angiogenesis-related gene expression and severity of PAH in PAH-SSc patients. Deciphering the role of genes involved in vascular remodeling and PAH development may reveal new treatment targets for this devastating disorder.
• Hong, S. B., Huang, Y., Moreno-Vinasco, L., Sammani, S., Moitra, J., Barnard, J. W., Ma, S. F., Mirzapoiazova, T., Evenoski, C., Reeves, R. R., Chiang, E. T., Lang, G. D., Husain, A. N., Dudek, S. M., Jacobson, J. R., Ye, S. Q., Lussier, Y. A., & Garcia, J. G. (2008). Essential role of pre-B-cell colony enhancing factor in ventilator-induced lung injury. American journal of respiratory and critical care medicine, 178(6), 605-17.
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  We previously demonstrated pre-B-cell colony enhancing factor (PBEF) as a biomarker in sepsis and sepsis-induced acute lung injury (ALI) with genetic variants conferring ALI susceptibility.
• Kamp, R., Sun, X., & Garcia, J. G. (2008). Making genomics functional: deciphering the genetics of acute lung injury. Proceedings of the American Thoracic Society, 5(3), 348-53.
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  Acute lung injury (ALI) is a common and frequently devastating illness characterized by acute hypoxemic respiratory failure, profound inflammation, and flooding of the alveoli. Despite recent advances in ALI care, the morbidity and mortality of ALI continues to be unacceptably high. ALI-inciting events (e.g., sepsis, trauma, aspiration, pneumonia) are quite common, yet only a fraction of patients develop the syndrome. This heterogeneity of patients presenting with ALI has sparked interest in identifying the role of genetic factors that contribute to ALI susceptibility and prognosis. Recent advances in high-throughput sequencing and expression technologies now provide the tools to perform large-scale genomic analyses in complex disorders such as ALI; gene expression profiling and pathway analysis provide further insight into previously described molecular pathways involved in the syndrome. In this article, we describe the use of genomewide association studies, ortholog in silico techniques, utility of consomic rat methods, and candidate gene approaches using expression profiling and pathway analyses. These methods have confirmed suspected ALI candidate genes (e.g., IL-6 and MIF), but more impressively have identified novel genes (e.g., GADD45alpha and PBEF) not previously suspected in ALI. The analysis of the molecular pathways (e.g., the cytoskeleton in vascular barrier regulation) has identified additional genes contributing to the development and severity of ALI (e.g., MLCK), thereby providing therapeutic targets in this devastating illness.
• Kolosova, I. A., Mirzapoiazova, T., Moreno-Vinasco, L., Sammani, S., Garcia, J. G., & Verin, A. D. (2008). Protective effect of purinergic agonist ATPgammaS against acute lung injury. American journal of physiology. Lung cellular and molecular physiology, 294(2), L319-24.
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  Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of acute respiratory failure associated with high morbidity and mortality. Although ALI/ARDS pathogenesis is only partly understood, pulmonary endothelium plays a major role by regulating lung fluid balance and pulmonary edema formation. Consequently, endothelium-targeted therapies may have beneficial effects in ALI/ARDS. Recently, attention has been given to the therapeutic potential of purinergic agonists and antagonists for the treatment of cardiovascular and pulmonary diseases. Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface P2Y receptors. We previously described ATP-induced endothelial cell (EC) barrier enhancement via a complex cell signaling and hypothesized endothelial purinoreceptors activation to exert anti-inflammatory barrier-protective effects. To test this hypothesis, we used a murine model of ALI induced by intratracheal administration of endotoxin/lipopolysaccharide (LPS) and cultured pulmonary EC. The nonhydrolyzed ATP analog ATPgammaS (50-100 muM final blood concentration) attenuated inflammatory response with decreased accumulation of cells (48%, P < 0.01) and proteins (57%, P < 0.01) in bronchoalveolar lavage and reduced neutrophil infiltration and extravasation of Evans blue albumin dye into lung tissue. In cell culture model, ATPgammaS inhibited junctional permeability induced by LPS. These findings suggest that purinergic receptor stimulation exerts a protective role against ALI by preserving integrity of endothelial cell-cell junctions.
• Ma, S. F., Flores, C., Wade, M. S., Dudek, S. M., Nicolae, D. L., Ober, C., & Garcia, J. G. (2008). A common cortactin gene variation confers differential susceptibility to severe asthma. Genetic epidemiology, 32(8), 757-66.
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  Genomic regions with replicated linkage to asthma-related phenotypes likely harbor multiple susceptibility loci with relatively minor effects on disease susceptibility. The 11q13 chromosomal region has repeatedly been linked to asthma with five genes residing in this region with reported replicated associations. Cortactin, an actin-binding protein encoded by the CTTN gene in 11q13, constitutes a key regulator of cytoskeletal dynamics and contractile cell machinery, events facilitated by interaction with myosin light chain kinase; encoded by MYLK, a gene we recently reported as associated with severe asthma in African Americans. To evaluate potential association of CTTN gene variation with asthma susceptibility, CTTN exons and flanking regions were re-sequenced in 48 non-asthmatic multiethnic samples, leading to selection of nine tagging polymorphisms for case-control association studies in individuals of European and African descent. After ancestry adjustments, an intronic variant (rs3802780) was significantly associated with severe asthma (odds ratio [OR]: 1.71; 95% confidence interval [CI]: 1.20-2.43; p=0.003) in a joint analysis. Further analyses evidenced independent and additive effects of CTTN and MYLK risk variants for severe asthma susceptibility in African Americans (accumulated OR: 2.93, 95% CI: 1.40-6.13, p=0.004). These data suggest that CTTN gene variation may contribute to severe asthma and that the combined effects of CTTN and MYLK risk polymorphisms may further increase susceptibility to severe asthma in African Americans harboring both genetic variants.
• Moitra, J., Evenoski, C., Sammani, S., Wadgaonkar, R., Turner, J. R., Ma, S. F., & Garcia, J. G. (2008). A transgenic mouse with vascular endothelial over-expression of the non-muscle myosin light chain kinase-2 isoform is susceptible to inflammatory lung injury: role of sexual dimorphism and age. Translational research : the journal of laboratory and clinical medicine, 151(3), 141-53.
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  We have generated genetically engineered mice that are uniquely susceptible to lipopolysaccharide (LPS)-induced and mechanical ventilation-induced lung injury in a sex-specific and age-specific manner. These mice express a nonmuscle isoform of the myosin light chain kinase gene (nmMLCK2) targeted to the endothelium. Homozygous mice have significantly reduced fecundity and litter survival until weaning, and they are initially growth delayed but eventually exceed the size of wild-type littermates. Mice at all ages show increased protein transport across the lung barrier; however, the phenotype is most discernible in 8-12-week-old male mice. When subjected to a clinically relevant LPS-induced lung injury model, 8-12-week-old young females and 30-36-week-old males seem to be the most significantly injured group. In contrast, 30-36-week-old males remain the most significantly injured group when mechanically ventilated at high tidal volumes, which is a clinically relevant model of mechanical stress lung injury. These data reveal that nmMLCK2 overexpression in the endothelium exacerbates lung injury in vivo in a sexually dimorphic and age-dependent manner.
• Moreno-Vinasco, L., Gomberg-Maitland, M., Maitland, M. L., Desai, A. A., Singleton, P. A., Sammani, S., Sam, L., Liu, Y., Husain, A. N., Lang, R. M., Ratain, M. J., Lussier, Y. A., & Garcia, J. G. (2008). Genomic assessment of a multikinase inhibitor, sorafenib, in a rodent model of pulmonary hypertension. Physiological genomics, 33(2), 278-91.
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  Pulmonary hypertension (PH) and cancer pathology share growth factor- and MAPK stress-mediated signaling pathways resulting in endothelial and smooth muscle cell dysfunction and angioproliferative vasculopathy. In this study, we assessed sorafenib, an antineoplastic agent and inhibitor of multiple kinases important in angiogenesis [VEGF receptor (VEGFR)-1-3, PDGF receptor (PDGFR)-beta, Raf-1 kinase] as a potential PH therapy. Two PH rat models were used: a conventional hypoxia-induced PH model and an augmented PH model combining dual VEGFR-1 and -2 inhibition (SU-5416, single 20 mg/kg injection) with hypoxia. In addition to normoxia-exposed control animals, four groups were maintained at 10% inspired O(2) fraction for 3.5 wk (hypoxia/vehicle, hypoxia/SU-5416, hypoxia/sorafenib, and hypoxia/SU-5416/sorafenib). Compared with normoxic control animals, rats exposed to hypoxia/SU-5416 developed hemodynamic and histological evidence of severe PH while rats exposed to hypoxia alone displayed only mild elevations in hemodynamic values (pulmonary vascular and right ventricular pressures). Sorafenib treatment (daily gavage, 2.5 mg/kg) prevented hemodynamic changes and demonstrated dramatic attenuation of PH-associated vascular remodeling. Compared with normoxic control rats, expression profiling (Affymetrix platform) of lung RNA obtained from hypoxia [false discovery rate (FDR) 6.5%]- and hypoxia/SU-5416 (FDR 1.6%)-challenged rats yielded 1,019 and 465 differentially regulated genes (fold change >1.4), respectively. A novel molecular signature consisting of 38 differentially expressed genes between hypoxia/SU-5416 and hypoxia/SU-5416/sorafenib (FDR 6.7%) was validated by either real-time RT-PCR or immunoblotting. Finally, immunoblotting studies confirmed the upregulation of the MAPK cascade in both PH models, which was abolished by sorafenib. In summary, sorafenib represents a novel potential treatment for severe PH with the MAPK cascade a potential canonical target.
• Neptune, E. R., Podowski, M., Calvi, C., Cho, J. H., Garcia, J. G., Tuder, R., Linnoila, R. I., Tsai, M. J., & Dietz, H. C. (2008). Targeted disruption of NeuroD, a proneural basic helix-loop-helix factor, impairs distal lung formation and neuroendocrine morphology in the neonatal lung. The Journal of biological chemistry, 283(30), 21160-9.
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  Despite the importance of airspace integrity in vertebrate gas exchange, the molecular pathways that instruct distal lung formation are poorly understood. Recently, we found that fibrillin-1 deficiency in mice impairs alveolar formation and recapitulates the pulmonary features of human Marfan syndrome. To further elucidate effectors involved in distal lung formation, we performed expression profiling analysis comparing the fibrillin-1-deficient and wild-type developing lung. NeuroD, a basic helix-loop-helix transcription factor, fulfilled the expression criteria for a candidate mediator of distal lung development. We investigated its role in murine lung development using genetically targeted NeuroD-deficient mice. We found that NeuroD deficiency results in both impaired alveolar septation and altered morphology of the pulmonary neuroendocrine cells. NeuroD-deficient mice had enlarged alveoli associated with reduced epithelial proliferation in the airway and airspace compartments during development. Additionally, the neuroendocrine compartment in these mice manifested an increased number of neuroepithelial bodies but a reduced number of solitary pulmonary neuroendocrine cells in the neonatal lung. Overexpression of NeuroD in a murine lung epithelial cell line conferred a neuroendocrine phenotype characterized by the induction of neuroendocrine markers as well as increased proliferation. These results support an unanticipated role for NeuroD in the regulation of pulmonary neuroendocrine and alveolar morphogenesis and suggest an intimate connection between the neuroendocrine compartment and distal lung development.
• Nonas, S., Birukova, A. A., Fu, P., Xing, J., Chatchavalvanich, S., Bochkov, V. N., Leitinger, N., Garcia, J. G., & Birukov, K. G. (2008). Oxidized phospholipids reduce ventilator-induced vascular leak and inflammation in vivo. Critical care (London, England), 12(1), R27.
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  Mechanical ventilation at high tidal volume (HTV) may cause pulmonary capillary leakage and acute lung inflammation resulting in ventilator-induced lung injury. Besides blunting the Toll-like receptor-4-induced inflammatory cascade and lung dysfunction in a model of lipopolysaccharide-induced lung injury, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exerts direct barrier-protective effects on pulmonary endothelial cells in vitro via activation of the small GTPases Rac and Cdc42. To test the hypothesis that OxPAPC may attenuate lung inflammation and barrier disruption caused by pathologic lung distension, we used a rodent model of ventilator-induced lung injury and an in vitro model of pulmonary endothelial cells exposed to pathologic mechanochemical stimulation.
• Sammani, S., Vigneswaran, W. T., Sammani, S., Moreno-vinasco, L., Mirzapoiazova, T., Jacobson, J. R., Garcia, J. G., & Bonde, P. (2008). Attenuation of Rodent Lung Ischemia Reperfusion Injury by Sphingosine 1 Phosphate. Journal of Organ Dysfunction, 4(2), 106-114. doi:10.1080/17471060701505289
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  Objective. Lung ischemia–reperfusion (IR) injury, a sequela of transplantation characterized by alveolar damage, edema and inflammation in donor lungs, remains a significant cause of transplant failure despite improvements in lung preservation techniques. We investigated the effects of sphingosine 1-phosphate (S1P), a potent vascular barrier-protective agent, in a rat model of IR injury. Material and methods. S1P (26.5 mg/kg, i.v.) or vehicle was administered 15 min prior to ischemia via pulmonary artery ligation (1 h) and subsequent reperfusion (2 h). Lung vascular permeability and inflammation were assessed. Results. Animals pretreated with S1P exhibited reduced bronchoalveolar lavage (BAL) inflammatory cells (32% decrease; p
• Singleton, P. A., Garcia, J. G., & Moss, J. (2008). Synergistic effects of methylnaltrexone with 5-fluorouracil and bevacizumab on inhibition of vascular endothelial growth factor-induced angiogenesis. Molecular cancer therapeutics, 7(6), 1669-79.
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  Many patients with cancer receive combinations of drug treatments that include 5-fluorouracil (5-FU) and bevacizumab. Therapeutic doses of 5-FU are often associated with unwanted side effects, and bevacizumab is costly. Therefore, we explored potential agents that can reduce the therapeutic concentration of these drugs. Our data indicate that methylnaltrexone (MNTX), a peripheral antagonist of the mu-opioid receptor, exerts a synergistic effect with 5-FU and bevacizumab on inhibition of vascular endothelial growth factor (VEGF)-induced human pulmonary microvascular endothelial cell (EC) proliferation and migration, two key components in cancer-associated angiogenesis. MNTX inhibited EC proliferation with an IC(50) of approximately 100 nmol/L. Adding 100 nmol/L MNTX to EC shifted the IC(50) of 5-FU from approximately 5 micromol/L to approximately 7 nmol/L. Further, adding 50 ng/mL MNTX shifted the IC(50) of bevacizumab on inhibition of EC migration from approximately 25 to approximately 6 ng/mL. These synergistic effects were not observed with naltrexone, a tertiary mu-opioid receptor antagonist. On a mechanistic level, we observed that treatment of human EC with MNTX, but not naltrexone, increased receptor protein tyrosine phosphatase mu activity, which was independent of mu-opioid receptor expression. Silencing receptor protein tyrosine phosphatase mu expression (small interfering RNA) in human EC inhibited both synergy between MNTX and bevacizumab or 5-FU and increased VEGF-induced tyrosine phosphorylation of Src and p190 RhoGAP with enhanced activation of Akt and the actin cytoskeletal regulatory protein, RhoA, whereas silencing Src, Akt, or RhoA blocked VEGF-induced angiogenic events. Therefore, addition of MNTX could potentially lower the therapeutic doses of 5-FU and bevacizumab, which could improve index.
• Singleton, P., & Garcia, J. G. (2008). Stretching the search for therapeutic targets in acute lung injury. Translational research : the journal of laboratory and clinical medicine, 152(6), 255-6.
• Szczepaniak, W. S., Zhang, Y., Hagerty, S., Crow, M. T., Kesari, P., Garcia, J. G., Choi, A. M., Simon, B. A., & McVerry, B. J. (2008). Sphingosine 1-phosphate rescues canine LPS-induced acute lung injury and alters systemic inflammatory cytokine production in vivo. Translational research : the journal of laboratory and clinical medicine, 152(5), 213-24.
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  S1P has been demonstrated to protect against the formation of lipopolysaccharide (LPS)-induced lung edema when administered concomitantly with LPS. In the current study, we sought to determine the effectiveness of S1P to attenuate lung injury in a translationally relevant canine model of ALI when administered as rescue therapy. Secondarily, we examined whether the attenuation of LPS-induced physiologic lung injury after administration of S1P was, at least in part, caused by an alteration in local and/or systemic inflammatory cytokine expression. We examined 18, 1-year-old male beagles prospectively in which we instilled bacterial LPS (2-4 mg/kg) intratracheally followed in 1 h with intravenous S1P (85 microg/kg) or vehicle and 8 h of high-tidal-volume mechanical ventilation. S1P attenuated the formation of Q(s)/Q(t) (32%), and both the presence of protein (72%) and neutrophils (95%) in BAL fluid compared with vehicle controls. Although lung tissue inflammatory cytokine production was found to vary regionally throughout the LPS-injured lung, S1P did not alter the expression pattern. Similarly, BAL cytokine production was not altered significantly by intravenous S1P in this model. Interestingly, S1P potentiated the LPS-induced systemic production of 3 inflammatory cytokines, TNF-alpha (6-fold), KC (1.2-fold), and IL-6 (3-fold), without resulting in end-organ dysfunction. In conclusion, intravenous S1P reduces inflammatory lung injury when administered as rescue therapy in our canine model of LPS-induced ALI. This improvement is observed in the absence of changes in local pulmonary inflammatory cytokine production and an augmentation of systemic inflammation.
• Wadgaonkar, R., Somnay, K., & Garcia, J. G. (2008). Thrombin induced secretion of macrophage migration inhibitory factor (MIF) and its effect on nuclear signaling in endothelium. Journal of cellular biochemistry, 105(5), 1279-88.
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  The procoagulant thrombin stimulates endothelial cells (EC) to undergo rapid cytoskeleton changes via signaling pathways that induce multiple phenotypic changes, including alterations in permeability, vasomotor tone, adhesion molecule synthesis, and leukocyte trafficking. We studied a novel role of thrombin's action on the endothelium that results in MIF secretion, which is linked to myosin light chain (MLC) and extracellular signal-regulated kinase (ERK(1/2))-dependent nuclear signaling. In bovine pulmonary artery EC (BPAEC), thrombin treatment induced intracellular MLC phosphorylation within 15 min, followed by a significant increase in MIF secretion within 30 min. Thrombin treatment induced biphasic ERK(1/2) phosphorylation with an early phase occurring at 15 min and a later phase at 120 min. To understand the role of MIF secretion in thrombin-induced biphasic activation of ERK(1/2), BPAE cells were treated with (i) recombinant MIF, and (ii) the medium collected from thrombin-treated BPAE cells. These studies demonstrated a sustained monophasic ERK(1/2) phosphorylation. Inhibition of MIF secretion by MIF siRNA or antisense-MIF treatment, along with a neutralizing antibody, attenuated the thrombin-induced second phase ERK phosphorylation, suggesting a direct involvement of MIF in the second phase of ERK(1/2) activation. Pretreatment of BPAE cells with an ERK kinase inhibitor and with antisense-MIF significantly inhibited thrombin-induced nuclear factor kappa (NF-kappaB) activation. These results indicate that MIF secretion and ERK phosphorylation both play a necessary role in thrombin induced NF-kappaB activation.
• Wang, T., Moreno-Vinasco, L., Huang, Y., Lang, G. D., Linares, J. D., Goonewardena, S. N., Grabavoy, A., Samet, J. M., Geyh, A. S., Breysse, P. N., Lussier, Y. A., Natarajan, V., & Garcia, J. G. (2008). Murine lung responses to ambient particulate matter: genomic analysis and influence on airway hyperresponsiveness. Environmental health perspectives, 116(11), 1500-8.
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  Asthma is a complex disease characterized by airway hyperresponsiveness (AHR) and chronic airway inflammation. Epidemiologic studies have demonstrated that exposures to environmental factors such as ambient particulate matter (PM), a major air pollutant, contribute to increased asthma prevalence and exacerbations.
• Wurfel, M. M., Gordon, A. C., Holden, T. D., Radella, F., Strout, J., Kajikawa, O., Ruzinski, J. T., Rona, G., Black, R. A., Stratton, S., Jarvik, G. P., Hajjar, A. M., Nickerson, D. A., Rieder, M., Sevransky, J., Maloney, J. P., Moss, M., Martin, G., Shanholtz, C., , Garcia, J. G., et al. (2008). Toll-like receptor 1 polymorphisms affect innate immune responses and outcomes in sepsis. American journal of respiratory and critical care medicine, 178(7), 710-20.
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  Polymorphisms affecting Toll-like receptor (TLR)-mediated responses could predispose to excessive inflammation during an infection and contribute to an increased risk for poor outcomes in patients with sepsis.
• Zhang, L. Q., Adyshev, D. M., Singleton, P., Li, H., Cepeda, J., Huang, S. Y., Zou, X., Verin, A. D., Tu, J., Garcia, J. G., & Ye, S. Q. (2008). Interactions between PBEF and oxidative stress proteins--a potential new mechanism underlying PBEF in the pathogenesis of acute lung injury. FEBS letters, 582(13), 1802-8.
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  Identification of pre-B-cell colony-enhancing factor (PBEF) interacting partners may reveal new molecular mechanisms of PBEF in the pathogenesis of acute lung injury (ALI). The interactions between PBEF and NADH dehydrogenase subunit 1(ND1), ferritin light chain and interferon induced transmembrane 3 (IFITM3) in human pulmonary vascular endothelial cells were identified and validated. ND1, ferritin and IFITM3 are involved in oxidative stress and inflammation. Overexpression of PBEF increased its interactions and intracellular oxidative stress, which can be attenuated by rotenone. The interaction modeling between PBEF and ND1 is consistent with the corresponding experimental finding. These interactions may underlie a novel role of PBEF in the pathogenesis of ALI.
• Zhang, L. Q., Ma, S. F., Grigoryev, D., Lavoie, T. L., Xiao, H. Q., Setterquist, R., Li, H., Jacobson, J., Garcia, J. G., & Ye, S. Q. (2008). Temporal gene expression analysis of human coronary artery endothelial cells treated with Simvastatin. Gene expression, 14(4), 229-39.
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  Increasing evidence indicates that the beneficial "pleiotropic" effects of statins on clinical events involve nonlipid mechanisms including the modification of blood vessel endothelial cell function. However, the involved molecular events and pathways are not completely understood. In the present study, Affymetrix microarrays were used to monitor the temporal gene expression of human coronary artery endothelial cells (HCAEC) treated with simvastatin (Sim) to gain insight into statins' direct effects on the endothelial function. We isolated and labeled mRNA from HCAEC treated with Sim for 0, 3, 6, 12, 24, and 48 h and hybridized these samples to Affymetrix GeneChip HG-U95Av2 to analyze the temporal gene expression profile. Out of 12,625 genes present on the HG-U95Av2 GeneChip, expression of 5,432 genes was detected. There were 1,475 of 5,432 genes that displayed the differential expression compared to baseline (0 h). Fifty-four genes were upregulated (< or = twofold) while 61 genes were downregulated ( > or = twofold) at 24-48 h after the Sim treatment. Many new target genes and pathways modulated by Sim were uncovered. This study indicates that many aspects of the pleiotropic effect of Sim on the endothelial cell function can be mediated by transcriptional control. Physiological function of 22% of 115 differentially expressed genes in Sim-treated HCAEC are currently unknown. These newly identified genes could be useful for new mechanistic study and new therapeutic modalities. Expressions of 13 out of 18 genes (> 70%) in the cell cycle/proliferation control process were significantly inhibited by the Sim treatment. CDC25B and ITGB4 gene expressions were validated by RT-PCR and Western blotting. Sim's inhibitory effect of on HCAEC growth was confirmed by the measurement of [3H]thymidine incorporation into the DNA synthesis. Further in-depth analysis of this effect may shed light on molecular mechanisms of Sim's beneficial inhibition of neointima formation in the atherosclerotic artery stenosis.
• Zhao, J., Schilling, A. B., Garcia, J. G., Dudek, S. M., Chiang, E. T., & Camp, S. M. (2008). Identification of novel in vitro protein kinase A phosphorylation sites on recombinant non-muscle myosin light chain kinase: Nano-liquid chromatography tandem mass spectrometry methodology. Journal of Organ Dysfunction, 5(4), 242-253. doi:10.3109/17471060802409712
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  Protein phosphorylation is a key event in endothelial cell signaling and barrier regulation. We previously demonstrated that the non-muscle myosin light chain kinase (nmMLCK) isoform present in endothelium (1914aa) is a multi-functional enzyme which drives the participation of the actin cytoskeleton in vascular barrier regulation, trafficking of inflammatory cells into the lung, and susceptibility to sepsis-induced acute lung injury. The activity of the nmMLCK isoform is differentially regulated by both Ser/Thr and Tyr phosphorylation; however, cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA)-mediated nmMLCK phosphorylation exerts paradoxical effects on kinase activity depending on the exact Ser/Thr sites. To address this conundrum, we mapped in vitro phosphorylation sites of nmMLCK catalyzed by the catalytic unit of PKA using data-dependent nano-liquid chromatography tandem mass spectrometry. High mass-accuracy MS and optimized biochemical protocols identified 26 novel nmMLCK1 PKA p...
• Desai, A. A., Moreno, L., Sammani, S., Sammani, S., Sam, L., Ratain, M. J., Moreno, L., Maitland, M. L., Ma, S., Lussier, Y. A., Liu, Y., Lang, R. M., Husain, A. N., Gomberg-maitland, M. M., Garcia, J. G., Desai, A. A., & Collins, K. A. (2007). 76 ATTENUATION OF A RODENT MODEL OF PULMONARY HYPERTENSION: A NOVEL ROLE FOR SORAFENIB, A MULTIKINASE INHIBITOR.. Journal of Investigative Medicine, 55(2), S361.2-S361. doi:10.1136/jim-55-02-76
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  Drawing from new drug discovery studies is the observation that severe pulmonary hypertension (PH) and cancer pathophysiology share common signal transduction pathways leading to abnormal smooth muscle and endothelial cell (EC) interactions and angioproliferative vasculopathy. Sorafenib (Sor), a chemotherapeutic agent in clinical trials for the treatment of renal cell cancer, is an inhibitor of multiple kinases, including Raf-1 kinase, MAPK, VEGFR-2, and VEGFR-3, genes implicated in angiogenesis, proliferation, and the inhibition of apoptosis. We therefore tested the hypothesis that Sor will attenuate the development of PH using an established rodent model of the disease. We performed two 3-week hypoxia (FiO2 10%) and SU5416 (a selective VEGFR-2 inhibitor known to dramatically augment hypoxia-induced PH) studies to induce PH in Dahl salt-sensitive rats (SS). Rat groups were normoxia/vehicle (Norm), hypoxia/vehicle (H), H-Su, hypoxia/sorafenib (H-Sor), and hypoxia/sorafenib/ SU5416 (H-Su-Sor). Except for Norm, all rats were kept in hypoxia, whereas the H-Su group received SU5416 at day 1 (20 mg/kg, sc) and Sor was gavaged daily (2.5 mg/kg). Echocardiography, pulmonary artery pressures (PAPs), right ventricular pressures (RVPs), and lung gene microarray analyses were assessed at 3 weeks. Our results showed that H-Su rats developed severe PH compared with Norm, rats in the H alone group had mildly elevated pressures compared with Norm, and no changes were seen in pressures, weights, or remodeling in the H-Sor or H-SU-Sor groups compared with Norm. The H-Su-Sor rats showed significant reductions in PAP (56%), RVP (55%), and RV hypertrophy (52%). Gene expression profiling data were compared with Norm using GCRMA normalization in R and SAM (> .639, MFC > 1.7). With false discovery rates (FDRs) of 5.1% and 0.7%, respectively, 356 and 293 genes were up- or down-regulated. Forty-seven of the 356 H genes were recapitulated from previous H studies in the rodent model. In addition, 45 genes were differentially expressed between H-Su and H-Su-Sor (FDR 12%), with ECM, cytoskeleton, and angiogenesis gene ontologies, and 81 genes were changed in the H and H-Su groups but not in the H-Su-Sor group. These studies suggest Sor as a powerful novel treatment in PH.
• Diette, G. B., Watson, H., Togias, A., Stockton, M. L., Shanholtz, C., Sevransky, J. E., Moss, M., Mathias, R. A., Maloney, J. P., Hansel, N. N., Grant, A. V., Garcia, J. G., Gao, P., Gao, L., Dunston, G. M., Diette, G. B., Chi, P. B., Brower, R. G., Beaty, T. H., & Barnes, K. C. (2007). Myosin Light Chain Kinase (MYLK) Variants that Confer Increased Risk of Sepsis and Acute Lung Injury are Associated with Asthma and Associated Phenotypes. The Journal of Allergy and Clinical Immunology, 119(1), S146-S147. doi:10.1016/j.jaci.2006.11.511
• Dudek, S. M., Camp, S. M., Chiang, E. T., Singleton, P. A., Usatyuk, P. V., Zhao, Y., Natarajan, V., & Garcia, J. G. (2007). Pulmonary endothelial cell barrier enhancement by FTY720 does not require the S1P1 receptor. Cellular signalling, 19(8), 1754-64.
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  Novel therapeutic strategies are needed to reverse the loss of endothelial cell (EC) barrier integrity that occurs during inflammatory disease states such as acute lung injury. We previously demonstrated potent EC barrier augmentation in vivo and in vitro by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P) via ligation of the S1P1 receptor. The S1P analogue, FTY720, similarly exerts barrier-protective vascular effects via presumed S1P1 receptor ligation. We examined the role of the S1P1 receptor in sphingolipid-mediated human lung EC barrier enhancement. Both S1P and FTY-induced sustained, dose-dependent barrier enhancement, reflected by increases in transendothelial electrical resistance (TER), which was abolished by pertussis toxin indicating Gi-coupled receptor activation. FTY-mediated increases in TER exhibited significantly delayed onset and intensity relative to the S1P response. Reduction of S1P1R expression (via siRNA) attenuated S1P-induced TER elevations whereas the TER response to FTY was unaffected. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated S1P1R phosphorylation on threonine residues. Inhibition of PI3 kinase activity attenuated S1P-mediated TER increases but failed to alter FTY-induced TER elevation. Finally, S1P, but not FTY, induced significant myosin light chain phosphorylation and dramatic actin cytoskeletal rearrangement whereas reduced expression of the cytoskeletal effectors, Rac1 and cortactin (via siRNA), attenuated S1P-, but not FTY-induced TER elevations. These results mechanistically characterize pulmonary vascular barrier regulation by FTY720, suggesting a novel barrier-enhancing pathway for modulating vascular permeability.
• Flores, C., Ma, S. F., Maresso, K., Ober, C., & Garcia, J. G. (2007). A variant of the myosin light chain kinase gene is associated with severe asthma in African Americans. Genetic epidemiology, 31(4), 296-305.
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  Asthma is a complex phenotype influenced by environmental and genetic factors for which severe irreversible structural airway alterations are more frequently observed in African Americans. In addition to a multitude of factors contributing to its pathobiology, increased amounts of myosin light chain kinase (MLCK), the central regulator of cellular contraction, have been found in airway smooth muscle from asthmatics. The gene encoding MLCK (MYLK) is located in 3q21.1, a region noted by a number of genome-wide studies to show linkage with asthma and asthma-related phenotypes. We studied 17 MYLK genetic variants in European and African Americans with asthma and severe asthma and identified a single non-synonymous polymorphism (Pro147Ser) that was almost entirely restricted to African populations and which was associated with severe asthma in African Americans. These results remained highly significant after adjusting for proportions of ancestry estimated using 30 unlinked microsatellites (adjusted odds ratio: 1.76 [95% confidence interval, CI: 1.17-2.65], p = 0.005). Since all common HapMap polymorphisms in approximately 500 kb contiguous regions have low-to-moderate linkage disequilibrium with Pro147Ser, we speculate that this polymorphism is causally related to the severe asthma phenotype in African Americans. The association of this polymorphism, located in the N-terminal region of the non-muscle MLCK isoform, emphasizes the potential importance of the vascular endothelium, a tissue in which MLCK is centrally involved in multiple aspects of the inflammatory response, in the pathogenesis of severe asthma. This finding also offers a possible genetic explanation for some of the more severe asthma phenotype observed in African American asthmatics.
• Gao, L., Flores, C., Fan-Ma, S., Miller, E. J., Moitra, J., Moreno, L., Wadgaonkar, R., Simon, B., Brower, R., Sevransky, J., Tuder, R. M., Maloney, J. P., Moss, M., Shanholtz, C., Yates, C. R., Meduri, G. U., Ye, S. Q., Barnes, K. C., & Garcia, J. G. (2007). Macrophage migration inhibitory factor in acute lung injury: expression, biomarker, and associations. Translational research : the journal of laboratory and clinical medicine, 150(1), 18-29.
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  The macrophage migration inhibitory factor (MIF), a pro-inflammatory cytokine central to the response to endotoxemia, is a putative biomarker in acute lung injury (ALI). To explore MIF as a molecular target and candidate gene in ALI, the MIF gene and protein expression were examined in murine and canine models of ALI (high tidal volume mechanical ventilation, endotoxin exposure) and in patients with either sepsis or sepsis-induced ALI. MIF gene expression and protein levels were significantly increased in each ALI model, with serum MIF levels significantly higher in patients with either sepsis or ALI compared with healthy controls (African- and European-descent). The association of 8 MIF gene polymorphisms (single-nucleotide polymorphisms (SNPs)) (within a 9.7-kb interval on chromosome 22q11.23) with the development of sepsis and ALI in European-descent and African-descent populations was studied next. Genotyping in 506 DNA samples (sepsis patients, sepsis-associated ALI patients, and healthy controls) revealed haplotypes located in the 3' end of the MIF gene, but not individual SNPs, associated with sepsis and ALI in both populations. These data, generated via functional genomic and genetic approaches, suggest that MIF is a relevant molecular target in ALI.
• Gao, L., Grant, A. V., Rafaels, N., Stockton-Porter, M., Watkins, T., Gao, P., Chi, P., Muñoz, M., Watson, H., Dunston, G., Togias, A., Hansel, N., Sevransky, J., Maloney, J. P., Moss, M., Shanholtz, C., Brower, R., Garcia, J. G., Grigoryev, D. N., , Cheadle, C., et al. (2007). Polymorphisms in the myosin light chain kinase gene that confer risk of severe sepsis are associated with a lower risk of asthma. The Journal of allergy and clinical immunology, 119(5), 1111-8.
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  Myosin light chain kinase (MYLK) is a multifunctional protein involved in regulation of airway hyperreactivity and other activities relevant to asthma.
• Grigoryev, D. N., Ma, S. F., Shimoda, L. A., Johns, R. A., Lee, B., & Garcia, J. G. (2007). Exon-based mapping of microarray probes: recovering differential gene expression signal in underpowered hypoxia experiment. Molecular and cellular probes, 21(2), 134-9.
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  There is an immense collection of underpowered Affymetrix gene array experiments. Although a majority of these experiments generated biologically feasible results, the considerable fraction of assays failed to identify expected transcriptional changes. There is an unused potential of Affymetrix probe-set redundancy for common exonic and UTR regions. We hypothesized that group analysis of multiple probe-sets which hybridize to the same exon or UTR will increase array discriminating power of transcriptional changes. To test this hypothesis, we analyzed Affymetrix mouse probe-sets that share the same exon using blocking feature of the Significance Analysis of Microarrays (SAM). Two-thousand two-hundred one exon-sharing probe-sets targeting 1011 transcripts were identified by mapping 36701 MG-U74v2 probe-sets to genomic alignments of 3,971,086 known mouse transcripts. Using the blocking feature of SAM with an underpowered (two microarrays per experimental condition) mouse hypoxia-induced pulmonary hypertension model, we identified 24 genes that were significantly (FDR
• Guo, Y., Singleton, P. A., Rowshan, A., Gucek, M., Cole, R. N., Graham, D. R., Van Eyk, J. E., & Garcia, J. G. (2007). Quantitative proteomics analysis of human endothelial cell membrane rafts: evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induced barrier enhancement. Molecular & cellular proteomics : MCP, 6(4), 689-96.
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  Endothelial cell barrier dysfunction results in the increased vascular permeability observed in inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Sphingosine 1-phosphate (S1P), a biologically active phosphorylated lipid growth factor released from activated platelets, enhances the endothelial cell barrier integrity in vitro and in vivo. To begin to identify the molecular mechanisms mediating S1P induced endothelial barrier enhancement, quantitative proteomics analysis (iTRAQ) was performed on membrane rafts isolated from human pulmonary artery endothelial cells in the absence or presence of S1P stimulation. Our results demonstrated that S1P mediates rapid and specific recruitment (1 microM, 5 min) of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKS-related protein (MRP) to membrane rafts. Western blot experiments confirmed these findings with both MARCKS and MRP. Finally, small interfering RNA-mediated silencing of MARCKS or MRP or both attenuates S1P-mediated endothelial cell barrier enhancement. These data suggest the regulation of S1P-mediated endothelial cell barrier enhancement via the cell specific localization of MARCKS and MRP and validate the utility of proteomics approaches in the identification of novel molecular targets.
• Hooper, W. C., Mensah, G. A., Haworth, S. G., Black, S. M., Garcia, J. G., & Langleben, D. (2007). Vascular endothelium summary statement V: Pulmonary hypertension and acute lung injury: public health implications. Vascular pharmacology, 46(5), 327-9.
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  Although relatively rare, pulmonary hypertension can be devastating for those individuals who are affected and has significant societal implications. The 2003 WHO classification separates PAH (idiopathic, specific disease linked) from pulmonary hypertension related to lung disease, thromboembolic disease, and pulmonary venous hypertension. Another form of pulmonary hypertension, persistent pulmonary hypertension (PPHN), occurs in the newborn. In general, PPHN is thought to be responsible for approximately 10% of admissions to neonatal intensive care units and can be a complicating factor in 5 of 1000 live births. Acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) are complex disorders that pose a significant threat to critically ill patients. They are usually related to direct lung injury or indirect injury from sepsis, trauma, and other disorders. Although these pulmonary disorders reflect distinct pathophysiologic mechanisms, current evidence strongly suggests that a common denominator underlying many of the established molecular and cellular elements is endothelial cell activation and dysfunction. This summary statement briefly summarizes the state of the science and suggests future avenues of public health research.
• Jacobson, J. R., & Garcia, J. G. (2007). Novel therapies for microvascular permeability in sepsis. Current drug targets, 8(4), 509-14.
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  Sepsis is characterized physiologically by an aberrant systemic inflammatory response and microvascular dysfunction. While appropriate antibiotics and supportive care are essential in the management of the septic patient, therapies targeting specific aspects of the pathophysiology could have a significant impact on the morbidity and mortality associated with both sepsis and its sequlea, including acute lung injury (ALI). We have characterized several mediators of endothelial cell (EC) barrier function that may serve as novel therapies for sepsis-induced microvascular dysfunction including simvastatin, adenosine triphosphate (ATP), sphingosine 1-phosphate (S1P), and activated protein C (APC). Notably, APC is already available for the treatment of severe sepsis, however, to date its mechanism of action has been unclear. While distinct in many ways, we have found that these agonists have in common the ability to induce dynamic rearrangement of the EC actin cytoskeleton that corresponds to barrier protection. In addition, we have extended our in vitro findings to relevant animal models of endotoxin-induced acute lung injury and have confirmed beneficial effects of both simvastatin and S1P which are associated with evidence of decreased vascular permeability in this setting. Moreover, our data also indicate that APC effects in sepsis may be largely due to augmentation of EC barrier function affecting decreased microvascular permeability. We speculate that the administration of direct modulators of EC barrier function and microvascular permeability, such as those described here, may ultimately become the standard of care for the septic patient.
• Ma, S., Kunznetsov, S., Garcia, J. G., Dudek, S. M., & Camp, S. M. (2007). 37 AN ACUTE LUNG INJURY-ASSOCIATED CORTACTIN POLYMORPHISM ALTERS PULMONARY ENDOTHELIAL CELL BARRIER FUNCTION.. Journal of Investigative Medicine, 55(2), S354.5-S354. doi:10.1136/jim-55-02-37
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  Introduction Acute lung injury (ALI) syndromes are highly morbid consequences of systemic inflammatory conditions such as sepsis. Inflammation-induced disruption during ALI of the endothelial cell (EC) barrier that lines the pulmonary vasculature results in leakage of fluid, protein, and cells into the airspaces of the lung, resulting in respiratory failure. We have previously described (Dudek et al. J Biol Chem 2004;279:24692-700) a critical role for the actin-binding protein cortactin in mediating EC cytoskeletal rearrangements that regulate in vitro barrier function. Methods/Results We now report immunofluorescence and coimmunoprecipitation data demonstrating increased association of cortactin with the junctional proteins β-catenin, focal adhesion kinase, and vinculin during the recovery phase following thrombin-induced permeability. Thus, cortactin interacts at critical cell-cell and cell-matrix junctional sites as the EC monolayer recovers after inflammation-induced disruption. As a translational approach building upon these in vitro observations, single-nucleotide polymorphism (SNP) discovery of the cortactin gene was performed using direct fluorescence-based resequencing. Cortactin SNP discovery in 36 patients, subdivided into those with sepsis-induced ALI, sepsis alone and healthy controls, identified 26 SNPs within the human cortactin gene, including a single novel coding variant. This SNP at amino acid position 484 results in a serine to asparagine change (Ser484Asn), which was enriched in patients with ALI and sepsis compared with controls. Since this S484N site is in close proximity to a critical p60src-targeted tyrosine residue (Y486), a major regulatory site of cortactin function, we generated wild-type and S484N mutant constructs for overexpression in human pulmonary ECs. These studies revealed greatly increased phosphorylation of cortactin at Y486 in the S484N mutant under baseline and EC barrier-altering conditions. In addition, overexpression of S484N in ECs inhibited the potent barrier-promoting effects of sphingosine 1-phosphate as well as delayed barrier recovery after the barrier disruption by thrombin. Summary Cortactin plays a critical role in mediating cytoskeletal and junctional protein rearrangements that regulate EC barrier function. Given that phosphorylation of Y486 modulates cortactin function, the S484N cortactin SNP may increase ALI susceptibility via this mechanism.
• Meyer, N. J., & Garcia, J. G. (2007). Wading into the genomic pool to unravel acute lung injury genetics. Proceedings of the American Thoracic Society, 4(1), 69-76.
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  Acute lung injury (ALI) is a common and often devastating illness characterized by acute hypoxemia, alveolar flooding, and an unacceptably high morbidity and mortality. Because only a fraction of the patients exposed to ALI-inciting events progress to development of the syndrome, there is significant interest in the identification of genetic factors potentially contributing to ALI susceptibility or prognosis. Two complementary strategies used to elucidate ALI genetics formulate the "candidate gene approach," whereby genes are identified by either global gene expression profiling in humans or animal models of ALI, often yielding highly conserved candidates across multiple species, or by related literature searches. Relevant variants or single nucleotide polymorphisms (single base pair substitutions) in these ALI candidate genes are tested for differences in allelic frequency for both ALI susceptibility and outcome between ALI cases and control patients at risk for ALI. This approach has yielded important variants in a number of genes (angiotensin converting enzyme, surfactant protein B, heat shock protein 70, pre-B-cell colony enhancing factor, myosin light chain kinase, and macrophage migration inhibitory factor) contributing toward an ALI phenotype. An alternative strategy not yet used in ALI genetic studies includes genomewide analyses to locate "hot" genomic segments harboring several hundred genes, with potential ALI candidate genes embedded within these segments. Overall, the detailing of specific ALI-associated polymorphisms will continue to provide new insights in the understanding of ALI pathogenesis, reveal novel molecular targets, and promote the development of individualized therapies to reduce morbidity and mortality from this devastating disease.
• Mirzapoiazova, T., Kolosova, I. A., Moreno, L., Sammani, S., Garcia, J. G., & Verin, A. D. (2007). Suppression of endotoxin-induced inflammation by taxol. The European respiratory journal, 30(3), 429-35.
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  The pathogenesis of acute lung injury includes transendothelial diapedesis of leukocytes into lung tissues and disruption of endothelial/epithelial barriers leading to protein-rich oedema. In vitro studies show that the microtubule network plays a role in the regulation of endothelial permeability as well as in neutrophil locomotion. It was hypothesised that the microtubule-stabilising agent, taxol, might attenuate inflammation and vascular leak associated with acute lung injury in vivo. The effect of intravenously delivered taxol was assessed using a model of murine lung injury induced by intratracheal lipopolysaccharide (LPS) administration. Parameters of lung injury and inflammation were assessed 18 h after treatment. Intravenously delivered taxol significantly reduced inflammatory histological changes in lung parenchyma and parameters of LPS-induced inflammation: infiltration of proteins and inflammatory cells into bronchoalveolar lavage fluid, lung myeloperoxidase activity, and extravasation of Evans blue-labelled albumin into lung tissue. Taxol alone (in the absence of LPS) had no appreciable effect on these parameters. In addition to lung proteins, intravenous taxol reduced accumulation of leukocytes in ascitic fluid in a model of LPS-induced peritonitis. Taken together, the present data demonstrate that microtubule stabilisation with taxol systemically attenuates lipopolysaccharide-induced inflammation and vascular leak.
• Moitra, J., Sammani, S., & Garcia, J. G. (2007). Re-evaluation of Evans Blue dye as a marker of albumin clearance in murine models of acute lung injury. Translational research : the journal of laboratory and clinical medicine, 150(4), 253-65.
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  Quantifying the amount of albumin conjugated to Evans Blue dye (EBA) fluxing across organ-specific vascular barriers is a popular technique to measure endothelial monolayer integrity in rodent and murine models of human diseases. We have re-evaluated this technique with a specific focus of assessing the commonly used turbidity correction factors. These factors, originally developed and required in a spectrophotometric assay to quantify Evans Blue (EB) in human infant or dog serum, produced negative numbers when applied to murine models of acute lung injury. We next sought to determine tissue-specific correction factors for murine tissues and experimentally derived such factors, which allow estimation of the amount of EB in formamide extracts of murine tissues as positive numbers. Utilization of a best fit correction factor in a lipopolysaccharide (LPS)-induced murine model of acute lung injury resulted in significantly increased sensitivity and repeatability of the EB dye tissue extravasation assay. This factor may be of significant utility in animal models of inflammatory injury.
• Nonas, S. A., Moreno-Vinasco, L., Vinasco, L. M., Ma, S. F., Jacobson, J. R., Desai, A. A., Dudek, S. M., Flores, C., Hassoun, P. M., Sam, L., Ye, S. Q., Moitra, J., Barnard, J., Grigoryev, D. N., Lussier, Y. A., & Garcia, J. G. (2007). Use of consomic rats for genomic insights into ventilator-associated lung injury. American journal of physiology. Lung cellular and molecular physiology, 293(2), L292-302.
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  Increasing evidence supports the contribution of genetic influences on susceptibility/severity in acute lung injury (ALI), a devastating syndrome requiring mechanical ventilation with subsequent risk for ventilator-associated lung injury (VALI). To identify VALI candidate genes, we determined that Brown Norway (BN) and Dahl salt-sensitive (SS) rat strains were differentially sensitive to VALI (tidal volume of 20 ml/kg, 85 breaths/min, 2 h) defined by bronchoalveolar lavage (BAL) protein and leukocytes. We next exploited differential sensitivities and phenotyped both the VALI-sensitive BN and the VALI-resistant SS rat strains by expression profiling coupled to a bioinformatic-intense candidate gene approach (Significance Analysis of Microarrays, i.e., SAM). We identified 106 differentially expressed VALI genes representing gene ontologies such as "transcription" and "chemotaxis/cell motility." We mapped the chromosomal location of the differentially expressed probe sets and selected consomic SS rats with single BN introgressions of chromosomes 2, 13, and 16 (based on the highest density of probe sets) while also choosing chromosome 20 (low probe sets density). VALI exposure of consomic rats with introgressions of BN chromosomes 13 and 16 resulted in significant increases in both BAL cells and protein (compared to parental SS strain), whereas introgression of BN chromosome 2 displayed a large increase only in BAL protein. Introgression of BN chromosome 20 had a minimal effect. These results suggest that genes residing on BN chromosomes 2, 13, and 16 confer increased sensitivity to high tidal volume ventilation. We speculate that the consomic-microarray-SAM approach is a time- and resource-efficient tool for the genetic dissection of complex diseases including VALI.
• Papaiahgari, S., Yerrapureddy, A., Hassoun, P. M., Garcia, J. G., Birukov, K. G., & Reddy, S. P. (2007). EGFR-activated signaling and actin remodeling regulate cyclic stretch-induced NRF2-ARE activation. American journal of respiratory cell and molecular biology, 36(3), 304-12.
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  Cyclic stretch (CS) associated with mechanical ventilation (MV) can cause excessive alveolar and endothelial distention, resulting in lung injury and inflammation. Antioxidant enzymes (AOEs) play a major role in suppressing these effects. The transcription factor Nrf2, via the antioxidant response element (ARE), alleviates pulmonary toxicant- and oxidant-induced oxidative stress by up-regulating the expression of several AOEs. Although gene expression profiling has revealed the induction of AOEs in the lungs of rodents exposed to MV, the mechanisms by which mechanical forces, such as CS, regulate the activation of Nrf2-dependent ARE-transcriptional responses are poorly understood. To mimic mechanical stress associated with MV, we have cultured pulmonary alveolar epithelial and endothelial cells on collagen I-coated BioFlex plates and subjected them to CS. CS exposure stimulated ARE-driven transcriptional responses and subsequent AOE expression. Ectopic expression of a dominant-negative Nrf2 suppressed the CS-stimulated ARE-driven responses. Our findings suggest that actin remodeling is necessary but not sufficient for high-level CS-induced ARE activation in both epithelial and endothelial cells. We also found that inhibition of EGFR activity by a pharmacologic agent ablated the CS-induced ARE transcriptional response in both cell types. Additional studies revealed that amphiregulin, an EGFR ligand, regulates this process. We further demonstrated that the PI3K-Akt pathway acts as the downstream effector of EGFR and regulates CS-induced ARE-activation in an oxidative stress-dependent manner. Collectively, these novel findings suggest that EGFR-activated signaling and actin remodeling act in concert to regulate the CS-induced Nrf2-ARE transcriptional response and subsequent AOE expression.
• Sammani, S., Moreno, L., Turner, J. R., Sammani, S., Moreno, L., Mirzapoiazova, T., Garcia, J. G., & Dudek, S. M. (2007). 52 A NOVEL MYOSIN LIGHT CHAIN KINASE INHIBITOR, PIK, PROTECTS FROM LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY.. Journal of Investigative Medicine, 55(2), S357.1-S357. doi:10.1136/jim-55-02-52
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  Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of acute respiratory failure that are associated with high morbidity and mortality. These disorders are characterized by a significant pulmonary inflammatory response resulting in injury to alveolar epithelial and endothelial barriers followed by pulmonary edema. Our prior studies demonstrated that myosin light chain kinase phosphorylation of myosin light chains (MLC) is associated with increases in both epithelial and endothelial barrier permeability. We previously have identified a novel oligopeptide, PIK, that inhibits MLC kinase in vitro, is membrane permeant, decreases intracellular MLC phosphorylation, and causes increased intestinal epithelial cell barrier function (Turner et al. Gastroenterology 2002). We hypothesized that PIK-mediated inhibition of MLC kinase activity would attenuate inflammation and vascular leak associated with acute lung injury. To test this hypothesis, we used a murine acute lung injury model with intratracheal administration of endotoxin/lipopolysaccharide (LPS, 2.5 mg/kg). Optimal PIK responses were observed at 125 μM PIK when tested over a range of PIK concentrations. PIK administered intravenously simultaneously with LPS resulted in significantly attenuated lung inflammation reflected by decreasing accumulation of bronchoalveolar lavage (BAL) proteins (25% reduction, p
• Sammani, S., Singleton, P. A., Sammani, S., Salgia, R., Moreno-vinasco, L., Moitra, J., Mirzapoiazova, T., Garcia, J. G., & Dudek, S. M. (2007). 89 LIPID RAFT REGULATION OF HEPATOCYTE GROWTH FACTOR/C-MET-MEDIATED VASCULAR INTEGRITY: ROLE OF CD44, TIAM1/RAC1, DYNAMIN 2, AND CORTACTIN.. Journal of Investigative Medicine, 55(2), S363.3-S363. doi:10.1136/jim-55-02-89
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  Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that protect vascular integrity have important therapeutic implications both in vivo and in vitro. We have previously shown that the binding of hepatocyte growth factor (HGF) to its cell surface receptor, c-Met, promotes Rac1-dependent increases in lung EC barrier function. Further examination of the regulatory mechanisms of HGF/c-Met-induced EC barrier enhancement revealed that HGF (25 ng/mL) promotes c-Met recruitment into specialized caveolin 1-enriched plasma membrane microdomains (lipid rafts). Abolishing lipid raft formation (MβCD) attenuated HGF-induced EC barrier enhancement. Within lipid rafts, HGF induced c-Met association with CD44 (a major glycoprotein receptor for hyaluronan). Silencing CD44 expression (siRNA) inhibited HGF-induced c-Met and Tiam1 (a Rac1 guanine nucleotide exchange factor) recruitment to lipid rafts, as well as the association of cortactin (an actin-binding regulatory protein) with dynamin 2 (a vesicular regulatory protein) within lipid raft structures. Silencing of either Tiam1 or dynamin 2 blocked HGF-induced Rac1 activation, cortactin recruitment to lipid rafts, and EC barrier enhancement. Silencing cortactin attenuated HGF-induced EC barrier regulation. Finally, HGF-mediated in vivo protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was inhibited in CD44 knockout mice. Taken together, these results suggest that lipid rafts are an essential regulator of HGF/c-Met-mediated barrier enhancement via a process involving CD44, Tiam1, Rac1, dynamin 2, and cortactin.
• Singleton, P. A., Moreno-Vinasco, L., Sammani, S., Wanderling, S. L., Moss, J., & Garcia, J. G. (2007). Attenuation of vascular permeability by methylnaltrexone: role of mOP-R and S1P3 transactivation. American journal of respiratory cell and molecular biology, 37(2), 222-31.
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  Endothelial cell (EC) barrier dysfunction (i.e., increased vascular permeability) is observed in inflammatory states, tumor angiogenesis, atherosclerosis, and both sepsis and acute lung injury. Therefore, agents that preserve vascular integrity have important clinical therapeutic implications. We examined the effects of methylnaltrexone (MNTX), a mu opioid receptor (mOP-R) antagonist, on human pulmonary EC barrier disruption produced by edemagenic agents including morphine, the endogenous mOP-R agonist DAMGO, thrombin, and LPS. Pretreatment of EC with MNTX (0.1 muM, 1 h) or the uncharged mOP-R antagonist naloxone attenuated morphine- and DAMGO-induced barrier disruption in vitro. However, MNTX, but not naloxone, pretreatment of EC inhibited thrombin- and LPS-induced barrier disruption, indicating potential mOP-R-independent effects of MNTX. In addition, intravenously delivered MNTX attenuated LPS-induced vascular hyperpermeability in the murine lung. We next examined the mechanistic basis for this MNTX barrier protection and observed that silencing of mOP-R attenuated the morphine- and DAMGO-induced EC barrier disruption, but not the permeability response to either thrombin or LPS. Because activation of the sphingosine 1-phosphate receptor, S1P(3), is key to a number of barrier-disruptive responses, we examined the role of this receptor in the permeability response to mOP-R ligation. Morphine, DAMGO, thrombin, and LPS induced RhoA/ROCK-mediated threonine phosphorylation of S1P(3), which was blocked by MNTX, suggesting S1P(3) transactivation. In addition, silencing of S1P(3) receptor expression (siRNA) abolished the permeability response to each edemagenic agonist. These results indicate that MNTX provides barrier protection against edemagenic agonists via inhibition of S1P(3) receptor activation and represents a potentially useful therapeutic agent for syndromes of increased vascular permeability.
• Singleton, P. A., Salgia, R., Moreno-Vinasco, L., Moitra, J., Sammani, S., Mirzapoiazova, T., & Garcia, J. G. (2007). CD44 regulates hepatocyte growth factor-mediated vascular integrity. Role of c-Met, Tiam1/Rac1, dynamin 2, and cortactin. The Journal of biological chemistry, 282(42), 30643-57.
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  The preservation of vascular endothelial cell (EC) barrier integrity is critical to normal vessel homeostasis, with barrier dysfunction being a feature of inflammation, tumor angiogenesis, atherosclerosis, and acute lung injury. Therefore, agents that preserve or restore vascular integrity have important therapeutic implications. In this study, we explored the regulation of hepatocyte growth factor (HGF)-mediated enhancement of EC barrier function via CD44 isoforms. We observed that HGF promoted c-Met association with CD44v10 and recruitment of c-Met into caveolin-enriched microdomains (CEM) containing CD44s (standard form). Treatment of EC with CD44v10-blocking antibodies inhibited HGF-mediated c-Met phosphorylation and c-Met recruitment to CEM. Silencing CD44 expression (small interfering RNA) attenuated HGF-induced recruitment of c-Met, Tiam1 (a Rac1 exchange factor), cortactin (an actin cytoskeletal regulator), and dynamin 2 (a vesicular regulator) to CEM as well as HGF-induced trans-EC electrical resistance. In addition, silencing Tiam1 or dynamin 2 reduced HGF-induced Rac1 activation, cortactin recruitment to CEM, and EC barrier regulation. We observed that both HGF- and high molecular weight hyaluronan (CD44 ligand)-mediated protection from lipopolysaccharide-induced pulmonary vascular hyperpermeability was significantly reduced in CD44 knock-out mice, thus validating these in vitro findings in an in vivo murine model of inflammatory lung injury. Taken together, these results suggest that CD44 is an important regulator of HGF/c-Met-mediated in vitro and in vivo barrier enhancement, a process with essential involvement of Tiam1, Rac1, dynamin 2, and cortactin.
• Usatyuk, P. V., Pendyala, S., Natarajan, V., Kamp, S. M., Kalari, S., Gorshkova, I., Garcia, J. G., & Dudek, S. M. (2007). Role of Phospholipase D in Hyperoxia-Mediated Myosin Light Chain Kinase Activation, Reactive Oxygen Species Formation and Cytoskeleton Organization. The FASEB Journal, 21(6).
• Usatyuk, P. V., Romer, L. H., He, D., Parinandi, N. L., Kleinberg, M. E., Zhan, S., Jacobson, J. R., Dudek, S. M., Pendyala, S., Garcia, J. G., & Natarajan, V. (2007). Regulation of hyperoxia-induced NADPH oxidase activation in human lung endothelial cells by the actin cytoskeleton and cortactin. The Journal of biological chemistry, 282(32), 23284-95.
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  Although the actin cytoskeleton has been implicated in the control of NADPH oxidase in phagocytosis, very little is known about the cytoskeletal regulation of endothelial NADPH oxidase assembly and activation. Here, we report a role for cortactin and the tyrosine phosphorylation of cortactin in hyperoxia-induced NADPH oxidase activation and ROS production in human pulmonary artery ECs (HPAECs). Exposure of HPAECs to hyperoxia for 3 h induced NADPH oxidase activation, as demonstrated by enhanced superoxide production. Hyperoxia also caused a thickening of the subcortical dense peripheral F-actin band and increased the localization of cortactin in the cortical regions and lamellipodia at cell-cell borders that protruded under neighboring cells. Pretreatment of HPAECs with the actin-stabilizing agent phallacidin attenuated hyperoxia-induced cortical actin thickening and ROS production, whereas cytochalasin D and latrunculin A enhanced basal and hyperoxia-induced ROS formation. In HPAECs, a 3-h hyperoxic exposure enhanced the tyrosine phosphorylation of cortactin and interaction between cortactin and p47(phox), a subcomponent of the EC NADPH oxidase, when compared with normoxic cells. Furthermore, transfection of HPAECs with cortactin small interfering RNA or myristoylated cortactin Src homology domain 3 blocking peptide attenuated ROS production and the hyperoxia-induced translocation of p47(phox) to the cell periphery. Similarly, down-regulation of Src with Src small interfering RNA attenuated the hyperoxia-mediated phosphorylation of cortactin tyrosines and blocked the association of cortactin with actin and p47(phox). In addition, the hyperoxia-induced generation of ROS was significantly lower in ECs expressing a tyrosine-deficient mutant of cortactin than in vector control or wild-type cells. These data demonstrate a novel function for cortactin and actin in hyperoxia-induced activation of NADPH oxidase and ROS generation in human lung endothelial cells.
• Villar, J., Theisen-toupal, J., Maresso, K., Ma, S., Garcia, J. G., Flores, C., & Devender, S. (2007). 40 A SHORT TANDEM REPEAT IN THE MACROPHAGE MIGRATION INHIBITORY FACTOR GENE PROMOTER IS ASSOCIATED WITH ACUTE LUNG INJURY SUSCEPTIBILITY IN A SPANISH POPULATION.. Journal of Investigative Medicine, 55(2), S355.1-S355. doi:10.1136/jim-55-02-40
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  Rationale Acute lung injury (ALI) is a complex inflammatory syndrome with presumed genetic predisposition. Macrophage migration inhibitory factor (MIF) potentially plays a major role in ALI pathogenesis and constitutes a candidate gene for ALI susceptibility. As the short tandem repeat (STR) 5-repeat allele at −794 of the MIF gene reported decreases MIF promoter activity, we examined this STR and the complete SNP variation of the MIF gene in association with susceptibility to ALI in a Spanish population. Methods DNA samples from 96 healthy controls and 80 severe septic patients were used to genotype the MIF STR and to sequence the entire gene and the ≈2 kb flanking regions. Polyphred 6.0 software was used to detect SNPs, and variation was examined for linkage disequilibrium (LD) using Haploview 3.32. Odds ratio (OR) and 95% confidence interval (CI) for association of individual polymorphisms were assessed with SNPstats. Haplotype associations were evaluated by a sliding-window approach. Results The 5-allele homozygous genotype at −794 STR was associated with an OR = 11.9 (95% CI 1.4-98.9, p = .01) after inclusion of covariates in the multiple logistic regression model. Sequencing revealed a total of 45 polymorphic loci (13 novel) that were also analyzed for association. None of them were significantly associated with ALI, either individually or in haplotypes. This result is congruent with the fact that neither SNPs nor haplotypes are in strong LD with the STR. Conclusions Our data suggest that the MIF gene is associated with susceptibility to ALI and support the causality of the STR locus. Genotyping of this STR is required for future association studies as the MIF SNPs examined were not in strong LD with the STR variation. Funded by Specialized Centers of Clinically Oriented Research P50 HL-073994.
• Zhao, J., Singleton, P. A., Natarajan, L., Garcia, J. G., & Dudek, S. M. (2007). 72 PROTEOME ANALYSIS OF SPHINGOSINE 1-PHOSPHATE-MEDIATED LIPID RAFTS IN HUMAN PULMONARY ENDOTHELIAL CELLS.. Journal of Investigative Medicine, 55(2), S360.3-S360. doi:10.1136/jim-55-02-72
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  Rationale Lipid rafts are detergent-insoluble plasma membrane microdomains implicated in membrane signaling and trafficking. We have demonstrated that the lysophospholipid sphingosine 1-phosphate (S1P) plays a critical regulatory role in the maintenance and enhancement of pulmonary vascular barrier function, which is dependent on lipid raft-mediated signaling events (Singleton et al. FASEB J 2005). In this study, we used two-dimensional electrophoresis analysis to determine the protein profiles in S1P-challenged lipid rafts of human pulmonary artery endothelial cells (HPAECs). Methods and Results HPAECs were treated with S1P (1 μM) for 5 minutes and solubilized with 1% Triton X-100 at 4°C. The Triton X-100 insoluble and light density fractions were collected after discontinuous OptiprepTM gradient centrifugation. The isolated lipid rafts pellets were resolubilized in 50 μL of isoelectric focusing (IEF) buffer (7 M urea/2 M thiourea, 4% CHAPS). IEF was carried out on a linear electrophoresis gel strip (pH 3-10, 7 cm), with the second dimension performed on 4 to 20% SDS-PAGE gel. Analytic gels were stained using Imperial blue (Pierce). We observed several novel protein bands in lipid rafts isolated from S1P versus control-treated cells (1 μM, 5 minutes). To identify these novel proteins, analytic gels were stained with Sypro Ruby and images were analyzed using PDQuest (Bio-Rad). Novel protein spots were sliced from the gel, and gel pieces were swelled in 20 ng/μL modified trypsin and incubated at 37°C overnight after protein reduction and alkylation. The digested peptides were extracted with 5% formic acid and 50% acetonitrile. Protein identification was performed on ABI 4700 Maldi TOF/TOF MS. Two-dimensional gel image analysis results indicated that about 50 protein spots (such as discoidin domain receptor tyrosine kinase, myosin light chain kinase, pp60src, phosphodiesterase 6C, etc.) have over a threefold change after S1P (1 μM, 5 minutes) treatment compared with control. Further, antiphosphotyrosine immunoblots of two-dimensional gels indicated that S1P increased tyrosine phosphorylation of over 20 proteins, including the actin regulatory protein cortactin. Conclusions Taken together, these results indicate that S1P induces recruitment of novel tyrosine kinases (ie, discoidin domain receptor tyrosine kinase, pp60src), with multiple tyrosine phosphorylation events in lipid rafts. These results suggest that recruitment of tyrosine kinases to lipid rafts may participate in S1P-induced signaling in HPAECs and could potentially play important roles in regulating S1P-induced pulmonary endothelial cells barrier function.
• Zhao, Y., Kalari, S. K., Usatyuk, P. V., Gorshkova, I., He, D., Watkins, T., Brindley, D. N., Sun, C., Bittman, R., Garcia, J. G., Berdyshev, E. V., & Natarajan, V. (2007). Intracellular generation of sphingosine 1-phosphate in human lung endothelial cells: role of lipid phosphate phosphatase-1 and sphingosine kinase 1. The Journal of biological chemistry, 282(19), 14165-77.
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  Sphingosine 1-phosphate (S1P) regulates diverse cellular functions through extracellular ligation to S1P receptors, and it also functions as an intracellular second messenger. Human pulmonary artery endothelial cells (HPAECs) effectively utilized exogenous S1P to generate intracellular S1P. We, therefore, examined the role of lipid phosphate phosphatase (LPP)-1 and sphingosine kinase1 (SphK1) in converting exogenous S1P to intracellular S1P. Exposure of (32)P-labeled HPAECs to S1P or sphingosine (Sph) increased the intracellular accumulation of [(32)P]S1P in a dose- and time-dependent manner. The S1P formed in the cells was not released into the medium. The exogenously added S1P did not stimulate the sphingomyelinase pathway; however, added [(3)H]S1P was hydrolyzed to [(3)H]Sph in HPAECs, and this was blocked by XY-14, an inhibitor of LPPs. HPAECs expressed LPP1-3, and overexpression of LPP-1 enhanced the hydrolysis of exogenous [(3)H]S1P to [(3)H]Sph and increased intracellular S1P production by 2-3-fold compared with vector control cells. Down-regulation of LPP-1 by siRNA decreased intracellular S1P production from extracellular S1P but had no effect on the phosphorylation of Sph to S1P. Knockdown of SphK1, but not SphK2, by siRNA attenuated the intracellular generation of S1P. Overexpression of wild type SphK1, but not SphK2 wild type, increased the accumulation of intracellular S1P after exposure to extracellular S1P. These studies provide the first direct evidence for a novel pathway of intracellular S1P generation. This involves the conversion of extracellular S1P to Sph by LPP-1, which facilitates Sph uptake, followed by the intracellular conversion of Sph to S1P by SphK1.
• Abdulnour, R. E., Peng, X., Finigan, J. H., Han, E. J., Hasan, E. J., Birukov, K. G., Reddy, S. P., Watkins, J. E., Kayyali, U. S., Garcia, J. G., Tuder, R. M., & Hassoun, P. M. (2006). Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways. American journal of physiology. Lung cellular and molecular physiology, 291(3), L345-53.
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  Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI.
• Barnes, K. C., Grant, A. V., Baltadzhieva, D., Zhang, S., Berg, T., Shao, L., Zambelli-Weiner, A., Anderson, W., Nelsen, A., Pillai, S., Yarnall, D. P., Dienger, K., Ingersoll, R. G., Scott, A. F., Fallin, M. D., Mathias, R. A., Beaty, T. H., Garcia, J. G., & Wills-Karp, M. (2006). Variants in the gene encoding C3 are associated with asthma and related phenotypes among African Caribbean families. Genes and immunity, 7(1), 27-35.
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  Proinflammatory and immunoregulatory products from C3 play a major role in phagocytosis, respiratory burst, and airways inflammation. C3 is critical in adaptive immunity; studies in mice deficient in C3 demonstrate that features of asthma are significantly attenuated in the absence of C3. To test the hypothesis that the C3 gene on chromosome 19p13.3-p13.2 contains variants associated with asthma and related phenotypes, we genotyped 25 single nucleotide polymorphism (SNP) markers distributed at intervals of approximately 1.9 kb within the C3 gene in 852 African Caribbean subjects from 125 nuclear and extended pedigrees. We used the multiallelic test in the family-based association test program to examine sliding windows comprised of 2-6 SNPs. A five-SNP window between markers rs10402876 and rs366510 provided strongest evidence for linkage in the presence of linkage disequilibrium for asthma, high log[total IgE], and high log[IL-13]/[log[IFN-gamma] in terms of global P-values (P = 0.00027, 0.00013, and 0.003, respectively). A three-SNP haplotype GGC for the first three of these markers showed best overall significance for the three phenotypes (P = 0.003, 0.007, 0.018, respectively) considering haplotype-specific tests. Taken together, these results implicate the C3 gene as a priority candidate controlling risk for asthma and allergic disease in this population of African descent.
• Barnes, K. C., Grant, A., Gao, P., Baltadjieva, D., Berg, T., Chi, P., Zhang, S., Zambelli-Weiner, A., Ehrlich, E., Zardkoohi, O., Brummet, M. E., Stockton, M., Watkins, T., Gao, L., Gittens, M., Wills-Karp, M., Cheadle, C., Beck, L. A., Beaty, T. H., , Becker, K. G., et al. (2006). Polymorphisms in the novel gene acyloxyacyl hydroxylase (AOAH) are associated with asthma and associated phenotypes. The Journal of allergy and clinical immunology, 118(1), 70-7.
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  The gene encoding acyloxyacyl hydroxylase (AOAH), an enzyme that hydrolyzes secondary fatty acyl chains of LPS, is localized on chromosome 7p14-p12, where evidence for linkage to total IgE (tIgE) concentrations and asthma has been previously reported.
• Birukova, A. A., Chatchavalvanich, S., Rios, A., Kawkitinarong, K., Garcia, J. G., & Birukov, K. G. (2006). Differential regulation of pulmonary endothelial monolayer integrity by varying degrees of cyclic stretch. The American journal of pathology, 168(5), 1749-61.
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  Ventilator-induced lung injury is a life-threatening complication of mechanical ventilation at high-tidal volumes. Besides activation of proinflammatory cytokine production, excessive lung distension directly affects blood-gas barrier and lung vascular permeability. To investigate whether restoration of pulmonary endothelial cell (EC) monolayer integrity after agonist challenge is dependent on the magnitude of applied cyclic stretch (CS) and how these effects are linked to differential activation of small GTPases Rac and Rho, pulmonary ECs were subjected to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of CS. Pathological CS enhanced thrombin-induced gap formation and delayed monolayer recovery, whereas physiological CS induced nearly complete EC recovery accompanied by peripheral redistribution of focal adhesions and cortactin after 50 minutes of thrombin. Consistent with differential effects on monolayer integrity, 18% CS enhanced thrombin-induced Rho activation, whereas 5% CS promoted Rac activation during the EC recovery phase. Rac inhibition dramatically attenuated restoration of monolayer integrity after thrombin challenge. Physiological CS preconditioning (5% CS, 24 hours) enhanced EC paracellular gap resolution after step-wise increase to 18% CS (30 minutes) and thrombin challenge. These results suggest a critical role for the CS amplitude and the balance between Rac and Rho in mechanochemical regulation of lung EC barrier.
• Bogatcheva, N. V., Birukova, A., Borbiev, T., Kolosova, I., Liu, F., Garcia, J. G., & Verin, A. D. (2006). Caldesmon is a cytoskeletal target for PKC in endothelium. Journal of cellular biochemistry, 99(6), 1593-605.
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  We have previously shown that treatment of bovine endothelial cell (EC) monolayers with phorbol myristate acetate (PMA) leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid-like structure, concomitant with the loss of endothelial barrier function. In the current work, we focused on caldesmon, a cytoskeletal protein, regulating actomyosin interaction. We hypothesized that protein kinase C (PKC) activation by PMA leads to the changes in caldesmon properties such as phosphorylation and cellular localization. We demonstrate here that PMA induces both myosin and caldesmon redistribution from cortical ring into the grid-like network. However, the initial step of PMA-induced actin and myosin redistribution is not followed by caldesmon redistribution. Co-immunoprecipitation experiments revealed that short-term PMA (5 min) treatment leads to the weakening of caldesmon ability to bind actin and, to the lesser extent, myosin. Prolonged incubation (15-60 min) with PMA, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid-like actin network formation. PMA stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK-kinase MEK fails to abolish grid-like structure formation, although reducing PMA-induced weakening of the cortical actin ring, whereas inhibition of PKC reverses PMA-induced cytoskeletal rearrangement. Our results suggest that PKC-dependent phosphorylation of caldesmon is involved in PMA-mediated complex cytoskeletal changes leading to the EC barrier compromise.
• Bogatcheva, N. V., Wang, P., Birukova, A. A., Verin, A. D., & Garcia, J. G. (2006). Mechanism of fluoride-induced MAP kinase activation in pulmonary artery endothelial cells. American journal of physiology. Lung cellular and molecular physiology, 290(6), L1139-45.
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  In this study, we demonstrate that challenge of endothelial cells (EC) with NaF, a recognized G protein activator and protein phosphatase inhibitor, leads to a significant Erk activation, with increased phosphorylation of the well-known Erk substrate caldesmon. Inhibition of the Erk MAPK, MEK, by U0126 produces a marked decrease in NaF-induced caldesmon phosphorylation. NaF transiently increases the activity of the MEK kinase known as Raf-1 (approximately 3- to 4-fold increase over basal level), followed by a sustained Raf-1 inhibition (approximately 3- to 4-fold decrease). Selective Raf-1 inhibitors (ZM-336372 and Raf-1 inhibitor 1) significantly attenuate NaF-induced Erk and caldesmon phosphorylation. Because we have previously shown that Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) participates in Erk activation in thrombin-challenged cells, we next explored if CaMKII is involved in NaF-induced EC responses. We found that in NaF-treated EC, CaMKII activity increases in a time-dependent manner with maximal activity at 10 min (approximately 4-fold increase over a basal level). Pretreatment with KN93, a specific CaMKII inhibitor, attenuates NaF-induced barrier dysfunction and Erk phosphorylation. The Rho inhibitor C3 exotoxin completely abolishes NaF-induced CaMKII activation. Collectively, these data suggest that sequential activation of Raf-1, MEK, and Erk is modulated by Rho-dependent CaMKII activation and represents important NaF-induced signaling response. Caldesmon phosphorylation occurring by an Erk-dependent mechanism in NaF-treated pulmonary EC may represent a link between NaF stimulation and contractile responses of endothelium.
• Chi, P. B., Duggal, P., Kao, W. H., Mathias, R. A., Grant, A. V., Stockton, M. L., Garcia, J. G., Ingersoll, R. G., Scott, A. F., Beaty, T. H., Barnes, K. C., & Fallin, M. D. (2006). Comparison of SNP tagging methods using empirical data: association study of 713 SNPs on chromosome 12q14.3-12q24.21 for asthma and total serum IgE in an African Caribbean population. Genetic epidemiology, 30(7), 609-19.
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  Few comparison studies have been performed on single nucleotide polymorphism (SNP) tagging methods to examine their consistency and effectiveness in terms of inferences about association with disease. We applied several SNP tagging methods to SNPs on chromosome 12q (n=713) and compared the utility of these methods to detect association for asthma and serum IgE levels among a sample of African Caribbean families from Barbados selected through asthmatic probands. We found that a high level of information regarding association is retained in Clayton's htSNP, Stram's TagSNP, and de Bakker's Tagger. We also found a high degree of consistency between TagSNP and Tagger. Using this set of 713 SNPs on chromosome 12q, our study provides insight towards analytic strategies for future studies of complex traits.
• Chiang, E. T., Persaud-Sawin, D. A., Kulkarni, S., Garcia, J. G., & Imani, F. (2006). Bluetongue virus and double-stranded RNA increase human vascular permeability: role of p38 MAPK. Journal of clinical immunology, 26(4), 406-16.
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  Endothelial cell (EC) involvement in viral hemorrhagic fevers has been clearly established. However, virally activated mechanisms leading to endothelial activation and dysfunction are not well understood. Several different potential mechanisms such as direct viral infection, alterations in procoagulant/anticoagulant balance, and increased cytokine production have been suggested. We utilized a model of EC barrier dysfunction and vascular endothelial leakage to explore the effect of bluetongue virus (BTV), a hemorrhagic fever virus of ruminants, on human lung endothelial cell barrier properties. Infection of human lung EC with BTV induced a significant and dose-dependent decrease in trans-endothelial electrical resistance (TER). Furthermore, decreases in TER occurred in conjunction with cytoskeletal rearrangement, suggesting a direct mechanism for viral infection-mediated endothelial barrier disruption. Interestingly, double-stranded RNA (dsRNA) mimicked the effects of BTV on endothelial barrier properties. Both BTV- and dsRNA-induced endothelial barrier dysfunction was blocked by treatment with a pharmacological inhibitor of p38 MAPK. The induction of vascular permeability by dsRNA treatment or BTV infection was concomitent with induction of inflammatory cytokines. Taken together, our data suggest that the presence of dsRNA during viral infections and subsequent activation of p38 MAPK is a potential molecular pathway for viral induction of hemorrhagic fevers. Collectively, our data suggest that inhibition of p38 MAPK may be a possible therapeutic approach to alter viral-induced acute hemorrhagic diseases.
• Flores, C., Ma, S. F., Maresso, K., Ahmed, O., & Garcia, J. G. (2006). Genomics of acute lung injury. Seminars in respiratory and critical care medicine, 27(4), 389-95.
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  Acute lung injury (ALI) is a complex syndrome involving the interplay of both environmental (such as the addition of mechanical ventilation) and genetic factors. Clinical models have identified risk factors for development and poor outcome but these strategies remain imprecise. To better understand the mechanisms of pathogenesis associated with mechanisms of ALI, candidate genes identified by global expression profiling or related literature searches are being explored for relevant polymorphisms (single base pair substitutions) that can affect both ALI susceptibility and outcome. This article summarizes several specific genetic association studies that have been conducted in ALI and reviews supporting data from in vitro and in vivo models of the disease and clinical observations. Although valuable information has been reported to date, intense analyses are needed in this developing discipline to assure significant clinical utility. The detailing of specific associated polymorphisms will continue to provide new insights in the understanding of disease pathogenesis, and promise to reveal novel molecular targets and personalized treatments to prevent the disease.
• Gao, L., Grant, A., Halder, I., Brower, R., Sevransky, J., Maloney, J. P., Moss, M., Shanholtz, C., Yates, C. R., Meduri, G. U., Shriver, M. D., Ingersoll, R., Scott, A. F., Beaty, T. H., Moitra, J., Ma, S. F., Ye, S. Q., Barnes, K. C., & Garcia, J. G. (2006). Novel polymorphisms in the myosin light chain kinase gene confer risk for acute lung injury. American journal of respiratory cell and molecular biology, 34(4), 487-95.
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  The genetic basis of acute lung injury (ALI) is poorly understood. The myosin light chain kinase (MYLK) gene encodes the nonmuscle myosin light chain kinase isoform, a multifunctional protein involved in the inflammatory response (apoptosis, vascular permeability, leukocyte diapedesis). To examine MYLK as a novel candidate gene in sepsis-associated ALI, we sequenced exons, exon-intron boundaries, and 2 kb of 5' UTR of the MYLK, which revealed 51 single-nucleotide polymorphisms (SNPs). Potential association of 28 MYLK SNPs with sepsis-associated ALI were evaluated in a case-control sample of 288 European American subjects (EAs) with sepsis alone, subjects with sepsis-associated ALI, or healthy control subjects, and a sample population of 158 African American subjects (AAs) with sepsis and ALI. Significant single locus associations in EAs were observed between four MYLK SNPs and the sepsis phenotype (P
• Garcia, J. G., & Moreno Vinasco, L. (2006). Genomic insights into acute inflammatory lung injury. American journal of physiology. Lung cellular and molecular physiology, 291(6), L1113-7.
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  Acute lung injury (ALI) is a devastating syndrome (usually associated with sepsis) that represents a major healthcare burden in the United States. We have focused our studies on unraveling the genetic underpinnings of this syndrome utilizing a candidate gene approach to identify novel genes for ALI susceptibility. Two novel genes identified by this approach include pre-B cell colony-enhancing factor (PBEF) and the gene for myosin light chain kinase (MLCK). PBEF protein levels were elevated in human bronchoalveolar lavage and serum samples from patients with ALI, and DNA sequencing identified two single nucleotide polymorphisms in the PBEF promoter (T-1001G, C-1543T) that were overrepresented in patients with sepsis-induced ALI. More recently, we found MLCK single polymorphisms and haplotypes to be associated with human ALI with unique variants observed in African-Americans with ALI. Thus genomic and genetic approaches represent powerful strategies in the identification of novel candidate genes and potential targets for ALI therapies.
• Garcia, J. G., Chatchavalvanich, S., Birukova, A. A., & Birukov, K. G. (2006). 17 SIGNAL TRANSDUCTION AND GENE EXPRESSION IN MAGNITUDE-DEPENDENT MECHANOCHEMICAL REGULATION OF LUNG ENDOTHELIAL BARRIER.. Journal of Investigative Medicine, 54(2), S346.4-S346. doi:10.2310/6650.2005.x0015.16
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  Ventilator-induced lung injury is associated with activation of inflammatory cytokines and excessive lung distention, which directly affects blood-gas barrier and lung vascular permeability. Small GTPases Rho and Rac play an essential role in agonist-mediated endothelial cell cytoskeletal remodeling and permeability, but their role in lung endothelial cell barrier regulation under mechanical stress is less understood. We studied magnitude-dependent effects of cyclic stretch on lung endothelial cell barrier restoration after thrombin challenge and linked these effects with differential activation of Rho and Rac. Consistent with differential effects of 5% cyclic stretch and 18% cyclic stretch on thrombin-induced endothelial cell monolayer disruption, a peak of thrombin-induced Rho activation at 5 minutes was enhanced by endothelial cell preconditioning at 18% cyclic stretch and was attenuated by 5% cyclic stretch. Endothelial cell preconditioning at physiological cyclic stretch (5%) induced more pronounced activation of Rac at 30 minutes compared to endothelial cell exposed to 18% cyclic stretch and caused nearly complete endothelial cell monolayer recovery observed after 50 minutes of thrombin stimulation, which was associated with peripheral accumulation of Rac effector cortactin. Promotion of endothelial cell barrier restoration by physiological cyclic stretch as well as cortactin peripheral accumulation was abolished by siRNA-based depletion of Rac expression or by Rac pharmacological inhibitor NSC-23766. Lung endothelial cell subjected to long-term cyclic stretch (18% elongation, 24 hours) increased expression of procontractile cellular proteins Rho, myosin light chain kinase, ZIP-kinase, and released extracellular bioactive molecules with barrier-disruptive properties. These results show involvement of Rac-mediated mechanisms in the barrier-promoting effects of physiologic cyclic stretch preconditioning and suggest direct link between amplitude-dependent activation of Rho- and Rac-mediated pathways and dynamic regulation of lung endothelial barrier by mechanochemical stimuli.
• Jacobson, J. R., Dudek, S. M., Singleton, P. A., Kolosova, I. A., Verin, A. D., & Garcia, J. G. (2006). Endothelial cell barrier enhancement by ATP is mediated by the small GTPase Rac and cortactin. American journal of physiology. Lung cellular and molecular physiology, 291(2), L289-95.
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  ATP is a physiologically relevant agonist released by various sources, including activated platelets, with complex effects mediated via activation of P(2) purinergic receptors. ATP-induced endothelial cell (EC) production of prostacyclin and nitric oxide is recognized, and EC barrier enhancement evoked by ATP has been described. ATP effects on EC barrier function and vascular permeability, however, remain poorly characterized. Although the mechanisms involved are unclear, we previously identified activation of the small GTPase Rac and translocation of cortactin, an actin-binding protein, as key to EC barrier augmentation induced by simvastatin and sphingosine 1-phosphate and therefore examined the role of these molecules in ATP-induced EC barrier enhancement. ATP induced rapid, dose-dependent barrier enhancement in human pulmonary artery EC as measured by transendothelial electrical resistance, with a peak effect appreciable at 25 min (39% increase, 10 microM) and persisting at 2 h. These effects were associated with rearrangement of the EC actin cytoskeleton, early myosin light chain phosphorylation, and spatially defined (cell periphery) translocation of both Rac and cortactin. ATP (10 microM)-treated EC demonstrated a significant increase in Rac activation relative to controls, with a maximal effect (approximately 4-fold increase) at 10 min. Finally, ATP-induced barrier enhancement was markedly attenuated by reductions of either Rac or cortactin (small interfering RNA) relative to controls. Our results suggest for the first time that ATP-mediated barrier protection is associated with cytoskeletal activation and is dependent on both Rac activation and cortactin.
• Jacobson, J. R., Garcia, J. G., & Chen, W. (2006). 63 ROLE OF RAC GUANOSINE TRIPHOSPHATASE IN SIMVASTATIN-MEDIATED ENDOTHELIAL CELL SIGNALING.. Journal of Investigative Medicine, 54(2), S354.1-S354. doi:10.2310/6650.2005.x0015.62
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  The statins are a class of HMG CoA-reductase inhibitors used clinically for their ability to reduce serum cholesterol levels via inhibition of the prenylation pathway. However, not all of their clinical benefits, including both vascular barrier protection and reduced superoxide generation, can be attributed to their lipid-lowering properties. One potential mechanism of these effects is via inhibition of geranylgeranylation, a covalent modification that allows translocation to the cell membrane and activation of the small GTPases Rho and Rac, mediators of cytoskeletal rearrangement. While statins inhibit Rho, we previously reported the paradoxical activation of Rac (Rac-GTP) in endothelial cell (EC) after prolonged treatment with simvastatin (5 μM, 16 hours). In this study, upon membrane fractionation and subsequent Western blotting, we report a 37% reduction in Rac translocation to the EC membrane by simvastatin (5 μM, 16 hours) relative to untreated control cells, consistent with the inhibition of geranylgeranylation and evidence of a separate mechanism of simvastatin-mediated Rac activation. In addition, as Rac is required for activation of the NADPH oxidase complex and subsequent superoxide anion generation, we examined the effect of simvastatin on the peripheral translocation of P47phox, an NADPH oxidase component. Simvastatin affected a 47% reduction in P47phox at the EC membrane relative to control cells. Finally, we measured transendothelial electrical resistance and quantified the effects of simvastatin pretreatment on sphingosine 1-phosphate-induced barrier enhancement, an event dependent on Rac. S1P-induced (1 μM) EC barrier function was increased by simvastatin (5 μM, 16 hours) 70% at peak effect relative to S1P-treated control cells. These data are consistent with a functional role for Rac in simvastatin-mediated EC barrier protection and further define the mechanism by which simvastatin is able to directly modulate EC.
• Moreno, L., Jacobson, J. R., Garcia, J. G., & Bonde, P. (2006). 71 SIMVASTATIN ATTENUATES ACUTE LUNG INJURY INDUCED BY ISCHEMIA-REPERFUSION IN RODENTS.. Journal of Investigative Medicine, 54(2), S355.3-S355. doi:10.2310/6650.2005.x0015.70
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  Ischemia-reperfusion (IR) lung injury, a common cause of lung transplant failure, is characterized by hypoxemia, alveolar damage, inflammation, and edema. As novel lung preservation techniques could have a significant clinical impact in this setting, we employed an animal model of IR injury to investigate the potential therapeutic role of simvastatin, an agent we have previously characterized as a potent vascular barrier protectant (Jacobson JR et al, 2004 and 2005). Ischemia was induced in anesthetized Sprague-Dawley rats by ligation of the left pulmonary artery for a period of 1 hour followed by 4 hours of reperfusion. Indices of inflammation and vascular leak, including bronchoalveolar lavage (BAL) cell counts and protein content and lung tissue myeloperoxidase activity, were then assessed. Similar to the protective effects we previously observed with sphingosine 1-phosphate in this model (Moreno L, 2004), BAL from animals pretreated with simvastatin (20 mg/kg, intraperitoneal injection, 16 hours prior to ischemia) demonstrated a reduced number of total cells (48.3% decrease), neutrophils (33% decrease), and albumin concentration (20.7% decrease) compared to controls. A single dose of simvastatin also resulted in reduced (49% decrease) lung tissue MPO. These data indicate that simvastastin significantly attenuates the protein leakage and inflammation associated with IR lung injury in our animal model. Ultimately, our results could have profound clinical implications as simvastatin treatment may represent a potential agent to reduce the incidence and severity of IR injury associated with lung transplantation. Funded by Parker B Francis Foundation.
• Moreno, L., Sammani, S., Verin, A. D., Sammani, S., Moreno, L., Mirzapoiazova, T., Kolosova, I. A., & Garcia, J. G. (2006). 27 PROTECTIVE EFFECT OF PURINERGIC AGONIST ATPgS AGAINST ACUTE LUNG INJURY.. Journal of Investigative Medicine, 54(2), S348.1-S348. doi:10.2310/6650.2005.x0015.26
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  Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of acute respiratory failure that are associated with high morbidity and mortality. These disorders are characterized by a significant pulmonary inflammatory response resulting in injury to alveolar epithelial and endothelial barriers of the lung and protein-rich pulmonary edema. The pharmacological treatment of ALI/ARDS is nonspecific and relies on good supportive care and control of initiating cause. ALI/ARDS pathogenesis is still only partly understood; however, pulmonary endothelium plays a major role by changing its barrier permeability, thus promoting pulmonary edema formation. Pulmonary endothelial functional and structural alterations are key components of increased vascular leak. Consequently, endothelium-related therapies may have beneficial effects in ALI/ARDS. Recently, much attention has been given to the therapeutic potential of purinergic agonists and antagonists for the treatment of cardiovascular and pulmonary diseases. Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface P2Y receptors. We have shown that ATP promotes endothelial cell (EC) barrier enhancement via a complex cell signaling. We hypothesize that activation of endothelial purinoreceptors would exert anti-inflammatory barrier-protective effect. To test this hypothesis we used two model systems: cultured pulmonary EC and murine model of ALI induced by intratracheal administration of endotoxin/lipopolysaccharide (LPS). In cell culture model, ATP inhibited intracellular gap formation and junctional permeability induced by inflammatory mediator thrombin. In murine model of ALI, nonhydrolyzed ATP analogue ATPgS (50 μM final blood concentration) administered intravenously attenuated inflammatory response by decreasing accumulation of cells (48%, p
• Moreno, L., Sammani, S., Verin, A. D., Sammani, S., Moreno, L., Mirzapoiazova, T., Kolosova, I. A., & Garcia, J. G. (2006). 3 SUPPRESSION OF LIPOPOLYSACCHARIDE-INDUCED ACUTE LUNG INJURY BY TAXOL.. Journal of Investigative Medicine, 54(2), S344.3-S344. doi:10.2310/6650.2005.x0015.2
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  Our prior study demonstrated that similar to actin cytoskeleton, mictotubule (MT) network is a key participant in the regulation of endothelial (EC) permeability. Disassembly of MT by pharmacological inhibitors nocodazole and vinblastine results in rearrangement of actin cytoskeleton, stress fiber formation, EC contraction, and increased permeability. MT-stabilizing agent, Taxol, prevented EC barrier dysfunction induced by MT inhibitors and significantly attenuated permeability induced by proinflammatory agonists such as thrombin in cell culture model. We hypothesized that Taxol may prevent inflammation and vascular leak associated with lung injury. The effect of Taxol was assessed employing a model of murine lung injury induced by intratracheal LPS administration (2.5 mg/kg). Our data demonstrate that intravenous Taxol (8.5 mg/kg) injected simultaneously with LPS administration dramatically reduced inflammatory histological changes in lung parenchyma, decreased infiltration of proteins (40%, p HL 58064; HL 60637.
• Moreno, L., Sammani, S., Verin, A. D., Sammani, S., Moreno, L., Moitra, J., Mirzapoiazova, T., Kolosova, I. A., & Garcia, J. G. (2006). PROTECTIVE EFFECT OF TAXOL ON LIPOPOLYSACCHARIDE (LPS)-INDUCED ACUTE LUNG INJURY. The FASEB Journal, 20(5).
• Moreno, L., Sammani, S., Wanderling, S., Sammani, S., Ratain, M. J., Moreno, L., Maitland, M. L., Lang, R. M., Lammertin, G., Husain, A. N., Gomberg-maitland, M., Garcia, J. G., & Collins, K. A. (2006). Abstract 1325: Selective Kinase Inhibition Attenuates Rodent Pulmonary Arterial Hypertension (PAH). Circulation, 114.
• Nonas, S. A., Miller, I., Garcia, J. G., Chatchavalvanich, S., Birukova, A. A., & Birukov, K. G. (2006). Protective effects of oxidized phospholipids on ventilator-induced lung injury*. Journal of Biomechanics, 39, S601. doi:10.1016/s0021-9290(06)85494-1
• Nonas, S. A., Miller, I., Garcia, J. G., Chatchavalvanich, S., Burdette, D. O., Birukova, A. A., & Birukov, K. G. (2006). 15 PROTECTIVE EFFECTS OF OXIDIZED PHOSPHOLIPIDS ON VENTILATOR-INDUCED LUNG INJURY.. Journal of Investigative Medicine, 54(2), S346.2-S346. doi:10.2310/6650.2005.x0015.14
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  Purpose of Study Acute lung injury (ALI) is a devastating syndrome characterized by pulmonary inflammation and vascular barrier dysfunction with protein-rich edema. Mechanical ventilation at high tidal volumes can worsen existing ALI and even cause ventilator-induced lung injury (VILI) de novo. Previous studies have demonstrated that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) enhances basal endothelial cell (EC) barrier properties and prevents acute lung inflammation and EC dysfunction in response to bacterial lipopolysaccharide (LPS) via direct Rac-Cdc42-mediated effects on EC cytoskeleton and via competitive inhibition of LPS binding to toll-like receptor 4 (TLR4). Methods Adult male Brown Norway rats (250-350 g) were ventilated at low tidal volume (LTV, 7 mL/kg) or high tidal volume (HTV, 20 mL/kg) for 2 hours, 85 breaths/min. A subset of animals received intravenous OxPAPC (1.5 mg/kg) at the start of mechanical ventilation. Bronchoalveolar lavage (BAL) cell count and protein concentration were measured as markers of inflammation and vascular permeability. IL-6, IL-1beta, and hyaluronan levels in BAL were quantitatively assessed by enzyme linked immunoabsorbance assays (ELISA). Results HTV caused a 70% increase in BAL total cell count (p HL75349, HL76259, HL58064.
• Nonas, S., Miller, I., Kawkitinarong, K., Chatchavalvanich, S., Gorshkova, I., Bochkov, V. N., Leitinger, N., Natarajan, V., Garcia, J. G., & Birukov, K. G. (2006). Oxidized phospholipids reduce vascular leak and inflammation in rat model of acute lung injury. American journal of respiratory and critical care medicine, 173(10), 1130-8.
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  Acute inflammation and vascular leak are cardinal features of acute lung injury and the acute respiratory distress syndrome. Nonspecific tissue inflammation and injury in response to infectious and noninfectious insults lead to oxidative stress and the generation of lipid oxidation products, which may inhibit the acute inflammatory response to bacterial components.
• Nonas, S., Natarajan, V., Miller, I. L., Leitinger, N., Kawkitinarong, K., Gorshkova, I., Garcia, J. G., Chatchavalvanich, S., Bochkov, V. N., & Birukov, K. G. (2006). 18 OXIDIZED PHOSPHOLIPIDS REDUCE VASCULAR LEAK AND INFLAMMATION IN A RAT MODEL OF ACUTE LUNG INJURY.. Journal of Investigative Medicine, 54(2), S346.5-S346. doi:10.2310/6650.2005.x0015.17
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  Acute inflammation and vascular leak are cardinal features of acute lung injury and the acute respiratory distress syndrome. Nonspecific tissue inflammation and injury in response to infectious and noninfectious insults lead to oxidative stress and the generation of lipid oxidation products, which, in turn, may inhibit the acute inflammatory response to bacterial components. In this study, we used animal and endothelial cell culture models of lipopolysaccharide-induced lung injury to test the hypothesis that oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphorylcholine may attenuate the acute lung inflammatory response to bacterial wall lipopolysaccharide (LPS) and enhance lung vascular barrier recovery. Rats received aerosolized LPS (5 mg/kg) or sterile water with concurrent intravenous oxidized 1-palmitoyl-2-arachidonoyl- sn -glycero-3-phosphorylcholine (0.5-6 mg/kg) or saline. At 18 hours, bronchoalveolar lavage was performed and the lungs were removed for histological analysis. Measurements of transendothelial electrical resistance and histochemical analysis of endothelial monolayer disruption were used as an in vitro model of LPS-induced lung barrier dysfunction. Intratracheal instillation of LPS induced lung injury with profound increases in bronchoalveolar lavage and tissue neutrophils, protein content, and production of inflammatory cytokines IL-6 and IL-1b. Intravenous injection of oxidized phospholipids markedly attenuated LPS-induced tissue inflammation, barrier disruption, and IL-6 and IL-1b production over a range of doses. In vitro, oxidized phospholipids attenuated LPS-induced endothelial permeability and reversed LPS-induced cytoskeletal remodeling and disruption of monolayer integrity. These studies demonstrate in vivo and in vitro protective effects of oxidized phospholipids on LPS-induced lung dysfunction.
• Romer, L. H., Birukov, K. G., & Garcia, J. G. (2006). Focal adhesions: paradigm for a signaling nexus. Circulation research, 98(5), 606-16.
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  The vascular wall contains intimal endothelium and medial smooth muscle that act as contiguous tissues with tight spatial and functional coordination in response to tonic and episodic input from the bloodstream and the surrounding parenchyma. Focal adhesions are molecular bridges between the intracellular and extracellular spaces that integrate a variety of environmental stimuli and mediate 2-way crosstalk between the extracellular matrix and the cytoskeleton. Focal adhesion components are targets for biochemical and mechanical stimuli that evoke crucial developmental and injury response mechanisms including cell growth, movement, and differentiation, and tailoring of the extracellular microenvironment. Focal adhesions provide the vascular wall constituents with flexible and specific tools for exchanging cues in a complex system. The molecular mechanisms that underlie these vital communications are detailed in this review with the goal of defining future targets for vascular tissue engineering and for the therapeutic modulation of disordered vascular growth, inflammation, thrombosis, and angiogenesis.
• Sammani, S., Sammani, S., Moitra, J., & Garcia, J. G. (2006). 37 A REEVALUATION OF EVANS BLUE DYE AS A MARKER OF ALBUMIN CLEARANCE IN MOUSE MODELS OF ACUTE LUNG INJURY.. Journal of Investigative Medicine, 54(2), S349.5-S349. doi:10.2310/6650.2005.x0015.36
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  Background Quantifying the amount of albumin conjugated to Evans Blue dye (Alb-EB) fluxing across organ barriers is a popular technique to measure intactness of the physical barrier in rodent models of a variety of diseases. We have reevaluated this technique in terms of the correction factors required in a spectrophotometric assay. Methods and Results Eight- to 10-week-old C57BL6/J mice received either pH-neutral water (controls) or LPS (treatment) by intratracheal instillation, and acute lung injury was allowed to develop for 24 hours. Both control and treatment mice were further injected with Alb-EB via the jugular vein, at doses of either 20 or 30 mg/kg body weight, at 30, 60, 120, or 180 minutes before termination of the LPS treatment (24 hours total). At the end of exposure, formamide extracts of lungs were prepared, and the centrifuged supernatants were measured at 620 and 740 nm in a spectrophotometer. Lungs from control mice that were not injected with Alb-EB were similarly extracted and measured. The linear regression equation between absorbances at 740 nm (X) and 620 nm (Y) in control lung extracts of animals that did not get Alb-EB was considered to be the tissue-specific correction factor. The observed absorbances of the control and treatment samples at 620 and 740 nm were then normalized using this factor. This tissue-specific correction resulted in control samples read as positive integers, as opposed to negative integers when using a serum correction factor commonly used in the literature. We also determined that adjusting the duration of the conjugated dye in circulation is critical for maximizing the signal-to-noise ratio. Conclusion The Evans Blue dye extravasation method to quantify barrier dysfunction can be improved in terms of repeatability and sensitivity by using tissue-specific correction factors and maximized signal-to-noise ratios, respectively. Funding: NIH/NHLBI SCORR #P50 HL 73994.
• Sammani, S., Ye, S. Q., Sammani, S., Rabb, H., Ma, S. F., Grigoryev, D. N., Garcia, J. G., & Barnes, K. C. (2006). 23 IDENTIFICATION OF ORGAN-SHARED AND STRAIN-INDEPENDENT CANDIDATE GENES DURING COMBINED VENTILATOR-ASSOCIATED LUNG AND KIDNEY INJURY.. Journal of Investigative Medicine, 54(2), S347.3-S347. doi:10.2310/6650.2005.x0015.22
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  Combined ventilator-induced lung injury (VILI) and acute renal failure (ARF) have 80% mortality in the ICU. We hypothesized that analysis of global genetic changes in lung and kidney tissues during developing of ALI will identify similar genomic responses to injury that could underlie injurious effects in both organs. We performed gene expression profiling of lung and kidney tissues in three different mouse strains in a model of VILI, speculating that key transcriptional changes would be tissue and strain independent. Three inbred mouse strains C57BL/6J (B), C3H/HeJ (H), and DBA/2J (D) were selected and young 4-week-old mice were mechanically ventilated (Vt = 17 mL/kg, 110 breaths/min) for 2 hours (n = 6-8) and compared to spontaneous ventilated (n = 6) mice. Analysis of bronchoalveolar lavage proteins demonstrated a significant (p 1.96 were considered significantly affected by VILI. This cross-tissue and cross-strain analysis identified 58 genes that behaved similarly in lung and kidney tissues during VILI. Gene ontology analysis of these genes revealed significant up-regulation of genes involved in response to stress (z = 2.51), including Mig-6 (5.97FCLung (L), 3.02FCKidney (K)) and Gadd-45{g (3.68FCL, 6.06FCK); and transcription activity (z = 4.04), including Ceb/pd} (3.97FCL, 3.74FCK) and Ceb/pb (2.87FCL, 4.58FCK). These data demonstrate that VILI activates specific transcription factors and stress responsive genes in both lung and kidney in all tested strains. Detailed studies of these pathways can reveal organ-shared VILI targets for the development of new therapeutic strategies for this devastating disorder.
• Sanchez, T., Singleton, P. A., Sanchez, T., Hla, T., Garcia, J. G., Dudek, S. M., Chiang, E. T., Camp, S. M., & Bittman, R. (2006). 55 NOVEL ANALOGS OF FTY720 PROMOTE PULMONARY VASCULAR BARRIER FUNCTION.. Journal of Investigative Medicine, 54(2), S352.4-S352. doi:10.2310/6650.2005.x0015.54
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  Rationale Modulation of pulmonary vascular barrier function is an important clinical goal given the devastating effects of vascular leak in ARDS. We and others have demonstrated that FTY720, an analog of the potent barrier-enhancing phospholipid sphingosine 1-phosphate (S1P), is also barrier protective through incompletely characterized mechanisms. We utilized various FTY analogs to better define FTY effects on endothelial cell (EC) barrier function. Methods/Results FTY (1 μM) significantly increased cultured human pulmonary artery EC barrier function in a sustained manner as measured by transendothelial electrical resistance (TER) but with a delayed onset and slower rate of TER rise relative to S1P (1 μM). Previous siRNA experiments have suggested that FTY does not exert its barrier-enhancing effects through the same S1P1R receptor required for S1P. We now report that FTY also increases TER in EC derived from embryonic S1P1R -/- mice, providing further support for a novel FTY barrier-enhancing pathway. In addition, S1P3R is not the responsible receptor as S1P3R siRNA did not alter FTY-induced TER increases. Increasing concentrations of phosphorylated FTY (0.1-50 μM) produced a faster TER elevation rate of onset than FTY itself but never reached the rate of rise observed with S1P. (R)-phosphonate and enephosphonate analogs of FTY (1-50 μM) produced rapid TER elevations similar to S1P, while the (S)-analogs were less potent or even barrier-disruptive. Both (R)- and (S)-regioisomers of FTY were barrier disruptive. Conclusion These results support a novel mechanism of FTY action and provide further insights into the vascular barrier enhancing effects of FTY720. HL58064, HL70013-01, FTY720 supplied by Novartis.
• Simon, B. A., Easley, R. B., Grigoryev, D. N., Ma, S. F., Ye, S. Q., Lavoie, T., Tuder, R. M., & Garcia, J. G. (2006). Microarray analysis of regional cellular responses to local mechanical stress in acute lung injury. American journal of physiology. Lung cellular and molecular physiology, 291(5), L851-61.
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  Human acute lung injury is characterized by heterogeneous tissue involvement, leading to the potential for extremes of mechanical stress and tissue injury when mechanical ventilation, required to support critically ill patients, is employed. Our goal was to establish whether regional cellular responses to these disparate local mechanical conditions could be determined as a novel approach toward understanding the mechanism of development of ventilator-associated lung injury. We utilized cross-species genomic microarrays in a unilateral model of ventilator-associated lung injury in anesthetized dogs to assess regional cellular responses to local mechanical conditions that potentially contribute pathogenic mechanisms of injury. Highly significant regional differences in gene expression were observed between lung apex/base regions as well as between gravitationally dependent/nondependent regions of the base, with 367 and 1,544 genes differentially regulated between these regions, respectively. Major functional groupings of differentially regulated genes included inflammation and immune responses, cell proliferation, adhesion, signaling, and apoptosis. Expression of genes encoding both acute lung injury-associated inflammatory cytokines and protective acute response genes were markedly different in the nondependent compared with the dependent regions of the lung base. We conclude that there are significant differences in the local responses to stress within the lung, and consequently, insights into the cellular responses that contribute to ventilator-associated lung injury development must be sought in the context of the mechanical heterogeneity that characterizes this syndrome.
• Singleton, P. A., Dudek, S. M., Ma, S. F., & Garcia, J. G. (2006). Transactivation of sphingosine 1-phosphate receptors is essential for vascular barrier regulation. Novel role for hyaluronan and CD44 receptor family. The Journal of biological chemistry, 281(45), 34381-93.
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  The role for hyaluronan (HA) and CD44 in vascular barrier regulation is unknown. We examined high and low molecular weight HA (HMW-HA, approximately 1,000 kDa; LMW-HA, approximately 2.5 kDa) effects on human transendothelial monolayer electrical resistance (TER). HMW-HA increased TER, whereas LMW-HA induced biphasic TER changes ultimately resulting in EC barrier disruption. HMW-HA induced the association of the CD44s isoform with, and AKT-mediated phosphorylation of, the barrier-promoting sphingosine 1-phosphate receptor (S1P1) within caveolin-enriched lipid raft microdomains, whereas LMW-HA induced brief CD44s association with S1P1 followed by sustained association of the CD44v10 isoform with, and Src and ROCK 1/2-mediated phosphorylation of, the barrier-disrupting S1P3 receptor. HA-induced EC cytoskeletal reorganization and TER alterations were abolished by either disruption of lipid raft formation, CD44 blocking antibody or siRNA-mediated reductions in expression of CD44 isoforms. Silencing S1P1, AKT1, or Rac1 blocked the barrier enhancing effects of HA whereas silencing S1P3, Src, ROCK1/2, or RhoA blocked the barrier disruption induced by LMW-HA. In summary, HA regulates EC barrier function through novel differential CD44 isoform interaction with S1P receptors, S1P receptor transactivation, and RhoA/Rac1 signaling to the EC cytoskeleton.
• Singleton, P. A., Garcia, J. G., & Dore, S. (2006). 77 NOVEL MECHANISM OF PROSTAGLANDIN E2-MEDIATED ENDOTHELIAL CELL BARRIER ENHANCEMENT: ROLE OF EPCR, S1P1 RECEPTOR, EPAC, RAP1, AF-6, AND PROFILIN.. Journal of Investigative Medicine, 54(2), S356.3-S356. doi:10.2310/6650.2005.x0015.76
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  Prostaglandin E2 (PGE2) is an important tissue-specific autocrine inflammatory mediator. In lung, unlike other tissues, PGE2 exerts an anti-inflammatory, tissue-reparative, and endothelial cell (EC) barrier protective response, which can have therapeutic potential in various pulmonary diseases. Therefore, we examined the mechanism of PGE2-induced pulmonary EC barrier regulation. Addition of PGE2 (100 nM, 5-30 minutes) to EC induced recruitment of EP2 and EP3 receptor (E prostanoid receptor subtypes), EPCR (endothelial cell protein C receptor), S1P1 receptor, Epac (cAMP-activated Rap1 exchange factor), Rap1 (small GTP binding protein), AF-6 (scaffolding protein that binds activated Rap1 and profilin), and profilin (actin polymerizing cytoskeletal protein) to caveolin-enriched microdomains (lipid rafts). Silencing EP2 (but not EP3), EPCR (siRNA) or inhibiting adenylyl cyclase activity (29,59-dideoxy-39-ATP) attenuated PGE2-induced recruitment of Epac and Rap1 to EC lipid rafts. Silencing S1P1 receptor inhibited PGE2-induced AF-6 and profilin recruitment to EC lipid rafts. Finally, inhibiting lipid raft formation (methyl-b-cyclodextrin), inhibiting adenylyl cyclase activity, or silencing EP2, EPCR, S1P1 receptor, Epac, Rap1, AF-6, or profilin attenuated PGE2-induced increased EC barrier function. Taken together, these data suggest that PGE2 ligation of EP2 receptor induces EPCR and S1P1 receptor signaling required for Epac, Rap1, AF-6, and profilin recruitment to lipid rafts during pulmonary EC barrier enhancement.
• Tar, K., Csortos, C., Czikora, I., Olah, G., Ma, S. F., Wadgaonkar, R., Gergely, P., Garcia, J. G., & Verin, A. D. (2006). Role of protein phosphatase 2A in the regulation of endothelial cell cytoskeleton structure. Journal of cellular biochemistry, 98(4), 931-53.
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  Our recently published data suggested the involvement of protein phosphatase 2A (PP2A) in endothelial cell (EC) barrier regulation (Tar et al. [2004] J Cell Biochem 92:534-546). In order to further elucidate the role of PP2A in the regulation of EC cytoskeleton and permeability, PP2A catalytic (PP2Ac) and A regulatory (PP2Aa) subunits were cloned and human pulmonary arterial EC (HPAEC) were transfected with PP2A mammalian expression constructs or infected with PP2A recombinant adenoviruses. Immunostaining of PP2Ac or of PP2Aa + c overexpressing HPAEC indicated actin cytoskeleton rearrangement. PP2A overexpression hindered or at least dramatically reduced thrombin- or nocodazole-induced F-actin stress fiber formation and microtubule (MT) dissolution. Accordingly, it also attenuated thrombin- or nocodazole-induced decrease in transendothelial electrical resistance indicative of barrier protection. Inhibition of PP2A by okadaic acid abolished its effect on agonist-induced changes in EC cytoskeleton; this indicates a critical role of PP2A activity in EC cytoskeletal maintenance. The overexpression of PP2A significantly attenuated thrombin- or nocodazole-induced phosphorylation of HSP27 and tau, two cytoskeletal proteins, which potentially could be involved in agonist-induced cytoskeletal rearrangement and in the increase of permeability. PP2A-mediated dephosphorylation of HSP27 and tau correlated with PP2A-induced preservation of EC cytoskeleton and barrier maintenance. Collectively, our observations clearly demonstrate the crucial role of PP2A in EC barrier protection.
• Verin, A. D., Liu, F., Kolosova, I. A., Garcia, J. G., Borbiev, T., Bogatcheva, N. V., & Birukova, A. A. (2006). 12 PHORBOL ESTER-INDUCED ENDOTHELIAL CYTOSKELETAL REMODELING.. Journal of Investigative Medicine, 54(2), S345.6-S345. doi:10.2310/6650.2005.x0015.11
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  We have previously shown that treatment of bovine endothelial cell monolayers with phorbol myristate acetate leads to the thinning of cortical actin ring and rearrangement of the cytoskeleton into a grid-like structure, concomitant with the loss of endothelial barrier function. Here we demonstrate that phorbol myristate acetate induces both myosin and caldesmon redistribution from cortical ring into the grid-like network. However, the initial step of actin and myosin redistribution is not followed by caldesmon. Coimmunoprecipitation experiments revealed that short-term (5 minutes) treatment with phorbol ester leads to the weakening of caldesmon ability to bind actin and myosin. Prolonged incubation with phorbol myristate acetate, however, strengthens caldesmon complexes with actin and myosin, which correlates with the grid-like actin network formation. Phorbol ester stimulation leads to an immediate increase in caldesmon Ser/Thr phosphorylation. This process occurs at sites distinct from the sites specific for ERK1/2 phosphorylation and correlates with caldesmon dissociation from the actomyosin complex. Inhibition of ERK-kinase MEK fails to abolish grid-like structure formation, although reducing weakening of the cortical actin ring, whereas inhibition of protein kinase C reverses phorbol ester-induced cytoskeletal rearrangement. Our results suggest that protein kinase C-dependent phosphorylation of caldesmon is involved in phorbol ester-mediated complex cytoskeletal changes leading to the endothelial cell barrier compromise.
• Villar, J., Maresso, K., Ma, S. F., Garcia, J. G., Gao, L., Flores, C., & Finigan, J. H. (2006). 59 A TWO-STEP TAGGING SINGLE NUCLEOTIDE POLYMORPHISM APPROACH FOR THE IDENTIFICATION OF GENETIC VARIANTS OF INTERLEUKIN-6 ASSOCIATED WITH ACUTE LUNG INJURY.. Journal of Investigative Medicine, 54(2), S353.3-S353. doi:10.2310/6650.2005.x0015.58
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  Rationale Interleukin-6 (IL-6) is a key proinflammatory cytokine in the inflammatory diseases including acute lung injury (ALI) and sepsis. Genetic associations of IL-6 variants with disease have focused on the -174 G>C single nucleotide polymorphism (SNP) within the IL-6 promoter. However, it is not clear whether these associations are due to linkage disequilibrium to flanking regions or to other SNPs of the gene. Methods Eighty-four healthy controls (H), 91 septic (S), and 64 ALI patients of European American descent (ED) were analyzed for association. A set of seven evenly distributed intergenic SNPs covering ≈74 kb plus the -174 SNP were chosen to evaluate the associations with the flanking regions of IL-6 gene. TagIT software (http://www.genome.duke.edu/resourses/computation/software>) was used to select IL-6 cosmopolitan tagging SNPs (tSNPs) from 23 ED and 24 African American descent (AD) individuals. SNP dropping with resampling method was used to predict the properties of tSNPs covering variation. Results Only the -174 G>C SNP was significantly associated with ALI (ALI vs H: OR = 0.28, 95% CI 0.08-0.96, p = .039; ALI vs S: OR = 0.26, 95% CI 0.08-0.87, p = .038). Additionally, a set of 14 cosmopolitan tSNPs were identified covering more than 85% of known and unknown ED or AD common variations in the IL-6 gene. Conclusion Our data demonstrate that variants in the IL-6 gene, but not flanking regions, are involved in susceptibility to ALI. The analysis of haplotypes defined by tSNPs within the IL-6 gene may confirm whether genetic variants other than -174 G>C SNP may be associated with ALI. This approach will allow us to delimitate the search for causal variants in the IL-6 gene. Funding Specialized Centers of Clinically Oriented Research P50 HL-073994 and Fundacion Canaria Dr. Manuel Morales.
• Wadgaonkar, R., Verin, A. D., Tar, K., Olah, G., Ma, S., Gergely, P., Garcia, J. G., Czikora, I., & Csortos, C. (2006). 43 THE ROLE OF PROTEIN PHOSPHATASE 2A IN THE REGULATION OF ENDOTHELIAL CELL CYTOSKELETON STRUCTURE.. Journal of Investigative Medicine, 54(2), S350.4-S350. doi:10.2310/6650.2005.x0015.42
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  Our recently published data suggested the involvement of protein phosphatase 2A (PP2A) in endothelial cell (EC) barrier regulation (Tar et al, 2004). In order to further elucidate the role of PP2A in the regulation of EC cytoskeleton and permeability, PP2A catalytic (PP2Ac) and A regulatory (PP2Aa) subunits were cloned and human pulmonary arterial EC (HPAEC) were transfected with PP2A mammalian expression constructs or infected with PP2A recombinant adenoviruses. Immunostaining of PP2Ac or of PP2Aa+c overexpressing HPAEC indicated actin cytoskeleton rearrangement. PP2A overexpression hindered or at least dramatically reduced thrombin- or nocodazole-induced F-actin stress fiber formation and microtubule (MT) dissolution. Accordingly, it also attenuated thrombin- or nocodazole-induced decrease in transendothelial electrical resistance indicative of barrier protection. Inhibition of PP2A by okadaic acid abolished its effect on agonist-induced changes in EC cytoskeleton; this indicates a critical role of PP2A activity in EC cytoskeletal maintenance. The overexpression of PP2A significantly attenuated thrombin- or nocodazole-induced phosphorylation of HSP27 and tau, two cytoskeletal proteins, which potentially could be involved in agonist-induced cytoskeletal rearrangement and in the increase of permeability. PP2A-mediated dephosphorylation of HSP27 and tau correlated with PP2A-induced preservation of EC cytoskeleton and barrier maintenance. Collectively, our observations clearly demonstrate the crucial role of PP2A in EC barrier protection. Grant sponsor: National Heart, Lung, and Blood Institutes; grant numbers: HL67307, HL68062, HL58064; grant sponsor: Hungarian Science Research Fund; grant number: OTKA T043133.
• Wei, C., Moreno-vinasco, L., Jacobson, J. R., Garcia, J. G., Gao, D., Chen, L., & Bonde, P. (2006). DNA damage and mismatch repair pathway in lung ischemia and reperfusion injury. Journal of Cardiothoracic Renal Research, 1(2), 147-152. doi:10.1016/j.jccr.2006.05.007
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  Summary Background Oxidative damage induced by reperfusion is responsible for increased morbidity and mortality following lung transplantation. A stable and deleterious DNA adduct, 8-oxogaunine (8-oxoG) results due to oxidative DNA damage. Mut-Y homologue (MYH) is a DNA repair enzyme promoting DNA reconstruction through the mismatch repair pathway to repair 8-oxoG lesion. We investigated the role of DNA mismatch repair pathway mediated by MYH in the setting of lung ischemia and reperfusion. Methods Left lungs of the adult Sprague Dawley rats were subjected to 1 h ischemia and 2 and 4 h reperfusion. Un-operated animals served as controls. Quantification of 8-oxoG was performed using immunohistochemistry (IHC) and MYH was analyzed by Western blot. Apoptosis was assessed by caspase-3 levels. Results Indices of inflammation and permeability were raised in both reperfusion groups. There was significant increase in DNA damage as reflected by positive 8-oxoG staining in 2 h (22% increase) and 4 h reperfusion (31% increase) compared to control ( p p Conclusion Reperfusion leads to increased DNA damage and down regulation of DNA mismatch repair pathway in a model of ischemia and reperfusion in lungs. Gene therapy targeted at this pathway may prove an attractive therapeutic intervention to reduce reperfusion injury in lung transplantation.
• West, E. E., Lavoie, T. L., Orens, J. B., Chen, E. S., Ye, S. Q., Finkelman, F. D., Garcia, J. G., & McDyer, J. F. (2006). Pluripotent allospecific CD8+ effector T cells traffic to lung in murine obliterative airway disease. American journal of respiratory cell and molecular biology, 34(1), 108-18.
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  Long-term success in lung transplantation is limited by obliterative bronchiolitis, whereas T cell effector mechanisms in this process remain incompletely understood. Using the mouse heterotopic allogeneic airway transplant model, we studied T cell effector responses during obliterative airways disease (OAD). Allospecific CD8+ IFN-gamma+ T cells were detected in airway allografts, with significant coexpression of TNF-alpha and granzyme B. Therefore, using IFN-gamma as a surrogate marker, we assessed the distribution and kinetics of extragraft allo-specific T cells during OAD. Robust allospecific IFN-gamma was produced by draining the lymph nodes, spleen, and lung mononuclear cells from allograft, but not isograft recipients by Day 14, and significantly decreased by Day 28. Although the majority of allospecific T cells were CD8+, allospecific CD4+ T cells were also detected in these compartments, with each employing distinct allorecognition pathways. An influx of pluripotent CD8+ effector cells with a memory phenotype were detected in the lung during OAD similar to those seen in the allografts and secondary lymphoid tissue. Antibody depletion of CD8+ T cells markedly reduced airway lumen obliteration and fibrosis at Day 28. Together, these data demonstrate that allospecific CD8+ effector T cells play an important role in OAD and traffic to the lung after heterotopic airway transplant, suggesting that the lung is an important immunologic site, and perhaps a reservoir, for effector cells during the rejection process.
• Wright, J. M., Merlo, C. A., Reynolds, J. B., Zeitlin, P. L., Garcia, J. G., Guggino, W. B., & Boyle, M. P. (2006). Respiratory epithelial gene expression in patients with mild and severe cystic fibrosis lung disease. American journal of respiratory cell and molecular biology, 35(3), 327-36.
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  Despite having identical cystic fibrosis transmembrane conductance regulator genotypes, individuals with DeltaF508 homozygous cystic fibrosis (CF) demonstrate significant variability in severity of pulmonary disease. This investigation used high-density oligonucleotide microarray analysis of nasal respiratory epithelium to investigate the molecular basis of phenotypic differences in CF by (1) identifying differences in gene expression between DeltaF508 homozygotes in the most severe 20th percentile of lung disease by forced expiratory volume in 1 s and those in the most mild 20th percentile of lung disease and (2) identifying differences in gene expression between DeltaF508 homozygotes and age-matched non-CF control subjects. Microarray results from 23 participants (12 CF, 11 non-CF) met the strict quality control guidelines and were used for final data analysis. A total of 652 of the 11,867 genes identified as present in 75% of the samples were significantly differentially expressed in one of the three disease phenotypes: 30 in non-CF, 53 in mild CF, and 569 in severe CF. An analysis of genes differentially expressed by severity of CF lung disease demonstrated significant upregulation in severe CF of genes involved in protein ubiquination (P < 0.04), mitochondrial oxidoreductase activity (P < 0.01), and lipid metabolism (P < 0.03). Analysis of genes with decreased expression in patients with CF compared with control subjects demonstrated significant downregulation of genes involved in airway defense (P < 0.047) and protein metabolism (P < 0.048). This study suggests that differences in CF lung phenotype are associated with differences in expression of genes involving airway defense, protein ubiquination, and mitochondrial oxidoreductase activity and identifies specific new candidate modifiers of the CF phenotype.
• Ye, S. Q., Ma, S. F., Grigoryev, D. N., Garcia, J. G., & Barnes, K. C. (2006). Gene Transcripts of Peripheral Blood as Potential Biomarkers for Allergic Changes in Upper Airways. The Journal of Allergy and Clinical Immunology, 117(2), S162. doi:10.1016/j.jaci.2005.12.647
• Ye, S. Q., Simon, B. A., Ma, S. F., Grigoryev, D. N., Garcia, J. G., & Easley, R. B. (2006). 33 BIOINFORMATIC CLASS DISCOVERY OF POTENTIAL BIOMARKERS IN VENTILATOR-ASSOCIATED LUNG INJURY.. Journal of Investigative Medicine, 54(2), S349.1-S349. doi:10.2310/6650.2005.x0015.32
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  Rationale The unilateral (one lung injured and the contralateral lung intact) model of ventilator-associated lung injury (VALI) allows us to utilize uninjured lung as the systemic VALI effects indicator. We speculated that regulation of genes in uninjured lung will be attributed to blood or lymph carrying effectors produced by an injured lung and the analysis of upstream bioprocesses governed by affected genes will lead to the effectors9 discovery. Methods Dogs (n = 4) were intubated, left lung lavaged, and then both lungs were either independently ventilated (total Vt = 15 mL/kg) for 6 hours (injured and uninjured) or immediately harvested (control). Lung mRNA (n = 16 for all three groups) was hybridized to HG_U133A and analyzed by SAM using interspecies probe adjustment. Genes with the lowest false discovery rate (q = 0.124%) that imposed fold change (FC) range from -3.52 to -1.26 and 1.22 to 6.96 with corresponding FC averages -1.59 and 2.01 were considered affected by VALI. Gene ontology filtering for receptor activity term was conducted by MAPPFinder. Results Our analyses revealed 22 receptor-related genes, of which 7 were growth factor receptors including EGFR (FC = -1.54), FGFR1 (FC = -1.59), FGFR2 (FC = -1.56), and PDGFR (FC = -1.30). The overall down-regulation of these receptors was concordant with decreased expression of their corresponding ligands in injured lung including EGF (FC = -1.39), FGF2 (FC = -1.39), PDGFA (FC = -1.41), and PDGFB (FC = -2.00). Fibroblast (Z = 6.17) and epidermal (Z = 4.49) growth factor receptor activity were the first and the third significantly (Z > 1.96) affected pathways. Conclusions Our approaches effectively identify a class of potential biomarkers in VALI. Further investigation of this study may elucidate systemic VALI effects and facilitate the development of new diagnostic tools. Funded by SCCOR U01 HL-073994.
• Zaborina, O., Garcia, J. G., Dudek, S. M., Chiang, E. T., Camp, S. M., & Alverdy, J. C. (2006). 51 HIGH-MOLECULAR-WEIGHT POLYETHYLENE GLYCOL PROTECTS THROMBIN-INDUCED ENDOTHELIAL BARRIER DYSFUNCTION BY INDUCING ACTIN CYTOSKELETON REARRANGEMENT, WHICH RESULTS IN ROBUST ENHANCEMENT OF ENDOTHELIAL CELL BARRIER INTEGRITY.. Journal of Investigative Medicine, 54(2), S351.6-S352. doi:10.2310/6650.2005.x0015.50
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  ARDS is characterized by sudden, life-threatening lung failure with diffuse alveolar infiltrate, reduced arterial oxygenation, and pulmonary edema. Acute lung injury (ALI) is the end result of common pathways initiated by a variety of local or systemic insults. Polyethylene glycol (PEG) compounds are inert and nontoxic polymers that act as a surrogate mucin lining providing protection against bacterial infections on intestinal epithelial cells. We hypothesized that PEG may provide similar protective effects on lung endothelium by attenuating endothelial cell (EC) activation that results in barrier dysfunction. This study examines the effects of a high-molecular-weight PEG (PEG15-20) on cultured human pulmonary microvessel EC from barrier disruptive agents. EC were cultured on microelectrodes and changes in transendothelial electrical resistances (TER) were measured to assess alterations in paracellular permeability. PEG induced a rapid, dose-dependent increase in TER similar to barrier-enhancing lipid such as sphingosine 1-phosphate (S1P). Optimal concentration of 7.5-9% PEG induced a robust increase in TER from 2,500 to 5,000 ohm, which was sustained for 40 hours. In comparison to S1P, which induced a 40% increase in resistance in 10-15 minutes, the maximum barrier enhancing effect of PEG was achieved in about 45 minutes, but with 100% increase in resistance. With a 2-fold increase in resistance, PEG is one of the most potent barrier-enhancing agents tested among all the barrier-enhancing agents, such as S1P, FTY720, phospho-FTY720, and HGF. Immunofluorescence data revealed that PEG altered the EC actin cytoskeleton to form a defined cortical actin ring that may help strengthen cell-cell junctional adhesion. PEG rapidly induced dephosphorylation of ERK and MLC as early as 1 minute and completely inhibited thrombin-induced ERK and MLC phosphorylation. More importantly, pretreatment with PEG for 1 hour attenuated thrombin-induced endothelial barrier dysfunction. In summary, PEG activates a rapid, actin-associated, barrier-enhancing signal transduction pathway in EC, which may have therapeutic potential to prevent and reverse pulmonary edema. Funded by HL058064 and GM062344.
• Zhang, L. Q., Ye, S. Q., Garcia, J. G., & Cepeda, J. (2006). 49 NOVEL ROLE OF PRE-B-CELL COLONY-ENHANCING FACTOR IN PULMONARY ENDOTHELIAL BARRIER REGULATION.. Journal of Investigative Medicine, 54(2), S351.4-S351. doi:10.2310/6650.2005.x0015.48
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  Rationale Our prior genomic and genetic studies identified pre-B-cell colony-enhancing factor (PBEF) as a potential novel biomarker in acute lung injury (ALI) (Ye et al, 2005). To elucidate the molecular mechanism underlying PBEF in the pathogenesis of ALI, we assessed the role of PBEF in in vitro vascular barrier regulation using confluent human pulmonary artery endothelial cell (HPAEC) or microvascular cells (HMVEC-L) monolayers since increased vascular permeability is a cardinal feature of ALI. Methods Inhibition of PBEF expression was achieved by the PBEF siRNAs. Overexpression of the PBEF gene was accomplished by adeno-PBEF. Endothelial cells (EC) were transfected with PBEF siRNA or infected with adeno-PBEF for 48 hours before treated with thrombin (0.1 U/mL) or S1P (1 mm) for various time. Parameters to evaluate endothelial cell barrier functions include transendothelial electric resistance (TER), actin staining, myosin light chain phosphorylation, calcium influx. Results Reductions in PBEF protein expression (> 70%) by siRNA significantly attenuated EC barrier dysfunction induced by the potent edemagenic agent, thrombin reflected by reductions in TER (60 to 70% reduction). Furthermore, PBEF siRNA blunted thrombin-mediated increases in increases in Ca 2+ entry, polymerized actin formation, and myosin light chain phosphorylation, events critical to the thrombin-mediated permeability response. Overexpression of PBEF by adeno-PBEF vector significantly increases EC permeability reflected by decreased TER in HMVEC-L by ≈30%, indicating that overexpression of PBEF results in dysfunction. Conclusions These in vitro observations strongly support that PBEF is critically involved in the endothelial cell barrier regulation. It encourages us to further explore the mechanistic insight into PBEF in the murine model of ALI. Funded by NHLBI SCCOR-Molecular Approaches to Ventilator-Associated Lung Injury (U01HL 073994).
• Zhang, S., Ilano, A., Grant, A. V., Garcia, J. G., Gao, L., Campbell, M., Beaty, T. H., & Barnes, K. C. (2006). Single Nucleotide Polymorphisms in Gene Encoding Myosin Light Chain Kinase (MLCK) are Associated with Increased Risk of Asthma. The Journal of Allergy and Clinical Immunology, 117(2), S185. doi:10.1016/j.jaci.2005.12.736
• Zhao, J., Singleton, P. A., Garcia, J. G., & Dudek, S. M. (2006). 50 SPHINGOSINE 1-PHOSPHATE DRAMATICALLY ALTERS THE HUMAN PULMONARY ARTERY ENDOTHELIAL CELLS' LIPID RAFT PROTEOME.. Journal of Investigative Medicine, 54(2), S351.5-S351. doi:10.2310/6650.2005.x0015.49
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  Rationale Lipid rafts are plasma membrane microdomains involved in dynamic membrane signaling and trafficking. Our previous studies (Singleton et al, FASEB J, 2005) have demonstrated that the platelet-derived lipid sphingosine 1-phosphate (S1P) plays a critical regulatory role in maintenance and enhancement of pulmonary vascular barrier function. S1P ligates the S1P receptor 1, which we have previously localized to lipid rafts of human pulmonary artery endothelial cells (HPAECs). Here we use 2-D electrophoresis analysis to characterize changes in tyrosine phosphoprotein content in lipid rafts of HPAECs challenged with S1P. Methods and Results HPAECs were challenged with 1 μM of S1P for 5 minutes, washed with cold PBS twice, and solublized with 1% Triton X-100 at 4°C. The Triton X-100 insoluble material was subjected to discontinuous Optiprep TM gradient centrigugation and the light density (lipid raft) fractions were collected. Isolated lipid rafts were resolubilized in 50 μL of 7 M urea/2 M thiourea and 50 mM DTT. Isoelectric focusing was carried out on a nonlinear electrophoresis gel strip (pH3-10, 7 cm) with the second dimension carried out on 4-20% SDS-PAGE gel. Analytical gels were stained using Imperial blue (Pierce) and Western blotting was performed using antiphosphotyrosine antibody. Protein levels were not significantly altered in lipid rafts isolated from control vs S1P-treated cells (1 μM, 5 minutes). The 2-D gel images revealed over 30 proteins recruited to lipid rafts after S1P treatment (5 minutes) with antiphosphotyrosine blots showing S1P to increase tyrosine phosphorylation of over 20 proteins, including the src kinase family and cortactin. Conclusions These results suggest that tyrosine kinases participate in S1P-induced signaling in lipid rafts and that 2-D proteomic analysis is a powerful tool for studying regulation of pulmonary endothelial cells barrier function. Supported by NIH grant HL58064.
• Zhao, Y., Saatian, B., Pendyala, S., Natarajan, V., Gorshkova, I., Garcia, J. G., & Berdyshev, E. V. (2006). 22 SPHINGOSINE-1-PHOSPHATE-MEDIATED ENDOTHELIAL CELL MOTILITY IS REGULATED BY S1P1 RECEPTOR, LIPID PHOSPHATE PHOSPHATASE-1, AND SPHINGOSINE KINASE 1.. Journal of Investigative Medicine, 54(2), S347.2-S347. doi:10.2310/6650.2005.x0015.21
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  The extracellular effects of S1P, a naturally occurring and platelet-derived bioactive lipid molecule, are mediated by S1P 1-5 family of G protein-coupled receptors. The exact role of different S1P receptors and signaling pathways regulating S1P-induced cell motility is not completely defined. Human pulmonary artery endothelial cells (HPAEC) expressed S1P 1 , S1P 3 , and S1P 4 receptors with a predominance of S1P 1 , as determined by RT PCR. Exposure of HPAEC to S1P resulted in a potent induction of cell motility as determined by a scratch wound healing assay and by monitoring changes in transendothelial electrical resistance of wounded HPAEC by ECIS. Inverse agonist of S1P 1 receptor, SB649146, and S1P 1 siRNA attenuated S1P-induced EC motility. Furthermore, overexpression of sphingosine kinase 1 wild type and S1P 1 wild type increased intracellular content of S1P as well DHS1P (DHS1P > S1P) with a concomitant loss of cell motility in response to exogenous S1P. Overexpression of lipid phosphate phosphatase (LPP)1 wild type that enhanced hydrolysis of exogenous S1P blocked S1P-induced cell motility. These results suggest that a balance between S1P 1 expression and intracellular content of S1P and DHS1P seems to be critical for maintaining cellular sensitivity to external S1P stimulation. Supported by NIH RO1s HL 79396 and 71152 to V.N.
• Berdyshev, E. V., Gorshkova, I. A., Garcia, J. G., Natarajan, V., & Hubbard, W. C. (2005). Quantitative analysis of sphingoid base-1-phosphates as bisacetylated derivatives by liquid chromatography-tandem mass spectrometry. Analytical biochemistry, 339(1), 129-36.
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  Sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (DHS1P) are important signaling sphingolipids. The presence of nanomolar levels of S1P and DHS1P in tissues, cells, and biological fluids requires a highly sensitive and selective assay method for their reliable detection and quantitation. Preliminary findings employing positive ion electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis indicated significant sample carryover from previous injections of authentic standards of S1P and DHS1P. This article details a negative ion ESI LC-MS/MS technique following modification of the zwitterionic nature of S1P and DHS1P via derivatization. A highly selective and sensitive LC-MS/MS technique capable of reliable detection of less than 50 fmol of the derivatives of S1P and DHS1P without significant sample carryover was developed. Standard curves for S1P and DHS1P are linear over wide ranges (0-300 pmol) of analyte concentrations with correlation coefficients (r2) greater than 0.995. The levels of S1P and DHS1P in human platelet poor plasma were 590.8+/-42.1 and 130.7+/-20.7 pmol/ml, respectively. The levels of S1P and DHS1P in fetal bovine serum were 141.7+/-4.6 and 0.6+/-0.2 pmol/ml, respectively. The addition of sphingosine (1 microM) to human pulmonary artery endothelial cells in culture resulted in a more than 20-fold increase in the cellular level of S1P, whereas the level of DHS1P was unchanged.
• Birukova, A. A., Birukov, K. G., Adyshev, D., Usatyuk, P., Natarajan, V., Garcia, J. G., & Verin, A. D. (2005). Involvement of microtubules and Rho pathway in TGF-beta1-induced lung vascular barrier dysfunction. Journal of cellular physiology, 204(3), 934-47.
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  Transforming growth factor-beta1 (TGF-beta1) is a cytokine critically involved in acute lung injury and endothelial cell (EC) barrier dysfunction. We have studied TGF-beta1-mediated signaling pathways and examined a role of microtubule (MT) dynamics in TGF-beta1-induced actin cytoskeletal remodeling and EC barrier dysfunction. TGF-beta1 (0.1-50 ng/ml) induced dose-dependent decrease in transendothelial electrical resistance (TER) in bovine pulmonary ECs, which was linked to increased actin stress fiber formation, myosin light chain (MLC) phosphorylation, EC retraction, and gap formation. Inhibitor of TGF-beta1 receptor kinase RI (5 microM) abolished TGF-beta1-induced TER decline, whereas inhibitor of caspase-3 zVAD (10 microM) was without effect. TGF-beta1-induced EC barrier dysfunction was linked to partial dissolution of peripheral MT meshwork and decreased levels of stable (acetylated) MT pool, whereas MT stabilization by taxol (5 microM) attenuated TGF-beta1-induced barrier dysfunction and actin remodeling. TGF-beta1 induced sustained activation of small GTPase Rho and its effector Rho-kinase; phosphorylation of myosin binding subunit of myosin specific phosphatase; MLC phosphorylation; EC contraction; and gap formation, which was abolished by inhibition of Rho and Rho-kinase, and by MT stabilization with taxol. Finally, elevation of intracellular cAMP induced by forskolin (50 microM) attenuated TGF-beta1-induced barrier dysfunction, MLC phosphorylation, and protected the MT peripheral network. These results suggest a novel role for MT dynamics in the TGF-beta1-mediated Rho regulation, EC barrier dysfunction, and actin remodeling.
• Birukova, A. A., Birukov, K. G., Gorshkov, B., Liu, F., Garcia, J. G., & Verin, A. D. (2005). MAP kinases in lung endothelial permeability induced by microtubule disassembly. American journal of physiology. Lung cellular and molecular physiology, 289(1), L75-84.
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  Lung endothelial barrier function is regulated by multiple signaling pathways, including mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK) 1/2 and p38. We have recently shown involvement of microtubule (MT) disassembly in endothelial cell (EC) barrier failure. In this study, we examined potential involvement of ERK1/2 and p38 MAPK in lung EC barrier dysfunction associated with MT disassembly. MT inhibitors nocodazole (0.2 microM) and vinblastine (0.1 microM) induced sustained activation of Ras-Raf-MEK1/2-ERK1/2 and MKK3/6-p38-MAPKAPK2 MAPK cascades in human and bovine pulmonary EC, as detected by phosphospecific antibodies and in MAPK activation assays. These effects were linked to increased permeability assessed by measurements of transendothelial electrical resistance and cytoskeletal remodeling analyzed by morphometric analysis of EC monolayers. MT stabilization by taxol (5 microM, 1 h) attenuated nocodazole-induced ERK1/2 and p38 MAPK activation and phosphorylation of p38 MAPK substrate 27-kDa heat shock protein and regulatory myosin light chains, the proteins involved in actin polymerization and actomyosin contraction. Importantly, only pharmacological inhibition of p38 MAPK by SB-203580 (20 microM, 1 h) attenuated nocodazole-induced MT depolymerization, actin remodeling, and EC barrier dysfunction, whereas the MEK/ERK1/2 inhibitor U0126 (5 microM, 1 h) exhibited no effect. These data suggest a direct link between p38 MAPK activation, remodeling of MT network, and EC barrier regulation.
• Fenton, J. W., Brezniak, D. V., Ofosu, F. A., Shen, G. X., Jacobson, J. R., & Garcia, J. G. (2005). Statins and thrombin. Current drug targets. Cardiovascular & haematological disorders, 5(2), 115-20.
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  L-Mevalonic acid is the distant precursor of cholesterol, in contrast to cholesterol, L-mevalonic acid, its distant precursor gives rise to farnesyl and geranylgeranyl pyrophosphates in relatively few metabolic steps. These isoprenyl pyrophophates covalently conjugate with specific G-proteins and serve as membrane anchors enabling them to carry out their function. Although farnesyl-proteins may participate in signal transduction, geranylgeranyl-proteins (e.g., Rho GTP binding proteins) are well known to downregulate signaling pathways by inhibiting L-mevalonic acid synthesis. Such inhibitors include 3-hydroxy-3-methylglutaryl CoA reductase inhibitors, drugs (statins) and isoprenoids of dietary origins, where Rho protein activation appears to be necessary for cellular-mediated thrombin generation. Thrombin and other proteases (e.g., coagulation factor Xa, tryptase) upregulate protease-activated receptor (PAR) synthesis and PAR activation promotes synthesis and expression of other proteins [e.g., tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1)]. With the PAR-1 activating peptide SSFLRNP, we found that either cerivastatin or atorvastatin mitigated platelet stimulation in a time- and dose-dependent manner, as predicted if a statin-mediated Rho pathway is required. We also found that simvastatin decreased prothrombin fragments F1+2 in plasma from type 2 diabetics, demonstrating that statins downregulate thrombin generation. Thus, independent of cholesterol, statins and dietary isoprenoids behave as inhibitors of TF-dependent thrombin generation. Because thrombin has multiple physiological functions, the 20 pleiotropic effects reported for statins may reflect a common mechanism for downregulation of thrombin-mediated events, in particular at the cellular level.
• Finigan, J. H., Dudek, S. M., Singleton, P. A., Chiang, E. T., Jacobson, J. R., Camp, S. M., Ye, S. Q., & Garcia, J. G. (2005). Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation. The Journal of biological chemistry, 280(17), 17286-93.
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  Increased endothelial cell (EC) permeability is central to the pathophysiology of inflammatory syndromes such as sepsis and acute lung injury (ALI). Activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, improves sepsis survival through an unknown mechanism. We hypothesized a direct effect of APC to both prevent increased EC permeability and to restore vascular integrity after edemagenic agonists. We measured changes in transendothelial electrical resistance (TER) and observed that APC produced concentration-dependent attenuation of TER reductions evoked by thrombin. We next explored known EC barrier-protective signaling pathways and observed dose-dependent APC-mediated increases in cortical myosin light chain (MLC) phosphorylation in concert with cortically distributed actin polymerization, findings highly suggestive of Rac GTPase involvement. We next determined that APC directly increases Rac1 activity, with inhibition of Rac1 activity significantly attenuating APC-mediated barrier protection to thrombin challenge. Finally, as these signaling events were similar to those evoked by the potent EC barrier-enhancing agonist, sphingosine 1-phosphate (S1P), we explored potential cross-talk between endothelial protein C receptor (EPCR) and S1P1, the receptors for APC and S1P, respectively. EPCR-blocking antibody (RCR-252) significantly attenuated both APC-mediated barrier protection and increased MLC phosphorylation. We next observed rapid, EPCR and PI 3-kinase-dependent, APC-mediated phosphorylation of S1P1 on threonine residues consistent with S1P1 receptor activation. Co-immunoprecipitation studies demonstrate an interaction between EPCR and S1P1 upon APC treatment. Targeted silencing of S1P1 expression using siRNA significantly reduced APC-mediated barrier protection against thrombin. These data suggest that novel EPCR ligation and S1P1 transactivation results in EC cytoskeletal rearrangement and barrier protection, components potentially critical to the improved survival of APC-treated patients with severe sepsis.
• Garcia, J. G. (2005). Searching for candidate genes in acute lung injury: SNPs, Chips and PBEF. Transactions of the American Clinical and Climatological Association, 116, 205-19; discussion 220.
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  Acute lung injury (ALI) is a devastating illness, occurring in the setting of sepsis, with genetic variations contributing to ALI susceptibility and severity. We utilized the "candidate gene approach" with extensive expression profiling in animal and human ALI models to identify novel candidate genes. We noted significant expression of pre-B-cell colony enhancing factor (PBEF), a gene not previously associated with lung pathophysiology. This finding was validated by molecular, biochemical and immunohistochemical approaches with increased levels of PBEF also detected in human BAL and serum. DNA sequencing identified two single nucleotide polymorphisms (SNPs) in the PBEF promoter (T-1001G, C-1543T), which were genotyped in a Caucasian cohort of sepsis-associated ALI patients. Carriers of the GC haplotype exhibited a 5.7-fold relative ALI risk compared to controls associated with increased PBEF promoter activity. These studies demonstrate the successful application of genomic technologies in the identification of novel candidate genes in complex lung disease.
• Garcia, J. G., & Ma, S. F. (2005). Polymerase chain reaction: a landmark in the history of gene technology. Critical care medicine, 33(12 Suppl), S429-32.
• Girgis, R. E., Ma, S. F., Ye, S., Grigoryev, D. N., Li, D., Hassoun, P. M., Tuder, R. M., Johns, R. A., & Garcia, J. G. (2005). Differential gene expression in chronic hypoxic pulmonary hypertension: effect of simvastatin treatment. Chest, 128(6 Suppl), 579S.
• Grigoryev, D. N., Ma, S. F., Simon, B. A., Irizarry, R. A., Ye, S. Q., & Garcia, J. G. (2005). In vitro identification and in silico utilization of interspecies sequence similarities using GeneChip technology. BMC genomics, 6, 62.
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  Genomic approaches in large animal models (canine, ovine etc) are challenging due to insufficient genomic information for these species and the lack of availability of corresponding microarray platforms. To address this problem, we speculated that conserved interspecies genetic sequences can be experimentally detected by cross-species hybridization. The Affymetrix platform probe redundancy offers flexibility in selecting individual probes with high sequence similarities between related species for gene expression analysis.
• Guo, Y., Ma, S. F., Grigoryev, D., Van Eyk, J., & Garcia, J. G. (2005). 1-DE MS and 2-D LC-MS analysis of the mouse bronchoalveolar lavage proteome. Proteomics, 5(17), 4608-24.
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  Bronchoalveolar lavage fluid (BALF) is a complex mixture of proteins, which represents a unique clinically useful sampling of the lower respiratory tract. Many proteomic technologies can be used to characterize complex biological mixtures; however, it is not yet clear which technology(s) provide more information regarding the number of proteins identified and sequence coverage. In this study, we initially compared two common proteomic approaches, 2-D LC microESI MS/MS and 1-DE followed by gel slice digestion, peptide extraction and peptide identification by MS in characterization of the mouse BALF proteome; secondly, we identified 297 unique proteins from the mouse BALF proteome, greatly expanded the BALF proteome by about threefold regardless of species.
• Irizarry, R. A., Warren, D., Spencer, F., Kim, I. F., Biswal, S., Frank, B. C., Gabrielson, E., Garcia, J. G., Geoghegan, J., Germino, G., Griffin, C., Hilmer, S. C., Hoffman, E., Jedlicka, A. E., Kawasaki, E., Martínez-Murillo, F., Morsberger, L., Lee, H., Petersen, D., , Quackenbush, J., et al. (2005). Multiple-laboratory comparison of microarray platforms. Nature methods, 2(5), 345-50.
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  Microarray technology is a powerful tool for measuring RNA expression for thousands of genes at once. Various studies have been published comparing competing platforms with mixed results: some find agreement, others do not. As the number of researchers starting to use microarrays and the number of cross-platform meta-analysis studies rapidly increases, appropriate platform assessments become more important. Here we present results from a comparison study that offers important improvements over those previously described in the literature. In particular, we noticed that none of the previously published papers consider differences between labs. For this study, a consortium of ten laboratories from the Washington, DC-Baltimore, USA, area was formed to compare data obtained from three widely used platforms using identical RNA samples. We used appropriate statistical analysis to demonstrate that there are relatively large differences in data obtained in labs using the same platform, but that the results from the best-performing labs agree rather well.
• Jacobson, J. R., Barnard, J. W., Grigoryev, D. N., Ma, S. F., Tuder, R. M., & Garcia, J. G. (2005). Simvastatin attenuates vascular leak and inflammation in murine inflammatory lung injury. American journal of physiology. Lung cellular and molecular physiology, 288(6), L1026-32.
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  Therapies to limit the life-threatening vascular leak observed in patients with acute lung injury (ALI) are currently lacking. We explored the effect of simvastatin, a 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor that mediates endothelial cell barrier protection in vitro, in a murine inflammatory model of ALI. C57BL/6J mice were treated with simvastatin (5 or 20 mg/kg body wt via intraperitoneal injection) 24 h before and again concomitantly with intratracheally administered LPS (2 microg/g body wt). Inflammatory indexes [bronchoalveolar lavage (BAL) myeloperoxidase activity and total neutrophil counts assessed at 24 h with histological confirmation] were markedly increased after LPS alone but significantly reduced in mice that also received simvastatin (20 mg/kg; approximately 35-60% reduction). Simvastatin also decreased BAL albumin (approximately 50% reduction) and Evans blue albumin dye extravasation into lung tissue (100%) consistent with barrier protection. Finally, the sustained nature of simvastatin-mediated lung protection was assessed by analysis of simvastatin-induced gene expression (Affymetrix platform). LPS-mediated lung gene expression was significantly modulated by simvastatin within a number of gene ontologies (e.g., inflammation and immune response, NF-kappaB regulation) and with respect to individual genes implicated in the development or severity of ALI (e.g., IL-6, Toll-like receptor 4). Together, these findings confirm significant protection by simvastatin on LPS-induced lung vascular leak and inflammation and implicate a potential role for statins in the management of ALI.
• Kittleson, M. M., Minhas, K. M., Irizarry, R. A., Ye, S. Q., Edness, G., Breton, E., Conte, J. V., Tomaselli, G., Garcia, J. G., & Hare, J. M. (2005). Gene expression analysis of ischemic and nonischemic cardiomyopathy: shared and distinct genes in the development of heart failure. Physiological genomics, 21(3), 299-307.
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  Cardiomyopathy can be initiated by many factors, but the pathways from unique inciting mechanisms to the common end point of ventricular dilation and reduced cardiac output are unclear. We previously described a microarray-based prediction algorithm differentiating nonischemic (NICM) from ischemic cardiomyopathy (ICM) using nearest shrunken centroids. Accordingly, we tested the hypothesis that NICM and ICM would have both shared and distinct differentially expressed genes relative to normal hearts and compared gene expression of 21 NICM and 10 ICM samples with that of 6 nonfailing (NF) hearts using Affymetrix U133A GeneChips and significance analysis of microarrays. Compared with NF, 257 genes were differentially expressed in NICM and 72 genes in ICM. Only 41 genes were shared between the two comparisons, mainly involved in cell growth and signal transduction. Those uniquely expressed in NICM were frequently involved in metabolism, and those in ICM more often had catalytic activity. Novel genes included angiotensin-converting enzyme-2 (ACE2), which was upregulated in NICM but not ICM, suggesting that ACE2 may offer differential therapeutic efficacy in NICM and ICM. In addition, a tumor necrosis factor receptor was downregulated in both NICM and ICM, demonstrating the different signaling pathways involved in heart failure pathophysiology. These results offer novel insight into unique disease-specific gene expression that exists between end-stage cardiomyopathy of different etiologies. This analysis demonstrates that transcriptome analysis offers insight into pathogenesis-based therapies in heart failure management and complements studies using expression-based profiling to diagnose heart failure of different etiologies.
• Kittleson, M. M., Minhas, K. M., Irizarry, R. A., Ye, S. Q., Edness, G., Breton, E., Conte, J. V., Tomaselli, G., Garcia, J. G., & Hare, J. M. (2005). Gene expression in giant cell myocarditis: Altered expression of immune response genes. International journal of cardiology, 102(2), 333-40.
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  Giant cell myocarditis is a rapidly progressive and often fatal condition without a clear etiology or treatment. A better understanding of giant cell myocarditis pathogenesis is critical to developing treatments to prevent progression and reverse damage. We compared the gene expression of giant cell myocarditis with that of nonfailing hearts.
• Kolosova, I. A., Mirzapoiazova, T., Adyshev, D., Usatyuk, P., Romer, L. H., Jacobson, J. R., Natarajan, V., Pearse, D. B., Garcia, J. G., & Verin, A. D. (2005). Signaling pathways involved in adenosine triphosphate-induced endothelial cell barrier enhancement. Circulation research, 97(2), 115-24.
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  Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell-cell junctions. Increases in cytosolic Ca2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically Galphaq and Galphai2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of Galphaq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA.
• Leitinger, N., Garcia, J. G., Bochkov, V. N., & Birukov, K. G. (2005). 20 MOLECULAR MECHANISMS OF LUNG ENDOTHELIAL BARRIER REGULATION BY OXIDIZED PHOSPHOLIPIDS. Journal of Investigative Medicine, 53(2), S360.2-S360. doi:10.2310/6650.2005.00206.19
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  Increased levels of oxidized phospholipids generated by oxidation of shed cell membranes released into local circulation have been recently detected in acute lung injury and cardiac ischemia. This study characterized signaling pathways and barrier regulation induced in human pulmonary artery endothelial cells (EC) by a bioactive component of cell membrane-derived oxidized phospholipids, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC). OxPAPC induced time-dependent activation of protein kinase C (PKC), protein kinase A (PKA), Raf/MEK1,2/Erk-1,2 MAP kinase cascade, JNK MAP-kinase, protein tyrosine phosphorylation, and small GTPases Rac and Cdcd42 without noticeable activation of Rho. These effects were specific to oxidized phospholipids, as non-oxidized species (PAPC, PLPC, DMPC) were without effect. OxPAPC caused sustained concentration-dependent increases in EC transendothelial resistance (TER) and alleviated increased permeability elicited by thrombin. This barrier-protective effect was linked to oxygenated, epoxyisoprostane-containing phospholipids, as confirmed by mass-spectrometric analysis of OxPAPC bioactive components. Pharmacological inhibitors of OxPAPC-induced signaling and depletion of small GTPases using siRNA approach showed direct involvement of PKC, PKA, Rac and Cdc42, but not MAP-kinase cascades and Rho GTPase in EC barrier-protective response. In addition, inhibition of PKC and PKA attenuated OxPAPC-induced activation of Rac, suggesting that PKC and PKA are upstream of Rac in OxPAPC-activated signaling cascade. These results demonstrate for the first time barrier-protective properties of cell membrane-derived oxidized phospholipids generated in acute lung or cardiac injury and suggest PKC-, PKA-, Cdc42- and Rac-dependent cytoskeletal mechanisms of the vascular barrier restoration.
• Ma, S. F., Grigoryev, D. N., Taylor, A. D., Nonas, S., Sammani, S., Ye, S. Q., & Garcia, J. G. (2005). Bioinformatic identification of novel early stress response genes in rodent models of lung injury. American journal of physiology. Lung cellular and molecular physiology, 289(3), L468-77.
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  Acute lung injury is a complex illness with a high mortality rate (>30%) and often requires the use of mechanical ventilatory support for respiratory failure. Mechanical ventilation can lead to clinical deterioration due to augmented lung injury in certain patients, suggesting the potential existence of genetic susceptibility to mechanical stretch (6, 48), the nature of which remains unclear. To identify genes affected by ventilator-induced lung injury (VILI), we utilized a bioinformatic-intense candidate gene approach and examined gene expression profiles from rodent VILI models (mouse and rat) using the oligonucleotide microarray platform. To increase statistical power of gene expression analysis, 2,769 mouse/rat orthologous genes identified on RG_U34A and MG_U74Av2 arrays were simultaneously analyzed by significance analysis of microarrays (SAM). This combined ortholog/SAM approach identified 41 up- and 7 downregulated VILI-related candidate genes, results validated by comparable expression levels obtained by either real-time or relative RT-PCR for 15 randomly selected genes. K-mean clustering of 48 VILI-related genes clustered several well-known VILI-associated genes (IL-6, plasminogen activator inhibitor type 1, CCL-2, cyclooxygenase-2) with a number of stress-related genes (Myc, Cyr61, Socs3). The only unannotated member of this cluster (n = 14) was RIKEN_1300002F13 EST, an ortholog of the stress-related Gene33/Mig-6 gene. The further evaluation of this candidate strongly suggested its involvement in development of VILI. We speculate that the ortholog-SAM approach is a useful, time- and resource-efficient tool for identification of candidate genes in a variety of complex disease models such as VILI.
• Manalo, D. J., Rowan, A., Lavoie, T., Natarajan, L., Kelly, B. D., Ye, S. Q., Garcia, J. G., & Semenza, G. L. (2005). Transcriptional regulation of vascular endothelial cell responses to hypoxia by HIF-1. Blood, 105(2), 659-69.
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  Hypoxia-inducible factor 1 (HIF-1) activates transcription of genes encoding angiogenic growth factors, which are secreted by hypoxic cells and stimulate endothelial cells, leading to angiogenesis. To determine whether HIF-1 also mediates cell-autonomous responses to hypoxia, we have compared gene expression profiles in arterial endothelial cells cultured under nonhypoxic versus hypoxic conditions and in nonhypoxic cells infected with adenovirus encoding beta-galactosidase versus a constitutively active form of HIF-1alpha (AdCA5). There were 245 gene probes that showed at least 1.5-fold increase in expression in response to hypoxia and in response to AdCA5; 325 gene probes showed at least 1.5-fold decrease in expression in response to hypoxia and in response to AdCA5. The largest category of genes down-regulated by both hypoxia and AdCA5 encoded proteins involved in cell growth/proliferation. Many genes up-regulated by both hypoxia and AdCA5 encoded cytokines/growth factors, receptors, and other signaling proteins. Transcription factors accounted for the largest group of HIF-1-regulated genes, indicating that HIF-1 controls a network of transcriptional responses to hypoxia in endothelial cells. Infection of endothelial cells with AdCA5 under nonhypoxic conditions was sufficient to induce increased basement membrane invasion and tube formation similar to the responses induced by hypoxia, indicating that HIF-1 mediates cell-autonomous activation of endothelial cells.
• McGlothlin, J. R., Gao, L., Lavoie, T., Simon, B. A., Easley, R. B., Ma, S. F., Rumala, B. B., Garcia, J. G., & Ye, S. Q. (2005). Molecular cloning and characterization of canine pre-B-cell colony-enhancing factor. Biochemical genetics, 43(3-4), 127-41.
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  During our previous attempt to search for the candidate genes to acute lung injury (ALI), we unexpectedly identified PBEF as the most highly upregulated gene in a canine model of ALI by crosshybridizing canine lung cRNA to the Affymetrix human gene chip HG-U133A. The result suggested that PBEF may be a potential biomarker in ALI. To extend and translate that finding, we have performed the molecular cloning and characterization of canine PBEF cDNA in this study. Deduced amino acid sequence alignment revealed that the PBEF gene is evolutionarily highly conserved, with the canine PBEF protein sequence 96% identical to human PBEF and 94% identical to both murine and rat PBEF counterparts. Canine PBEF protein was successfully expressed both by in vitro transcription coupled with translation in a cell-free system and by transfection of canine PBEF cDNA into the human lung type II alveolar adenocarcinoma cell line A549. The expressed canine PBEF protein was visualized by either an anti-V5 tag peptide polyclonal antibody or an anti-canine PBEF peptide polyclonal antibody. RT-PCR assay indicates that canine PBEF is expressed in canine lung, brain, heart, liver, spleen, kidney, pancreas, and muscle, with liver showing the highest expression,followed by muscle. Isolation of the canine PBEF cDNA and expression of its recombinant protein may provide molecular tools to study the molecular mechanism of ALI in the canine model and to elucidate the potential role of PBEF as an ALI biomarker.
• McVerry, B. J., & Garcia, J. G. (2005). In vitro and in vivo modulation of vascular barrier integrity by sphingosine 1-phosphate: mechanistic insights. Cellular signalling, 17(2), 131-9.
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  Sphingosine 1-phosphate (S1P), a biologically active lipid growth factor, induces robust endothelial cell activation resulting in cellular locomotion, vascular maturation and angiogenesis. Recent work by our laboratory has demonstrated S1P to enhance the cellular barrier function of the vascular endothelium. S1P-induced modulation of vascular permeability is effected through profound cytoskeletal reorganization initiated by cell surface receptor-mediated G protein activation and downstream signaling via the Rho family of small GTPases. The details of the downstream signaling mechanism remain an active area of in vitro investigation. Translational investigation suggests a profound impact of S1P administration in the modulation of edema formation in disease state manifest as acute inflammatory lung injury in which increased vascular permeability is a hallmark feature. These data support an exciting potential therapeutic role for S1P in vascular barrier enhancement necessary for the treatment of critically ill patients.
• Mirzapoiazova, T., Kolosova, I. A., Romer, L., Garcia, J. G., & Verin, A. D. (2005). The role of caldesmon in the regulation of endothelial cytoskeleton and migration. Journal of cellular physiology, 203(3), 520-8.
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  The actin- and myosin-binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non-muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l-CaD cloned from bovine endothelium. Wild-type CaD (WT-CaD) and truncated mutants lacking either the N-terminal myosin-binding site or the C-terminal domain 4b (containing actin- and calmodulin-binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N-terminal myosin-binding site, resulted in decreased affinity to both the detergent-insoluble cytoskeleton and soluble actin. Recombinant WT-CaD co-localized with acto-myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin-binding site are essential for proper localization of CaD in EC. Overexpression of WT-CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD-transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h-CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK-mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling.
• Moreno-vinasco, L., Jacobson, J. R., Garcia, J. G., & Bonde, P. (2005). 56 ATTENUATION OF RODENT LUNG ISCHEMIA/REPERFUSION INJURY BY SPHINGOSINE 1-PHOSPHATE. Journal of Investigative Medicine, 53(2), S366.2-S366. doi:10.2310/6650.2005.00206.55
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  Ischemia/reperfusion (I/R) injury is a common sequelae of lung transplantation occurring in donor lungs within hours and associated with significant morbidity and mortality. I/R injury is characterized by alveolar damage, edema, and inflammation. Despite improvements in lung preservation techniques, I/R injury remains a significant cause of lung transplant failure and novel therapeutic interventions are desperately needed. We employed a rodent model to investigate a potential therapeutic role of sphingosine 1-phosphate (S1P), a potent vascular barrier protective product of platelets, in I/R lung injury. Sprague Dawley rats were subjected to thoracotomy and pulmonary artery ligation (1 hr) followed by a period of reperfusion (2 hr). S1P (1 μm final, i.v) was administered either 15 min prior to ischemia or at the time of reperfusion. Indices of lung vascular permeability and inflammation were assessed including bronchoalveolar lavage (BAL) cell counts, differentials and albumin concentrations. Myeloperoxidase activity (MPO) was measured in left lung homogenates as a reflection of parenchymal leukocyte infiltration. BAL fluid from I/R animals pre-treated with S1P contained decreased total number of cells (32.4% decrease, p = 0.05), neutrophils (63.3% decrease p = 0.04) and albumin concentration (57.2% decrease p = 0.04) compared to controls. In addition, S1P administered prior to ischemia resulted in decreased (63.0% decrease p = 0.04) lung tissue MPO. Our findings suggest that S1P significantly attenuates vascular permeability and inflammation associated with I/R injury. Ultimately, our results may have profound clinical implications as S1P may serve as a useful adjunct to current preservation approaches employed during lung transplantation allowing for improved patient outcomes. Funded by HL 073994
• Nakashima, S., Soong, T. R., Fox-Talbot, K., Qian, Z., Rahimi, S., Wasowska, B. A., Rohde, C. A., Chen, S., Garcia, J. G., & Baldwin, W. M. (2005). Impact of MHC class II incompatibility on localization of mononuclear cell infiltrates to the bronchiolar compartment of orthotopic lung allografts. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 5(4 Pt 1), 694-701.
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  Chronic pathological changes in transplanted lungs are unique because they center on the airways. We examined the relative role of MHC class I and II antigens in causing bronchial pathology in orthotopic lung transplants to rats maintained on cyclosporin A (CsA). Transplants mismatched for MHC class II antigens had significantly more peri-bronchiolar infiltrates than MHC class I incompatible transplants. No significant increase in infiltrates was found in lung transplants incompatible for MHC class I plus II antigens compared to MHC class II antigens alone. Immunohistochemistry demonstrated that MHC class II antigen expression was confined to macrophages in MHC class I incompatible transplants, but was upregulated on bronchial epithelium in transplants with MHC class II incompatibilities. Vascular endothelium was notably devoid of MHC class II antigen expression in all transplants. However, both peri-bronchial and peri-vascular infiltrates were frequently cuffed by alveolar macrophages and type II pneumocytes that expressed MHC class II antigens. PCR analysis demonstrated that IFN-gamma and regulated on activation, normal T cells expressed and secreted (RANTES) were upregulated in MHC class II incompatible transplants. Thus, MHC class II incompatible orthotopic lung transplants in rats maintained on CsA immunosuppression undergo a bronchiolcentric upregulation of alloantigens.
• Natarajan, V., Garcia, J. G., Dudek, S. M., Chiang, E. T., & Camp, S. M. (2005). 24 PROTEIN KINASE C DELTA MEDIATES THROMBIN-INDUCED ENDOTHELIAL BARRIER DYSFUNCTION THROUGH ACTIN STRESS FIBER FORMATION. Journal of Investigative Medicine, 53(2), S360.6-S360. doi:10.2310/6650.2005.00206.23
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  Pulmonary endothelial cells (EC) maintain vascular barrier integrity through dynamic regulation of the actin cytoskeleton. In response to an inflammatory agonist, such as thrombin, endothelial barrier dysfunction occurs in part through actin stress fiber formation and myosin light chain (MLC) phosphorylation. Protein kinase C (PKCs) belong to a family of serine/threonine kinases, which have been implicated in barrier regulation, but the role of specific PKC isoforms in mediating thrombin-induced barrier dysfunction in human pulmonary endothelium is unclear. PKCδ is highly expressed in human lung EC and is activated in response to thrombin. Using pharmacological (rottlerin) and molecular (AdDN PKCδ) approaches to inhibit PKCδ, we assessed the role of PKCδ in regulating stress fiber formation, MLC phosphorylation, and barrier function. Inhibition of PKCδ attenuated both thrombin-induced actin stress fiber formation and endothelial barrier dysfunction, but not MLC phosphorylation. Since actin stress fiber formation and MLC phosphorylation are downstream effectors of RhoA via mDia and Rho kinase signaling pathways, respectively, we examined the relationship between RhoA and PKCδ. Overexpression of constitutive active RhoA resulted in PKCδ activation and stress fiber formation, both of which can be blocked with rottlerin treatment. In addition, Y-27632 (10 μM), a Rho kinase inhibitor, blocked thrombin-induced MLC phosphorylation and attenuated endothelial barrier dysfunction suggesting PKCδ does not signal through RhoA-Rho kinase pathway. Since PKCδ inhibition improved barrier restoration upon thrombin challenge, we next examined the effect of PKCδ inhibition on the barrier-enhancing agent, sphingosine 1-phosphate (S1P), which induces a rapid and sustained endothelial barrier enhancement. PKCδ inhibition delineates cortical actin formation and augments the S1P response. Taken together, our data demonstrate that PKCδ is a downstream target of RhoA and plays a barrier disruptive role in mediating thrombin-induced stress fiber formation and negatively regulating the barrier-enhancing response of S1P.
• Nonas, S. A., Finigan, J. H., Gao, L., & Garcia, J. G. (2005). Functional genomic insights into acute lung injury: role of ventilators and mechanical stress. Proceedings of the American Thoracic Society, 2(3), 188-94.
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  Acute lung injury (ALI) is a complex and devastating illness, often occurring in the setting of sepsis and trauma. Despite recent advances in the understanding and treatment of ALI, pathogenic mechanisms and genetic modifiers in ALI remain incompletely understood. Furthermore, there has been increasing interest in the identification of genetic variations that contribute to ALI susceptibility and severity in order to gain unique insights into ALI pathogenesis and to design novel treatment strategies. However, the sporadic nature of ALI and the lack of family-based cohort studies preclude conventional genomic approaches such as linkage mapping (or "positional cloning"). We have used a "candidate gene approach" with extensive gene expression profiling studies in animal (rat, murine, canine) and human models of ALI to identify potential ALI candidate genes associated with sepsis and ventilator-associated lung injury. These studies, when combined with innovative in silico bioinformatics approaches, revealed both novel (pre--B-cell colony enhancing factor, myosin light chain kinase) and previously identified (interleukin 6, macrophage migration inhibitory factor) gene candidates. Subsequent single nucleotide polymorphism discovery and genotyping studies revealed polymorphisms that demonstrate an influence on ALI susceptibility in patients. These studies indicate that the candidate gene approach is a robust strategy to provide novel insights into the genetic basis of ALI, and the identification of potentially novel therapeutic targets.
• Nonas, S. A., Miller, I. L., & Garcia, J. G. (2005). 34 IDENTIFICATION OF RAT STRAIN-SPECIFIC DIFFERENCES IN SUSCEPTIBILITY TO VENTILATION INDUCED LUNG INJURY. Journal of Investigative Medicine, 53(2), S362.4-S362. doi:10.2310/6650.2005.00206.33
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  Rationale/Methods Little is known regarding the genetic predisposition to the increased vascular permeability and lung inflammation that are cardinal features of acute lung injury (ALI) and mechanical ventilation-induced lung injury (VILI). The comparison of strain-specific responses to high volume mechanical ventilation in rodent models of lung injury is useful in understanding genetic predisposition to ALI/VILI in humans. In the present study, we identified strain-specific differences in susceptibility to high volume mechanical ventilation using an in vivo rat model. Two strains of highly inbred rats, Dahl Salt Sensitive (SS) and Brown Norway (BN), underwent either spontaneous ventilation or high tidal volume ventilation (20 mL/kg, 85 bpm) for two hours. Vascular permeability was measured by tissue Evan9s Blue dye (EBD) deposition and bronchoalveolar lavage fluid (BALF) protein concentrations. Lung inflammation was assessed with BALF cell count and differential. Results There were no significant differences in baseline BALF cell count/differential, BALF protein, or tissue EBD deposition between the two strains. High volume ventilation had no significant effect on BALF cell count or differential in either strain. However, the BN strain rats had a significant increase in both BALF protein (235% increase, p Conclusion Using an in vivo rodent model, we have identified strain-specific differences in susceptibility to VILI that strongly support an important genetic contribution to the development of increased vascular permeability that occurs during ALI. Ongoing genomic studies using parental and consomic rats will allow for the identification of quantitative trait loci (QTLs) and candidate genes important in determining both susceptibility and mechanisms of VILI. NHLBI HL07534.
• Peng, X., Abdulnour, R. E., Sammani, S., Ma, S. F., Han, E. J., Hasan, E. J., Tuder, R., Garcia, J. G., & Hassoun, P. M. (2005). Inducible nitric oxide synthase contributes to ventilator-induced lung injury. American journal of respiratory and critical care medicine, 172(4), 470-9.
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  Inducible nitric oxide synthase (iNOS) has been implicated in the development of acute lung injury. Recent studies indicate a role for mechanical stress in iNOS and endothelial NOS (eNOS) regulation.
• Shikata, Y., Rios, A., Kawkitinarong, K., DePaola, N., Garcia, J. G., & Birukov, K. G. (2005). Differential effects of shear stress and cyclic stretch on focal adhesion remodeling, site-specific FAK phosphorylation, and small GTPases in human lung endothelial cells. Experimental cell research, 304(1), 40-9.
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  Regulation of endothelial cell (EC) permeability by bioactive molecules is associated with specific patterns of cytoskeletal and cell contact remodeling. A role for mechanical factors such as shear stress (SS) and cyclic stretch (CS) in cytoskeletal rearrangements and regulation of EC permeability becomes increasingly recognized. This paper examined redistribution of focal adhesion (FA) proteins, site-specific focal adhesion kinase (FAK) phosphorylation, small GTPase activation and barrier regulation in human pulmonary EC exposed to laminar shear stress (15 dyn/cm2) or cyclic stretch (18% elongation) in vitro. SS caused peripheral accumulation of FAs, whereas CS induced randomly distributed FAs attached to the ends of newly formed stress fibers. SS activated small GTPase Rac without effects on Rho, whereas 18% CS activated without effect on Rac. SS increased transendothelial electrical resistance (TER) in EC monolayers, which was further elevated by barrier-protective phospholipid sphingosine 1-phosphate. Finally, SS induced FAK phosphorylation at Y576, whereas CS induced FAK phosphorylation at Y397 and Y576. These results demonstrate for the first time differential effects of SS and CS on Rho and Rac activation, FA redistribution, site-specific FAK phosphorylation, and link them with SS-mediated barrier enhancement. Thus, our results suggest common signaling and cytoskeletal mechanisms shared by mechanical and chemical factors involved in EC barrier regulation.
• Singelton, P., Jacobson, J. R., Garcia, J. G., & Dudek, S. M. (2005). 29 ENDOTHELIAL CELL BARRIER ENHANCEMENT BY ADENOSINE TRIPHOSPHATE IS MEDIATED BY THE SMALL GUANOSINE TRIPHOSPHATASE RAC. Journal of Investigative Medicine, 53(2), S361.5-S361. doi:10.2310/6650.2005.00206.28
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  Adenosine triphosphate (ATP) is a physiologically relevant agonist released by activated platelets with complex effects mediated via activation of P2 receptors. Although ATP-induced endothelial cell (EC) production of prostacyclin and nitric oxide is recognized, its effects on EC barrier function and vascular permeability remain poorly characterized. Although we recently reported EC barrier enhancement and cytoskeletal activation by ATP the mechanisms involved remains unclear. We have previously identified activation of the small guanosine triphosphatase (GTPase) Rac as key to EC barrier augmentation (simvastatin, sphingosine 1-phosphate) and therefore examined the role of Rac in ATP-induced EC barrier enhancement. Human pulmonary artery EC, grown to confluence overlying gold microelectrodes, were treated with ATP (1-100 μM) or vehicle and transendothelial electrical resistance (TER) measured (n = 3). EC were also grown to ˜ 50% confluence and treated with ATP (10 μM, 1-20 min) and the p21 (Rac) binding domain of PAK1 fused to GST used to precipitate activated Rac GTP complexes followed by Western blotting for Rac and quantification by densitometry. Finally, EC were transfected with Rac siRNA and TER again measured in response to ATP (10 μM). ATP induced rapid, dose-dependent barrier enhancement (TER) with a maximal effect appreciable at 30 min (˜ 38% increase, 10 μM), persisting at 2 h. ATP-treated EC (10 μM) demonstrated a significant increase in Rac activation relative to control with a maximal effect (˜ 4-fold increase) at 10 min (n = 3, p
• Singleton, P. A., Dudek, S. M., Chiang, E. T., & Garcia, J. G. (2005). Regulation of sphingosine 1-phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by S1P1 receptor, PI3 kinase, Tiam1/Rac1, and alpha-actinin. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 19(12), 1646-56.
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  Endothelial cell (EC) barrier dysfunction results in increased vascular permeability observed in inflammation, tumor angiogenesis, and atherosclerosis. The platelet-derived phospholipid sphingosine-1-phosphate (S1P) decreases EC permeability in vitro and in vivo and thus has obvious therapeutic potential. We examined S1P-mediated human pulmonary artery EC signaling and barrier regulation in caveolin-enriched microdomains (CEM). Immunoblotting from S1P-treated EC revealed S1P-mediated rapid recruitment (1 microM, 5 min) to CEMs of the S1P receptors S1P1 and S1P3, p110 PI3 kinase alpha and beta catalytic subunits, the Rac1 GEF, Tiam1, and alpha-actinin isoforms 1 and 4. Immunoprecipitated p110 PI3 kinase catalytic subunits from S1P-treated EC exhibited PIP3 production in CEMs. Immunoprecipitation of S1P receptors from CEM fractions revealed complexes containing Tiam1 and S1P1. PI3 kinase inhibition (LY294002) attenuated S1P-induced Tiam1 association with S1P1, Tiam1/Rac1 activation, alpha-actinin-1/4 recruitment, and EC barrier enhancement. Silencing of either S1P1 or Tiam1 expression resulted in the loss of S1P-mediated Rac1 activation and alpha-actinin-1/4 recruitment to CEM. Finally, silencing S1P1, Tiam1, or both alpha-actinin isoforms 1/4 inhibits S1P-induced cortical F-actin rearrangement and S1P-mediated barrier enhancement. Taken together, these results suggest that S1P-induced recruitment of S1P1 to CEM fractions promotes PI3 kinase-mediated Tiam1/Rac1 activation required for alpha-actinin-1/4-regulated cortical actin rearrangement and EC barrier enhancement.
• Singleton, P. A., Natarajan, V., Garcia, J. G., Dudek, S. M., Chiang, E. T., & Camp, S. M. (2005). 22 FTY720 ENHANCEMENT OF PULMONARY ENDOTHELIAL BARRIER FUNCTION IS S1P1 RECEPTOR INDEPENDENT. Journal of Investigative Medicine, 53(2), S360.4-S360. doi:10.2310/6650.2005.00206.21
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  Rationale Disruption of pulmonary vascular integrity occurs during inflammatory disease processes such as acute lung injury and sepsis, and as a result, regulation of this barrier is an important clinical objective. We have described rapid endothelial cell (EC) barrier enhancement by the platelet-derived phospholipid, sphingosine 1-phosphate (S1P), mediated via S1P1 receptor (S1P1R) ligation. FTY720, an immunosuppressive drug currently in phase III clinical trials, is phosphorylated in vivo to form a structural analogue of S1P. We and others have demonstrated that FTY720 also induces EC barrier enhancement, and we now explore the role of S1P1R ligation in this process. Methods/Results FTY (1 μM) significantly increased cultured human pulmonary artery EC barrier function in a sustained manner as measured by transendothelial electrical resistance (TER), with a delayed onset and slower rate of rise relative to S1P (1 μM). Lipid extraction and 2-phase thin layer chromatography studies revealed that phosphorylation of FTY is not required for barrier enhancement. Both S1P- and FTY-induced barrier enhancement were abolished by pretreatment with pertussis toxin; however, reduction of S1P1R expression via siRNA significantly inhibited S1P-, but not FTY-induced TER elevation. Both S1P and FTY rapidly (within 5 min) induced S1P1R accumulation in membrane lipid rafts, but only S1P stimulated threonine phosphorylation of S1P1R. Inhibition of this phosphorylation by preincubation with the PI3 kinase inhibitor LY294002 (10 μM) blocked S1P-, but not FTY-induced TER elevation. S1P, but not FTY, induced significant phosphorylation of ERK and myosin light chains within 5 min as detected by Western blotting and dramatic actin cytoskeletal rearrangement. Reduction of downstream effectors Rac1 or cortactin via siRNA strategies attenuated S1P-, but not FTY-induced TER elevation. Conclusion These results indicate that FTY720 augments EC barrier function through a novel, S1P1R-independent signaling pathway. HL58064, HL70013-01, FTY720 supplied by Novartis.
• Wadgaonkar, R., Dudek, S. M., Zaiman, A. L., Linz-McGillem, L., Verin, A. D., Nurmukhambetova, S., Romer, L. H., & Garcia, J. G. (2005). Intracellular interaction of myosin light chain kinase with macrophage migration inhibition factor (MIF) in endothelium. Journal of cellular biochemistry, 95(4), 849-58.
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  The endothelial cell Ca2+/calmodulin (CaM)-dependent myosin light chain kinase isoform (EC MLCK) is a multifunctional contractile effector involved in vascular barrier regulation, leukocyte diapedesis, apoptosis, and angiogenesis. The EC MLCK isoform and its splice variants contain a unique N-terminal sequence not present in the smooth muscle MLCK isoform (SM MLCK), which allows novel upregulation of MLCK activation by signaling cascades including p60src. The yeast two-hybrid assay system using the entire EC MLCK1 N-terminus (922 aa) as bait, identified additional stable MLCK binding partners including the 12 KDa macrophage migration inhibitory factor (MIF). This finding was confirmed by cross immunoprecipitation assays under non-denaturing conditions and by GST pull down experiments using GST-N-terminal MLCK (#1-923) and MLCK N-terminal deletion mutants in TNFalpha- and thrombin-stimulated endothelium. This EC MLCK-MIF interaction was shown biochemically and by immunofluorescent microscopy to be enhanced in TNFalpha- and thrombin-stimulated endothelium, both of which induce increased MLCK activity. Thrombin induced the colocalization of an epitope-tagged, full-length MIF fusion protein with phosphorylated MLC along peripheral actin stress fibers. Together these studies suggest that the novel interaction between MIF and MLCK may have important implications for the regulation of both non-muscle cytoskeletal dynamics as well as pathobiologic vascular events that involve MLCK.
• Wadgaonkar, R., Linz-McGillem, L., Zaiman, A. L., & Garcia, J. G. (2005). Endothelial cell myosin light chain kinase (MLCK) regulates TNFalpha-induced NFkappaB activity. Journal of cellular biochemistry, 94(2), 351-64.
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  Tumor necrosis factor (TNFalpha-) generates both apoptotic and survival signals with endothelial cell (EC) survival dependent on nuclear factor kappa-B (NFkappaB) activation, a regulator of anti-apoptotic genes. We previously demonstrated that increased EC contractility, rearrangement of the actin cytoskeleton, and increased myosin light chain (MLC) phosphorylation occurs as a consequence of TNFalpha-induced activation of EC MLC kinase (EC MLCK) and is required for bovine lung EC apoptosis. As the association between MLCK and pro-survival signals such as NFkappaB activation is unknown, we studied the role of MLCK in the regulation of NFkappaB-dependent transactivation in bovine pulmonary artery EC. Both TNFalpha-induced increase in NFkappaB dependent transactivation measured by NFkappaB luciferase reporter assay (approximately fivefold) and nuclear translocation of NFkappaB were significantly inhibited by MLCK-selective inhibitors, KT5926 (60% inhibition of luciferase activity) and ML7 (50% decrease). Furthermore, our data revealed that inhibition of MLCK attenuated the TNFalpha-induced IkappaB phosphorylation, translocation of p65, NFkappaB-DNA binding, and NFkappaB transcriptional activity. Molecular approaches to either reduce EC MLCK expression (AdV EC MLCK antisense construct) or to reduce kinase activity (kinase-dead EC MLCK ATPdel mutant) produced similar attenuation of the TNFalpha-induced NFkappaB response. In contrast, a constitutively active MLCK mutant (EC MLCK1745) enhanced TNFalpha-induced luciferase activity. Together, these novel observations indicate that TNFalpha-induced cytoskeletal rearrangement driven by MLCK activity is necessary for TNFalpha-dependent NFkappaB activation and amplification of pro-survival signals.
• Wang, P., Verin, A. D., Taylor, A. D., Ma, S. F., Kolosova, I. A., Garcia, J. G., & Adyshev, D. M. (2005). 18 THE ROLE OF CPI-17 IN ENDOTHELIAL CELL CYTOSKELETAL ORGANIZATION. Journal of Investigative Medicine, 53(2), S359.6-S359. doi:10.2310/6650.2005.00206.17
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  We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial cell (EC) cytoskeletal remodeling. In smooth muscles protein kinase C (PKC) has been proposed to mediate inhibition of MLCP in response to various agonists via phosphorylation/activation of CPI-17, a specific inhibitor of MLCP. We found that similar to smooth muscle, human lung microvascular endothelial cells (HLMVEC) expressed CPI-17. However, the properties and role of CPI-17 in endothelium are unknown. Ectopic expression of CPI-17 in pulmonary artery EC had no effect on the cytoskeleton under non-stimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions and MLC phosphorylation. Similar to PMA, inflammatory agonist histamine caused activation of CPI-17. Depletion of endogenous CPI-17 in HLMVC significantly attenuated histamine-induced increase in endothelial permeability. Together these data indicate a tight link between CPI-17 activation and changes in EC cytoskeleton. Up to now, no protein partners that bind to CPI-17 have been identified. To further evaluate a role of CPI-17 in EC cytoskeleton we employed BacterioMatch Two-Hybrid System (Stratagene) using Escherichia coli as a host for screening of human lung cDNA library and detection putative CPI-17 binding proteins. Sequence analysis of obtained clones revealed a potential interaction of CPI-17 with 30 proteins, including several key cytoskeletal proteins: alpha spectrin, plakoglobin, plectin and gelsolin. Collectively, these data indicate critical involvement of CPI-17 in the regulation of EC cytoskeleton. HL 58064; HL 67307; HL 68062; AHA and ALA of Maryland.
• Ye, S. Q., Simon, B. A., Maloney, J. P., Zambelli-Weiner, A., Gao, L., Grant, A., Easley, R. B., McVerry, B. J., Tuder, R. M., Standiford, T., Brower, R. G., Barnes, K. C., & Garcia, J. G. (2005). Pre-B-cell colony-enhancing factor as a potential novel biomarker in acute lung injury. American journal of respiratory and critical care medicine, 171(4), 361-70.
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  Although the pathogenic and genetic basis of acute lung injury (ALI) remains incompletely understood, the identification of novel ALI biomarkers holds promise for unique insights. Expression profiling in animal models of ALI (canine and murine) and human ALI detected significant expression of pre-B-cell colony-enhancing factor (PBEF), a gene not previously associated with lung pathophysiology. These results were validated by real-time polymerase chain reaction and immunohistochemistry studies, with PBEF protein levels significantly increased in both bronchoalveolar lavage fluid and serum of ALI models and in cytokine- or cyclic stretch-activated lung microvascular endothelium. We genotyped two PBEF single-nucleotide polymorphisms (SNPs) in a well characterized sample of white patients with sepsis-associated ALI, patients with severe sepsis, and healthy subjects and observed that carriers of the haplotype GC from SNPs T-1001G and C-1543T had a 7.7-fold higher risk of ALI (95% confidence interval 3.01-19.75, p < 0.001). The T variant from the SNP C-1543T resulted in a significant decrease in the transcription rate (1.8-fold; p < 0.01) by the reporter gene assay. Together, these results strongly indicate that PBEF is a potential novel biomarker in ALI and demonstrate the successful application of robust genomic technologies in the identification of candidate genes in complex lung disease.
• Ye, S. Q., Singleton, P. A., Garcia, J. G., Finigan, J. H., Dudek, S. M., Chiang, E. T., & Camp, S. M. (2005). 45 ACTIVATED PROTEIN C ATTENUATES THROMBIN-INDUCED BARRIER DYSFUNCTION IN HUMAN PULMONARY ARTERY ENDOTHELIAL CELLS VIA AN ENDOTHELIAL PROTEIN C RECEPTOR-DEPENDENT MECHANISM. Journal of Investigative Medicine, 53(2), S364.3-S364. doi:10.2310/6650.2005.00206.44
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  Rationale Acute lung injury (ALI) is a frequent complication of sepsis in which disruption of the pulmonary endothelial cell (EC) monolayer leads to increased vascular permeability and life-threatening pulmonary edema. Activated protein C (APC) reduces mortality in sepsis; however, the mechanism through which it exerts its protective effect remains undefined. We hypothesized that APC, via interaction with the endothelial protein C receptor (EPCR), and the EC cytoskeletal protein phosphorylated myosin light chain (phosphoMLC), enhances EC barrier function. Methods Transendothelial electrical resistance (TER) was measured in confluent human pulmonary artery EC monolayers cells treated with APC alone and with the addition of thrombin(0.2 U/mL). Activation of MLC via phosphorylation was assessed using Western blot analysis. Results APC dose-dependently attenuated the thrombin-induced reductions in TER at 1 hr and Western blot analysis of APC (1 μg/mL)-challenged EC demonstrated a 3 fold increase in phosphoMLC (5 min) compared to control. Addition of an EPCR blocking antibody abolished APC-induced barrier-protection and increases in phosphoMLC, while incubation with a control antibody did not alter APC-mediated changes in TER or phosphoMLC levels. Incubation with an antibody against the thrombin receptor, PAR1, had no effect on APC-mediated increases in phosphoMLC. Conclusion APC significantly reduces thrombin-induced barrier dysfunction in ECs via an EPCR-dependent mechanism. APC-mediated alterations in the cytoskeletal protein phosphoMLC was EPCR-dependent but independent of an interaction of APC with PAR1. Modulation of EC barrier function may play a crucial role in the improved survival in patients with sepsis and ALI treated with APC.
• Ye, S. Q., Zhang, L. Q., Adyshev, D., Usatyuk, P. V., Garcia, A. N., Lavoie, T. L., Verin, A. D., Natarajan, V., & Garcia, J. G. (2005). Pre-B-cell-colony-enhancing factor is critically involved in thrombin-induced lung endothelial cell barrier dysregulation. Microvascular research, 70(3), 142-51.
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  Prior genomic and genetic studies identified pre-B-cell colony-enhancing factor (PBEF) as a novel candidate gene and biomarker in acute lung injury (ALI). As increased vascular permeability is a cardinal feature of ALI, we assessed the role of PBEF in in vitro vascular barrier regulation using confluent human pulmonary artery endothelial cell (HPAEC) monolayers. Reductions in PBEF protein expression (>70%) by siRNA significantly attenuated EC barrier dysfunction induced by the potent edemagenic agent, thrombin, reflected by reductions in transendothelial electric resistance (TER, approximately 60% reduction). Furthermore, PBEF siRNA blunted thrombin-mediated increases in Ca(2+) entry, polymerized actin formation, and myosin light chain phosphorylation, events critical to the thrombin-mediated permeability response. Finally, PBEF siRNA also significantly inhibited thrombin-stimulated increase of IL-8 secretion in HPAEC, a chemokine known to induce actin fiber formation and intercellular gap formation of endothelial cells. Taken together, these studies demonstrate that PBEF may be required for complete expression of the thrombin-induced inflammatory response and reveal potentially novel role for PBEF in the regulation of EC Ca(2+)-dependent cytoskeletal rearrangement and endothelial barrier dysfunction. Ongoing studies will continue to address the molecular mechanisms by which PBEF contributes to ALI susceptibility.
• Atwood, C. W., McCrory, D., Garcia, J. G., Abman, S. H., Ahearn, G. S., & , A. C. (2004). Pulmonary artery hypertension and sleep-disordered breathing: ACCP evidence-based clinical practice guidelines. Chest, 126(1 Suppl), 72S-77S.
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  The objective of this article is to review the available data on the relationship between sleep-disordered breathing (SDB) and pulmonary arterial hypertension (PAH), with a focus on the prevalence of SDB in patients with idiopathic PAH (IPAH); the prevalence of PAH in patients with SDB; and the effects of SDB treatment on PAH. The evidence to date suggests that PAH may occur in the setting of SDB, although the prevalence is low. However, pulmonary hypertension (PH) in SDB is most strongly associated with other risk factors, such as left-sided heart disease, parenchymal lung disease, nocturnal desaturation, and obesity. The limited data available also suggest that SDB is uncommon in patients with IPAH. Treatment of SDB with continuous positive airway pressure may lower pulmonary artery pressures when the degree of PH is mild.
• Birukov, K. G., Bochkov, V. N., Birukova, A. A., Kawkitinarong, K., Rios, A., Leitner, A., Verin, A. D., Bokoch, G. M., Leitinger, N., & Garcia, J. G. (2004). Epoxycyclopentenone-containing oxidized phospholipids restore endothelial barrier function via Cdc42 and Rac. Circulation research, 95(9), 892-901.
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  After an acute phase of inflammation or injury, restoration of the endothelial barrier is important to regain vascular integrity and to prevent edema formation. However, little is known about mediators that control restoration of endothelial barrier function. We show here that oxidized phospholipids that accumulate at sites of inflammation and tissue damage are potent regulators of endothelial barrier function. Oxygenated epoxyisoprostane-containing phospholipids, but not fragmented oxidized phospholipids, exhibited barrier-protective effects mediated by small GTPases Cdc42 and Rac and their cytoskeletal, focal adhesion, and adherens junction effector proteins. Oxidized phospholipid-induced cytoskeletal rearrangements resulted in a unique peripheral actin rim formation, which was mimicked by coexpression of constitutively active Cdc42 and Rac, and abolished by coexpression of dominant-negative Rac and Cdc42. Thus, oxidative modification of phospholipids during inflammation leads to the formation of novel regulators that may be critically involved in restoration of vascular barrier function.
• Birukov, K. G., Leitinger, N., Bochkov, V. N., & Garcia, J. G. (2004). Signal transduction pathways activated in human pulmonary endothelial cells by OxPAPC, a bioactive component of oxidized lipoproteins. Microvascular research, 67(1), 18-28.
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  The bioactive component of mildly oxidized low-density lipoproteins, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), activates tissue factor expression and monocyte adhesion to endothelial cells (EC) from systemic circulation, but blocks expression of inflammatory adhesion molecules (VCAM, E-selectin) and neutrophil adhesion associated with EC acute inflammatory response to bacterial lypopolysacharide (LPS). Due to constant exposure to oxygen free radicals, lipids in the injured lung are especially prone to oxidative modification and increased OxPAPC generation. In this study, we focused on OxPAPC-mediated intracellular signaling mechanisms that lead to physiological responses in pulmonary endothelial cells. Our results demonstrate that OxPAPC treatment activated in a time-dependent fashion protein kinase C (PKC), protein kinase A (PKA), Raf/MEK1,2/Erk-1,2 MAP kinase cascade, JNK MAP kinase and transient protein tyrosine phosphorylation in human pulmonary artery endothelial cells (HPAEC), whereas nonoxidized PAPC was without effect. Pharmacological inhibition of PKC and tyrosine kinases blocked activation of Erk-1,2 kinase cascade upstream of Raf. OxPAPC did not affect myosin light chain (MLC) phosphorylation, but increased phosphorylation of cofillin, a molecular regulator of actin polymerization. Finally, OxPAPC induced p60Src-dependent tyrosine phosphorylation of focal adhesion proteins paxillin and FAK. Our results suggest a critical involvement of PKC and tyrosine phosphorylation in OxPAPC-induced activation of Erk-1,2 MAP kinase cascade associated with regulation of specific gene expression, and demonstrate rapid phosphorylation of cytoskeletal proteins, which indicates OxPAPC-induced EC remodeling.
• Birukova, A. A., Birukov, K. G., Smurova, K., Adyshev, D., Kaibuchi, K., Alieva, I., Garcia, J. G., & Verin, A. D. (2004). Novel role of microtubules in thrombin-induced endothelial barrier dysfunction. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 18(15), 1879-90.
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  Disturbances in endothelial cell (EC) barrier regulation are critically dependent upon rearrangements of EC actin cytoskeleton. However, the role of microtubule (MT) network in the regulation of EC permeability is not well understood. We examined involvement of MT remodeling in thrombin-induced EC permeability and explored MT regulation by heterotrimeric G12/13 proteins and by small GTPase Rho. Thrombin induced phosphorylation of MT regulatory protein tau at Ser409 and Ser262 and peripheral MT disassembly, which was linked to increased EC permeability. MT stabilization by taxol attenuated thrombin-induced permeability, actin remodeling, and paracellular gap formation and diminished thrombin-induced activation of Rho and Rho-kinase. Expression of activated Galpha12/13 subunits involved in thrombin-mediated signaling or their effector p115RhoGEF involved in Rho activation caused MT disassembly, whereas p115RhoGEF-specific negative regulator RGS preserved MT from thrombin-induced disassembly. Consistent with these results, expression of activated RhoA and Rho-kinase induced MT disassembly. Conversely, thrombin-induced disassembly of peripheral MT network was attenuated by expression of dominant negative RhoA and Rho-kinase mutants or by pharmacological inhibition of Rho-kinase. Collectively, our data demonstrate for the first time a critical involvement of MT disassembly in thrombin-induced EC barrier dysfunction and indicate G-protein-dependent mechanisms of thrombin-induced MT alteration.
• Birukova, A. A., Liu, F., Garcia, J. G., & Verin, A. D. (2004). Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly. American journal of physiology. Lung cellular and molecular physiology, 287(1), L86-93.
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  Cross talk between the actin cytoskeleton and the microtubule (MT) network plays a critical role in regulation of endothelial permeability. We have previously demonstrated that MT disruption by nocodazole results in increases in MLC phosphorylation, actomyosin contraction, cell retraction, and paracellular gap formation, cardinal features of endothelial barrier dysfunction (Verin AD, Birukova A, Wang P, Liu F, Becker P, Birukov K, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 281: L565-L574, 2001; Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva A, Garcia JG, and Verin AD. J Cell Physiol. In press.). Although activation of PKA opposes barrier-disrupting effects of edemagenic agents on confluent EC monolayers, information about the molecular mechanisms of PKA-mediated EC barrier protection is limited. Our results suggest that MT disassembly alters neither intracellular cAMP levels nor PKA enzymatic activity; however, elevation of cAMP levels and PKA activation by either cholera toxin or forskolin dramatically attenuates the decline in transendothelial electrical resistance induced by nocodazole in human pulmonary EC. Barrier-protective effects of PKA on EC were associated with PKA-mediated inhibition of nocodazole-induced stress fiber formation, Rho activation, phosphorylation of myosin phosphatase regulatory subunit at Thr696, and decreased MLC phosphorylation. In addition, forskolin pretreatment attenuated MT disassembly induced by nocodazole. These results suggest a critical role for PKA activity in stabilization of MT cytoskeleton and provide a novel mechanism for cAMP-mediated regulation of Rho-induced actin cytoskeletal remodeling, actomyosin contraction, and EC barrier dysfunction induced by MT disassembly.
• Birukova, A. A., Smurova, K., Birukov, K. G., Kaibuchi, K., Garcia, J. G., & Verin, A. D. (2004). Role of Rho GTPases in thrombin-induced lung vascular endothelial cells barrier dysfunction. Microvascular research, 67(1), 64-77.
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  Thrombin-induced barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contraction, and gap formation. Phosphorylation of regulatory myosin light chains (MLC) is a key mechanism of endothelial cell (EC) contraction and barrier dysfunction, which is triggered by Ca(2+)/calmodulin-dependent MLC kinase (MLCK) and Rho-associated kinase (Rho-kinase). The role of MLCK in EC barrier regulation has been previously described; however, Rho-mediated pathway in thrombin-induced pulmonary EC dysfunction is not yet precisely characterized. Here, we demonstrate that thrombin-induced decreases in transendothelial electrical resistance (TER) indicating EC barrier dysfunction are universal for human and bovine pulmonary endothelium, and involve membrane translocation and direct activation of small GTPase Rho and its downstream target Rho-kinase. Transient Rho membrane translocation coincided with translocation of upstream Rho activator, guanosine nucleotide exchange factor p115-RhoGEF. Rho mediated activation of downstream target, Rho-kinase induced phosphorylation of the EC MLC phosphatase (MYPT1) at Thr(686) and Thr(850), resulting in MYPT1 inactivation, accumulation of diphospho-MLC, actin remodeling, and cell contraction. The specific Rho-kinase inhibitor, Y27632, abolished MYPT1 phosphorylation, MLC phosphorylation, significantly attenuated stress fiber formation and thrombin-induced TER decrease. Furthermore, expression of dominant-negative Rho and Rho-kinase abolished thrombin-induced stress fiber formation and MLC phosphorylation. Our data, which provide comprehensive analysis of Rho-mediated signal transduction in pulmonary EC, demonstrate involvement of guanosine nucleotide exchange factor, p115-RhoGEF, in thrombin-mediated Rho regulation, and suggest Rho, Rho-kinase, and MYPT1 as potential pharmacological and gene therapy targets critical for prevention of thrombin-induced EC barrier disruption and pulmonary edema associated with acute lung injury.
• Birukova, A. A., Smurova, K., Birukov, K. G., Usatyuk, P., Liu, F., Kaibuchi, K., Ricks-Cord, A., Natarajan, V., Alieva, I., Garcia, J. G., & Verin, A. D. (2004). Microtubule disassembly induces cytoskeletal remodeling and lung vascular barrier dysfunction: role of Rho-dependent mechanisms. Journal of cellular physiology, 201(1), 55-70.
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  Barrier dysfunction of pulmonary endothelial monolayer is associated with dramatic cytoskeletal reorganization, activation of actomyosin contractility, and gap formation. The linkage between the microtubule (MT) network and the contractile cytoskeleton has not been fully explored, however, clinical observations suggest that intravenous administration of anti-cancer drugs and MT inhibitors (such as the vinca alkaloids) can lead to the sudden development of pulmonary edema in breast cancer patients. In this study, we investigated the crosstalk between MT and actomyosin cytoskeleton and characterized specific molecular mechanisms of endothelial cells (EC) barrier dysfunction induced by MT inhibitor nocodazole (ND). Our results demonstrate that MT disassembly by ND induced rapid decreases in transendothelial electrical resistance (TER) and actin cytoskeletal remodeling, indicating EC barrier dysfunction. These effects involved ND-induced activation of Rho GTPase. Rho-mediated activation of its downstream target, Rho-kinase, induced phosphorylation of Rho-kinase effector EC MLC phosphatase (MYPT1) at Thr(696) and Thr(850) resulting in MYPT1 inactivation. Phosphatase inhibition leaded to accumulation of diphospho-MLC, which induced acto-myosin polymerization, stress fiber formation and gap formation. Inhibition of Rho-kinase by Y27632 abolished ND-induced MYPT1 phosphorylation, MLC phosphorylation, and stress fiber formation. In addition, MT preservation via the MT stabilizer paclitaxel, Rho inhibition (via C3 exotoxin, or dominant negative (DN)-Rho, or DN-Rho-kinase) attenuated ND-induced TER decreases, stress fiber formation and MLC phosphorylation. Collectively, our results demonstrate a leading role for Rho-dependent mechanisms in crosstalk between the MT and actomyosin cytoskeleton, and suggest Rho-kinase and MYPT1 as major Rho effectors mediating pulmonary EC barrier disruption in response to ND-induced MT disassembly.
• Borbiev, T., Birukova, A., Liu, F., Nurmukhambetova, S., Gerthoffer, W. T., Garcia, J. G., & Verin, A. D. (2004). p38 MAP kinase-dependent regulation of endothelial cell permeability. American journal of physiology. Lung cellular and molecular physiology, 287(5), L911-8.
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  We have previously shown that thrombin induces endothelial cell barrier dysfunction via cytoskeleton activation and contraction and have determined the important role of endothelial cell myosin light chain kinase (MLCK) in this process. In the present study we explored p38 MAP kinase as a potentially important enzyme in thrombin-mediated endothelial cell contractile response and permeability. Thrombin induces significant p38 MAP kinase activation in a time-dependent manner with maximal effect at 30 min, which correlates with increased phosphorylation of actin- and myosin-binding protein, caldesmon. Both SB-203580 and dominant negative p38 adenoviral vector significantly attenuated thrombin-induced declines in transendothelial electrical resistance. Consistent with these data SB-203580 decreased actin stress fiber formation produced by thrombin in endothelium. In addition, dominant negative p38 had no effect on thrombin-induced myosin light chain diphosphorylation. Thrombin-induced total and site-specific caldesmon phosphorylation (Ser789) as well as dissociation of caldesmon-myosin complex were attenuated by SB-203580 pretreatment. These results suggest the involvement of p38 MAP kinase activities and caldesmon phosphorylation in the MLCK-independent regulation of thrombin-induced endothelial cell permeability.
• Cummings, R., Zhao, Y., Jacoby, D., Spannhake, E. W., Ohba, M., Garcia, J. G., Watkins, T., He, D., Saatian, B., & Natarajan, V. (2004). Protein kinase Cdelta mediates lysophosphatidic acid-induced NF-kappaB activation and interleukin-8 secretion in human bronchial epithelial cells. The Journal of biological chemistry, 279(39), 41085-94.
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  Lysophosphatidic acid (LPA), a potent bioactive lipid, elicits many of its biological actions via the specific G-protein-coupled receptors LPA1, LPA2, LPA3, and LPA4. Recently, we have shown that LPA-induced transactivation of platelet-derived growth factor receptor-beta is regulated by phospholipase D2 in human bronchial epithelial cells (HBEpCs) (Wang, L., Cummings, R. J., Zhao, Y., Kazlauskas, A., Sham, J., Morris, A., Brindley, D. N., Georas, S., and Natarajan, V. (2003) J. Biol. Chem. 278, 39931-39940). Here, we report that protein kinase Cdelta (PKCdelta) mediates LPA-induced NF-kappaB transcription and interleukin-8 (IL-8) secretion in HBEpCs. Treatment of HBEpCs with LPA increased both IL-8 gene and protein expression, which was coupled to Gi and G(12/13) proteins. LPA caused a marked activation of NF-kappaB in HBEpCs as determined by IkappaB phosphorylation and of NF-kappaB nuclear translocation and a strong induction of NF-kappaB promoter-mediated luciferase activity. Furthermore, LPA-activated PKCdelta and the LPA-mediated activation of NF-kappaB and IL-8 production were attenuated by overexpression of dominant-negative PKCdelta and rottlerin. Intratracheal administration of LPA in mice resulted in elevated levels of macrophage inflammatory protein-2, a murine homolog of IL-8, and an influx of neutrophils in the bronchoalveolar lavage fluid. These results demonstrate for the first time that LPA is a potent stimulator of IL-8 production in HBEpCs, which involves PKCdelta/NF-kappaB signaling pathways.
• Dudek, S. M., Jacobson, J. R., Chiang, E. T., Birukov, K. G., Wang, P., Zhan, X., & Garcia, J. G. (2004). Pulmonary endothelial cell barrier enhancement by sphingosine 1-phosphate: roles for cortactin and myosin light chain kinase. The Journal of biological chemistry, 279(23), 24692-700.
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  We recently reported the critical importance of Rac GTPase-dependent cortical actin rearrangement in the augmentation of pulmonary endothelial cell (EC) barrier function by sphingosine 1-phosphate (S1P). We now describe functional roles for the actin-binding proteins cortactin and EC myosin light chain kinase (MLCK) in mediating this response. Antisense down-regulation of cortactin protein expression significantly inhibits S1P-induced barrier enhancement in cultured human pulmonary artery EC as measured by transendothelial electrical resistance (TER). Immunofluorescence studies reveal rapid, Rac-dependent translocation of cortactin to the expanded cortical actin band following S1P challenge, where colocalization with EC MLCK occurs within 5 min. Adenoviral overexpression of a Rac dominant negative mutant attenuates TER elevation by S1P. S1P also induces a rapid increase in cortactin tyrosine phosphorylation (within 30 s) critical to subsequent barrier enhancement, since EC transfected with a tyrosine-deficient mutant cortactin exhibit a blunted TER response. Direct binding of EC MLCK to the cortactin Src homology 3 domain appears essential to S1P barrier regulation, since cortactin blocking peptide inhibits both S1P-induced MLC phosphorylation and peak S1P-induced TER values. These data support novel roles for the cytoskeletal proteins cortactin and EC MLCK in mediating lung vascular barrier augmentation evoked by S1P.
• Grigoryev, D. N., Finigan, J. H., Hassoun, P., & Garcia, J. G. (2004). Science review: searching for gene candidates in acute lung injury. Critical care (London, England), 8(6), 440-7.
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  Acute lung injury (ALI) is a complex and devastating illness, often occurring within the setting of sepsis, and carries an annual mortality rate of 30-50%. Although the genetic basis of ALI has not been fully established, an increasing body of evidence suggests that genetic predisposition contributes to disease susceptibility and severity. Significant difficulty exists, however, in defining the exact nature of these genetic factors, including large phenotypic variance, incomplete penetrance, complex gene-environment interactions, and strong potential for locus heterogeneity. We utilized the candidate gene approach and an ortholog gene database to provide relevant gene ontologies and insights into the genetic basis of ALI. We employed a Medline search of selected basic and clinical studies in the English literature and studies sponsored by the HopGene National Institutes of Health sponsored Program in Genomic Applications. Extensive gene expression profiling studies in animal models of ALI (rat, murine, canine), as well as in humans, were performed to identify potential candidate genes http://www.hopkins-genomics.org/. We identified a number of candidate genes for ALI, with blood coagulation and inflammation gene ontologies being the most highly represented. The candidate gene approach coupled with extensive gene profiling and novel bioinformatics approaches is a valuable way to identify genes that are involved in ALI.
• Grigoryev, D. N., Ma, S. F., Irizarry, R. A., Ye, S. Q., Quackenbush, J., & Garcia, J. G. (2004). Orthologous gene-expression profiling in multi-species models: search for candidate genes. Genome biology, 5(5), R34.
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  Microarray-driven gene-expression profiles are generally produced and analyzed for a single specific experimental model. We have assessed an analytical approach that simultaneously evaluates multi-species experimental models within a particular biological condition using orthologous genes as linkers for the various Affymetrix microarray platforms on multi-species models of ventilator-associated lung injury. The results suggest that this approach may be a useful tool in the evaluation of biological processes of interest and selection of process-related candidate genes.
• Jacobson, J. R., Dudek, S. M., Birukov, K. G., Ye, S. Q., Grigoryev, D. N., Girgis, R. E., & Garcia, J. G. (2004). Cytoskeletal activation and altered gene expression in endothelial barrier regulation by simvastatin. American journal of respiratory cell and molecular biology, 30(5), 662-70.
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  The statins, a class of HMG-CoA reductase inhibitors, directly affect multiple vascular processes via inhibition of geranylgeranylation, a covalent modification essential for Rho GTPase interaction with cell membrane-bound activators. We explored simvastatin effects on endothelial cell actomyosin contraction, gap formation, and barrier dysfunction produced by the edemagenic agent, thrombin. Human pulmonary artery endothelial cells exposed to prolonged simvastatin treatment (5 microM, 16 h) demonstrated significant reductions in thrombin-induced (1 U/ml) barrier dysfunction ( approximately 70% inhibition) with accelerated barrier recovery, as measured by transendothelial resistance. Furthermore, simvastatin attenuated basal and thrombin-stimulated (1 U/ml, 5 min) myosin light chain diphosphorylation and stress fiber formation while dramatically increasing peripheral immunostaining of actin and cortactin, an actin-binding protein, in conjunction with increased Rac GTPase activity. As both simvastatin-induced Rac activation and barrier protection were delayed (maximal after 16 h), we assessed the role of gene expression and protein translation in the simvastatin response. Simultaneous treatment with cycloheximide (10 microg/ml, 16 h) abolished simvastatin-mediated barrier protection. Robust alterations were noted in the expression of cytoskeletal proteins (caldesmon, integrin beta4), thrombin regulatory elements (PAR-1, thrombomodulin), and signaling genes (guanine nucleotide exchange factors) in response to simvastatin by microarray analysis. These novel observations have broad clinical implications in numerous vascular pathobiologies characterized by alterations in vascular integrity including inflammation, angiogenesis, and acute lung injury.
• Jacobson, J. R., Garcia, J. G., & Dudek, S. M. (2004). 28 ENDOTHELIAL CELL BARRIER ENHANCEMENT AND CYTOSKELETAL REARRANGEMENT BY ADENOSINE TRIPHOSPHATE.. Journal of Investigative Medicine, 52(Suppl 2), S350.6-S350. doi:10.1136/jim-52-suppl2-28
• Kawkitinarong, K., Linz-McGillem, L., Birukov, K. G., & Garcia, J. G. (2004). Differential regulation of human lung epithelial and endothelial barrier function by thrombin. American journal of respiratory cell and molecular biology, 31(5), 517-27.
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  Lung epithelial and endothelial barrier dysfunction is critical to the physiologic derangement observed in acute lung injury, but remains poorly understood. We utilized human alveolar epithelial (A549) and endothelial cells (EC) to study cytoskeletal remodeling, myosin light chain (MLC) phosphorylation and barrier regulation evoked by the edemagenic agent, thrombin. Thrombin-challenged human EC monolayers demonstrated increased MLC phosphorylation, actin stress fiber formation and loss of barrier integrity reflected by decreased transmonolayer electrical resistance (TER). In contrast, thrombin produced prominent circumferential localization of actin fibers, increased MLC phosphorylation and increased TER across epithelial monolayers, consistent with barrier protection. Reductions in MLC phosphorylation induced by cell pretreatment with pharmacological inhibitors of MLC kinase (ML-7) and Rho kinase (Y-27632) significantly attenuated thrombin-mediated TER changes and MLC phosphorylation in both lung cell types. Thrombin-produced, time-dependent activation of Rho GTPase in both epithelial and EC, whereas Rac GTPase activation was observed only in A549 cells. Molecular inhibition of Rac activity by adenoviral transfer of dominant-negative Rac mutant abolished thrombin-induced TER increases in alveolar epithelial cells. Finally, A549 cells, but not endothelium, demonstrated increased levels of tight junction proteins (ZO-1 and occludin) after thrombin at the cell-cell interface areas linked to thrombin-elicited barrier protection. These results demonstrate differential pulmonary endothelial and alveolar epithelial barrier regulation via unique actomyosin remodeling and cytoskeletal interactions with tight junction complexes, which confer selective barrier responses to edemagenic stimuli.
• Kittleson, M. M., Ye, S. Q., Irizarry, R. A., Minhas, K. M., Edness, G., Conte, J. V., Parmigiani, G., Miller, L. W., Chen, Y., Hall, J. L., Garcia, J. G., & Hare, J. M. (2004). Identification of a gene expression profile that differentiates between ischemic and nonischemic cardiomyopathy. Circulation, 110(22), 3444-51.
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  Gene expression profiling refines diagnostic and prognostic assessment in oncology but has not yet been applied to myocardial diseases. We hypothesized that gene expression differentiates ischemic and nonischemic cardiomyopathy, demonstrating that gene expression profiling by clinical parameters is feasible in cardiology.
• Kolosova, I. A., Ma, S. F., Adyshev, D. M., Wang, P., Ohba, M., Natarajan, V., Garcia, J. G., & Verin, A. D. (2004). Role of CPI-17 in the regulation of endothelial cytoskeleton. American journal of physiology. Lung cellular and molecular physiology, 287(5), L970-80.
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  We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin AD, Patterson CE, Day MA, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 269: L99-L108, 1995). The molecular mechanisms of endothelial MLCP regulation, however, are not completely understood. In this study we found that, similar to smooth muscle, lung microvascular endothelial cells expressed specific endogenous inhibitor of MLCP, CPI-17. To elucidate the role of CPI-17 in the regulation of endothelial cytoskeleton, full-length CPI-17 plasmid was transiently transfected into pulmonary artery endothelial cells, where the background of endogenous protein is low. CPI-17 had no effect on cytoskeleton under nonstimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions, and MLC phosphorylation compared with untransfected cells. Inflammatory agonist histamine and, to a much lesser extent, thrombin were capable of activating CPI-17. Histamine caused stronger CPI-17 phosphorylation than thrombin. Inhibitory analysis revealed that PKC more significantly contributes to agonist-induced CPI-17 phosphorylation than Rho-kinase. Dominant-negative PKC-alpha abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC-alpha isoform is most likely responsible for CPI-17 activation in the endothelium. Depletion of endogenous CPI-17 in lung microvascular endothelial cell significantly attenuated histamine-induced increase in endothelial permeability. Together these data suggest the potential importance of PKC/CPI-17-mediated pathway in histamine-triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise.
• Leitinger, N., Garcia, J. G., Bochkov, V. N., & Birukov, K. G. (2004). 2 NOVEL ROLE OF OXIDIZED PHOSPHOLIPIDS IN HUMAN PULMONARY ENDOTHELIAL BARRIER REGULATION.. Journal of Investigative Medicine, 52(Suppl 2), S346.2-S346. doi:10.1136/jim-52-suppl2-02
• Li, Y., Uruno, T., Haudenschild, C., Dudek, S. M., Garcia, J. G., & Zhan, X. (2004). Interaction of cortactin and Arp2/3 complex is required for sphingosine-1-phosphate-induced endothelial cell remodeling. Experimental cell research, 298(1), 107-21.
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  Sphingosine-1-phosphate (S1P) induces capillary formation of endothelial cells on Matrigel in accompany with actin assembly and accumulation of cortactin and Arp2/3 complex at the cell-leading edge. Suppression of cortactin expression with a cortactin antisense oligo significantly impaired S1P-induced capillary formation, migration of endothelial cells, and actin assembly at the cell periphery. Overexpression of wild-type cortactin tagged by green fluorescent protein (GFP) increased the S1P-induced tube formation and cell motility, whereas the cells overexpressing the mutant formed poorly capillary network and became less motile in response to S1P. Analysis of distribution in Triton X-100 insoluble fractions demonstrated that the cortactin mutant inhibited the association of wild-type cortactin and Arp2/3 complex with the actin-enriched complex. Furthermore, actin polymerization at and distribution of Arp2/3 complex as well as endogenous cortactin into the cell-leading edge mediated by S1P was disturbed. These data suggest that the interaction between cortactin and Arp2/3 complex plays an important role in S1P-mediated remodeling of endothelial cells.
• Linz-McGillem, L. A., Moitra, J., & Garcia, J. G. (2004). Cytoskeletal rearrangement and caspase activation in sphingosine 1-phosphate-induced lung capillary tube formation. Stem cells and development, 13(5), 496-508.
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  Angiogenesis is a multistep process involving the endothelial cell (EC) cytoskeleton in migration, proliferation, and barrier stabilization. Although precise intracellular pathways by which angiogenic tube formation occurs remain poorly understood, we speculated that interactions between the cytoskeleton and apoptosis are involved and explored cytoskeletal rearrangement and caspase activation in human lung microvascular EC capillary-like tube formation induced by sphingosine 1-phosphate (Sph 1-P) and vascular endothelial growth factor (VEGF). Sph 1-P and VEGF enhance tube formation quantified by a Tube Immaturity Index (TII) generated from the ratio of cell number to tube length, with concomitant morphologic and actomyosin network changes. Angiogenesis was temporally grouped into three stages with early changes characterized by cortical actin localization, whereas midstage tube development demonstrated elongated EC with peripheral actin labeling with transcellular stress fibers. Late tube formation was characterized by broad actin distribution and presence of caspase-positive EC. Phosphorylated MLC immunoreactivity was present at all stages, suggesting that coordinate Rho kinase and MLCK involvement is important to Sph 1-P-induced cell motility; however, chemical inhibition of either MLCK or Rho kinase failed to alter early tube formation. To address whether gaps created by apoptosis expand the lumen, Sph 1-P-induced tubes were differentiated in the presence of caspase inhibitor z-Val-Ala-Asp-fluoromethylketone (zVAD-FMK). Capillary-like tube maturation, but not length, was decreased by zVAD-FMK treatment. These studies suggest that Sph 1-P may induce EC tube formation by regulating early cytoskeletal rearrangement, whereas EC apoptosis within capillary-like tubes is necessary for late stage Sph 1-P-induced tube maturation and lumen formation.
• McVerry, B. J., & Garcia, J. G. (2004). Endothelial cell barrier regulation by sphingosine 1-phosphate. Journal of cellular biochemistry, 92(6), 1075-85.
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  Disruption of vascular barrier integrity markedly increases permeability to fluid and solute and is the central pathophysiologic mechanism of many inflammatory disease processes, including sepsis and acute lung injury (ALI). Dynamic control of the endothelial barrier involves complex signaling to the endothelial cytoskeleton and to adhesion complexes between neighboring cells and between cells and the underlying matrix. Sphingosine 1-phosphate (S1P), a biologically active lipid generated by hydrolysis of membrane lipids in activated platelets, organizes actin into a strong cortical ring and strengthens both intercellular and cell-matrix adherence. The mechanisms by which S1P increases endothelial barrier integrity remain the focus of intense basic research. The downstream structural changes induced by S1P interact to decrease vascular permeability to fluid and solute, which translates into a reduction lung edema formation in animal models of ALI, thus suggesting a potentially life-saving therapeutic role for vascular barrier modulation in critically ill patients.
• McVerry, B. J., Peng, X., Hassoun, P. M., Sammani, S., Simon, B. A., & Garcia, J. G. (2004). Sphingosine 1-phosphate reduces vascular leak in murine and canine models of acute lung injury. American journal of respiratory and critical care medicine, 170(9), 987-93.
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  Excessive mechanical stress is a key component of ventilator-associated lung injury, resulting in profound vascular leak and an intense inflammatory response. To extend our in vitro observations concerning the barrier-protective effects of the lipid growth factor sphingosine 1-phosphate (Sph 1-P), we assessed the ability of Sph 1-P to prevent regional pulmonary edema accumulation in clinically relevant rodent and canine models of acute lung injury induced by combined intrabronchial endotoxin administration and high tidal volume mechanical ventilation. Intravenously delivered Sph 1-P significantly attenuated both alveolar and vascular barrier dysfunction while significantly reducing shunt formation associated with lung injury. Whole lung computed tomographic image analysis demonstrated the capability of Sph 1-P to abrogate significantly the accumulation of extravascular lung water evoked by 6-hour exposure to endotoxin. Axial density profiles and vertical density gradients localized the Sph 1-P response to transitional zones between aerated and consolidated lung regions. Together, these results indicate that Sph 1-P represents a novel therapeutic intervention for the prevention of pulmonary edema related to inflammatory injury and increased vascular permeability.
• Mcgillem, L. L., & Garcia, J. G. (2004). 33 ROLE OF CASPASE ACTIVATION AND APOPTOSIS IN SPHINGOSINE 1-P-INDUCED HUMAN ENDOTHELIAL CELL ANGIOGENESIS AND TUBE MATURATION.. Journal of Investigative Medicine, 52(Suppl 2), S351.4-S351. doi:10.1136/jim-52-suppl2-33
• Newman, J. H., Fanburg, B. L., Archer, S. L., Badesch, D. B., Barst, R. J., Garcia, J. G., Kao, P. N., Knowles, J. A., Loyd, J. E., McGoon, M. D., Morse, J. H., Nichols, W. C., Rabinovitch, M., Rodman, D. M., Stevens, T., Tuder, R. M., Voelkel, N. F., Gail, D. B., & , N. H. (2004). Pulmonary arterial hypertension: future directions: report of a National Heart, Lung and Blood Institute/Office of Rare Diseases workshop. Circulation, 109(24), 2947-52.
• Peng, X., Hassoun, P. M., Sammani, S., McVerry, B. J., Burne, M. J., Rabb, H., Pearse, D., Tuder, R. M., & Garcia, J. G. (2004). Protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury. American journal of respiratory and critical care medicine, 169(11), 1245-51.
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  Our prior in vitro studies indicate that sphingosine 1-phosphate (S1P), a phospholipid angiogenic factor, produces endothelial cell barrier enhancement through ligation of endothelial differentiation gene family receptors. We hypothesized that S1P may reduce the vascular leak associated with acute lung injury and found that S1P infusion produced a rapid and significant reduction in lung weight gain (more than 50%) in the isolated perfused murine lung. The effect of S1P was next assessed in a murine model of LPS-mediated microvascular permeability and inflammation with marked increases in parameters of lung injury at both 6 and 24 hours after intratracheal LPS. Each parameter assessed was significantly reduced by intravenous S1P (1 microM final) and in selected experiments by the S1P analogue FTY720 (0.1 mg/kg, intraperitoneally) delivered 1 hour after LPS. S1P produced an approximately 40-50% reduction in LPS-mediated extravasation of Evans blue dye albumin, bronchoalveolar lavage protein content, and lung tissue myeloperoxidase activity (reflecting phagocyte infiltration). Consistent with systemic barrier enhancement, S1P significantly decreased Evans blue dye albumin extravasation and myeloperoxidase content in renal tissues of LPS-treated mice. These studies indicate that S1P significantly decreases pulmonary/renal vascular leakage and inflammation in a murine model of LPS-mediated acute lung injury and may represent a novel therapeutic strategy for vascular barrier dysfunction.
• Romer, L. H., Linz-mcgillem, L. A., Garcia, J. G., Dudek, S. M., & Birukov, K. G. (2004). 11 REGULATORY ROLES FOR CORTACTIN AND MYOSIN LIGHT CHAIN KINASE IN SPHINGOSINE 1-PHOSPHATE-MEDIATED PULMONARY ENDOTHELIAL BARRIER REGULATION.. Journal of Investigative Medicine, 52(Suppl 2), S347.5-S347. doi:10.1136/jim-52-suppl2-11
• Simon, B. A., Mcverry, B. J., & Garcia, J. G. (2004). 38 PHINGOSINE 1-PHOSPHATE REDUCES REGIONAL ALVEOLAR EDEMA FORMATION IN A CANINE MODEL OF LPS-MEDIATED ACUTE LUNG INJURY.. Journal of Investigative Medicine, 52(Suppl 2), S352.3-S352. doi:10.1136/jim-52-suppl2-38
• Tar, K., Birukova, A. A., Csortos, C., Bakó, E., Garcia, J. G., & Verin, A. D. (2004). Phosphatase 2A is involved in endothelial cell microtubule remodeling and barrier regulation. Journal of cellular biochemistry, 92(3), 534-46.
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  We have recently shown that microtubule (MT) inhibitor, nocodazole (2-5 microM) significantly increases endothelial cells (EC) actomyosin contraction and permeability indicating the importance of MT in maintaining the EC barrier (Verin et al. [2001]: Cell Mol Physiol 281:L565-L574). Okadaic acid (OA, 2-5 nM), a powerful inhibitor of protein phosphatase 2A (PP2A), significantly potentiates the effect of submaximal concentrations of nocodazole (50-200 nM) on transendothelial electrical resistance (TER) suggesting the involvement of PP2A activity in the MT-mediated EC barrier regulation. Immunofluorescent staining of EC revealed that in control cells PP2A distributes in a pattern similar to MT. Consistent with these results, we demonstrated that significant amounts of PP2A were present in MT-enriched EC fractions indicating tight association of PP2A with MT in endothelium. Treatment of EC with OA leads to disappearance of MT-like PP2A staining suggesting dissociation of PP2A from the MT network. Next, we examined the effect of PP2A inhibition on phosphorylation status of MT-associated protein tau, which in its unphosphorylated form promotes MT assembly. OA caused significant increases in tau phosphorylation confirming that tau is a substrate for PP2A in endothelium. Immunofluorescent experiments demonstrated that the OA-induced increases in tau phosphorylation strongly correlated with translocation of phospho-tau to cell periphery and disassembly of peripheral MT. These results suggest the involvement of PP2A-mediated tau dephosphorylation in alteration of EC MT structure and highlight the potential importance of PP2A in the regulation of EC the MT cytoskeleton and barrier function.
• Vorob'ev, I. A., Verin, A. D., Smurova, K. M., Ia, V., Garcia, J. G., Biriukova, A. A., & Alieva, I. B. (2004). Reorganization of microtubule system in pulmonary endothelial cells in response to thrombin treatment.. Tsitologiia, 46(8), 695-703.
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  Thrombin induces rapid and reversible increase of endothelial (EC) barrier permeability associated with actin cytoskeleton remodeling and contraction. The role of microtubules (Mts) in EC barrier regulation compared with actin systems is poorly understood. In this work we studied pathways of Mt and actin regulation in response to thrombin treatment in cultured EC, and the involvement of trimeric G-proteins and in this process. Cells were treated with thrombin, and further analysed using immunofluorescent staining of actin and Mts, digital microscopy and morphometric analysis. In normal cells actin network consists of thin bundles basically located in the cell periphery, Mt density decreases from the cell center to the cell edge. Thrombin (25 nM) induced endothelial dysfunction associated with a rapid (within 5 min) decrease of peripheral Mt network and a slower actin stress fiber formation in the cytoplasm. Pretreatment with Pertussis toxin, which is Gi protein inhibitor, attenuated thrombin-induced stress fiber formation and Mt disassembly. Overexpression of activated G12, G13, Gi and Gq proteins, which are involved in thrombin receptor-mediated signaling, resulted in increasing stress fibers thickness and density and complete Mt disassembly. From the results obtained we suggest that thrombin regulates actin cytoskeleton of EC using local Mt depolymerization at the cell edge.
• Wadgaonkar, R., Pierce, J. W., Somnay, K., Damico, R. L., Crow, M. T., Collins, T., & Garcia, J. G. (2004). Regulation of c-Jun N-terminal kinase and p38 kinase pathways in endothelial cells. American journal of respiratory cell and molecular biology, 31(4), 423-31.
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  The rapid and transient induction of E-selectin gene expression by inflammatory tumor necrosis factor (TNF)-alpha in endothelial cells is mediated by signaling pathways which involve c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) kinase pathways. To explore this regulation, we first observed that in the continuous presence of cytokine TNF, activation of JNK-1 in both nuclear and cytoplasmic compartments peaked at 15-30 min, with activity returning to uninduced levels by 60 min. Phosphorylation of both the p38 kinase and its molecular target, the nuclear transcription factor, activating transcription factor-2, were transient after TNF-alpha or interleukin (IL)-1beta induction. However, cycloheximide treatment prolonged the TNF-alpha-induced JNK-1 kinase activity beyond 60 min, suggesting that protein synthesis is required to limit this signaling cascade. We investigated the possible role of the dual-specificity phosphatases MAPK phosphatase (MKP)-1 and MKP-2 in limiting cytokine-induced MAPK signaling. Maximum induction of MKP-1 mRNA and nuclear protein levels by TNF-alpha or IL-1beta were noted at 60 min and their expression correlated with the termination of JNK kinase activity, whereas nuclear levels of MKP-2 were not significantly affected by treatment with TNF-alpha or IL-1beta. Transient overexpression of MKP-1 demonstrated significant specific inhibition of E-selectin promoter activity consistent with a regulatory role for dual-specificity phosphatases. Inhibition of MKP-1 expression through the use of small interfering RNAs prolonged the cytokine-induced p38 and JNK kinase phosphorylation. Our results suggest that endogenous inhibitors of the MAPK cascade, such as the dual-specificity phosphatases like MKP-1 may be important for the postinduction repression of MAPK activity and E-selectin transcription in endothelial cells. Thus, these inhibitors may play an important role in limiting the inflammatory effects of TNF-alpha and IL-1beta.
• Ye, S. Q., Garcia, J. G., Finigan, J. H., & Dudek, S. M. (2004). 72 ACTIVATED PROTEIN C ATTENUATES THROMBIN-INDUCED BARRIER DYSFUNCTION IN HUMAN PULMONARY ARTERY ENDOTHELIAL CELLS.. Journal of Investigative Medicine, 52(Suppl 2), S358.1-S358. doi:10.1136/jim-52-suppl2-72
• Zambelli-weiner, A., Ye, S. Q., Munoz, M., Lorenzo, M., Grant, A. V., Garcia, J. G., Gao, L., Ehrlich, E., Burki, M., Beaty, T. H., & Barnes, K. C. (2004). Association between variants in the gene encoding myosin light chain kinase (MLCK) and asthma and atopy. The Journal of Allergy and Clinical Immunology, 113(2), S206. doi:10.1016/j.jaci.2004.01.187
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  Abstract Rationale Asthma is characterized by lower airway smooth muscle contraction. Smooth muscle contractility is regulated by pathways involving the phosphorylation of myosin light chain 20 by myosin light chain kinase (MLCK), an enzyme whose expression has been observed to be augmented in severe asthmatics compared to milder patients. Recently we identified 19 single nucleotide polymorphisms (SNPs) in the NH 2 -terminus of the human endothelial cell MLCK gene, three of which lead to an amino acid change (c.36C>A, c.439T>C, c.797C>T). The present study tested for the presence of linkage disequilibrium (LD) between each of these three MLCK polymorphisms and the asthma phenotype as well as total serum IgE (tIgE) levels in a family-based study of asthma in Barbados. Methods Genomic DNA was extracted from peripheral blood samples of 743 subjects (in 125 pedigrees) and screened for the MLCK SNPs using PCR-based restriction enzyme assays. The Family-Based Association Test was used to assess association in the presence of linkage. Results We found evidence for linkage and disequilibrium to both asthma and an apparent quantitative trait locus (QTL) controlling tIgE levels for all 3 SNPs. When analyzing asthma itself, the heterozygous genotype was more common than expected under the null hypothesis, suggesting this genotype is at higher risk. When tIgE levels were examined, the heterozygotes showed higher tIgE than expected under a co-dominant model ( P ≤0.001). Conclusions These findings suggest a role for variants in the endothelial cell MLCK gene in a sample of multiplex asthmatic families from Barbados.
• Zhang, Y., Zhang, Y., Zerbe, L., Xu, Y., Worthen, G. S., Whitsett, J. A., Wesselkamper, S. C., Wert, S. E., Weaver, T. E., Tsukada, K., Tomlinson, C. R., Tichelaar, J. W., Taveira-dasilva, A., Stearman, R. S., Steagall, W. K., Silverman, E. K., Schwartz, D. A., Sartor, M. A., Regev, A., , Redline, S., et al. (2004). Functional genomics of lung disease. Second annual Pittsburgh International Lung Conference, October 2003.. American journal of respiratory cell and molecular biology, 31(2 Suppl), S1-81. doi:10.1165/rcmb.2004-0071su
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  Jeffrey A. Whitsett, Cindy J. Bachurski, Kathleen C. Barnes, Paul A. Bunn, Jr., Lisa M. Case, Donald N. Cook, Denise Crooks, Mark W. Duncan, Lori Dwyer-Nield, Robert C. Elston, Michael B. Fessler, Wilbur A. Franklin, Nir Friedman, Joe G. N. Garcia, Mark W. Geraci, Connie Glasgow, Stephan W. Glasser, William D. Hardie, Lisa M. Henning, Gary L. Johnson, Kamon Kawkitinarong, Robert L. Keith, Thomas R. Korfhagen, George D. Leikauf, Stephen B. Liggett, Kenneth C. Malcolm, Alvin M. Malkinson, Thomas J. Mariani, Susan A. McDowell, Dennis W. McGraw, Mario Medvedovic, Joel Moss, Lawrence M. Nogee, Stephanie Nonas, Gustavo Pacheco-Rodriguez, Lyle J. Palmer, David G. Peters, Daniel R. Prows, Susan Redline, Aviv Regev, Maureen A. Sartor, David A. Schwartz, Edwin K. Silverman, Wendy K. Steagall, Robert S. Stearman, Angelo Taveira-DaSilva, Jay W. Tichelaar, Craig R. Tomlinson, Katsuya Tsukada, Timothy E. Weaver, Susan E. Wert, Scott C. Wesselkamper, G. S. Worthen, Yan Xu, Laura Zerbe, Yi Zhang, Yingze Zhang, Augustine M. K. Choi, and Naftali Kaminski
• Becker, P. M., Kazi, A. A., Wadgaonkar, R., Pearse, D. B., Kwiatkowski, D., & Garcia, J. G. (2003). Pulmonary vascular permeability and ischemic injury in gelsolin-deficient mice. American journal of respiratory cell and molecular biology, 28(4), 478-84.
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  Gelsolin is a potent actin filament regulatory protein that controls cytoskeletal assembly and disassembly. Because cellular gelsolin deficiency leads to pronounced actin stress fiber formation and defective chemotaxis, and similar cytoskeletal remodeling results in endothelial barrier dysfunction, we hypothesized that gelsolin deficient mice would exhibit increased vascular permeability. To test this hypothesis, we compared baseline lung lavage (BAL) protein concentration, wet/dry weight ratio, and osmotic reflection coefficient for albumin (sigma alb) in gelsolin-deficient (gsn-/-) and C57BL/6 (wild-type) mice. In addition, we assessed lung permeability in response to ischemia by evaluating BAL protein concentration after 4, 8, or 24 h of left pulmonary arterial (LPA) occlusion, and lung wet/dry weight ratio and histology after 24 h of LPA occlusion, in gsn-/- and wild-type animals, as compared with control and sham-operated mice. Baseline measurements revealed that BAL protein concentration was 18-fold higher in gsn-/- than in wild-type mice, whereas sigma alb averaged 0.62 + 0.15 in wild-type, as compared with 0.31 + 0.05 in gsn-/- animals, indicating that gelsolin deficiency caused increased pulmonary vascular permeability. Ischemia increased lung permeability (BAL protein and lung wet/dry weight) in both wild-type and gsn-/- mice. However, whereas the fold-increase in BAL protein concentration was less in gsn-/- mice (2- to 4-fold) as compared with wild-type (22- to 34-fold), the duration of ischemia-induced permeability changes was prolonged. Lung wet/dry weight and gross histology following ischemia were comparable in wild-type and gsn-/- animals. These data suggest that gelsolin significantly contributes to maintenance of vascular barrier function in the lung.
• Birukov, K. G., Jacobson, J. R., Flores, A. A., Ye, S. Q., Birukova, A. A., Verin, A. D., & Garcia, J. G. (2003). Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch. American journal of physiology. Lung cellular and molecular physiology, 285(4), L785-97.
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  Ventilator-induced lung injury syndromes are characterized by profound increases in vascular leakiness and activation of inflammatory processes. To explore whether excessive cyclic stretch (CS) directly causes vascular barrier disruption or enhances endothelial cell sensitivity to edemagenic agents, human pulmonary artery endothelial cells (HPAEC) were exposed to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of strain. CS produced rapid (10 min) increases in myosin light chain (MLC) phosphorylation, activation of p38 and extracellular signal-related kinase 1/2 MAP kinases, and actomyosin remodeling. Acute (15 min) and chronic (48 h) CS markedly enhanced thrombin-induced MLC phosphorylation (2.1-fold and 3.2-fold for 15-min CS at 5 and 18% elongation and 2.1-fold and 3.1-fold for 48-h CS at 5 and 18% elongation, respectively). HPAEC preconditioned at 18% CS, but not at 5% CS, exhibited significantly enhanced thrombin-induced reduction in transendothelial electrical resistance but did not affect barrier protective effect of sphingosine-1-phosphate (0.5 microM). Finally, expression profiling analysis revealed a number of genes, including small GTPase rho, apoptosis mediator ZIP kinase, and proteinase activated receptor-2, to be regulated by CS in an amplitude-dependent manner. Thus our study demonstrates a critical role for the magnitude of CS in regulation of agonist-mediated pulmonary endothelial cell permeability and strongly suggests phenotypic regulation of HPAEC barrier properties by CS.
• Bogatcheva, N. V., Dudek, S. M., Garcia, J. G., & Verin, A. D. (2003). Mitogen-activated protein kinases in endothelial pathophysiology. Journal of investigative medicine : the official publication of the American Federation for Clinical Research, 51(6), 341-52.
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  Endothelial cells continuously respond to extracellular stimuli such as chemical signals produced by circulating blood elements or mechanical forces such as shear stress. Proinflammatory cytokines, mitogens, reactive oxygen species, and shear stress trigger signal molecules to initiate multiple intracellular pathways, which often converge at mitogen-activated protein (MAP) kinase activation. The MAP kinase superfamily represents a burgeoning area of clinical investigation for treatment of various inflammatory and oncologic diseases and plays an essential role in mediating response to infection, ischemia/reperfusion injury, and vessel healing and remodeling through regulation of such diverse phenomena as endothelial cell proliferation, migration, apoptosis, and endothelial barrier function. The downstream effects of MAP kinase activation include modulation of gene expression via up-regulation of various transcription factors. In addition to these sustained effects, MAP kinases coordinate more immediate responses that affect dynamic cytoskeletal rearrangements necessary for cell migration and regulation of barrier function. This review discusses the important regulatory roles of MAP kinases in the vital physiologic functions of endothelium, focusing mainly on the role of MAP kinases in the maintenance of endothelial barrier.
• Borbiev, T., Verin, A. D., Birukova, A., Liu, F., Crow, M. T., & Garcia, J. G. (2003). Role of CaM kinase II and ERK activation in thrombin-induced endothelial cell barrier dysfunction. American journal of physiology. Lung cellular and molecular physiology, 285(1), L43-54.
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  We have previously shown that thrombin-induced endothelial cell barrier dysfunction involves cytoskeletal rearrangement and contraction, and we have elucidated the important role of endothelial cell myosin light chain kinase and the actin- and myosin-binding protein caldesmon. We evaluated the contribution of calmodulin (CaM) kinase II and extracellular signal-regulated kinase (ERK) activation in thrombin-mediated bovine pulmonary artery endothelial cell contraction and barrier dysfunction. Similar to thrombin, infection with a constitutively active adenoviral alpha-CaM kinase II construct induced significant ERK activation, indicating that CaM kinase II activation lies upstream of ERK. Thrombin-induced ERK-dependent caldesmon phosphorylation (Ser789) was inhibited by either KN-93, a specific CaM kinase II inhibitor, or U0126, an inhibitor of MEK activation. Immunofluorescence microscopy studies revealed phosphocaldesmon colocalization within thrombin-induced actin stress fibers. Pretreatment with either U0126 or KN-93 attenuated thrombin-mediated cytoskeletal rearrangement and evoked declines in transendothelial electrical resistance while reversing thrombin-induced dissociation of myosin from nondenaturing caldesmon immunoprecipitates. These results strongly suggest the involvement of CaM kinase II and ERK activities in thrombin-mediated caldesmon phosphorylation and both contractile and barrier regulation.
• Cappola, T. P., Cope, L., Cernetich, A., Barouch, L. A., Minhas, K., Irizarry, R. A., Parmigiani, G., Durrani, S., Lavoie, T., Hoffman, E. P., Ye, S. Q., Garcia, J. G., & Hare, J. M. (2003). Deficiency of different nitric oxide synthase isoforms activates divergent transcriptional programs in cardiac hypertrophy. Physiological genomics, 14(1), 25-34.
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  Decreased nitric oxide synthase (NOS) activity induces left ventricular hypertrophy (LVH), but the transcriptional pathways mediating this effect are unknown. We hypothesized that specific NOS isoform deletion (NOS3 or NOS1) would activate different transcriptional programs in LVH. We analyzed cardiac expression profiles (Affymetrix MG-U74A) from NOS-/- mice using robust multi-array average (RMA). Of 12,422 genes analyzed, 47 genes were differentially expressed in NOS3-/- and 67 in NOS1(-/-) hearts compared with wild type (WT). Only 16 showed similar changes in both NOS-/- strains, most notably decreased heat-shock proteins (HSP10, 40, 70, 86, 105). Hypertrophied NOS1-/- hearts had unique features, including decreased myocyte-enriched calcineurin interacting protein and paradoxical downregulation of fetal isoforms (alpha-skeletal actin and brain natriuretic peptide). Cluster analyses demonstrated that NOS1 deletion caused more pronounced changes in the myocardial transcriptome than did NOS3 deletion, despite similar cardiac phenotypes. These findings suggest that the transcriptional basis for LVH varies depending on the inciting biochemical stimulus. In addition, NOS isoforms appear to play distinct roles in modulating cardiac structure.
• Dudek, S. M., & Garcia, J. G. (2003). Rho family of guanine exchange factors (GEFs) in cellular activation: who's dancing? And with whom?. Circulation research, 93(9), 794-5.
• Dull, R. O., Dinavahi, R., Schwartz, L., Humphries, D. E., Berry, D., Sasisekharan, R., & Garcia, J. G. (2003). Lung endothelial heparan sulfates mediate cationic peptide-induced barrier dysfunction: a new role for the glycocalyx. American journal of physiology. Lung cellular and molecular physiology, 285(5), L986-95.
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  The endothelial glycocalyx is believed to play a major role in microvascular permeability. We tested the hypothesis that specific components of the glycocalyx, via cytoskeletal-mediated signaling, actively participate in barrier regulation. With the use of polymers of arginine and lysine as a model of neutrophil-derived inflammatory cationic proteins, we determined size- and dose-dependent responses of cultured bovine lung microvascular endothelial cell permeability as assessed by transendothelial electrical resistance (TER). Polymers of arginine and lysine >11 kDa produced maximal barrier dysfunction as demonstrated by a 70% decrease in TER. Monomers of l-arginine and l-lysine did not alter barrier function, suggesting a cross-linking requirement of cell surface "receptors". To test the hypothesis that glycosaminoglycans (GAGs) are candidate receptors for this response, we used highly selective enzymes to remove specific GAGs before polyarginine (PA) treatment and examined the effect on TER. Heparinase III attenuated PA-induced barrier dysfunction by 50%, whereas heparinase I had no effect. To link changes in barrier function with structural alterations, we examined actin organization and syndecan localization after PA. PA induced actin stress fiber formation and clustering of syndecan-1 and syndecan-4, which were significantly attenuated by heparinase III. PA-induced cytoskeletal rearrangement and barrier function did not involve myosin light chain kinase (MLCK) or p38 MAPK, as ML-7, a specific MLCK inhibitor, or SB-20358, a p38 MAPK inhibitor, did not alter PA-induced barrier dysfunction. In summary, lung endothelial cell heparan sulfate proteoglycans are key participants in inflammatory cationic peptide-induced signaling that links cytoskeletal reorganization with subsequent barrier dysfunction.
• Garcia, J. G., & D'amore, P. (2003). Microvascular Research: Editorial. Microvascular Research, 65(1), 1. doi:10.1016/s0026-2862(02)00022-5
• Girgis, R. E., Li, D., Zhan, X., Garcia, J. G., Tuder, R. M., Hassoun, P. M., & Johns, R. A. (2003). Attenuation of chronic hypoxic pulmonary hypertension by simvastatin. American journal of physiology. Heart and circulatory physiology, 285(3), H938-45.
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  The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) have been shown to improve multiple normal endothelial cell functions and inhibit vascular wall cell proliferation. We hypothesized that one such agent, simvastatin, would attenuate chronic hypoxic pulmonary hypertension. Male adult Sprague-Dawley rats were exposed (14 days) to normoxia (N), normoxia plus once-a-day administered simvastatin (20 mg/kg ip) (NS), hypoxia (10% inspired O2 fraction) (H), or hypoxia plus simvastatin (HS). Mean pulmonary artery pressure, measured in anesthetized, ventilated rats with an open-chest method, was reduced from 25 +/- 2 mmHg in H to 18 +/- 1 in HS (P < 0.001) but did not reach normoxic values (12 +/- 1 mmHg). Similarly, right ventricular/left ventricular plus interventricular septal weight was reduced from 0.53 +/- 0.02 in the H group to 0.36 +/- 0.02 in the HS group (P < 0.001). The increased hematocrit in H (0.65 +/- 0.02) was prevented by simvastatin treatment (0.51 +/- 0.01, P < 0.001). Hematocrit was similar in N versus NS. Alveolar vessel muscularization and medial thickening of vessels 50-200 microM in diameter induced by hypoxia were also significantly attenuated in the HS animals. Lung endothelial nitric oxide synthase (eNOS) protein expression in the HS group was less than H (P < 0.01) but was similar in N versus NS. We conclude that simvastatin treatment potently attenuates chronic hypoxic pulmonary hypertension and polycythemia in rats and inhibits vascular remodeling. Enhancement of lung eNOS expression does not appear to be involved in mediating this effect.
• Jacobson, J. R., & Garcia, J. G. (2003). Genomics made functional in ventilator-associated lung injury. American journal of respiratory and critical care medicine, 168(9), 1023-5.
• Parinandi, N. L., Kleinberg, M. A., Usatyuk, P. V., Cummings, R. J., Pennathur, A., Cardounel, A. J., Zweier, J. L., Garcia, J. G., & Natarajan, V. (2003). Hyperoxia-induced NAD(P)H oxidase activation and regulation by MAP kinases in human lung endothelial cells. American journal of physiology. Lung cellular and molecular physiology, 284(1), L26-38.
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  Hyperoxia increases reactive oxygen species (ROS) production in vascular endothelium; however, the mechanisms involved in ROS generation are not well characterized. We determined the role and regulation of NAD(P)H oxidase in hyperoxia-induced ROS formation in human pulmonary artery endothelial cells (HPAECs). Exposure of HPAECs to hyperoxia for 1, 3, and 12 h increased the generation of superoxide anion, which was blocked by diphenyleneiodonium but not by rotenone or oxypurinol. Furthermore, hyperoxia enhanced NADPH- and NADH-dependent and superoxide dismutase- or diphenyleneiodonium-inhibitable ROS production in HPAECs. Immunohistocytochemistry and Western blotting revealed the presence of gp91, p67 phox, p22 phox, and p47 phox subcomponents of NADPH oxidase in HPAECs. Transfection of HPAECs with p22 phox antisense plasmid inhibited hyperoxia-induced ROS production. Exposure of HPAECs to hyperoxia activated p38 MAPK and ERK, and inhibition of p38 MAPK and MEK1/2 attenuated the hyperoxia-induced ROS generation. These results suggest a role for MAPK in regulating hyperoxia-induced NAD(P)H oxidase activation in HPAECs.
• Petrache, I., Birukov, K., Zaiman, A. L., Crow, M. T., Deng, H., Wadgaonkar, R., Romer, L. H., & Garcia, J. G. (2003). Caspase-dependent cleavage of myosin light chain kinase (MLCK) is involved in TNF-alpha-mediated bovine pulmonary endothelial cell apoptosis. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 17(3), 407-16.
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  Cytoskeletal proteins are key participants in the cellular progression to apoptosis. Our previous work demonstrated the critical dependence of actomyosin rearrangement and MLC phosphorylation in TNF-alpha-induced endothelial cell apoptosis. As these events reflect the activation of the multifunctional endothelial cell (EC) MLCK isoform, we assessed the direct role of EC MLCK in the regulation of TNF-alpha-induced apoptosis. Bovine pulmonary artery endothelial cells expressing either an adenovirus encoding antisense MLCK cDNA (Ad.GFP-AS MLCK) or a dominant/negative EC MLCK construct (EC MLCK-ATPdel) resulted in marked reductions in MLCK activity and TNF-alpha-mediated apoptosis. In contrast, a constitutively active EC MLCK lacking the carboxyl-terminal autoinhibitory domains (EC MLCK-1745) markedly enhanced the apoptotic response to TNF-alpha. Immunostaining in GFP-EC MLCK-expressing cells revealed colocalization of caspase 8 and EC MLCK along actin stress fibers after TNF-alpha. TNF-alpha induced the caspase-dependent cleavage of EC MLCK-1745 in transfected endothelial cells, which was confirmed by mass spectroscopy with in vitro cleavage by caspase 3 at LKKD (D1703). The resulting MLCK fragments displayed significant calmodulin-independent kinase activity. These studies convincingly demonstrate that novel interactions between the apoptotic machinery and EC MLCK exist that regulate the endothelial contractile apparatus in TNF-alpha-induced apoptosis.
• Petrache, I., Birukova, A., Ramirez, S. I., Garcia, J. G., & Verin, A. D. (2003). The role of the microtubules in tumor necrosis factor-alpha-induced endothelial cell permeability. American journal of respiratory cell and molecular biology, 28(5), 574-81.
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  Tumor necrosis factor (TNF)-alpha, a major proinflammatory cytokine, triggers endothelial cell activation and barrier dysfunction which are implicated in the pathogenesis of pulmonary edema associated with acute lung injury syndromes. The mechanisms of TNF-alpha-induced vascular permeability are not completely understood. Our initial experiments demonstrated that TNF-alpha-induced decreases in transendothelial electrical resistance across human pulmonary artery endothelial cells are independent of myosin light chain phosphorylation catalyzed by either myosin light chain kinase or Rho kinase. We next assessed the involvement of another cytoskeletal component, the tubulin-based microtubule network, and found TNF-alpha to induce a decrease in stable tubulin content and partial dissolution of peripheral microtubule network as evidenced by anti-acetylated tubulin and anti-beta-tubulin immunofluorescent staining, respectively. Microtubule-stabilizing agents, paclitaxel and epothilone B, significantly attenuated TNF-alpha-induced decreases in transendothelial electrical resistance, inhibited the cytokine-induced increases in actin stress fibers, formation of intercellular gap, and restored the TNF-alpha-compromised vascular endothelial (VE)-cadherin-based cell-cell junctions. Importantly, neither TNF-alpha nor paclitaxel treatment was associated with endothelial cell apoptosis. Inhibition of p38 mitogen-activated protein kinase by SB203580 significantly attenuated TNF-alpha-induced microtubule destabilization, actin rearrangement, and endothelial barrier dysfunction. These results strongly suggest the involvement of microtubule rearrangement in TNF-alpha-induced endothelial cell permeability via p38 mitogen-activated protein kinase activation.
• Petrache, I., Crow, M. T., Neuss, M., & Garcia, J. G. (2003). Central involvement of Rho family GTPases in TNF-alpha-mediated bovine pulmonary endothelial cell apoptosis. Biochemical and biophysical research communications, 306(1), 244-9.
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  In our recent studies, we defined a critical role for increased levels of myosin light chain (MLC) phosphorylation, a regulatory event in the interaction between actin and myosin in TNF-alpha-induced pulmonary endothelial cell actomyosin rearrangement and apoptosis. The Rho GTPase effector, Rho kinase is an important signaling effector governing levels of MLC phosphorylation which contributes to plasma membrane blebbing in several models of apoptosis. In this study, we directly assessed the role of Rho kinase in TNF-alpha-induced endothelial cell microfilament rearrangement and apoptosis. Inhibition of RhoA GTPase activity by the overexpression of dominant negative RhoA attenuates TNF-alpha-triggered stress fiber formation, consistent with Rho activation as a key event in TNF-alpha-induced cytoskeletal rearrangement. Furthermore, pharmacologic inhibition of Rho kinase as well as dominant negative RhoA overexpression dramatically reduced TNF-alpha-induced bovine endothelial apoptosis reflected by nucleosomal fragmentation as well as caspase 7, 3, and 8 activation. These results indicate that Rho kinase-dependent cytoskeletal rearrangement is critical for early apoptotic events, possibly in the assembly of the death-inducing signaling complex leading to initiator and effector caspase activation, and suggest a novel role for Rho GTPases in endothelial cell apoptosis.
• Schaphorst, K. L., Chiang, E., Jacobs, K. N., Zaiman, A., Natarajan, V., Wigley, F., & Garcia, J. G. (2003). Role of sphingosine-1 phosphate in the enhancement of endothelial barrier integrity by platelet-released products. American journal of physiology. Lung cellular and molecular physiology, 285(1), L258-67.
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  In vitro and in vivo evidence indicates that circulating platelets affect both vascular integrity and hemostasis. How platelets enhance the permeability barrier of the vascular endothelium is not well understood. We measured the effect of isolated human platelets on human pulmonary artery endothelial cell (EC) barrier integrity by monitoring transmonolayer electrical resistance. EC barrier function was significantly increased by the addition of platelets ( approximately 40% maximum, 2.5 x 106 platelets/ml). Platelet supernatants, derived from 2.5 x 106 platelets/ml, reproduced the barrier enhancement and reversed the barrier dysfunction produced by the edemagenic agonist thrombin, which implicates a soluble barrier-promoting factor. The barrier-enhancing effect of platelet supernatants was heat stable but was attenuated by either charcoal delipidation (suggesting a vasoactive lipid mediator) or pertussis toxin, implying involvement of a Gialpha-coupled receptor signal transduction pathway. Sphingosine-1-phosphate (S1P), a sphingolipid that is released from activated platelets, is known to ligate G protein-coupled EC differentiation gene (EDG) receptors, increase EC electrical resistance, and reorganize the actin cytoskeleton (Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, and English D. J Clin Invest 108: 689-701, 2001). Infection of EC with an adenoviral vector expressing an antisense oligonucleotide directed against EDG-1 but not infection with control vector attenuated the barrier-enhancing effect of both platelet supernatants and S1P. These results indicate that a major physiologically relevant vascular barrier-protective mediator produced by human platelets is S1P.
• Shikata, Y., Birukov, K. G., & Garcia, J. G. (2003). S1P induces FA remodeling in human pulmonary endothelial cells: role of Rac, GIT1, FAK, and paxillin. Journal of applied physiology (Bethesda, Md. : 1985), 94(3), 1193-203.
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  Sphingosine 1-phosphate (S1P) enhances human pulmonary endothelial monolayer integrity via Rac GTPase-dependent formation of a cortical actin ring (Garcia et al. J Clin Invest 108: 689-701, 2001). The mechanisms underlying this response are not well understood but may involve rapid redistribution of focal adhesions (FA) as attachment sites for actin filaments. We evaluate the effects of S1P on the redistribution of paxillin, FA kinase (FAK), and the G protein-coupled receptor kinase-interacting proteins (GITs). S1P induced Rac GTPase activation and cortical actin ring formation at physiological concentrations (0.5 microM), whereas 5 microM S1P caused prominent stress fiber formation and activation of Rho and Rac GTPases. S1P (0.5 microM) stimulated the tyrosine phosphorylation of FAK Y(576), and paxillin was linked to FA disruption and redistribution to the cell periphery. Furthermore, S1P induced a transient association of GIT1 with paxillin and redistribution of the GIT2-paxillin complex to the cell cortical area without affecting GIT2-paxillin association. These results suggest a role of FA rearrangement in S1P-mediated barrier enhancement via Rac- and GIT-mediated processes.
• Shikata, Y., Birukov, K. G., Birukova, A. A., Verin, A., & Garcia, J. G. (2003). Involvement of site-specific FAK phosphorylation in sphingosine-1 phosphate- and thrombin-induced focal adhesion remodeling: role of Src and GIT. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 17(15), 2240-9.
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  Sphingosine-1 phosphate (S1P) and thrombin are agents with profound but divergent effects on vascular endothelial cell (EC) barrier properties. We have previously reported that S1P-induced focal adhesion (FA) remodeling involves interactions between focal adhesion kinase (FAK), paxillin, and G-protein-coupled receptor kinase-interacting proteins GIT1 and GIT2 and suggested a critical involvement of focal adhesions in the EC barrier regulation. In this study, we examined redistribution of FA proteins (FAK, paxillin, GIT1, and GIT2) and site-specific FAK tyrosine phosphorylation in human pulmonary artery endothelial cells stimulated with thrombin. In contrast to S1P, which we have shown to induce peripheral translocation of FA proteins associated with cortical actin ring formation, thrombin caused the redistribution of FA proteins to the ends of the newly formed massive stress fibers. S1P and thrombin induced distinct patterns of FAK site-specific phosphorylation with the FAK Y576 phosphorylation site targeted by SIP challenge and phosphorylation of three FAK sites (Y397, Y576, and Y925) in response to thrombin stimulation. Pharmacological inhibition of Src with Src-specific inhibitor PP2 abolished S1P-induced translocation of FA proteins, cortical actin ring formation, and FAK [Y576] phosphorylation. However, PP2 failed to alter thrombin-induced morphological changes and exhibited only partial inhibition of FAK site-specific tyrosine phosphorylation. These observations highlight the differential mechanisms of focal adhesion protein complex remodeling and FAK activation by S1P and thrombin and link differential FA remodeling to EC barrier regulation.
• Simon, B. A., Mcverry, B. J., & Garcia, J. G. (2003). Sphingosine-1-phosphate Attenuates Endotoxin-induced Lung Injury in a Canine Mode. Chest, 124(4), 181S. doi:10.1378/chest.124.4_meetingabstracts.181s-a
• Usatyuk, P. V., Fomin, V. P., Shi, S., Garcia, J. G., Schaphorst, K., & Natarajan, V. (2003). Role of Ca2+ in diperoxovanadate-induced cytoskeletal remodeling and endothelial cell barrier function. American journal of physiology. Lung cellular and molecular physiology, 285(5), L1006-17.
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  Diperoxovanadate (DPV), a potent inhibitor of protein tyrosine phosphatases and activator of tyrosine kinases, alters endothelial barrier function via signaling pathways that are incompletely understood. One potential pathway is Src kinase-mediated tyrosine phosphorylation of proteins such as cortactin that regulate endothelial cell (EC) cytoskeleton assembly. As DPV modulates endothelial cell signaling via protein tyrosine phosphorylation, we determined the role of DPV-induced intracellular free calcium concentration ([Ca2+]i) in activation of Src kinase, cytoskeletal remodeling, and barrier function in bovine pulmonary artery endothelial cells (BPAECs). DPV in a dose- and time-dependent fashion increased [Ca2+]i, which was partially blocked by the calcium channel blockers nifedipine and Gd3+. Treatment of cells with thapsigargin released Ca2+ from the endoplasmic reticulum, and subsequent addition of DPV caused no further change in [Ca2+]i. These data suggest that DPV-induced [Ca2+]i includes Ca release from the endoplasmic reticulum and Ca influx through store-operated calcium entry. Furthermore, DPV induced an increase in protein tyrosine phosphorylation, phosphorylation of Src and cortactin, actin remodeling, and altered transendothelial electrical resistance in BPAECs. These DPV-mediated effects were significantly attenuated by BAPTA (25 microM), a chelator of [Ca2+]i. Immunofluorescence studies reveal that the DPV-mediated colocalization of cortactin with peripheral actin was also prevented by BAPTA. Chelation of extracellular Ca2+ by EGTA had marginal effects on DPV-induced phosphorylation of Src and cortactin; actin stress fibers formation, however, affected EC barrier function. These data suggest that DPV-induced changes in [Ca2+]i regulate endothelial barrier function using signaling pathways that involve Src and cytoskeleton remodeling.
• Wadgaonkar, R., Nurmukhambetova, S., Zaiman, A. L., & Garcia, J. G. (2003). Mutation analysis of the non-muscle myosin light chain kinase (MLCK) deletion constructs on CV1 fibroblast contractile activity and proliferation. Journal of cellular biochemistry, 88(3), 623-34.
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  Smooth muscle myosin light chain kinase (MLCK) is a multifunctional molecule composed of an N-terminal actin binding domain, a central kinase domain, and C-terminal calmodulin- and myosin-binding domains. We previously cloned and characterized a novel MLCK isoform from endothelial cells (EC MLCK) consisting of 1,914 amino acids displaying a higher molecular weight (210 kDa) and a novel-amino-terminal stretch of 922 amino acids not shared by the smooth muscle isoform (smMLCK, 150 kDa). To further define the role of specific EC MLCK motifs in endothelial and non-muscle cells, we constructed two epitope-tagged EC MLCK deletion mutants in mammalian expression vectors lacking either the C-terminal auto-inhibitory and calmodulin-binding domain (EC MLCK1745) or the ATP-binding site (EC MLCKATPdel). Expression of EC MLCK1745 in CV1 fibroblasts showed increased basal actin stress fiber formation, which was markedly enhanced after tumor necrosis factor (TNF-alpha) or thrombin treatment. Distribution of EC MLCK1745 was largely confined to stress fibers, cortical actin filaments, and focal adhesion contacts, and co-localized with myosin light chains (MLCs) diphosphorylated on Ser(19) and Thr(18). In contrast, immunofluorescence staining demonstrated that EC MLCKATPdel abolished thrombin- and TNFalpha-induced stress fiber formation and MLC phosphorylation, suggesting this kinase-dead mutant functions as a dominant-negative MLCK construct, thereby confirming the role of EC MLCK in stress fiber formation. Finally, we compared the serum-stimulated growth rate of mutant MLCK-transfected fibroblasts to sham controls, and found EC MLCK1745 to augment thymidine incorporation whereas EC MLCKATPdel reduced CV1 growth rates. These data demonstrate the necessary role for MLCK in driving the contractile apparatus via MLC phosphorylation, which can alter fibroblast growth and contractility.
• Zhao, Z., Yatish, A. J., Wong, G. W., Tewari, M., Suresh, S., Surendranath, V., Steen, H., Shivashankar, H. N., Shanker, K., Schiemann, W. P., Saravana, R., Roepstorff, P., Rashmi, B. P., Ramya, M. A., Pevsner, J., Peri, S., Pandey, A., Padma, N., Niranjan, V., , Navarro, J. D., et al. (2003). Development of human protein reference database as an initial platform for approaching systems biology in humans.. Genome research, 13(10), 2363-71. doi:10.1101/gr.1680803
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  Human Protein Reference Database (HPRD) is an object database that integrates a wealth of information relevant to the function of human proteins in health and disease. Data pertaining to thousands of protein-protein interactions, posttranslational modifications, enzyme/substrate relationships, disease associations, tissue expression, and subcellular localization were extracted from the literature for a nonredundant set of 2750 human proteins. Almost all the information was obtained manually by biologists who read and interpreted >300,000 published articles during the annotation process. This database, which has an intuitive query interface allowing easy access to all the features of proteins, was built by using open source technologies and will be freely available at http://www.hprd.org to the academic community. This unified bioinformatics platform will be useful in cataloging and mining the large number of proteomic interactions and alterations that will be discovered in the postgenomic era.
• Birukov, K. G., Birukova, A. A., Dudek, S. M., Verin, A. D., Crow, M. T., Zhan, X., DePaola, N., & Garcia, J. G. (2002). Shear stress-mediated cytoskeletal remodeling and cortactin translocation in pulmonary endothelial cells. American journal of respiratory cell and molecular biology, 26(4), 453-64.
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  Hemodynamic forces in the form of shear stress (SS) and mechanical strain imposed by circulating blood are recognized factors involved in the control of systemic endothelial cell (EC) cytoskeletal structure and function. However, the effects of acute SS on pulmonary endothelium have not been precisely characterized, nor the mechanism of rapid SS-induced EC cytoskeletal rearrangement understood. We exposed bovine and human pulmonary EC monolayers to laminar SS (10 dynes/cm2) in a parallel plate flow chamber and observed increased actin stress fiber formation 15 min after application of flow. Acute SS-induced pronounced cortical cytoskeletal rearrangement characterized by myosin light chain kinase (MLCK)- and Rho-associated kinase (RhoK)-dependent accumulation of diphosphorylated regulatory myosin light chains (MLC) in the cortical actin ring, junctional protein tyrosine phosphorylation, and transient peripheral translocation of cortactin, an actin-binding protein involved in the regulation of actin polymerization. SS-induced cortactin translocation was independent of Erk-1,2 MAP kinase, p60(Src), MLCK, or RhoK activities, and unaffected by overexpression of a cortactin mutant lacking four major p60(Src) phosphorylation sites. However, both SS-induced transient cortactin translocation and cytoskeletal reorientation in response to sustained (24 h) SS was abolished in cells overexpressing either dominant negative Rac 1 or a dominant negative construct of its downstream target, p21-activated kinase (PAK)-1. Our results suggest a potential role for cortactin in the SS-induced EC cortical cytoskeletal remodeling and demonstrate a novel mechanism of Rac GTPase-dependent regulation of the pulmonary endothelial cytoskeleton by SS.
• Bogatcheva, N. V., Garcia, J. G., & Verin, A. D. (2002). Molecular mechanisms of thrombin-induced endothelial cell permeability. Biochemistry. Biokhimiia, 67(1), 75-84.
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  Confluent endothelium serves as a selective barrier between the vascular space of blood vessels and underlying tissues. Compromised barrier function of the endothelium in response to inflammation mediators, such as thrombin, is accompanied by reversible cell rounding and interendothelial gap formation. Endothelial barrier integrity substantially depends on the cytoskeleton, which ensures actin stress fiber formation and via actomyosin-driven contraction regulates cell shape and adhesion. Recent studies have shown the sequence of events that mediate signal transduction in endothelial cells. Binding of thrombin with its receptor initiates activation of heterotrimeric G-proteins, which, in turn, entails a decrease in cAMP level in the cell, increase in intracellular Ca2+ and diacylglycerol concentration, and activation of the small G-protein Rho. Phosphorylation of myosin light chains as a result of activation of myosin light chain kinase and inactivation of myosin phosphatases stimulates stress fiber formation and triggers actomyosin contraction. In addition, thrombin-induced rearrangement in the endothelial cytoskeleton is regulated by Ca2+/calmodulin-dependent protein kinase, protein kinase C, and tyrosine protein kinases. This review focuses on presently known biochemical mechanisms of cell response to thrombin and their role in endothelial barrier dysfunction.
• Bogatcheva, N. V., Garcia, J. G., & Verin, A. D. (2002). Role of tyrosine kinase signaling in endothelial cell barrier regulation. Vascular pharmacology, 39(4-5), 201-12.
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  Phosphorylation of proteins on tyrosine acts as a reversible and specific trigger mechanism, forming or disrupting regulatory connections between proteins. Tyrosine kinases and phosphatases participate in multiple cellular processes, and considerable evidence now supports a role for tyrosine phosphorylation in vascular permeability. A semipermeable barrier between the vascular compartment and the interstitium is maintained by the integrity of endothelial monolayer, controlling movement of fluids, macromolecules and leucocytes. Barrier function is regulated by the adjustment of paracellular gaps between endothelial cells (ECs) by two antagonistic forces, centripetal cytoskeletal tension and opposing cell-cell and cell-matrix adhesion forces. Both cytoskeletal filaments and adhesion sites are intimately linked in complex machinery which is regulated by multiple signaling events including protein phosphorylation and/or protein translocation to specific intracellular positions. Tyrosine kinases occupy key positions in the mechanism controlling cell responses mediated through various cell surface receptors, which use tyrosine phosphorylation to transduce extracellular signal.
• Cummings, R. J., Parinandi, N. L., Zaiman, A., Wang, L., Usatyuk, P. V., Garcia, J. G., & Natarajan, V. (2002). Phospholipase D activation by sphingosine 1-phosphate regulates interleukin-8 secretion in human bronchial epithelial cells. The Journal of biological chemistry, 277(33), 30227-35.
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  Sphingosine 1-phosphate (S1P), a potent bioactive sphingolipid, has been implicated in many critical cellular events, including a regulatory role in the pathogenesis of airway inflammation. We investigated the participation of S1P as an inflammatory mediator by assessing interleukin-8 (IL-8) secretion and phospholipase D (PLD) activation in human bronchial epithelial cells (Beas-2B). S1P(1), S1P(3), S1P(4), S1P(5), and weak S1P(2) receptors were detected in Beas-2B and primary human bronchial epithelial cells. S1P stimulated a rapid activation of PLD, which was nearly abolished by pertussis toxin (PTX) treatment, consistent with S1P receptor/G(i) protein coupling. S1P also markedly induced Beas-2B secretion of IL-8, a powerful neutrophil chemoattractant and activator, in a PTX-sensitive manner. This S1P-mediated response was dependent on transcription as indicated by a strong induction of IL-8 promoter-mediated luciferase activity in transfected Beas-2B cells and a complete inhibition by actinomycin D. Beas-2B exposure to 1-butanol, which converts the PLD-generated phosphatidic acid (PA) to phosphatidylbutanol by a transphosphatidylation reaction, significantly attenuated the S1P-induced IL-8 secretion, indicating the involvement of PLD-derived PA in the signaling pathway. Inhibition of 12-O-tetradecanoyl-phorbol-13-acetate-stimulated IL-8 production by 1-butanol further strengthened this observation. Blocking protein kinase C and Rho kinase also attenuated S1P-induced IL-8 secretion. Our data suggest that PLD-derived PA, protein kinase C, and Rho are important signaling components in S1P-mediated IL-8 secretion by human bronchial epithelial cells.
• Dudek, S. M., Birukov, K. G., Zhan, X., & Garcia, J. G. (2002). Novel interaction of cortactin with endothelial cell myosin light chain kinase. Biochemical and biophysical research communications, 298(4), 511-9.
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  Inflammatory mediators such as thrombin evoke increases in vascular permeability through activation of endothelial contractile mechanisms which involve increased levels of MLC phosphorylation catalyzed by Ca(2+)/calmodulin-dependent myosin light chain kinase (MLCK). We previously noted that the high molecular weight endothelial MLCK isoform (EC MLCK) is stably associated with a complex containing p60(src) and 80kDa cortactin, an actin-binding protein and known p60(src) target. In this study we have utilized in vitro binding assays to confirm specific interaction between EC MLCK and cortactin. Tyrosine phosphorylation of either EC MLCK (Y(464), Y(471)) or cortactin (Y(421), Y(466), and Y(482)) by p60(src) significantly increased this direct association. Site-specific antibody and peptide studies subsequently confirmed EC MLCK AA #972-979 and 1019-1025 as sites of cortactin interaction. EC MLCK-cortactin interaction in vitro failed to modulate MLCK enzymatic activity but appeared to inhibit EC MLCK binding to F-actin, while EC MLCK abolished cortactin-mediated augmentation of Arp2/3-stimulated actin polymerization. These data suggest that cortactin-EC MLCK interaction may be a novel determinant of endothelial cortical actin-based cytoskeletal rearrangement.
• Dull, R. O., & Garcia, J. G. (2002). Leukocyte-induced microvascular permeability: how contractile tweaks lead to leaks. Circulation research, 90(11), 1143-4.
• English, D., Brindley, D. N., Spiegel, S., & Garcia, J. G. (2002). Lipid mediators of angiogenesis and the signalling pathways they initiate. Biochimica et biophysica acta, 1582(1-3), 228-39.
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  Investigations carried out over the past 3 years have implicated a key role for sphingosine 1-phosphate (SPP) in angiogenesis and blood vessel maturation. SPP is capable of inducing almost every aspect of angiogenesis and vessel maturation in vitro, including endothelial cell chemotaxis, survival, proliferation, capillary morphogenesis and adherence antigen deployment, as well as stabilizing developing endothelial cell monolayers and recruitment of smooth muscle cells to maturing vessels. Acting in conjunction with protein angiogenic factors, SPP induces prolific vascular development in many established models of angiogenesis in vivo. Thus, SPP is a unique, potent and multifaceted angiogenic agent. While SPP induces angiogenic effects by ligating members of the endothelial differentiation gene (EDG) G-protein-coupled family of receptors, recent studies suggest that endogenously produced SPP may also account for the ability of tyrosine kinase receptors to induce cell migration. Thus, SPP provides a clear link between tyrosine kinase and G-protein-coupled receptor agonists involved in the angiogenic response. However, the mechanisms by which SPP exerts its effects on vascular cells remain unclear, conflicting and controversial. Precise definition of the signalling pathways by which SPP induces specific aspects of the angiogenic response promises to lead to new and effective therapeutic approaches to regulate angiogenesis at sites of tissue damage, neoplastic transformation and inflammation. This review will trace the discovery of SPP as a novel angiogenic factor as it outlines present information on the signalling pathways by which SPP induces its effects on cells of the developing vascular bed.
• Garcia, J. G., Wang, P., Schaphorst, K. L., Becker, P. M., Borbiev, T., Liu, F., Birukova, A., Jacobs, K., Bogatcheva, N., & Verin, A. D. (2002). Critical involvement of p38 MAP kinase in pertussis toxin-induced cytoskeletal reorganization and lung permeability. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 16(9), 1064-76.
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  Bordetella pertussis is an important cause of infection in humans worldwide, with full expression of the syndrome associated with characteristic increases in lung permeability and airway edema. The exact cellular mechanisms by which pertussis toxin (PTX) exerts pulmonary toxicity remain unknown, but may involve its ability to ADP-ribosylate-specific G-proteins. We determined that PTX directly and reproducibly reduced lung endothelial and epithelial cell barrier function in vitro and in vivo assessed by decreases in transmonolayer electrical resistance (TER) and isolated perfused lung preparations. Alterations in lung permeability began approximately 30 min after PTX and were dependent on intrinsic ADP-ribosyltransferase activity, as neither the cell binding beta-oligomer subunit or a genetically engineered PTX mutant (devoid of ADP-ribosyltransferase activity) altered TER. PTX-induced barrier dysfunction was associated with mild increases in F-actin stress fiber formation and causally linked to p38 MAP kinase activities. PTX-mediated p38 MAP kinase activation did not involve either p42/p44 ERK, p60src, Rho family of GTPases, or phosphatidylinositol-3' kinase pathways. PTX-mediated decreases in TER were temporally linked to phosphorylation of the actin binding proteins Hsp27 and caldesmon, known substrates for the Ser/Thr kinase MAPKAP2, whose activity is regulated by p38 MAP kinase. In addition to defining novel signaling pathways involved in PTX-induced respiratory pathophysiology, these data suggest that the direct cell-activating effects of PTX be carefully considered as a potential limitation to its use as a tool in signal transduction analysis.
• Harvey, K., Siddiqui, R. A., Sliva, D., Garcia, J. G., & English, D. (2002). Serum factors involved in human microvascular endothelial cell morphogenesis. The Journal of laboratory and clinical medicine, 140(3), 188-98.
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  Our previous studies have demonstrated that lipid and protein angiogenic factors operate in tandem to induce optimal angiogenic responses in vivo. This study was undertaken to clarify the nature of the substances in human serum that are responsible for its remarkable ability to promote capillary morphogenesis in vitro. The ability of dilute (2%) human serum to promote the morphogenic differentiation of human dermal microvascular endothelial cells on Matrigel supports was depleted by more than 50% by treatment of the serum with activated charcoal, a procedure that effectively removes biologically active lipid growth factors. The remainder of the activity within serum was lost on heating to 60 degrees C for 60 minutes, indicating the involvement of a protein in the response. The ability of charcoal-treated serum to promote capillary morphogenesis was completely restored by the addition of sphingosine 1-phosphate (SPP, 500 nmol/L), but other lipids thought to be released into serum during clotting were ineffective. In addition, basic fibroblast growth factor (bFGF) effectively restored the ability of heat-treated serum to promote endothelial cell morphogenesis, but other protein growth factors, including vascular endothelial growth factor and platelet-derived growth factor, were ineffective. Together, SPP and bFGF were as effective as whole serum in promoting capillary morphogenesis. Responses to purified SPP were entirely sensitive to the effects of preexposure of the cells to pertussis toxin, whereas responses to bFGF were entirely pertussis toxin-resistant. Consistent with our hypothesis that two distinct factors in serum play a role in promoting capillary morphogenesis, responses induced by serum were inhibited approximately 50% by preexposure of endothelial cells to pertussis toxin. We conclude that platelet-released SPP acts in conjunction with circulating bFGF to promote capillary formation by microvascular endothelial cells. Lipid and protein growth factors apparently exert complementary roles in the angiogenic response, as demonstrated by their ability to promote chemotaxis, angiogenic differentiation, and angiogenesis in vivo.
• Harvey, K., Welch, Z., Kovala, A. T., Garcia, J. G., & English, D. (2002). Comparative analysis of in vitro angiogenic activities of endothelial cells of heterogeneous origin. Microvascular research, 63(3), 316-26.
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  Endothelial cells are dynamic participants in many aspects of host defense, innate immunity, inflammation, angiogenesis, and vasculogenesis, but the interpretation of studies of their responses is often clouded by the source of the cells under observation. Thus, it is not clear which endothelial cell type should be utilized in in vitro studies to clarify the basis and physiological relevance of essential processes, including chemotactic migration and morphogenic differentiation. In this study, we compared responses of endothelial cells of a variety of origins, as well as an immortalized cell line, using both protein growth factors and biologically active lipid mediators as agonists. While cells of divergent origin displayed marked differences in the extent of their responsiveness, with a few notable exceptions, their pattern of responsiveness to receptor-dependent stimuli was remarkably similar. Moreover, even the immortalized endothelial cell line Py-4-l migrated in a pattern consistent to that seen with primary cells in culture although the immortalized cells failed to form capillarylike structures under any of the conditions tested. We conclude that although the immortalized endothelial cell line Py-4-l is not appropriate for investigations of endothelial cell morphogenic responses, cultured cells from other sources, including arteries, veins, and capillaries, often provide qualitatively similar results to divergent metabolic stimuli.
• Li, D., Tuder, R. M., Li, D., Johns, R. A., Girgis, R. E., & Garcia, J. G. (2002). Attenuation of hypoxic pulmonary hypertension in rats by the HMG-CoA reductase inhibitor, simvastatin. Journal of Heart and Lung Transplantation, 21(1), 149. doi:10.1016/s1053-2498(01)00695-7
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  Pulmonary arterial hypertension (PAH) is characterized by excessive vasoconstriction and vascular wall thickening which is frequently progressive leading to right heart failure and death. Current therapeutic options are limited. The HMG-CoA reductase inhibitors, statins, have been shown to have several beneficial effects in the systemic circulation beyond their cholesterollowering action, including improvements in endothelial cell function and inhibition of vascular smooth muscle cell proliferation. We hypothesized that these actions would attenuate hypoxic pulmonary hypertension in rats. Adult, male Sprague-Dawley rats were exposed to 4 days of 10% FiO2 (H) vs normoxia (N) and treated with pre-activated simvastatin 20 mg/kg/d sq (HS, NS) vs vehicle (N 4 in each group). H had signficantly greater right ventricular/LV septum and lung to body weight ratios and decreased proportion of non-muscularinized vessels 80 microns on smooth muscle alpha-actin stained sections, compared with N (0.36 /-0.005 vs 0.24 /0.006, 7.6 /0.16 mg/gm vs 5.5 /0.12, and 30 /4%vs 62 /2%, respectively; P 0.001 for all). Treatment with simvastatin (HS) was associated with 10% (0.32 /0.003) and 14% (6.5 /0.2 mg/gm) reduction in the first 2 indices and a 20% (49 /1.7%) increase in the latter (P 0.05 for all 3) compared with H. Conclusion: This preliminary data suggests that simvastatin is capable of attenuating hypoxic pulmonary hypertension and may play a role in the treatment of PAH.
• Liu, F., Schaphorst, K. L., Verin, A. D., Jacobs, K., Birukova, A., Day, R. M., Bogatcheva, N., Bottaro, D. P., & Garcia, J. G. (2002). Hepatocyte growth factor enhances endothelial cell barrier function and cortical cytoskeletal rearrangement: potential role of glycogen synthase kinase-3beta. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 16(9), 950-62.
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  The stabilization of endothelial cell (EC) barrier function within newly formed capillaries is a critical feature of angiogenesis. We examined human lung EC barrier regulation elicited by hepatocyte growth factor (HGF), a recognized angiogenic factor and EC chemoattractant. HGF rapidly and dose-dependently elevated transendothelial electrical resistance (TER) of EC monolayers (>50% increase at 100 ng/ml), with immunofluorescence microscopic evidence of both cytoplasmic actin stress fiber dissolution and strong augmentation of the cortical actin ring. HGF rapidly stimulated phosphatidylinositol 3'-kinase, ERK, p38 mitogen-activated protein kinase, and protein kinase C activities. Pharmacological inhibitor studies demonstrated each pathway to be intimately involved in HGF-induced increases in TER, cortical actin thickening, and phosphorylation of the Ser/Thr glycogen synthase kinase-3beta (GSK-3beta), a potential target for the HGF barrier-promoting response. GSK-3beta phosphorylation was strongly correlated with reductions in both HGF-induced TER and enhanced beta-catenin immunoreactivity observed at cell-cell junctions. Our data suggest a model in which HGF-mediated EC cytoskeletal rearrangement and barrier enhancement depend critically on the activation of a complex kinase cascade that converges at GSK-3beta to increase the availability of beta-catenin, thereby enhancing endothelial junctional integrity and vascular barrier function.
• Becker, P. M., Verin, A. D., Booth, M. A., Liu, F., Birukova, A., & Garcia, J. G. (2001). Differential regulation of diverse physiological responses to VEGF in pulmonary endothelial cells. American journal of physiology. Lung cellular and molecular physiology, 281(6), L1500-11.
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  The mechanisms responsible for the divergent physiological responses of endothelial cells to vascular endothelial growth factor (VEGF) are incompletely understood. We hypothesized that VEGF elicits increased endothelial permeability and cell migration via differential activation of intracellular signal transduction pathways. To test this hypothesis, we established a model of VEGF-induced endothelial barrier dysfunction and chemotaxis with bovine pulmonary endothelial cells. We compared the effects of VEGF on transendothelial electrical resistance (TER), actin cytoskeletal remodeling, and chemotaxis of lung endothelial cells and then evaluated the role of the mitogen-activated protein kinases (MAPKs) p38 and extracellular signal-regulated kinase (ERK)1/2 in VEGF-mediated endothelial responses. The dose response of pulmonary arterial and lung microvascular endothelial cells to VEGF differed when barrier regulation and chemotaxis were evaluated. Inhibition of tyrosine kinase, phosphoinositol 3-kinase, or p38 MAPK significantly attenuated VEGF-mediated TER, F-actin remodeling, and chemotaxis. VEGF-mediated decreased TER was also significantly attenuated by inhibition of ERK1/2 MAPK but not by inhibition of fetal liver kinase-1 (flk-1) or Src kinase. In contrast, VEGF-mediated endothelial migration was not attenuated by ERK1/2 inhibition but was abolished by inhibition of either flk-1 or Src kinase. These data suggest potential mechanisms by which VEGF may differentially mediate physiological responses in vivo.
• Birukov, K. G., Csortos, C., Marzilli, L., Dudek, S., Ma, S. F., Bresnick, A. R., Verin, A. D., Cotter, R. J., & Garcia, J. G. (2001). Differential regulation of alternatively spliced endothelial cell myosin light chain kinase isoforms by p60(Src). The Journal of biological chemistry, 276(11), 8567-73.
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  The Ca(2+)/calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210-214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch (amino acids 436-505) that contains potentially important consensus sites for phosphorylation by p60(Src) kinase (Lazar, V., and Garcia, J. G. (1999) Genomics 57, 256-267). We have now found that both recombinant EC MLCK splice variants exhibit comparable enzymatic activities but a 2-fold reduction of V(max), and a 2-fold increase in K(0.5 CaM) when compared with the SM MLCK isoform, whereas K(m) was similar in the three isoforms. However, only EC MLCK-1 is readily phosphorylated by purified p60(Src) in vitro, resulting in a 2- to 3-fold increase in EC MLCK-1 enzymatic activity (compared with EC MLCK-2 and SM MLCK).