- Associate Professor
- Scientific Director, Pulmonary and Endothelial Research Core
- Associate Professor, Medicine
- Associate Professor, Physiological Sciences - GIDP
- Associate Professor, Clinical Translational Sciences
- Ph.D. Pharmaceutical Sciences
- University of South Carolina, Columbia, South Carolina, United States
- Synthesis, Pharmacology and Pharmaceutical Analysis of MS23, a Dual Action Antihypertensive Agent.
- Faculty Service Recognition Award
- UA COMP, Fall 2020
- Central Society for Clinical and Translational Research, Spring 2015
No activities entered.
ResearchCTS 900 (Spring 2021)
DissertationCTS 920 (Fall 2020)
DissertationPS 920 (Spring 2020)
ResearchCTS 900 (Spring 2020)
DissertationPS 920 (Fall 2019)
ResearchCTS 900 (Fall 2019)
DissertationPS 920 (Spring 2019)
Individualized Science WritingCTS 585 (Spring 2019)
ResearchCTS 900 (Spring 2019)
DissertationPS 920 (Fall 2018)
ResearchCTS 900 (Fall 2018)
DissertationPS 920 (Spring 2018)
DissertationPS 920 (Fall 2017)
DissertationPS 920 (Spring 2017)
Individualized Science WritingCTS 585 (Spring 2017)
ResearchPS 900 (Spring 2017)
DissertationPS 920 (Fall 2016)
ResearchCTS 900 (Fall 2016)
ResearchPS 900 (Fall 2016)
ResearchPS 900 (Spring 2016)
- Bime, C., Bime, C., Gurguis, C. I., Gurguis, C. I., Hecker, L., Hecker, L., Wang, T., Wang, T., Desai, A., Desai, A., Garcia, J. G., & Garcia, J. G. (2016). MicroRNAs in inflammatory lung diseases. Translating MicroRNAs to the clinic. Elsevier.
- Bime, C., Gurguis, C. I., Hecker, L., Wang, T., Desai, A., & Garcia, J. G. (2016). MicroRNAs in inflammatory lung diseases.. In Translating MicroRNAs to the clinic(pp 135-177). Elsevier.
- Feng, A., Ma, W., Faraj, R., Kelly, G. T., Black, S. M., Fallon, M. B., & Wang, T. (2021). Identification of S1PR3 gene signature involved in survival of sepsis patients. BMC medical genomics, 14(1), 43.More infoSepsis is a life-threatening complication of infection that rapidly triggers tissue damage in multiple organ systems and leads to multi-organ deterioration. Up to date, prognostic biomarkers still have limitations in predicting the survival of patients with sepsis. We need to discover more prognostic biomarkers to improve the sensitivity and specificity of the prognosis of sepsis patients. Sphingosine-1-phosphate (S1P) receptor 3 (S1PR3), as one of the S1P receptors, is a prospective prognostic biomarker regulating sepsis-relevant events, including compromised vascular integrity, antigen presentation, and cytokine secretion. Until now, no S1PR3-related prognostic gene signatures for sepsis patients have been found.
- Lu, Q., Zemskov, E. A., Sun, X., Wang, H., Yegambaram, M., Wu, X., Garcia-Flores, A., Song, S., Tang, H., Kangath, A., Cabanillas, G. Z., Yuan, J. X., Wang, T., Fineman, J. R., & Black, S. M. (2021). Activation of the mechanosensitive Ca channel TRPV4 induces endothelial barrier permeability via the disruption of mitochondrial bioenergetics. Redox biology, 38, 101785.More infoMechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS), a refractory lung disease with an unacceptable high mortality rate. Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The specific mechanisms involved in VILI-induced pulmonary capillary leakage, a key pathologic feature of VILI are still far from resolved. The mechanoreceptor, transient receptor potential cation channel subfamily V member 4, TRPV4 plays a key role in the development of VILI through unresolved mechanism. Endothelial nitric oxide synthase (eNOS) uncoupling plays an important role in sepsis-mediated ARDS so in this study we investigated whether there is a role for eNOS uncoupling in the barrier disruption associated with TRPV4 activation during VILI. Our data indicate that the TRPV4 agonist, 4α-Phorbol 12,13-didecanoate (4αPDD) induces pulmonary arterial endothelial cell (EC) barrier disruption through the disruption of mitochondrial bioenergetics. Mechanistically, this occurs via the mitochondrial redistribution of uncoupled eNOS secondary to a PKC-dependent phosphorylation of eNOS at Threonine 495 (T495). A specific decoy peptide to prevent T495 phosphorylation reduced eNOS uncoupling and mitochondrial redistribution and preserved PAEC barrier function under 4αPDD challenge. Further, our eNOS decoy peptide was able to preserve lung vascular integrity in a mouse model of VILI. Thus, we have revealed a functional link between TRPV4 activation, PKC-dependent eNOS phosphorylation at T495, and EC barrier permeability. Reducing pT495-eNOS could be a new therapeutic approach for the prevention of VILI.
- Wang, H., Sun, X., Lu, Q., Zemskov, E. A., Yegambaram, M., Wu, X., Wang, T., Tang, H., & Black, S. M. (2021). The mitochondrial redistribution of eNOS is involved in lipopolysaccharide induced inflammasome activation during acute lung injury. Redox biology, 41, 101878.More infoAcute lung injury (ALI) is a devastating clinical syndrome with no effective therapies. Inflammasome activation has been reported to play a critical role in the initiation and progression of ALI. The molecular mechanisms involved in regulating the activation of inflammasome in ALI remains unresolved, although increases in mitochondrial derived reactive oxygen species (mito-ROS) are involved. Our previous work has shown that the mitochondrial redistribution of uncoupled eNOS impairs mitochondrial bioenergetics and increases mito-ROS generation. Thus, the focus of our study was to determine if lipopolysaccharide (LPS)-mediated inflammasome activation involves the mitochondrial redistribution of uncoupled eNOS. Our data show that the increase in mito-ROS involved in LPS-mediated inflammasome activation is associated with the disruption of mitochondrial bioenergetics in human lung microvascular endothelial cells (HLMVEC) and the mitochondrial redistribution of eNOS. These effects are dependent on RhoA-ROCK signaling and are mediated via increased phosphorylation of eNOS at Threonine (T)-495. A derivative of the mitochondrial targeted Szeto-Schiller peptide (SSP) attached to the antioxidant Tiron (T-SSP), significantly attenuated LPS-mediated mito-ROS generation and inflammasome activation in HLMVEC. Further, T-SSP attenuated mitochondrial superoxide production in a mouse model of sepsis induced ALI. This in turn significantly reduced the inflammatory response and attenuated lung injury. Thus, our findings show that the mitochondrial redistribution of uncoupled eNOS is intimately involved in the activation of the inflammatory response in ALI and implicate attenuating mito-ROS as a therapeutic strategy in humans.
- Wang, T., Yegambaram, M., Gross, C., Sun, X., Lu, Q., Wang, H., Wu, X., Kangath, A., Tang, H., Aggarwal, S., & Black, S. M. (2021). RAC1 nitration at Y IS involved in the endothelial barrier disruption associated with lipopolysaccharide-mediated acute lung injury. Redox biology, 38, 101794.More infoAcute lung injury (ALI), a devastating illness induced by systemic inflammation e.g., sepsis or local lung inflammation e.g., COVID-19 mediated severe pneumonia, has an unacceptably high mortality and has no effective therapy. ALI is associated with increased pulmonary microvascular hyperpermeability and alveolar flooding. The small Rho GTPases, RhoA and Rac1 are central regulators of vascular permeability through cytoskeleton rearrangements. RhoA and Rac1 have opposing functional outcome: RhoA induces an endothelial contractile phenotype and barrier disruption, while Rac1 stabilizes endothelial junctions and increases barrier integrity. In ALI, RhoA activity is increased while Rac1 activity is reduced. We have shown that the activation of RhoA in lipopolysaccharide (LPS)-mediated ALI, is dependent, at least in part, on a single nitration event at tyrosine (Y). Thus, the purpose of this study was to determine if the inhibition of Rac1 is also dependent on its nitration. Our data show that Rac1 inhibition by LPS is associated with its nitration that mass spectrometry identified as Y, within the switch I region adjacent to the nucleotide-binding site. Using a molecular modeling approach, we designed a nitration shielding peptide for Rac1, designated NipR2 (nitration inhibitor peptide for the Rho GTPases 2), which attenuated the LPS-induced nitration of Rac1 at Y, preserves Rac1 activity and attenuates the LPS-mediated disruption of the endothelial barrier in human lung microvascular endothelial cells (HLMVEC). Using a murine model of ALI induced by intratracheal installation of LPS we found that NipR2 successfully prevented Rac1 nitration and Rac1 inhibition, and more importantly attenuated pulmonary inflammation, reduced lung injury and prevented the loss of lung function. Together, our data identify a new post-translational mechanism of Rac1 inhibition through its nitration at Y. As NipR2 also reduces sepsis induced ALI in the mouse lung, we conclude that Rac1 nitration is a therapeutic target in ALI.
- Wu, X., Sun, X., Sharma, S., Lu, Q., Yegambaram, M., Hou, Y., Wang, T., Fineman, J. R., & Black, S. M. (2021). Arginine recycling in endothelial cells is regulated BY HSP90 and the ubiquitin proteasome system. Nitric oxide : biology and chemistry, 108, 12-19.More infoDespite the saturating concentrations of intracellular l-arginine, nitric oxide (NO) production in endothelial cells (EC) can be stimulated by exogenous arginine. This phenomenon, termed the "arginine paradox" led to the discovery of an arginine recycling pathway in which l-citrulline is recycled to l-arginine by utilizing two important urea cycle enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL). Prior work has shown that ASL is present in a NO synthetic complex containing hsp90 and endothelial NO synthase (eNOS). However, it is unclear whether hsp90 forms functional complexes with ASS and ASL and if it is involved regulating their activity. Thus, elucidating the role of hsp90 in the arginine recycling pathway was the goal of this study. Our data indicate that both ASS and ASL are chaperoned by hsp90. Inhibiting hsp90 activity with geldanamycin (GA), decreased the activity of both ASS and ASL and decreased cellular l-arginine levels in bovine aortic endothelial cells (BAEC). hsp90 inhibition led to a time-dependent decrease in ASS and ASL protein, despite no changes in mRNA levels. We further linked this protein loss to a proteasome dependent degradation of ASS and ASL via the E3 ubiquitin ligase, C-terminus of Hsc70-interacting protein (CHIP) and the heat shock protein, hsp70. Transient over-expression of CHIP was sufficient to stimulate ASS and ASL degradation while the over-expression of CHIP mutant proteins identified both TPR- and U-box-domain as essential for ASS and ASL degradation. This study provides a novel insight into the molecular regulation l-arginine recycling in EC and implicates the proteasome pathway as a possible therapeutic target to stimulate NO signaling.
- Fang, F., Ge, Q., Li, R., Lv, J., Zhang, Y., Feng, A., Kelly, G. T., Wang, H., Wang, X., Song, C., Wang, T., & Qian, Z. (2020). LPS restores protective immunity in macrophages against Mycobacterium tuberculosis via autophagy. Molecular immunology, 124, 18-24.More infoAutophagy has been identified as an important immune regulatory mechanism. Recent studies have linked macrophage autophagy with innate immune responses against Mycobacterium tuberculosis (M. tuberculosis), which can survive within macrophages by blocking fusion of the phagosome with lysosomes. These findings suggest that autophagy is a regulatable cellular mechanism of M. tuberculosis defense in macrophages. Transcriptomic profiles in human blood in TB patients suggest that M. tuberculosis affects autophagy related pathways. In order to better understand the role of macrophage autophagy in enhancing protective immunity against M. tuberculosis, in this study, we investigate the effects of the autophagy activators rapamycin and LPS in macrophage autophagy and immunity against M. tuberculosis. We confirm that rapamycin and LPS induce autophagy in M. tuberculosis infected THP-1-derived macrophages or PMA primed THP-1 macrophages [THP-1(A)]. LPS restores M. tuberculosis-inhibited IL-12 synthesis and secretion in THP-1(A) cells via autophagy. Similarly, autophagy activators increase IL-12 synthesis and secretion in THP-1(A) cells. These studies demonstrate the importance of autophagy in M. tuberculosis elimination in macrophages and may lead to novel therapies for tuberculosis and other bacterial infections.
- Feng, A., Rice, A. D., Zhang, Y., Kelly, G. T., Zhou, T., & Wang, T. (2020). S1PR1-Associated Molecular Signature Predicts Survival in Patients with Sepsis. Shock (Augusta, Ga.), 53(3), 284-292.More infoSepsis is a potentially life-threatening complication of an underlying infection that quickly triggers tissue damage in multiple organ systems. To date, there are no established useful prognostic biomarkers for sepsis survival prediction. Sphingosine-1-phosphate (S1P) and its receptor S1P receptor 1 (S1PR1) are potential therapeutic targets and biomarkers for sepsis, as both are active regulators of sepsis-relevant signaling events. However, the identification of an S1PR1-related gene signature for prediction of survival in sepsis patients has yet to be identified. This study aims to find S1PR1-associated biomarkers which could predict the survival of patients with sepsis using gene expression profiles of peripheral blood to be used as potential prognostic and diagnostic tools.
- Gu, W., Shi, J., Liu, H., Zhang, X., Zhou, J. J., Li, M., Zhou, D., Li, R., Lv, J., Wen, G., Zhu, S., Qi, T., Li, W., Wang, X., Wang, Z., Zhu, H., Zhou, C., Knox, K. S., Wang, T., , Chen, Q., et al. (2020). Peripheral blood non-canonical small non-coding RNAs as novel biomarkers in lung cancer. Molecular cancer, 19(1), 159.More infoOne unmet challenge in lung cancer diagnosis is to accurately differentiate lung cancer from other lung diseases with similar clinical symptoms and radiological features, such as pulmonary tuberculosis (TB). To identify reliable biomarkers for lung cancer screening, we leverage the recently discovered non-canonical small non-coding RNAs (i.e., tRNA-derived small RNAs [tsRNAs], rRNA-derived small RNAs [rsRNAs], and YRNA-derived small RNAs [ysRNAs]) in human peripheral blood mononuclear cells and develop a molecular signature composed of distinct ts/rs/ysRNAs (TRY-RNA). Our TRY-RNA signature precisely discriminates between control, lung cancer, and pulmonary TB subjects in both the discovery and validation cohorts and outperforms microRNA-based biomarkers, which bears the diagnostic potential for lung cancer screening.
- Kelly, G. T., Faraj, R., Dai, Z., Cress, A. E., & Wang, T. (2020). A mutation found in esophageal cancer alters integrin β4 mRNA splicing. Biochemical and biophysical research communications, 529(3), 726-732.More infoIntegrin β4 (CD104, mRNA: ITGβ4) contributes to anchoring cells to the extracellular matrix and is regulated in many cancer types where it contributes to tumor progression. One splice variant, integrin β4E, is poorly characterized. We extracted several mutations from tumor samples within ITGB4 near the splice site that controls ITGβ4E production, and computational analysis predicted six of these would alter splicing to alter ITGβ4E abundance. One of these mutations, from an esophageal squamous cell carcinoma sample, was predicted to increase splicing toward ITGβ4E. We verified this effect using a minigene, and observed that integrin β4E slows esophageal squamous cell migration while other variants enhance migration, demonstrating that integrin β4E regulation through mutations may contribute to esophageal squamous cell tumorigenesis.
- Ornatowski, W., Lu, Q., Yegambaram, M., Garcia, A. E., Zemskov, E. A., Maltepe, E., Fineman, J. R., Wang, T., & Black, S. M. (2020). Complex interplay between autophagy and oxidative stress in the development of pulmonary disease. Redox biology, 36, 101679.More infoThe autophagic pathway involves the encapsulation of substrates in double-membraned vesicles, which are subsequently delivered to the lysosome for enzymatic degradation and recycling of metabolic precursors. Autophagy is a major cellular defense against oxidative stress, or related conditions that cause accumulation of damaged proteins or organelles. Selective forms of autophagy can maintain organelle populations or remove aggregated proteins. Dysregulation of redox homeostasis under pathological conditions results in excessive generation of reactive oxygen species (ROS), leading to oxidative stress and the associated oxidative damage of cellular components. Accumulating evidence indicates that autophagy is necessary to maintain redox homeostasis. ROS activates autophagy, which facilitates cellular adaptation and diminishes oxidative damage by degrading and recycling intracellular damaged macromolecules and dysfunctional organelles. The cellular responses triggered by oxidative stress include the altered regulation of signaling pathways that culminate in the regulation of autophagy. Current research suggests a central role for autophagy as a mammalian oxidative stress response and its interrelationship to other stress defense systems. Altered autophagy phenotypes have been observed in lung diseases such as chronic obstructive lung disease, acute lung injury, cystic fibrosis, idiopathic pulmonary fibrosis, and pulmonary arterial hypertension, and asthma. Understanding the mechanisms by which ROS regulate autophagy will provide novel therapeutic targets for lung diseases. This review highlights our current understanding on the interplay between ROS and autophagy in the development of pulmonary disease.
- Sun, X., Lu, Q., Yegambaram, M., Kumar, S., Qu, N., Srivastava, A., Wang, T., Fineman, J. R., & Black, S. M. (2020). TGF-β1 attenuates mitochondrial bioenergetics in pulmonary arterial endothelial cells via the disruption of carnitine homeostasis. Redox biology, 36, 101593.More infoTransforming growth factor beta-1 (TGF-β1) signaling is increased and mitochondrial function is decreased in multiple models of pulmonary hypertension (PH) including lambs with increased pulmonary blood flow (PBF) and pressure (Shunt). However, the potential link between TGF-β1 and the loss of mitochondrial function has not been investigated and was the focus of our investigations. Our data indicate that exposure of pulmonary arterial endothelial cells (PAEC) to TGF-β1 disrupted mitochondrial function as determined by enhanced mitochondrial ROS generation, decreased mitochondrial membrane potential, and disrupted mitochondrial bioenergetics. These events resulted in a decrease in cellular ATP levels, decreased hsp90/eNOS interactions and attenuated shear-mediated NO release. TGF-β1 induced mitochondrial dysfunction was linked to a nitration-mediated activation of Akt1 and the subsequent mitochondrial translocation of endothelial NO synthase (eNOS) resulting in the nitration of carnitine acetyl transferase (CrAT) and the disruption of carnitine homeostasis. The increase in Akt1 nitration correlated with increased NADPH oxidase activity associated with increased levels of p47, p67, and Rac1. The increase in NADPH oxidase was associated with a decrease in peroxisome proliferator-activated receptor type gamma (PPARγ) and the PPARγ antagonist, GW9662, was able to mimic the disruptive effect of TGF-β1 on mitochondrial bioenergetics. Together, our studies reveal for the first time, that TGF-β1 can disrupt mitochondrial function through the disruption of cellular carnitine homeostasis and suggest that stimulating carinitine homeostasis may be an avenue to treat pulmonary vascular disease.
- Wu, F., Chen, X., Zhai, L., Wang, H., Sun, M., Song, C., Wang, T., & Qian, Z. (2020). CXCR2 antagonist attenuates neutrophil transmigration into brain in a murine model of LPS induced neuroinflammation. Biochemical and biophysical research communications, 529(3), 839-845.More infoSepsis-associated encephalopathy (SAE) is a devastating neurological complication of sepsis with intolerable high motility. SAE is accompanied with brain vascular injury, endothelial hyperpermeability, and neutrophil infiltration into the brain tissue, key inflammatory processes leading to further brain edema and neuronal cell apoptosis. Recent studies from us and others suggest that the chemokine receptor C-X-C Motif Chemokine Receptor 2 (CXCR2) is crucial for neutrophil recruitment during SAE. Here we use CXCR2 antagonist SB225002 to characterize the role of CXCR2 in brain infiltration of neutrophil in a murine model of SAE. Systemic administration of high-dose LPS (10 mg/kg) induced evident neutrophil infiltration into the cerebral cortex in wild-type mice. However, CXCR2 antagonist SB225002 markedly attenuated neutrophil infiltration into brain. The CXCR2 expression on neutrophils in the peripheral circulation was dramatically downregulated in response to this LPS dose, and endothelial CXCR2 was significantly upregulated, suggesting endothelial but not neutrophil CXCR2 plays a more important role in neutrophil infiltration into brain. Strikingly, although these CXCR2 antagonist SB225002 treated mice displayed reduced neutrophil infiltration, no change in neutrophil rolling and adhesion was observed. Furthermore, we confirmed that CXCR2 agonist CXCL1 induced a marked increase in actin stress fiber synthesis and paracellular gap formation in cultured cerebral endothelial cells, which is attenuated by SB225002. Thus, these results demonstrate a selective role for endothelial CXCR2 to regulate cerebral vascular permeability and neutrophil transmigration in high-dose LPS induced neuroinflammation, and also suggest a therapeutic potential of CXCR2 antagonist SB225002 in SAE.
- Feng, A., Rice, A., Zhang, Y., Zhou, T., & Wang, T. (2019). S1P receptor 1 associated gene signature predicts survival in sepsis patients.. Shock.
- Johnson Kameny, R., Datar, S. A., Boehme, J. B., Morris, C., Zhu, T., Goudy, B. D., Johnson, E. G., Galambos, C., Raff, G. W., Sun, X., Wang, T., Chiacchia, S. R., Lu, Q., Black, S. M., Maltepe, E., & Fineman, J. R. (2019). Ovine Models of Congenital Heart Disease and the Consequences of Hemodynamic Alterations for Pulmonary Artery Remodeling. American journal of respiratory cell and molecular biology, 60(5), 503-514.More infoThe natural history of pulmonary vascular disease associated with congenital heart disease (CHD) depends on associated hemodynamics. Patients exposed to increased pulmonary blood flow (PBF) and pulmonary arterial pressure (PAP) develop pulmonary vascular disease more commonly than patients exposed to increased PBF alone. To investigate the effects of these differing mechanical forces on physiologic and molecular responses, we developed two models of CHD using fetal surgical techniques: ) left pulmonary artery (LPA) ligation primarily resulting in increased PBF and ) aortopulmonary shunt placement resulting in increased PBF and PAP. Hemodynamic, histologic, and molecular studies were performed on control, LPA, and shunt lambs as well as pulmonary artery endothelial cells (PAECs) derived from each. Physiologically, LPA, and to a greater extent shunt, lambs demonstrated an exaggerated increase in PAP in response to vasoconstricting stimuli compared with controls. These physiologic findings correlated with a pathologic increase in medial thickening in pulmonary arteries in shunt lambs but not in control or LPA lambs. Furthermore, in the setting of acutely increased afterload, the right ventricle of control and LPA but not shunt lambs demonstrates ventricular-vascular uncoupling and adverse ventricular-ventricular interactions. RNA sequencing revealed excellent separation between groups via both principal components analysis and unsupervised hierarchical clustering. In addition, we found hyperproliferation of PAECs from LPA lambs, and to a greater extent shunt lambs, with associated increased angiogenesis and decreased apoptosis in PAECs derived from shunt lambs. A further understanding of mechanical force-specific drivers of pulmonary artery pathology will enable development of precision therapeutics for pulmonary hypertension associated with CHD.
- Kelly, G. T., Faraj, R., Zhang, Y., Maltepe, E., Fineman, J. R., Black, S. M., & Wang, T. (2019). Pulmonary Endothelial Mechanical Sensing and Signaling, a Story of Focal Adhesions and Integrins in Ventilator Induced Lung Injury. Frontiers in physiology, 10, 511.More infoPatients with critical illness such as acute lung injury often undergo mechanical ventilation in the intensive care unit. Though lifesaving in many instances, mechanical ventilation often results in ventilator induced lung injury (VILI), characterized by overdistension of lung tissue leading to release of edemagenic agents, which further damage the lung and contribute to the mortality and progression of pulmonary inflammation. The endothelium is particularly sensitive, as VILI associated mechanical stress results in endothelial cytoskeletal rearrangement, stress fiber formation, and integrity loss. At the heart of these changes are integrin tethered focal adhesions (FAs) which participate in mechanosensing, structure, and signaling. Here, we present the known roles of FA proteins including c-Src, talin, FAK, paxillin, vinculin, and integrins in the sensing and response to cyclic stretch and VILI associated stress. Attention is given to how stretch is propagated from the extracellular matrix through integrins to talin and other FA proteins, as well as signaling cascades that include FA proteins, leading to stress fiber formation and other cellular responses. This unifying picture of FAs aids our understanding in an effort to prevent and treat VILI.
- 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.More infoSphingosine 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.More infoIncreased 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.
- Zemskov, E., Lu, Q., Ornatowski, W., Klinger, C., Desai, A., Maltepe, E., Yuan, J., Wang, T., Fineman, J., & Black, S. (2019). Biomechanical Forces and Oxidative Stress: Implications for Pulmonary Vascular Disease.. Antioxidants and Redox Signaling.
- Zemskov, E., Lu, Q., Wojciech Ornatowski, W., Klinger, C. N., Desai, A. A., Maltepe, E., Yuan, J. X., Wang, T., Fineman, J. R., & Black, S. (2019). BIOMECHANICAL FORCES AND OXIDATIVE STRESS: IMPLICATIONS FOR PULMONARY VASCULAR DISEASE. Antioxidants & redox signaling.More infoAs our fundamental understanding of the underlying causes of lung disease has increased it has become evident that oxidative stress plays a critical role. Oxidative stress in the cell is characterized by excessive generation of reactive oxygen species (ROS). Superoxide (O2-) and hydrogen peroxide (H2O2) are the main ROS involved in the regulation of cellular metabolism. A number of cells in the lung both produce, and respond to, ROS. These include vascular endothelial- (ECs) and smooth muscle (SMCs) cells, fibroblasts, and epithelial cells as well as the cells involved in the inflammatory response, including macrophages, neutrophils, eosinophils. The redox system is involved in multiple aspects of cell metabolism and cell homeostasis. However, dysregulation of the cellular redox system has consequential effects on cell signaling pathways that are intimately involved in disease progression. The lung is exposed to biomechanical forces (fluid shear stress, cyclic stretch, and pressure) due to the passage of blood through the pulmonary vessels and the distension of the lungs during the breathing cycle. Cells within the lung respond to these forces by activating signal transduction pathways that alter their redox state with both physiologic and pathologic consequences. Here, we will discuss the intimate relationship between biomechanical forces and redox signaling and its role in the development of pulmonary disease.
- 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.More infoGenetic 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.More infoOne 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.
- 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.More infoNRF2 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.
- Lv, J., Zhang, X., Wang, C., Wang, H., Wang, T., & Qian, Z. (2018). Hydrogen peroxide promotes the activation of preeclampsia peripheral T cells. Innate immunity, 24(4), 203-209.More infoPreeclampsia (PE) is a pregnancy disorder with a high mortality rate. Patients with PE exhibit systemic high oxidative stress status and inflammatory immune activation. This study aims to define the role of HO in the activation of neutrophils and T lymphocytes in PE patients. CD3/HLA-DR cells in blood from PE patients are remarkably increased compared with those of normal non-pregnancies or normal pregnancies, while the percentage of CD3/CD62L cells is significantly reduced in PE patients compared to normal pregnancies. Furthermore, CD62L levels in granulocytes of periphery blood of PE patients are significantly higher than non-pregnancies, but significantly lower than normal pregnancies. To characterize the effects of intracellular reactive oxygen species (ROS) on T lymphocyte activation in PE patients, PBMCs from normal pregnancies were challenged with HO, and intracellular ROS levels in neutrophil granulocytes, as well as T cell surface marker levels, have been determined. We confirm that HO exposure increases intracellular ROS levels in neutrophil granulocytes, and increases the proportion of CD3/HLA-DR cells, but does not alter the percentage of CD3/CD62L cells in PBMCs. Our study has confirmed dysregulated CD3/HLA-DR and CD3/CD62L T lymphocytes in PE patient peripheral blood, and the dysregulative effects of HO on T lymphocyte activation, suggesting a novel mechanism of immune activation in PE.
- 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.More infoPseudogenes 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.
- 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.More infoProfound 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.More infoNicotinamide 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.
- Qian, Z., Liu, H., Li, M., Shi, J., Li, N., Zhang, Y., Zhang, X., Lv, J., Xie, X., Bai, Y., Ge, Q., Ko, E. A., Tang, H., Wang, T., Wang, X., Wang, Z., Zhou, T., & Gu, W. (2018). Potential Diagnostic Power of Blood Circular RNA Expression in Active Pulmonary Tuberculosis. EBioMedicine, 27, 18-26.More infoCircular RNAs (circRNAs) are a class of novel RNAs with important biological functions, and aberrant expression of circRNAs has been implicated in human diseases. However, the feasibility of using blood circRNAs as disease biomarkers is largely unknown.
- 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.
- Siegler, J. H., Siegler, J. H., Garcia, J. G., Garcia, J. G., Wang, T., Wang, T., Casanova, N., Casanova, N., Gonzalez-Garay, M. L., Gonzalez-Garay, M. L., Karnes, J. H., Karnes, J. H., Ayshiev, D., Ayshiev, D., Sun, X., Sun, X., Lynn, H. D., & Lynn, H. D. (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.
- Siegler, J. H., Siegler, J. H., Garcia, J. G., Garcia, J. G., Wang, T., Wang, T., Casanova, N., Casanova, N., Gonzalez-Garay, M. L., Gonzalez-Garay, M. L., Karnes, J. H., Karnes, J. H., Ayshiev, D., Ayshiev, D., Sun, X., Sun, X., Lynn, H. D., & Lynn, H. D. (2018). Single nucleotide polymorphisms (SNPs) in the MYLKP1 pseudogene are associated with increased colon cancer risk in African Americans. PLOS ONE.More infoWe 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
- Xu, T., Zhu, A., Sun, M., Lv, J., Qian, Z., Wang, X., Wang, T., & Wang, H. (2018). Quantitative assessment of HLA-DQ gene polymorphisms with the development of hepatitis B virus infection, clearance, liver cirrhosis, and hepatocellular carcinoma. Oncotarget, 9(1), 96-109.More infoHepatitis B is one of the most common infectious diseases, which leads to public health problems in the world, especially in Asian counties. In recent years, extensive human genetic association studies have been carried out to identify susceptible genes and genetic polymorphisms to understand the genetic contributions to the disease progression of HBV infection. gene variations have been reported to be associated with HBV infection/clearance, disease progression and the development of hepatitis B-related complications, including liver cirrhosis (LC) and hepatocellular carcinoma (HCC). However, the results are either inconclusive or controversial. Therefore, to derive a more precise estimation of the association, a meta-analysis was performed. Our data revealed that the alleles , and were significantly associated with decreased risk of HBV infection and HBV natural clearance. Logistic regression analyses showed that alleles significantly increased HBV infection clearance, and decreased HBV natural clearance. However, and were not associated with development of cirrhosis. The polymorphisms ( and ) were associated with a decreased HBV-related HCC risk in all genetic models, but increased the risk of HBV-related HCC. In addition, no significant association was observed between polymorphism and HBV-related HCC. These stratified analyses were limited due to relatively modest size of correlational studies. In future, further investigation on a large population and different ethnicities are warranted. Our findings contribute to the personalized care and prognosis in hepatitis B.
- Zhou, T., Xie, X., Li, M., Shi, J., Zhou, J. J., Knox, K. S., Wang, T., Chen, Q., & Gu, W. (2018). Rat BodyMap transcriptomes reveal unique circular RNA features across tissue types and developmental stages. RNA (New York, N.Y.), 24(11), 1443-1456.More infoCircular RNAs (circRNAs) are a novel class of regulatory RNAs. Here, we present a comprehensive investigation of circRNA expression profiles across 11 tissues and four developmental stages in rats, along with cross-species analyses in humans and mice. Although the expression of circRNAs is positively correlated with that of cognate mRNAs, highly expressed genes tend to splice a larger fraction of circular transcripts. Moreover, circRNAs exhibit higher tissue specificity than cognate mRNAs. Intriguingly, while we observed a monotonic increase of circRNA abundance with age in the rat brain, we further discovered a dynamic, age-dependent pattern of circRNA expression in the testes that is characterized by a dramatic increase with advancing stages of sexual maturity and a decrease with aging. The age-sensitive testicular circRNAs are highly associated with spermatogenesis, independent of cognate mRNA expression. The tissue/age implications of circRNAs suggest that they present unique physiological functions rather than simply occurring as occasional by-products of gene transcription.
- 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.More infoThe 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.
- Lv, J., He, X., Wang, H., Wang, Z., Kelly, G. T., Wang, X., Chen, Y., Wang, T., & Qian, Z. (2017). TLR4-NOX2 axis regulates the phagocytosis and killing of Mycobacterium tuberculosis by macrophages. BMC pulmonary medicine, 17(1), 194.More infoMacrophages stand at the forefront of both innate and adapted immunity through their capacities to recognize, engulf, and eliminate foreign particles, and to stimulate adapted immune cells. They are also involved in controlling pro- and anti-inflammatory pathways. Macrophage activity against Mycobacterium tuberculosis (M. tuberculosis) has been shown to involve Toll-like receptor (TLR) activation and ROS production. Previous studies have shown that lipopolysaccharide (LPS), through TLR4, could activate macrophages, improve their bactericidal ROS production, and facilitate anti-infective immune responses. We sought to better understand the role of the TLR4-NOX2 axis in macrophage activation during M. tuberculosis infection.
- Palumbo, S., Shin, Y. J., Ahmad, K., Desai, A., Quijada, H., Mohamed, M., Knox, A., Colson, B., Wang, T., Garcia, J. G., & Hecker, L. (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.More infoAcute 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.
- Wang, T., Gross, C., Desai, A., Zemskov, E., Wu, X., Garcia, J. G., Jacobson, J. R., Garcia, A. N., Yuan, J., & Black, S. (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.More infoMechanical 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.
- 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.More infoSarcoidosis 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.
- 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.More infoSepsis-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.
- Elangovan, V. R., Camp, S. M., Kelly, G. T., Adyshev, D., Sun, X., Garcia, J. G., Wang, T., Black, S., & Desai, A. (2016). Endotoxin- and mechanical stress-induced epigenetic changes in the regulation of the nicotinamide phosphoribosyltransferase promoter. Pulmonary circulation, 6(4), 539-544.More infoMechanical 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.
- 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. (2016). Hyper-activation of pp60(Src) limits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury. Free radical biology & medicine, 102, 217-228.More infoThe 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(Src) 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(Src) (Y527FSrc) mutant and attenuated by over-expression of dominant negative pp60(Src) mutant or reducing pp60(Src) expression. LPS induces both RhoA nitration and endothelial barrier disruption and these events were attenuated when pp60(Src) 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-(Src)-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(Src)-mediated inhibition. We conclude that pp60(Src) 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. (2016). Mechanical Stress and Single Nucleotide Variants Regulate Alternative Splicing of the MYLK Gene. American journal of respiratory cell and molecular biology.More infoThe non-muscle myosin light chain kinase isoform (nmMLCK), encoded by the MYLK gene, is a vital participant in regulating vascular barrier responses to mechanical and inflammatory stimuli. We identified that MYLK is alternatively spliced yielding functionally distinct nmMLCK splice variants including nmMLCK2, a splice variant highly expressed in vascular endothelial cells (ECs) and associated with reduced EC barrier integrity. We previously demonstrated that the nmMLCK2 variant lacks exon 11 that encodes a key regulatory region containing two differentially-phosphorylated tyrosine residues (Y464, Y471) that influence vascular barrier function during inflammation. In this study, we utilized minigene constructs and RT-PCR to interrogate biophysical factors (mechanical stress) and genetic variants (MYLK 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 these variants to alter exon 11 donor and acceptor sites for alternative splicing, computational predictions that were confirmed by minigene studies. 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, 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.
- Qian, Z., Lv, J., Kelly, G. T., Wang, H., Zhang, X., Gu, W., Yin, X., Wang, T., & Zhou, T. (2016). Expression of nuclear factor, erythroid 2-like 2-mediated genes differentiates tuberculosis. Tuberculosis (Edinburgh, Scotland), 99, 56-62.More infoDuring infection and host defense, nuclear factor, erythroid 2-like 2 (Nrf2) dependent signaling is an efficient antioxidant defensive mechanism used by host cells to control the destructive effects of reactive oxygen species. This allows for effective defense responses against microbes while minimizing oxidative injury to the host cell itself. As a central regulator of antioxidant genes, Nrf2 has gained great attention in its pivotal role in infection, especially in tuberculosis (TB), the top infectious disease killer worldwide. To elucidate the genes potentially regulated by Nrf2 in TB, we conducted a meta-analysis on published gene expression datasets. Firstly, we compared the global gene expression profiles between control and Nrf2-deficient human cells. The differentially expressed genes were deemed as "Nrf2-mediated genes". Next, the whole blood gene expression pattern of TB patients was compared with that of healthy controls, pneumonia patients, and lung cancer patients. We found that the genes deregulated in TB significantly overlap with the Nrf2-mediated genes. Based on the intersection of Nrf2-mediated and TB-regulated genes, we identified an Nrf2-mediated 17-gene signature, which reflects a cluster of gene ontology terms highly related to TB physiology. We demonstrated that the 17-gene signature can be used to distinguish TB patients from healthy controls and patients with latent TB infection, pneumonia, or lung cancer. Also, the Nrf2-mediated gene signature can be used as an indicator of the anti-TB therapeutic response. More importantly, we confirmed that the predictive power of the Nrf2-mediated 17-gene signature is significantly better than the random gene sets selected from the human transcriptome. Also, the 17-gene signature performs even better than the random gene signatures selected from TB-associated genes. Our study confirms the central role of Nrf2 in TB pathogenesis and provides a novel and useful diagnostic method to differentiate TB patients from other human subjects.
- 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.More infoAsymmetric 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.
- Szilágyi, K. L., Liu, C., Zhang, X., Wang, T., Fortman, J. D., Zhang, W., & Garcia, J. G. (2016). 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.More infoAcute 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
- 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.More infoMechanical 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.
- Wang, T., Gross, C., Desai, A., Zemskov, E., Wu, X., Garcia, A. N., Jacobson, J. R., Yuan, J. X., Garcia, J. G., & Black, S. M. (2016). Endothelial Cell Signaling and Ventilator-Induced Lung Injury (VILI): Molecular Mechanisms, Genomic Analyses & Therapeutic Targets. American journal of physiology. Lung cellular and molecular physiology, ajplung.00231.2016.More infoMechanical 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 remain 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: i) the key role of the endothelium in the pathogeneiss of VILI; ii) the transcription factors that relay the effects of excessive mechanical stress in the endothelium; ii) the mechanical stress-induced post-translational modifications (PTMs) that influence key signaling pathways involved in VILI responses in the endothelium; iii) the genetic and epigenetic regulation of key target genes in the endothelium that are involved in VILI responses; and iv) the need for novel therapeutic strategies for VILI that can preserve endothelial barrier function.
- 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.More infoRadiotherapy 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.
- 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.More infoProtein 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.
- de la Vega, M. R., Dodson, M., Gross, C., Manzour, H., 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.More infoAcute 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.
- 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.More infoVentilator-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.
- Gu, W., Gurguis, C. I., Zhou, J. J., Zhu, Y., Ko, E., Ko, J., Wang, T., & Zhou, T. (2015). Functional and Structural Consequence of Rare Exonic Single Nucleotide Polymorphisms: One Story, Two Tales. Genome biology and evolution, 7(10), 2929-40.More infoGenetic variation arising from single nucleotide polymorphisms (SNPs) is ubiquitously found among human populations. While disease-causing variants are known in some cases, identifying functional or causative variants for most human diseases remains a challenging task. Rare SNPs, rather than common ones, are thought to be more important in the pathology of most human diseases. We propose that rare SNPs should be divided into two categories dependent on whether the minor alleles are derived or ancestral. Derived alleles are less likely to have been purified by evolutionary processes and may be more likely to induce deleterious effects. We therefore hypothesized that the rare SNPs with derived minor alleles would be more important for human diseases and predicted that these variants would have larger functional or structural consequences relative to the rare variants for which the minor alleles are ancestral. We systematically investigated the consequences of the exonic SNPs on protein function, mRNA structure, and translation. We found that the functional and structural consequences are more significant for the rare exonic variants for which the minor alleles are derived. However, this pattern is reversed when the minor alleles are ancestral. Thus, the rare exonic SNPs with derived minor alleles are more likely to be deleterious. Age estimation of rare SNPs confirms that these potentially deleterious SNPs are recently evolved in the human population. These results have important implications for understanding the function of genetic variations in human exonic regions and for prioritizing functional SNPs in genome-wide association studies of human diseases.
- 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.More infoNuclear 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.
- 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.More infoThe 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.More infoDespite 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.
- 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.More infoMyosin 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.
- Zhou, T., Wang, T., & Garcia, J. G. (2015). A nonmuscle myosin light chain kinase-dependent gene signature in peripheral blood mononuclear cells is linked to human asthma severity and exacerbation status. Pulmonary circulation, 5(2), 335-8.More infoAsthma is increasingly recognized as a heterogeneous disease influenced by complex genetic and environmental contributions. Myosin light chain kinase (MLCK; gene symbol, MYLK), especially the nonmuscle isoform nmMLCK, is a cytoskeleton protein known to be related to human asthma susceptibility and severity, findings confirmed in preclinical models of asthmatic inflammation. In this study, we define the central capacity for a nmMLCK-influenced gene signature in human peripheral blood mononuclear cells to predict human asthma severity and exacerbation status. We refined this signature from a list of nmMLCK-influenced genes identified in lung tissues of nmMLCK knockout mice exposed to inflammatory stimuli (ventilator-induced lung injury), with subsequent identification of nmMLCK-influenced genes in a list of human asthma severity-related genes expressed in blood. The enriched nmMLCK-influenced gene signature successfully predicted human asthma severity and exacerbation status in both discovery and validation human asthma cohorts. These findings validate the central role played by nmMLCK in asthma susceptibility, severity, and exacerbation and further provide novel gene signatures as effective asthma biomarkers for severity, exacerbation, and prognosis.
- 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.More infoPatients 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.
- Gu, W., Li, M., Xu, Y., Wang, T., Ko, J., & Zhou, T. (2014). The impact of RNA structure on coding sequence evolution in both bacteria and eukaryotes. BMC evolutionary biology, 14, 87.More infoMany studies have found functional RNA secondary structures are selectively conserved among species. But, the effect of RNA structure selection on coding sequence evolution remains unknown. To address this problem, we systematically investigated the relationship between nucleotide conservation level and its structural sensitivity in four model organisms, Escherichia coli, yeast, fly, and mouse.
- Gu, W., Xu, Y., Xie, X., Wang, T., Ko, J., & Zhou, T. (2014). The role of RNA structure at 5' untranslated region in microRNA-mediated gene regulation. RNA (New York, N.Y.), 20(9), 1369-75.More infoRecent studies have suggested that the secondary structure of the 5' untranslated region (5' UTR) of messenger RNA (mRNA) is important for microRNA (miRNA)-mediated gene regulation in humans. mRNAs that are targeted by miRNA tend to have a higher degree of local secondary structure in their 5' UTR; however, the general role of the 5' UTR in miRNA-mediated gene regulation remains unknown. We systematically surveyed the secondary structure of 5' UTRs in both plant and animal species and found a universal trend of increased mRNA stability near the 5' cap in mRNAs that are regulated by miRNA in animals, but not in plants. Intra-genome comparison showed that gene expression level, GC content of the 5' UTR, number of miRNA target sites, and 5' UTR length may influence mRNA structure near the 5' cap. Our results suggest that the 5' UTR secondary structure performs multiple functions in regulating post-transcriptional processes. Although the local structure immediately upstream of the start codon is involved in translation initiation, RNA structure near the 5' cap site, rather than the structure of the full-length 5' UTR sequences, plays an important role in miRNA-mediated gene regulation.
- 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.More infoWe 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.
- Sun, X., Elangovan, V. R., Mapes, B., Camp, S. M., Sammani, S., Saadat, L., Ceco, E., Ma, S., 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.More infoIncreased 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.
- 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.More infoEffective 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., 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.More infoMyosin 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.
- 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.More infoNicotinamide 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.More infoThe 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
- 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.More infoParticulate 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., 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.More infoThe 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., 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.More infoExposure 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.More infoExposure 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.
- Youn, J. Y., Wang, T., Blair, J., Laude, K. M., Oak, J., McCann, L. A., Harrison, D. G., & Cai, H. (2012). Endothelium-specific sepiapterin reductase deficiency in DOCA-salt hypertension. American journal of physiology. Heart and circulatory physiology, 302(11), H2243-9.More infoThe endothelial nitric oxide synthase (eNOS) requires tetrahydrobiopterin (H(4)B) as a cofactor and, in its absence, produces superoxide (O(2)(·-)) rather than nitric oxide (NO(·)), a condition referred to as eNOS uncoupling. DOCA-salt-induced hypertension is associated with H(4)B oxidation and uncoupling of eNOS. The present study investigated whether administration of sepiapterin or H(4)B recouples eNOS in DOCA-salt hypertension. Bioavailable NO(·) detected by electron spin resonance was markedly reduced in aortas of DOCA-salt hypertensive mice. Preincubation with sepiapterin (10 μmol/l for 30 min) failed to improve NO(·) bioavailability in hypertensive aortas while it augmented NO(·) production from control vessels, implicating a hypertension-associated deficiency in sepiapterin reductase (SPR), the rate-limiting enzyme for sepiapterin conversion to H(4)B. Indeed, a decreased SPR expression was observed in aortic endothelial cells, but not in endothelium-denuded aortic remains, implicating an endothelium-specific SPR deficiency. Administration of hypertensive aortas with H(4)B (10 μmol/l, 30 min) partially restored vascular NO(·) production. Combined administration of H(4)B and the NADPH oxidase inhibitor apocynin (100 μmol/l, 30 min) fully restored NO(·) bioavailability while reducing O(2)(·-) production. In angiotensin II-induced hypertension, however, aortic endothelial SPR expression was not affected. In summary, administration of sepiapterin is not effective in recoupling eNOS in DOCA-salt hypertension, due to an endothelium-specific loss in SPR, whereas coadministration of H(4)B and apocynin is highly efficient in recoupling eNOS. This is consistent with our previous observations that in angiotensin II hypertension, endothelial deficiency in dihydrofolate reductase is alternatively responsible for uncoupling of eNOS. Taken together, these data indicate that strategies specifically targeting at different H(4)B metabolic enzymes might be necessary in restoring eNOS function in different types of hypertension.
- Ma, S., 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.More infoThe 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.More infoNovel 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.More infoClinically 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.
- 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.More infoAcute 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.More infoThe 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.
- Sammani, S., Park, K., 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.More infoLung 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.
- 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.More infoThe 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.
- 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.More infoEpidemiologic 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.
- Gao, L., Pung, Y., Zhang, J., Chen, P., Wang, T., Li, M., Meza, M., Toro, L., & Cai, H. (2009). Sepiapterin reductase regulation of endothelial tetrahydrobiopterin and nitric oxide bioavailability. American journal of physiology. Heart and circulatory physiology, 297(1), H331-9.More infoSepiapterin reductase (SPR) catalyzes the final step of tetrahydrobiopterin (H(4)B) biosynthesis and the first step of H(4)B regeneration from an exogenous precursor sepiapterin. Despite the potential significance of SPR in regulating H(4)B-dependent nitric oxide (NO(*)) production, the endothelium-specific sequence and functions of SPR remain elusive. We first cloned endothelial SPR cDNA from bovine aortic endothelial cells (Genebank: DQ978331). In cells transiently transfected with SPR gene, SPR activity (HPLC) was dramatically increased by 19-fold, corresponding to a significant increase in endothelial H(4)B content (HPLC) and NO(*) production (electron spin resonance). In vivo delivery of SPR gene significantly increased vascular SPR protein expression (mouse vs. bovine antibodies to differentiate endogenous vs. exogenous), activity, H(4)B content, and NO(*) production, as well as NO(*)-dependent vasorelaxation. In endothelial cells transfected with small interfering RNA specific for SPR, approximately 87% of mRNA were attenuated (real-time quantitative RT-PCR), corresponding to a significant reduction in SPR protein expression and activity, which was associated with decreases in both intracellular H(4)B content and NO(*) level. Exogenous administration of sepiapterin to endothelial cells significantly upregulated H(4)B and NO(*) levels, which were attenuated by SPR RNA interference (RNAi). H(4)B-stimulated increase in NO(*) production, however, was SPR RNAi independent. GTP cyclohydrolase 1 expression and activity, as well as dihydrofolate reductase expression, were not affected by SPR RNAi, whereas dihydrofolate reductase activity was significantly downregulated. These data represent the first to study endothelial SPR functionally and clearly demonstrate an important role of endothelial SPR in modulating H(4)B and NO(*) bioavailability.
- Xia, B., Wang, T., Fox, L. M., & Wang, D. (2009). HPLC/MS/MS analysis of 3-carbamyl-4-methylpyrrole analog MNP001, a highly potent antihypertensive agent, in rat plasma. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 877(20-21), 1867-72.More infoChemically synthesized 3-carbamyl-4-methylpyrroles were characterized as a group of antihypertensive agents with dual-targeting mechanism to simultaneously inhibit type 4 phosphodiesterase (PDE4) and L-type calcium channels. A 5-butyl analog of the pyrrole family, MNP001, was found to have high potency in reducing animal blood pressure and heart rate. A method for measuring MNP001 using high performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS/MS) was developed. The calibration curve for MNP001 showed good linearity with the value of correlation coefficient greater than 0.987 over the range of 0.25-500 ng/mL. The results for inter-day and intra-day precision as well as accuracy were acceptable according to the criteria established by FDA. The lower limit of quantification was 0.25 ng/mL. This method was quick, sensitive and sufficient for in vivo pharmacokinetic and pharmacodynamic studies on this novel antihypertensive pyrrole compound.
- Youn, J., Wang, T., & Cai, H. (2009). An ezrin/calpain/PI3K/AMPK/eNOSs1179 signaling cascade mediating VEGF-dependent endothelial nitric oxide production. Circulation research, 104(1), 50-9.More infoCalpain was recently reported to mediate vascular endothelial growth factor (VEGF)-induced angiogenesis. In the present study, we investigated detailed molecular mechanisms. VEGF (100 ng/mL) induced a marked increase in endothelial cell production of NO(*), specifically detected by electron spin resonance. This response was abolished by inhibition of calpain with N-acetyl-leucyl-leucyl-norleucinal (ALLN) or Calpeptin. Both also diminished membrane-specific calpain activation by VEGF, which was intriguingly attenuated by silencing ezrin with RNA interference. A rapid membrane colocalization of calpain and ezrin occurred as short as 10 minutes after VEGF stimulation. AKT, AMP-dependent kinase (AMPK), and endothelial nitric oxide synthase (eNOS)(s1179) phosphorylations in VEGF-stimulated endothelial cells were markedly enhanced, which were however significantly attenuated by either ALLN, Calpeptin, or ezrin small interfering RNA, as well as by Wortmannin or compound C (respectively for phosphatidylinositol 3-kinase [PI3K] or AMPK). The latter 3 also abolished VEGF induction of NO(*). These data indicate that AMPK and AKT are both downstream of PI3K and that AKT activation is partially dependent on AMPK. The interrelationship between AMPK and AKT, although known to be individually important in mediating VEGF activation of eNOS, is clearly characterized. Furthermore, AMPK/AKT/eNOS(s1179) was found downstream of a calpain/ezrin membrane interaction. These data no doubt provide new insights into the long mystified signaling gap between VEGF receptors and PI3K/AKT or AMPK-dependent eNOS activation. In view of the well-established significance of VEGF-dependent angiogenesis, these findings might have broad and important implications in cardiovascular pathophysiology.
- 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.More infoParticulate 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.
- 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.More infoAsthma 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.
- Wang, T., & Wang, D. (2006). High performance liquid chromatographic analysis of MS23 piperidine analog MSP001 in rat plasma. Journal of pharmaceutical and biomedical analysis, 42(5), 607-12.More infoMSP001 is a newly synthesized piperidine analog of the lead antihypertensive compound MS23 that dually targets cAMP-specific type 4 phosphodiesterase and L-type calcium channels. We validated an analytical protocol for MSP001 in rat plasma using high performance liquid chromatographic method. A C18 column and a phosphate/acetonitrile buffer were used to perform the chromatographic separation. UV detection was carried out at 307 nm, a wavelength at which an absorption peak was detected for this group of compounds. The calibration curve for MSP001 was linear in the range from 25 to 10,000 ng/ml. The limit of quantification (LOQ) was 25 ng/ml. The results demonstrate that this method has high linearity (R=0.99995), compound specificity, and acceptable precision/accuracy. The protocol is suitable for in vivo pharmacokinetic studies on the compound.
- Wang, T., Fox, L. M., & Wang, D. (2006). Validation of HPLC analysis method of a novel antihypertensive agent MS23 in rat plasma. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 830(1), 13-7.More infoMS23 is a vasodilator with unique dual action pharmacological profile to inhibit type 4 PDE and antagonize L-type calcium channels. We validated an analytical protocol for MS23 in rat plasma using high performance liquid chromatography (HPLC). A C18 column and a phosphate/acetonitrite buffer were used for chromatographic separation. UV detection was performed at 307 nm. The calibration curve for MS23 was linear in the range from 50 to 10,000 ng/ml. The limit of quantification (LOQ) was 50 ng/ml. The results demonstrate that the method has linearity (R = 0.9989), specificity, and acceptable precision/accuracy. This method is simple, economic, and sufficient for in vivo pharmacokinetic studies on the compound.
- Wang, D., & Wang, T. (2005). Novel approaches to using PDE4 inhibitors for antihypertensive therapy. Current opinion in investigational drugs (London, England : 2000), 6(3), 283-8.More infoPhosphodiesterase (PDE)4 inhibitors are effective vasodilators. In addition to the treatment of asthma and other inflammatory diseases, such as chronic obstructive pulmonary disease, wide-spectrum PDE4 inhibitors may also be developed for use in antihypertensive therapy, especially in patients with impaired renal function and elevated pulmonary arterial pressure. Rational approaches to overcoming the intrinsic emetogenic response associated with PDE4 inhibition are discussed, and a dual-action drug development that incorporates L-type Ca2+ channel antagonism and selective PDE4 inhibition is proposed. The potential for research and development of such a dual-action agent should eventually provide healthcare professionals with a new category of therapeutic options with which to attempt to combat hypertension.