Ruslan Rafikov

Interests

Research

• Heme scavengers for Sickle Cell disease and other hemolytic disorders• Akt signaling and anaplerosis in pulmonary arterial hypertension• Heme mediated dual epithelial-endothelial barrier dysfunction

Courses

Scholarly Contributions

Journals/Publications

• Ahmed, M., Zaghloul, N., Zimmerman, P., Casanova, N. G., Sun, X., Song, J. H., Hernon, V. R., Sammani, S., Rischard, F., Rafikova, O., Rafikov, R., Makino, A., Kempf, C. L., Camp, S. M., Wang, J., Desai, A. A., Lussier, Y., Yuan, J. X., & Garcia, J. G. (2021). Endothelial eNAMPT drives EndMT and preclinical PH: rescue by an eNAMPT-neutralizing mAb. Pulmonary circulation, 11(4), 20458940211059712.
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  Pharmacologic interventions to halt/reverse the vascular remodeling and right ventricular dysfunction in pulmonary arterial hypertension (PAH) remains an unmet need. We previously demonstrated extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a DAMP (damage-associated molecular pattern protein) contributing to PAH pathobiology via TLR4 ligation. We examined the role of endothelial cell (EC)-specific eNAMPT in experimental PH and an eNAMPT-neutralizing mAb as a therapeutic strategy to reverse established PH. Hemodynamic/echocardiographic measurements and tissue analyses were performed in Sprague Dawley rats exposed to 10% hypoxia/Sugen (three weeks) followed by return to normoxia and weekly intraperitoneal delivery of the eNAMPT mAb (1 mg/kg). WT C57BL/6J mice and conditional EC-cNAMPT mice were exposed to 10% hypoxia (three weeks). Biochemical and RNA sequencing studies were performed on rat PH lung tissues and human PAH PBMCs. Hypoxia/Sugen-exposed rats exhibited multiple indices of severe PH (right ventricular systolic pressure, Fulton index), including severe vascular remodeling, compared to control rats. PH severity indices and plasma levels of eNAMPT, IL-6, and TNF- were all significantly attenuated by eNAMPT mAb neutralization. Compared to hypoxia-exposed WT mice, cNAMPT KO mice exhibited significantly reduced PH severity and evidence of EC to mesenchymal transition (EndMT). Finally, biochemical and RNAseq analyses revealed eNAMPT mAb-mediated rectification of dysregulated inflammatory signaling pathways (TLR/NF-κB, MAP kinase, Akt/mTOR) and EndMT in rat PH lung tissues and human PAH PBMCs. These studies underscore EC-derived eNAMPT as a key contributor to PAH pathobiology and support the eNAMPT/TLR4 inflammatory pathway as a highly druggable therapeutic target to reduce PH severity and reverse PAH.
• Bermudez, T., Sammani, S., Song, J. H., Hernon, V. R., Kempf, C. L., Garcia, A. N., Burt, J., Hufford, M., Camp, S. M., Cress, A. E., Desai, A. A., Natarajan, V., Jacobson, J. R., Dudek, S. M., Cancio, L. C., Alvarez, J., Rafikov, R., Li, Y., Zhang, D. D., , Casanova, N. G., et al. (2022). eNAMPT neutralization reduces preclinical ARDS severity via rectified NFkB and Akt/mTORC2 signaling. Scientific reports, 12(1), 696.
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  Despite encouraging preclinical data, therapies to reduce ARDS mortality remains a globally unmet need, including during the COVID-19 pandemic. We previously identified extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a novel damage-associated molecular pattern protein (DAMP) via TLR4 ligation which regulates inflammatory cascade activation. eNAMPT is tightly linked to human ARDS by biomarker and genotyping studies in ARDS subjects. We now hypothesize that an eNAMPT-neutralizing mAb will significantly reduce the severity of ARDS lung inflammatory lung injury in diverse preclinical rat and porcine models. Sprague Dawley rats received eNAMPT mAb intravenously following exposure to intratracheal lipopolysaccharide (LPS) or to a traumatic blast (125 kPa) but prior to initiation of ventilator-induced lung injury (VILI) (4 h). Yucatan minipigs received intravenous eNAMPT mAb 2 h after initiation of septic shock and VILI (12 h). Each rat/porcine ARDS/VILI model was strongly associated with evidence of severe inflammatory lung injury with NFkB pathway activation and marked dysregulation of the Akt/mTORC2 signaling pathway. eNAMPT neutralization dramatically reduced inflammatory indices and the severity of lung injury in each rat/porcine ARDS/VILI model (~ 50% reduction) including reduction in serum lactate, and plasma levels of eNAMPT, IL-6, TNFα and Ang-2. The eNAMPT mAb further rectified NFkB pathway activation and preserved the Akt/mTORC2 signaling pathway. These results strongly support targeting the eNAMPT/TLR4 inflammatory pathway as a potential ARDS strategy to reduce inflammatory lung injury and ARDS mortality.
• Rafikov, R., Rischard, F., Vasilyev, M., Varghese, M. V., Yuan, J. X., Desai, A. A., Garcia, J. G., & Rafikova, O. (2022). Cytokine profiling in pulmonary arterial hypertension: the role of redox homeostasis and sex. Translational research : the journal of laboratory and clinical medicine, 247, 1-18.
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  Pulmonary arterial hypertension (PAH) is a fatal disease with a well-established sexual dimorphism. Activated inflammatory response and altered redox homeostasis, both known to manifest in a sex-specific manner, are implicated in the pathogenic mechanisms involved in PAH development. This study aimed to evaluate the impact of sex and plasma redox status on circulating cytokine profiles. Plasma oxidation-reduction potential (ORP), as a substitute measure of redox status, was analyzed in male and female Group 1 PAH and healthy subjects. The profiles of 27 circulating cytokines were compared in 2 PAH groups exhibiting the highest and lowest quartile for plasma ORP, correlated with clinical parameters, and used to predict patient survival. The analysis of the PAH groups with the highest and lowest ORP revealed a correlation between elevated cytokine levels and increased oxidative stress in females. In contrast, in males, cytokine expressions were increased in the lower oxidative environment (except for IL-1b). Correlations of the increased cytokine expressions with PAH severity were highly sex-dependent and corresponded to the increase in PAH severity in males and less severe PAH in females. Machine learning algorithms trained on the combined cytokine and redox profiles allowed the prediction of PAH mortality with 80% accuracy. We conclude that the profile of circulating cytokines in PAH patients is redox- and sex-dependent, suggesting the vital need to stratify the patient cohort subjected to anti-inflammatory therapies. Combined cytokine and/or redox profiling showed promising value for predicting the patients' survival.
• Yoval-Sánchez, B., Ansari, F., James, J., Niatsetskaya, Z., Sosunov, S., Filipenko, P., Tikhonova, I. G., Ten, V., Wittig, I., Rafikov, R., & Galkin, A. (2022). Redox-dependent loss of flavin by mitochondria complex I is different in brain and heart. Redox biology, 51, 102258.
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  Pathologies associated with tissue ischemia/reperfusion (I/R) in highly metabolizing organs such as the brain and heart are leading causes of death and disability in humans. Molecular mechanisms underlying mitochondrial dysfunction during acute injury in I/R are tissue-specific, but their details are not completely understood. A metabolic shift and accumulation of substrates of reverse electron transfer (RET) such as succinate are observed in tissue ischemia, making mitochondrial complex I of the respiratory chain (NADH:ubiquinone oxidoreductase) the most vulnerable enzyme to the following reperfusion. It has been shown that brain complex I is predisposed to losing its flavin mononucleotide (FMN) cofactor when maintained in the reduced state in conditions of RET both in vitro and in vivo. Here we investigated the process of redox-dependent dissociation of FMN from mitochondrial complex I in brain and heart mitochondria. In contrast to the brain enzyme, cardiac complex I does not lose FMN when reduced in RET conditions. We proposed that the different kinetics of FMN loss during RET is due to the presence of brain-specific long 50 kDa isoform of the NDUFV3 subunit of complex I, which is absent in the heart where only the canonical 10 kDa short isoform is found. Our simulation studies suggest that the long NDUFV3 isoform can reach toward the FMN binding pocket and affect the nucleotide affinity to the apoenzyme. For the first time, we demonstrated a potential functional role of tissue-specific isoforms of complex I, providing the distinct molecular mechanism of I/R-induced mitochondrial impairment in cardiac and cerebral tissues. By combining functional studies of intact complex I and molecular structure simulations, we defined the critical difference between the brain and heart enzyme and suggested insights into the redox-dependent inactivation mechanisms of complex I during I/R injury in both tissues.
• James, J., Zemskova, M., Eccles, C. A., Varghese, M. V., Niihori, M., Barker, N. K., Luo, M., Mandarino, L. J., Langlais, P. R., Rafikova, O., & Rafikov, R. (2021). Single Mutation in the NFU1 Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells. Arteriosclerosis, Thrombosis, and Vascular Biology.
• Varghese, M. V., James, J., Rafikova, O., & Rafikov, R. (2021). Glucose-6-phosphate dehydrogenase deficiency contributes to metabolic abnormality and pulmonary hypertension. American journal of physiology. Lung cellular and molecular physiology, 320(4), L508-L521.
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  We have previously reported that several patients with idiopathic pulmonary hypertension (PH) had different types of G6PD deficiency. However, the role of G6PD in PH is multifactorial because G6PD is involved in controlling oxidative stress, metabolic switch, and red blood cell fragility. To delineate the contribution of G6PD to PH pathogenesis, we utilized a mouse line with decreased expression of G6PD (10% from wild-type level). We confirmed that mice with G6PD deficiency develop spontaneous pulmonary hypertension with pulmonary artery and right heart remodeling. G6PD deficiency resulted in increased free hemoglobin and activation of the p38 pathway, which we recently reported induces the development of PH in the sugen/hypoxia model via endothelial barrier dysfunction. Metabolomics analysis of G6PD deficient mice indicates the switch to alternative metabolic fluxes that feed into the pentose phosphate pathway (PPP), resulting in the upregulation of oxidative stress, fatty acid pathway, and reduction in pyruvate production. Thus, G6PD deficiency did not reduce PPP flux that is important for proliferation but activated collateral pathways at the cost of increased oxidative stress. Indeed, we found the upregulation of myo-inositol oxidase, reduction in GSH/GSSG ratio, and increased nitration in the lungs of G6PD-deficient mice. Increased oxidative stress also results in the activation of PI3K, ERK1/2, and AMPK that contribute to the proliferation of pulmonary vasculature. Therefore, G6PD deficiency has a multimodal effect, including hemolysis, metabolic reprogramming, and oxidative stress leading to the PH phenotype in mice.
• James, J., Srivastava, A., Varghese, M. V., Eccles, C. A., Zemskova, M., Rafikova, O., & Rafikov, R. (2020). Heme induces rapid endothelial barrier dysfunction via the MKK3/p38MAPK axis. Blood, 136(6), 749-754.
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  Several studies demonstrate that hemolysis and free heme in circulation cause endothelial barrier dysfunction and are associated with severe pathological conditions such as acute respiratory distress syndrome, acute chest syndrome, and sepsis. However, the precise molecular mechanisms involved in the pathology of heme-induced barrier disruption remain to be elucidated. In this study, we investigated the role of free heme in the endothelial barrier integrity and mechanisms of heme-mediated intracellular signaling of human lung microvascular endothelial cells (HLMVECs). Heme, in a dose-dependent manner, induced a rapid drop in the endothelial barrier integrity of HLMVECs. An investigation into barrier proteins revealed that heme primarily affected the tight junction proteins zona occludens-1, claudin-1, and claudin-5, which were significantly reduced after heme exposure. The p38MAPK/HSP27 pathway, involved in the regulation of endothelial cytoskeleton remodeling, was also significantly altered after heme treatment, both in HLMVECs and mice. By using a knockout (KO) mouse for MKK3, a key regulator of the p38MAPK pathway, we showed that this KO effectively decreased heme-induced endothelial barrier dysfunction. Taken together, our results indicate that targeting the p38MAPK pathway may represent a crucial treatment strategy in alleviating hemolytic diseases.
• James, J., Valuparampil Varghese, M., Vasilyev, M., Langlais, P. R., Tofovic, S. P., Rafikova, O., & Rafikov, R. (2020). Complex III Inhibition-Induced Pulmonary Hypertension Affects the Mitochondrial Proteomic Landscape. International journal of molecular sciences, 21(16).
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  The mitochondria play a vital role in controlling cell metabolism and regulating crucial cellular outcomes. We previously demonstrated that chronic inhibition of the mitochondrial complex III in rats by Antimycin A (AA) induced sustained pulmonary vasoconstriction. On the metabolic level, AA-induced mitochondrial dysfunction resulted in a glycolytic shift that was reported as the primary contributor to pulmonary hypertension pathogenesis. However, the regulatory proteins driving this metabolic shift with complex III inhibition are yet to be explored. Therefore, to delineate the mechanisms, we followed changes in the rat lung mitochondrial proteome throughout AA treatment. Rats treated with AA for up to 24 days showed a disturbed mitochondrial proteome with significant changes in 28 proteins ( < 0.05). We observed a time-dependent decrease in the expression of key proteins that regulate fatty acid oxidation, the tricarboxylic acid cycle, the electron transport chain, and amino acid metabolism, indicating a correlation with diminished mitochondrial function. We also found a significant dysregulation in proteins that controls the protein import machinery and the clearance and detoxification of oxidatively damaged peptides via proteolysis and mitophagy. This could potentially lead to the onset of mitochondrial toxicity due to misfolded protein stress. We propose that chronic inhibition of mitochondrial complex III attenuates mitochondrial function by disruption of the global mitochondrial metabolism. This potentially aggravates cellular proliferation by initiating a glycolytic switch and thereby leads to pulmonary hypertension.
• James, J., Zemskova, M., Eccles, C. A., Varghese, M. V., Niihori, M., Barker, N. K., Luo, M., Mandarino, L. J., Langlais, P. R., Rafikova, O., & Rafikov, R. (2020). Single Mutation in the Gene Metabolically Reprograms Pulmonary Artery Smooth Muscle Cells. Arteriosclerosis, thrombosis, and vascular biology, ATVBAHA120314655.
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  NFU1 is a mitochondrial iron-sulfur scaffold protein, involved in iron-sulfur assembly and transfer to complex II and LAS (lipoic acid synthase). Patients with the point mutation NFU1 and CRISPR/CAS9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-generated rats develop mitochondrial dysfunction leading to pulmonary arterial hypertension. However, the mechanistic understanding of pulmonary vascular proliferation due to a single mutation in NFU1 remains unresolved. Approach and Results: Quantitative proteomics of isolated mitochondria showed the entire phenotypic transformation of NFU1 rats with a disturbed mitochondrial proteomic landscape, involving significant changes in the expression of 208 mitochondrial proteins. The NFU1 mutation deranged the expression pattern of electron transport proteins, resulting in a significant decrease in mitochondrial respiration. Reduced reliance on mitochondrial respiration amplified glycolysis in pulmonary artery smooth muscle cell (PASMC) and activated GPD (glycerol-3-phosphate dehydrogenase), linking glycolysis to oxidative phosphorylation and lipid metabolism. Decreased PDH (pyruvate dehydrogenase) activity due to the lipoic acid shortage is compensated by increased fatty acid metabolism and oxidation. PASMC became dependent on extracellular fatty acid sources due to upregulated transporters such as CD36 and CPT (carnitine palmitoyltransferase)-1. Finally, the NFU1 mutation produced a dysregulated antioxidant system in the mitochondria, leading to increased reactive oxygen species levels. PASMC from NFU1 rats showed apoptosis resistance, increased anaplerosis, and attained a highly proliferative phenotype. Attenuation of mitochondrial reactive oxygen species by mitochondrial-targeted antioxidant significantly decreased PASMC proliferation.
• Rafikov, R., Coletta, D. K., Mandarino, L. J., & Rafikova, O. (2020). Pulmonary Arterial Hypertension Induces a Distinct Signature of Circulating Metabolites. Journal of clinical medicine, 9(1).
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  Pulmonary arterial hypertension (PAH) is an incurable, progressive disorder, and the early diagnosis and treatment of PAH are associated with increased survival [...].
• Rafikov, R., Rafikova, O., Tofovic, S. P., Langlais, P. R., Vasilyev, M., Varghese, M. V., & James, J. (2020). Complex III Inhibition-Induced Pulmonary Hypertension Affects the Mitochondrial Proteomic Landscape. International Journal of Molecular Sciences.
• Rafikova, O., James, J., Eccles, C. A., Kurdyukov, S., Niihori, M., Varghese, M. V., & Rafikov, R. (2020). Early progression of pulmonary hypertension in the monocrotaline model in males is associated with increased lung permeability. Biology of sex differences, 11(1), 11.
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  The mechanisms involved in pulmonary hypertension (PH) development in patients and pre-clinical models are poorly understood. PH has a well-established sex dimorphism in patients with increased frequency of PH in females, and more severe disease with poor survival prognosis in males. Previously, we found that heme signaling plays an essential role in the development phase of the Sugen/Hypoxia (SU/Hx) model. This study is focused on the elucidation of sex differences in mechanisms of PH development related to heme action at the early stage of the monocrotaline (MCT) PH model.
• Valuparampil Varghese, M., James, J., Eccles, C. A., Niihori, M., Rafikova, O., & Rafikov, R. (2020). Inhibition of Anaplerosis Attenuated Vascular Proliferation in Pulmonary Arterial Hypertension. Journal of clinical medicine, 9(2).
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  Vascular remodeling is considered a key event in the pathogenesis of pulmonary arterial hypertension (PAH). However, mechanisms of gaining the proliferative phenotype by pulmonary vascular cells are still unresolved. Due to well-established pyruvate dehydrogenase (PDH) deficiency in PAH pathogenesis, we hypothesized that the activation of another branch of pyruvate metabolism, anaplerosis, via pyruvate carboxylase (PC) could be a key contributor to the metabolic reprogramming of the vasculature. In sugen/hypoxic PAH rats, vascular proliferation was found to be accompanied by increased activation of Akt signaling, which upregulated membrane Glut4 translocation and caused upregulation of hexokinase and pyruvate kinase-2, and an overall increase in the glycolytic flux. Decreased PDH activity and upregulation of PC shuttled more pyruvate to oxaloacetate. This results in the anaplerotic reprogramming of lung vascular cells and their subsequent proliferation. Treatment of sugen/hypoxia rats with the PC inhibitor, phenylacetic acid 20 mg/kg, starting after one week from disease induction, significantly attenuated right ventricular systolic pressure, Fulton index, and pulmonary vascular cell proliferation. PC inhibition reduced the glycolytic shift by attenuating Akt-signaling, glycolysis, and restored mitochondrial pyruvate oxidation. Our findings suggest that targeting PC mediated anaplerosis is a potential therapeutic intervention for the resolution of vascular remodeling in PAH.
• Varghese, M. V., Niihori, M., Eccles, C. A., Kurdyukov, S., James, J., Rafikova, O., & Rafikov, R. (2020). Antioxidant-Conjugated Peptide Attenuated Metabolic Reprogramming in Pulmonary Hypertension. Antioxidants (Basel, Switzerland), 9(2).
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  Pulmonary arterial hypertension (PAH) is a chronic cardiopulmonary disorder instigated by pulmonary vascular cell proliferation. Activation of Akt was previously reported to promote vascular remodeling. Also, the irreversible nitration of Y350 residue in Akt results in its activation. NitroAkt was increased in PAH patients and the SU5416/Hypoxia (SU/Hx) PAH model. This study investigated whether the prevention of Akt nitration in PAH by Akt targeted nitroxide-conjugated peptide (NP) could reverse vascular remodeling and metabolic reprogramming. Treatment of the SU/Hx model with NP significantly decreased nitration of Akt in lungs, attenuated right ventricle (RV) hypertrophy, and reduced RV systolic pressure. In the PAH model, Akt-nitration induces glycolysis by activation of the glucose transporter Glut4 and lactate dehydrogenase-A (LDHA). Decreased G6PD and increased GSK3β in SU/Hx additionally shunted intracellular glucose via glycolysis. The increased glycolytic rate upregulated anaplerosis due to activation of pyruvate carboxylase in a nitroAkt-dependent manner. NP treatment resolved glycolytic switch and activated collateral pentose phosphate and glycogenesis pathways. Prevention of Akt-nitration significantly controlled pyruvate in oxidative phosphorylation by decreasing lactate and increasing pyruvate dehydrogenases activities. Histopathological studies showed significantly reduced pulmonary vascular proliferation. Based on our current observation, preventing Akt-nitration by using an Akt-targeted nitroxide-conjugated peptide could be a useful treatment option for controlling vascular proliferation in PAH.
• Zemskova, M., Kurdyukov, S., James, J., McClain, N., Rafikov, R., & Rafikova, O. (2020). Sex-specific stress response and HMGB1 release in pulmonary endothelial cells. PloS one, 15(4), e0231267.
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  Women are known to be associated with a higher susceptibility to pulmonary arterial hypertension (PAH). In contrast, male PAH patients have a worse survival prognosis. In this study, we investigated whether the contribution of sex goes beyond the effects of sex hormones by comparing the ability of isolated male and female pulmonary endothelial cells to respire, proliferate and tolerate the stress. Mouse lung endothelial cells (MLEC) were isolated from the lungs of male and female 3-week old mice. Male MLEC showed an increased basal mitochondrial respiration rate, elevated maximal respiration, a significantly greater level of mitochondrial polarization, and a higher rate of proliferation. Exposure of cells to hypoxia (2% of O2 for 24 hours) induced a strong apoptotic response in female but not male MLEC. In contrast, treatment with mitochondrial respiratory Complex III inhibitor Antimycin A (AA, 50μM) mediated severe necrosis specifically in male MLEC, while female cells again responded primarily by apoptosis. The same effect with female cells responding to the stress by apoptosis and male cells responding by necrosis was confirmed in starved pulmonary endothelial cells isolated from human donors. Elevated necrosis seen in male cells was associated with a significant release of damage-associated alarmin, HMGB1. No stimuli induced a significant elevation of HMGB1 secretion in females. We conclude that male cells appear to be protected against mild stress conditions, such as hypoxia, possibly due to increased mitochondrial respiration. In contrast, they are more sensitive to impaired mitochondrial function, to which they respond by necrotic death. Necrosis in male vascular cells releases a significant amount of HMGB1 that could contribute to the pro-inflammatory phenotype known to be associated with the male gender.
• Zemskova, M., McClain, N., Niihori, M., Varghese, M. V., James, J., Rafikov, R., & Rafikova, O. (2020). Necrosis-Released HMGB1 (High Mobility Group Box 1) in the Progressive Pulmonary Arterial Hypertension Associated With Male Sex. Hypertension (Dallas, Tex. : 1979), 76(6), 1787-1799.
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  Damage-associated molecular patterns, such as HMGB1 (high mobility group box 1), play a well-recognized role in the development of pulmonary arterial hypertension (PAH), a progressive fatal disease of the pulmonary vasculature. However, the contribution of the particular type of vascular cells, type of cell death, or the form of released HMGB1 in PAH remains unclear. Moreover, although male patients with PAH show a higher level of circulating HMGB1, its involvement in the severe PAH phenotype reported in males is unknown. In this study, we aimed to investigate the sources and active forms of HMGB1 released from damaged vascular cells and their contribution to the progressive type of PAH in males. Our results showed that HMGB1 is released by either pulmonary artery human endothelial cells or human pulmonary artery smooth muscle cells that underwent necrotic cell death, although only human pulmonary artery smooth muscle cells produce HMGB1 during apoptosis. Moreover, only human pulmonary artery smooth muscle cell death induced a release of dimeric HMGB1, found to be mitochondrial reactive oxygen species dependent, and TLR4 (toll-like receptor 4) activation. The modified Sugen/Hypoxia rat model replicates the human sexual dimorphism in PAH severity (right ventricle systolic pressure in males versus females 54.7±2.3 versus 44.6±2 mm Hg). By using this model, we confirmed that necroptosis and necrosis are the primary sources of circulating HMGB1 in the male rats, although only necrosis increased circulation of HMGB1 dimers. Attenuation of necrosis but not apoptosis or necroptosis prevented TLR4 activation in males and blunted the sex differences in PAH severity. We conclude that necrosis, through the release of HMGB1 dimers, predisposes males to a progressive form of PAH.
• Cho, S. G., Xiao, X., Wang, S., Gao, H., Rafikov, R., Black, S., Huang, S., Ding, H. F., Yoon, Y., Kirken, R. A., Yin, X. M., Wang, H. G., & Dong, Z. (2019). Bif-1 Interacts with Prohibitin-2 to Regulate Mitochondrial Inner Membrane during Cell Stress and Apoptosis. Journal of the American Society of Nephrology : JASN, 30(7), 1174-1191.
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  Mitochondria are dynamic organelles that undergo fission and fusion. During cell stress, mitochondrial dynamics shift to fission, leading to mitochondrial fragmentation, membrane leakage, and apoptosis. Mitochondrial fragmentation requires the cleavage of both outer and inner membranes, but the mechanism of inner membrane cleavage is unclear. Bif-1 and prohibitin-2 may regulate mitochondrial dynamics.
• Niihori, M., Eccles, C. A., Kurdyukov, S., Zemskova, M., Varghese, M. V., Stepanova, A. A., Galkin, A., Rafikov, R., & Rafikova, O. (2019). Rats with Human Mutation of NFU1 Develop Pulmonary Hypertension. American journal of respiratory cell and molecular biology.
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  NFU1 is a mitochondrial protein involved in iron-sulfur clusters biosynthesis, and its genetic modification is associated with disorders of mitochondrial energy metabolism. Patients with autosome recessive inheritance of NFU1 mutation G208C have reduced activity of the respiratory chain complex II, lipoic-acid dependent enzymes, and develop pulmonary arterial hypertension (PAH) in ~ 70% of cases. We investigated whether rats with a human mutation in NFU1 are also predisposed to PAH development. A point mutation in rat NFU1G206C (human G208C) was introduced through CRISP/Cas9 genome editing. Hemodynamic data, tissue samples, and fresh mitochondria were collected and analyzed. NFU1G206C rats showed an increased right ventricular (RV) pressure, RV hypertrophy and high levels of pulmonary artery remodeling. Computed tomography and angiography of the pulmonary vasculature indicated severe angioobliterative changes in NFU1G206C rats. Importantly, the penetrance of PAH phenotype was found to be more prevalent in females than in males, replicating the established gender difference within PAH patients. Male and female homozygote rats exhibited a decreased expression and activity of mitochondrial complex II, and markedly decreased pyruvate dehydrogenase activity and lipoate binding. The limited development of PAH in males correlated with the preserved levels of oligomeric NFU1, increased expression of ISCU, an alternative branch of iron-sulfur assembly system, and increased complex IV activity. Thus, the male sex has additional plasticity to overcome the iron-sulfur cluster deficiency. Our work describes a novel, humanized rat model of NFU1 deficiency showed mitochondrial dysfunction similar to patients and developed PAH with the same gender dimorphism.
• Rafikov, R., Al Ghouleh, I., & Rafikova, O. (2019). Focus on Early Events: Pathogenesis of Pulmonary Arterial Hypertension Development. Antioxidant Redox Signaling.
• Rafikov, R., James, J., McClain, N., Tofovic, S. P., & Rafikova, O. (2019). Role of Gender in Regulation of Redox Homeostasis in Pulmonary Arterial Hypertension. Antioxidants (Basel, Switzerland), 8(5).
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  Pulmonary arterial hypertension (PAH) is one of the diseases with a well-established gender dimorphism. The prevalence of PAH is increased in females with a ratio of 4:1, while poor survival prognosis is associated with the male gender. Nevertheless, the specific contribution of gender in disease development and progression is unclear due to the complex nature of the PAH. Oxidative and nitrosative stresses are important contributors in PAH pathogenesis; however, the role of gender in redox homeostasis has been understudied. This review is aimed to overview the possible sex-specific mechanisms responsible for the regulation of the balance between oxidants and antioxidants in relation to PAH pathobiology.
• Rafikov, R., McBride, M. L., Zemskova, M., Kurdyukov, S., McClain, N., Niihori, M., Langlais, P. R., & Rafikova, O. (2019). INOSITOL MONOPHOSPHATASE 1 (IMPA1) AS A NOVEL INTERACTING PARTNER OF RAGE IN PULMONARY HYPERTENSION. American journal of physiology. Lung cellular and molecular physiology.
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  Pulmonary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vascular remodeling. The receptor for advanced glycation endproducts (RAGE) plays an important role in PAH by promoting proliferation of pulmonary vascular cells. RAGE is also known to mediate activation of Akt signaling, although the particular molecular mechanism remains unknown. This study aimed to identify the interacting partner of RAGE that could facilitate RAGE-mediated Akt activation and vascular remodeling in PAH. The progressive angioproliferative PAH was induced in 24 female Sprague-Dawley rats (n=8/group) that were randomly assigned to develop PAH for one, two or five weeks (right ventricle peak systolic pressure (RVPSP) 56.5±3.2mmHg, 63.6±1.6mmHg and 111.1±4.5mmHg respectively vs. 22.9±1.1mmHg in Controls). PAH triggered early and late episodes of apoptosis in rat lungs accompanied by RAGE activation. Mass spectrometry analysis has identified IMPA1 as a novel PAH-specific interacting partner of RAGE. The proximity ligation assay (PLA) confirmed the formation of RAGE/IMPA1 complex in the pulmonary artery wall. Activation of IMPA1 in response to increased glucose-6-phosphate (G6P) is known to play a critical role in inositol synthesis and recycling. Indeed, we confirmed a 3-fold increase of G6P (p=0.0005) levels in lungs of PAH rats starting from week 1 that correlated with accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), membrane translocation of PI3K, and a 3-fold increase in membrane Akt levels (p=0.02) and Akt phosphorylation. We conclude that the formation of the newly discovered RAGE/IMPA1 complex could be responsible for the stimulation of inositol pathways and activation of Akt signaling in PAH.
• Rafikov, R., Nair, V., Sinari, S., Babu, H., Sullivan, J. C., Yuan, J. X., Desai, A. A., & Rafikova, O. (2019). Gender Difference in Damage-Mediated Signaling Contributes to Pulmonary Arterial Hypertension. Antioxidants & redox signaling, 31(13), 917-932.
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  Pulmonary arterial hypertension (PAH) is a progressive lethal disease with a known gender dimorphism. Female patients are more susceptible to PAH, whereas male patients have a lower survival rate. Initial pulmonary vascular damage plays an important role in PAH pathogenesis. Therefore, this study aimed at investigating the role of gender in activation of apoptosis/necrosis-mediated signaling pathways in PAH. The media collected from pulmonary artery endothelial cells (PAECs) that died by necrosis or apoptosis were used to treat naive PAECs. Necrotic cell death stimulated phosphorylation of toll-like receptor 4, accumulation of interleukin 1 beta, and expression of E-selectin in a redox-dependent manner; apoptosis did not induce any of these effects. In the animal model of severe PAH, the necrotic marker, high mobility group box 1 (HMGB1), was visualized in the pulmonary vascular wall of male but not female rats. This vascular necrosis was associated with male-specific redox changes in plasma, activation of the same inflammatory signaling pathway seen in response to necrosis , and an increased endothelial-leukocyte adhesion in small pulmonary arteries. In PAH patients, gender-specific changes in redox homeostasis correlated with the prognostic marker, B-type natriuretic peptide. Males had also shown elevated circulating levels of HMGB1 and pro-inflammatory changes. This study discovered the role of gender in the initiation of damage-associated signaling in PAH and highlights the importance of the gender-specific approach in PAH therapy. In PAH, the necrotic cell death is augmented in male patients compared with female patients. Factors released from necrotic cells could alter redox homeostasis and stimulate inflammatory signaling pathways.
• Rafikova, O., Al Ghouleh, I., & Rafikov, R. (2019). Focus on Early Events: Pathogenesis of Pulmonary Arterial Hypertension Development. Antioxidants & redox signaling, 31(13), 933-953.
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  Pulmonary arterial hypertension (PAH) is a progressive disease of the lung vasculature characterized by the proliferation of all vascular wall cell types, including endothelial, smooth muscle, and fibroblasts. The disease rapidly advances into a form with extensive pulmonary vascular remodeling, leading to a rapid increase in pulmonary vascular resistance, which results in right heart failure. Most current research in the PAH field has been focused on the late stage of the disease, largely due to an urgent need for patient treatment options in clinics. Further, the pathobiology of PAH is multifaceted in the advanced disease, and there has been promising recent progress in identifying various pathological pathways related to the late clinical picture. Early stage PAH still requires additional attention from the scientific community, and although the survival of patients with early diagnosis is comparatively higher, the disease develops in patients asymptomatically, making it difficult to identify and treat early. There are several reasons to focus on the early stage of PAH. First, the complexity of late stage disease, owing to multiple pathways being activated in a complex system with intra- and intercellular signaling, leads to an unclear picture of the key contributors to the pathobiology. Second, an understanding of early pathophysiological events can increase the ability to identify PAH patients earlier than what is currently possible. Third, the prompt diagnosis of PAH would allow for the therapy to start earlier, which has proved to be a more successful strategy, and it ensures better survival in PAH patients.
• Rafikova, O., Desai, A., Yuan, J., Sullivan, J., Babu, H., Sinari, S., Nair, V., & Rafikov, R. (2019). Gender Difference in Damage-Mediated Signaling Contributes to Pulmonary Arterial Hypertension. Antioxidant Redox Signaling.
• Rafikova, O., Langlais, P. R., Niihori, M., McClain, N., Kurdyukov, S., Zemskova, M., McBride, M. L., & Rafikov, R. (2019). INOSITOL MONOPHOSPHATASE 1 (IMPA1) AS A NOVEL INTERACTING PARTNER OF RAGE IN PULMONARY HYPERTENSION.. American Journal of Physiology - Lung Cellular and Molecular Physiology.
• Rafikova, O., Rafikov, R., Galkin, A., Stepanova, A. A., Varghese, M. V., Zemskova, M., Kurdyujov, S., Eccles, C., & Niihori, M. (2019). Rats with Human Mutation of NFU1 Develop Pulmonary Hypertension. Am J Respir Cell Mol Biol.
• Rafikova, O., Tofovic, S. S., McClain, N., James, J., & Rafikov, R. (2019). Role of Gender in Regulation of Redox Homeostasis in Pulmonary Arterial Hypertension. Antioxidants.
• Kurdyukov, S., Eccles, C. A., Desai, A. A., Gonzalez-Garay, M., Yuan, J. X., Garcia, J. G., Rafikova, O., & Rafikov, R. (2018). New cases of Glucose-6-Phosphate Dehydrogenase deficiency in Pulmonary Arterial Hypertension. PloS one, 13(8), e0203493.
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  Pulmonary Arterial Hypertension (PAH) is a fatal disorder with limited treatment options and reduced life expectancy after diagnosis. Complex genetic backgrounds in PAH complicates identification of causative mutations that is essential for an understanding of the disease diagnostics and etiology especially for idiopathic PAH (iPAH). Hemolysis has been implicated as contributing to the pathobiology of PAH. Glucose-6-Phosphate Dehydrogenase (G6PD) expression and activity define erythrocyte's antioxidant capacity, and its decrease contributes to erythrocyte fragility. As G6PD deficiency was previously reported in a limited number of PAH cases, we tested whether iPAH patients exhibit underlying G6PD alterations in erythrocytes. A cohort of 22 PAH patients and 8 non-PAH patients were recruited for this study. DNA isolated from Peripheral Blood Mononuclear Cells (PBMC) was used for detection of mutations in the coding region of the G6PD gene. RNA isolated from PBMC was used for determination of G6PD mRNA expression level. G6PD activity was measured in Red Blood Cell (RBC) pellets. Three patients had missense mutations in G6PD (Val291Met, Asn126Asp, Asp194Glu), however, only one mutation (Val291Met) results in a severe G6PD deficiency. A single patient with mutation (Asn126Asp) showed a 21% decrease in G6PD activity, two subjects showed G6PD deficiency without mutations, and one patient had a decreased level of G6PD mRNA and reduced enzyme levels. This study demonstrates that a moderate decrease in G6PD activity is associated with PAH. Screening for G6PD activity and mutations in the G6PD gene may provide early detection of individuals predisposed to PAH.
• Rafikova, O., Srivastava, A., Desai, A. A., Rafikov, R., & Tofovic, S. P. (2018). Recurrent inhibition of mitochondrial complex III induces chronic pulmonary vasoconstriction and glycolytic switch in the rat lung. Respiratory research, 19(1), 69.
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  Pulmonary arterial hypertension (PAH) is a fatal disease; however, the mechanisms directly involved in triggering and the progression of PAH are not clear. Based on previous studies that demonstrated a possible role of mitochondrial dysfunction in the pathogenesis of PAH, we investigated the effects of chronic inhibition of mitochondrial function in vivo in healthy rodents.
• Rafikova, O., Williams, E. R., McBride, M. L., Zemskova, M., Srivastava, A., Nair, V., Desai, A. A., Langlais, P. R., Zemskov, E., Simon, M., Mandarino, L. J., & Rafikov, R. (2018). Hemolysis-induced Lung Vascular Leakage Contributes to the Development of Pulmonary Hypertension. American journal of respiratory cell and molecular biology, 59(3), 334-345.
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  Although hemolytic anemia-associated pulmonary hypertension (PH) and pulmonary arterial hypertension (PAH) are more common than the prevalence of idiopathic PAH alone, the role of hemolysis in the development of PAH is poorly characterized. We hypothesized that hemolysis independently contributes to PAH pathogenesis via endothelial barrier dysfunction with resulting perivascular edema and inflammation. Plasma samples from patients with and without PAH (both confirmed by right heart catheterization) were used to measure free hemoglobin (Hb) and its correlation with PAH severity. A sugen (50 mg/kg)/hypoxia (3 wk)/normoxia (2 wk) rat model was used to elucidate the role of free Hb/heme pathways in PAH. Human lung microvascular endothelial cells were used to study heme-mediated endothelial barrier effects. Our data indicate that patients with PAH have increased levels of free Hb in plasma that correlate with PAH severity. There is also a significant accumulation of free Hb and depletion of haptoglobin in the rat model. In rats, perivascular edema was observed at early time points concomitant with increased infiltration of inflammatory cells. Heme-induced endothelial permeability in human lung microvascular endothelial cells involved activation of the p38/HSP27 pathway. Indeed, the rat model also exhibited increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling, there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF-1a during the first 2 weeks of PH regardless of hypoxic conditions. Our data suggest that hemolysis may play a significant role in PAH pathobiology.
• 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. (2017). LPS Induced Acute Lung Injury Involves the NF-κB-mediated Downregulation of SOX18. American journal of respiratory cell and molecular biology.
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  One of the early events in the progression of lipopolysaccharide (LPS)-mediated acute lung injury (ALI) 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-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, Claudin-5. Thus, the purpose of this study was to determine if down-regulation of the Sox18-Claudin-5 axis plays a role in the pulmonary endothelial barrier disruption associated with LPS exposure. Our data indicate that both Sox18 and Claudin-5 expression is decreased in two models of in vivo LPS exposure (intraperitoneal, intratracheal). A similar down-regulation was observed in cultured human lung microvascular endothelial cells (HLMVECs) exposed to LPS. Sox18 over-expression in HLMVECs or in the mouse lung attenuated the LPS-mediated vascular barrier disruption. Conversely, reduced Claudin-5 expression (siRNA) reduced the HLMVEC barrier protective effects of Sox18 over-expression. The mechanism by which LPS decreases Sox18 expression was identified as transcriptional repression through binding of p65 NF-kB to a Sox18 promoter sequence located between -1082 and -1073 bp with peroxynitrite contributing to LPS-mediated NF-kB activation. We conclude that NFkB-dependent decreases in the Sox18-Claudin 5 axis is essentially involved in the disruption of human EC barrier integrity associated with LPS-mediated ALI.
• Kruse, R., Krantz, J., Barker, N., Coletta, R. L., Rafikov, R., Luo, M., Højlund, K., Mandarino, L. J., & Langlais, P. R. (2017). Characterization of the CLASP2 Protein Interaction Network Identifies SOGA1 as a Microtubule-Associated Protein. Molecular & cellular proteomics : MCP, 16(10), 1718-1735.
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  CLASP2 is a microtubule-associated protein that undergoes insulin-stimulated phosphorylation and co-localization with reorganized actin and GLUT4 at the plasma membrane. To gain insight to the role of CLASP2 in this system, we developed and successfully executed a streamlined interactome approach and built a CLASP2 protein network in 3T3-L1 adipocytes. Using two different commercially available antibodies for CLASP2 and an antibody for epitope-tagged, overexpressed CLASP2, we performed multiple affinity purification coupled with mass spectrometry (AP-MS) experiments in combination with label-free quantitative proteomics and analyzed the data with the bioinformatics tool Significance Analysis of Interactome (SAINT). We discovered that CLASP2 coimmunoprecipitates (co-IPs) the novel protein SOGA1, the microtubule-associated protein kinase MARK2, and the microtubule/actin-regulating protein G2L1. The GTPase-activating proteins AGAP1 and AGAP3 were also enriched in the CLASP2 interactome, although subsequent AGAP3 and CLIP2 interactome analysis suggests a preference of AGAP3 for CLIP2. Follow-up MARK2 interactome analysis confirmed reciprocal co-IP of CLASP2 and revealed MARK2 can co-IP SOGA1, glycogen synthase, and glycogenin. Investigating the SOGA1 interactome confirmed SOGA1 can reciprocal co-IP both CLASP2 and MARK2 as well as glycogen synthase and glycogenin. SOGA1 was confirmed to colocalize with CLASP2 and with tubulin, which identifies SOGA1 as a new microtubule-associated protein. These results introduce the metabolic function of these proposed novel protein networks and their relationship with microtubules as new fields of cytoskeleton-associated protein biology.
• Kumar, S., Sun, X., Noonepalle, S. K., Lu, Q., Zemskov, E., Wang, T., Aggarwal, S., Gross, C., Sharma, S., Desai, A. A., Hou, Y., Dasarathy, S., Qu, N., Reddy, V., Lee, S. G., Cherian-Shaw, M., Yuan, J. X., Catravas, J. D., Rafikov, R., , Garcia, J. G., et al. (2017). Hyper-activation of pp60(Src) limits nitric oxide signaling by increasing asymmetric dimethylarginine levels during acute lung injury. Free radical biology & medicine, 102, 217-228.
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  The molecular mechanisms by which the endothelial barrier becomes compromised during lipopolysaccharide (LPS) mediated acute lung injury (ALI) are still unresolved. We have previously reported that the disruption of the endothelial barrier is due, at least in part, to the uncoupling of endothelial nitric oxide synthase (eNOS) and increased peroxynitrite-mediated nitration of RhoA. The purpose of this study was to elucidate the molecular mechanisms by which LPS induces eNOS uncoupling during ALI. Exposure of pulmonary endothelial cells (PAEC) to LPS increased pp60(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.
• Rafikova, O., & Rafikov, R. (2017). Pulmonary arterial hypertension: are we close to the success?. Journal of clinical respiratory medicine, 1(1), 1-2.
• Sakipov, S., Rafikova, O., Kurnikova, M. G., & Rafikov, R. (2017). Molecular mechanisms of bio-catalysis of heme extraction from hemoglobin. Redox biology, 11, 516-523.
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  Red blood cell hemolysis in sickle cell disease (SCD) releases free hemoglobin. Extracellular hemoglobin and its degradation products, free heme and iron, are highly toxic due to oxidative stress induction and decrease in nitric oxide availability. We propose an approach that helps to eliminate extracellular hemoglobin toxicity in SCD by employing a bacterial protein system that evolved to extract heme from extracellular hemoglobin. NEAr heme Transporter (NEAT) domains from iron-regulated surface determinant proteins from Staphylococcus aureus specifically bind free heme as well as facilitate its extraction from hemoglobin. We demonstrate that a purified NEAT domain fused with human haptoglobin β-chain is able to remove heme from hemoglobin and reduce heme content and peroxidase activity of hemoglobin. We further use molecular dynamics (MD) simulations to resolve molecular pathway of heme transfer from hemoglobin to NEAT, and to elucidate molecular mechanism of such heme transferring process. Our study is the first of its kind, in which simulations are employed to characterize the process of heme leaving hemoglobin and subsequent rebinding with a NEAT domain. Our MD results highlight important amino acid residues that facilitate heme transfer and will guide further studies for the selection of best NEAT candidate to attenuate free hemoglobin toxicity.
• Chen, F., Wang, Y., Rafikov, R., Haigh, S., Zhi, W. B., Kumar, S., Doulias, P. T., Rafikova, O., Pillich, H., Chakraborty, T., Lucas, R., Verin, A. D., Catravas, J. D., She, J. X., Black, S. M., & Fulton, D. J. (2016). RhoA S-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to G(+)-bacterial toxins. Biochemical pharmacology.
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  Disruption of the endothelial barrier in response to Gram positive (G(+)) bacterial toxins is a major complication of acute lung injury (ALI) and can be further aggravated by antibiotics which stimulate toxin release. The integrity of the pulmonary endothelial barrier is mediated by the balance of disruptive forces such as the small GTPase RhoA, and protective forces including endothelium-derived nitric oxide (NO). How NO protects against the barrier dysfunction is incompletely understood and our goal was to determine whether NO and S-nitrosylation can modulate RhoA activity and whether this mechanism is important for G(+) toxin-induced microvascular permeability. We found that the G(+) toxin listeriolysin-O (LLO) increased RhoA activity and that NO and S-NO donors inhibit RhoA activity. RhoA was robustly S-nitrosylated as determined by biotin-switch and mercury column analysis. MS revealed that three primary cysteine residues are S-nitrosylated including cys16, cys20 and cys159. Mutation of these residues to serine diminished S-nitrosylation to endogenous NO and mutant RhoA was less sensitive to inhibition by S-NO. G(+)-toxins stimulated the denitrosylation of RhoA which was not mediated by S-nitrosoglutathione reductase (GSNOR), thioredoxin (TRX) or thiol-dependent enzyme activity but was instead stimulated directly by elevated calcium levels. Calcium-promoted the direct denitrosylation of WT but not mutant RhoA and mutant RhoA adenovirus was more effective than WT in disrupting the barrier integrity of human lung microvascular endothelial cells. In conclusion, we reveal a novel mechanism by which NO and S-nitrosylation reduces RhoA activity which may be of significance in the management of pulmonary endothelial permeability induced by G(+)-toxins.
• Chen, Q., Varga, M., Wang, X., Haddad, D. J., An, S., Medzikovic, L., Derakhshandeh, R., Kostyushev, D. S., Zhang, Y., Clifford, B. T., Luu, E., Danforth, O. M., Rafikov, R., Gong, W., Black, S. M., Suchkov, S. V., Fineman, J. R., Heiss, C., Aschbacher, K., , Yeghiazarians, Y., et al. (2016). Overexpression of Nitric Oxide Synthase Restores Circulating Angiogenic Cell Function in Patients With Coronary Artery Disease: Implications for Autologous Cell Therapy for Myocardial Infarction. Journal of the American Heart Association, 5(1).
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  Circulating angiogenic cells (CACs) are peripheral blood cells whose functional capacity inversely correlates with cardiovascular risk and that have therapeutic benefits in animal models of cardiovascular disease. However, donor age and disease state influence the efficacy of autologous cell therapy. We sought to determine whether age or coronary artery disease (CAD) impairs the therapeutic potential of CACs for myocardial infarction (MI) and whether the use of ex vivo gene therapy to overexpress endothelial nitric oxide (NO) synthase (eNOS) overcomes these defects.
• Indik, J. H., Nair, V., Rafikov, R., Nyotowidjojo, I. S., Bisla, J., Kansal, M., Parikh, D. S., Robinson, M., Desai, A., Oberoi, M., Gupta, A., Abbasi, T., Khalpey, Z., Patel, A. R., Lang, R. M., Dudley, S. C., Choi, B. R., Garcia, J. G., Machado, R. F., & Desai, A. A. (2016). Associations of Prolonged QTc in Sickle Cell Disease. PloS one, 11(10), e0164526.
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  Sudden death is a leading cause of mortality in sickle cell disease, implicating ventricular tachyarrhythmias. Prolonged QTc on an electrocardiogram (ECG), commonly seen with myocardial ischemia, is a known risk for polymorphic ventricular tachycardia (VT). We hypothesized that prolonged QTc is associated with mortality in sickle cell disease. ECG were analyzed from a cohort of 224 sickle patients (University of Illinois at Chicago, UIC) along with available laboratory, and echocardiographic findings, and from another cohort of 38 patients (University of Chicago, UC) for which cardiac MRI and free heme values were also measured. In the UIC cohort, QTc was potentially related to mortality with a hazard ratio (HR) of 1.22 per 10ms, (P = 0.015), and a HR = 3.19 (P = 0.045) for a QTc>480ms. In multivariate analyses, QTc remained significantly associated with survival after adjusting for inpatient ECG status (HR 1.26 per 10ms interval, P = 0.010) and genotype status [HR 1.21 per 10ms interval, P = 0.037). QTc trended toward association with mortality after adjusting for both LDH and hydroxyurea use (HR 1.21 per 10ms interval, P = 0.062) but was not significant after adjusting for TRV. In univariate analyses, QTc was related to markers of hemolysis including AST (P = 0.031), hemoglobin (P = 0.014), TR velocity (P = 0.036), higher in inpatients (P
• Rafikova, O., Meadows, M. L., Kinchen, J. M., Mohney, R. P., Maltepe, E., Desai, A. A., Yuan, J. X., Garcia, J. G., Fineman, J. R., Rafikov, R., & Black, S. M. (2016). Metabolic Changes Precede the Development of Pulmonary Hypertension in the Monocrotaline Exposed Rat Lung. PloS one, 11(3), e0150480.
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  There is increasing interest in the potential for metabolic profiling to evaluate the progression of pulmonary hypertension (PH). However, a detailed analysis of the metabolic changes in lungs at the early stage of PH, characterized by increased pulmonary artery pressure but prior to the development of right ventricle hypertrophy and failure, is lacking in a preclinical animal model of PH. Thus, we undertook a study using rats 14 days after exposure to monocrotaline (MCT), to determine whether we could identify early stage metabolic changes prior to the manifestation of developed PH. We observed changes in multiple pathways associated with the development of PH, including activated glycolysis, increased markers of proliferation, disruptions in carnitine homeostasis, increased inflammatory and fibrosis biomarkers, and a reduction in glutathione biosynthesis. Further, our global metabolic profile data compare favorably with prior work carried out in humans with PH. We conclude that despite the MCT-model not recapitulating all the structural changes associated with humans with advanced PH, including endothelial cell proliferation and the formation of plexiform lesions, it is very similar at a metabolic level. Thus, we suggest that despite its limitations it can still serve as a useful preclinical model for the study of PH.
• Rafikova, O., Rafikov, R., Kangath, A., Qu, N., Aggarwal, S., Sharma, S., Desai, J., Fields, T., Ludewig, B., Yuan, J. X., Jonigk, D., & Black, S. M. (2016). Redox regulation of epidermal growth factor receptor signaling during the development of pulmonary hypertension. Free radical biology & medicine, 95, 96-111.
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  The development of pulmonary hypertension (PH) involves the uncontrolled proliferation of pulmonary smooth muscle cells via increased growth factor receptor signaling. However, the role of epidermal growth factor receptor (EGFR) signaling is controversial, as humans with advanced PH exhibit no changes in EGFR protein levels and purpose of the present study was to determine whether there are post-translational mechanisms that enhance EGFR signaling in PH. The EGFR inhibitor, gefinitib, significantly attenuated EGFR signaling and prevented the development of PH in monocrotaline (MCT)-exposed rats, confirming the contribution of EGFR activation in MCT induced PH. There was an early MCT-mediated increase in hydrogen peroxide, which correlated with the binding of the active metabolite of MCT, monocrotaline pyrrole, to catalase Cys377, disrupting its multimeric structure. This early oxidative stress was responsible for the oxidation of EGFR and the formation of sodium dodecyl sulfate (SDS) stable EGFR dimers through dityrosine cross-linking. These cross-linked dimers exhibited increased EGFR autophosphorylation and signaling. The activation of EGFR signaling did not correlate with pp60(src) dependent Y845 phosphorylation or EGFR ligand expression. Importantly, the analysis of patients with advanced PH revealed the same enhancement of EGFR autophosphorylation and covalent dimer formation in pulmonary arteries, while total EGFR protein levels were unchanged. As in the MCT exposed rat model, the activation of EGFR in human samples was independent of pp60(src) phosphorylation site and ligand expression. This study provides a novel molecular mechanism of oxidative stress stimulated covalent EGFR dimerization via tyrosine dimerization that contributes into development of PH.
• Sun, X., Kellner, M., Desai, A. A., Wang, T., Lu, Q., Kangath, A., Qu, N., Klinger, C., Fratz, S., Yuan, J. X., Jacobson, J. R., Garcia, J. G., Rafikov, R., Fineman, J. R., & Black, S. M. (2016). Asymmetric Dimethylarginine Stimulates Akt1 Phosphorylation via Heat Shock Protein 70-Facilitated Carboxyl-Terminal Modulator Protein Degradation in Pulmonary Arterial Endothelial Cells. American journal of respiratory cell and molecular biology, 55(2), 275-87.
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  Asymmetric dimethylarginine (ADMA) induces the mitochondrial translocation of endothelial nitric oxide synthase (eNOS) through the nitration-mediated activation of Akt1. However, it is recognized that the activation of Akt1 requires phosphorylation events at threonine (T) 308 and serine (S) 473. Thus, the current study was performed to elucidate the potential effect of ADMA on Akt1 phosphorylation and the mechanisms that are involved. Exposure of pulmonary arterial endothelial cells to ADMA enhanced Akt1 phosphorylation at both threonine 308 and Ser473 without altering Akt1 protein levels, phosphatase and tensin homolog activity, or membrane Akt1 levels. Heat shock protein (Hsp) 90 plays a pivotal role in maintaining Akt1 activity, and our results demonstrate that ADMA decreased Hsp90-Akt1 interactions, but, surprisingly, overexpression of a dominant-negative Hsp90 mutant increased Akt1 phosphorylation. ADMA exposure or overexpression of dominant-negative Hsp90 increased Hsp70 levels, and depletion of Hsp70 abolished ADMA-induced Akt1 phosphorylation. ADMA decreased the interaction of Akt1 with its endogenous inhibitor, carboxyl-terminal modulator protein (CTMP). This was mediated by the proteasomal-dependent degradation of CTMP. The overexpression of CTMP attenuated ADMA-induced Akt1 phosphorylation at Ser473, eNOS phosphorylation at Ser617, and eNOS mitochondrial translocation. Finally, we found that the mitochondrial translocation of eNOS in our lamb model of pulmonary hypertension is associated with increased Akt1 and eNOS phosphorylation and reduced Akt1-CTMP protein interactions. In conclusion, our data suggest that CTMP is directly involved in ADMA-induced Akt1 phosphorylation in vitro and in vivo, and that increasing CTMP levels may be an avenue to treat pulmonary hypertension.
• Gross, C. M., Rafikov, R., Kumar, S., Aggarwal, S., Ham, P. B., Meadows, M. L., Cherian-Shaw, M., Kangath, A., Sridhar, S., Lucas, R., & Black, S. M. (2015). Endothelial nitric oxide synthase deficient mice are protected from lipopolysaccharide induced acute lung injury. PloS one, 10(3), e0119918.
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  Lipopolysaccharide (LPS) derived from the outer membrane of gram-negative bacteria induces acute lung injury (ALI) in mice. This injury is associated with lung edema, inflammation, diffuse alveolar damage, and severe respiratory insufficiency. We have previously reported that LPS-mediated nitric oxide synthase (NOS) uncoupling, through increases in asymmetric dimethylarginine (ADMA), plays an important role in the development of ALI through the generation of reactive oxygen and nitrogen species. Therefore, the focus of this study was to determine whether mice deficient in endothelial NOS (eNOS-/-) are protected against ALI. In both wild-type and eNOS-/- mice, ALI was induced by the intratracheal instillation of LPS (2 mg/kg). After 24 hours, we found that eNOS-/-mice were protected against the LPS mediated increase in inflammatory cell infiltration, inflammatory cytokine production, and lung injury. In addition, LPS exposed eNOS-/- mice had increased oxygen saturation and improved lung mechanics. The protection in eNOS-/- mice was associated with an attenuated production of NO, NOS derived superoxide, and peroxynitrite. Furthermore, we found that eNOS-/- mice had less RhoA activation that correlated with a reduction in RhoA nitration at Tyr34. Finally, we found that the reduction in NOS uncoupling in eNOS-/- mice was due to a preservation of dimethylarginine dimethylaminohydrolase (DDAH) activity that prevented the LPS-mediated increase in ADMA. Together our data suggest that eNOS derived reactive species play an important role in the development of LPS-mediated lung injury.
• Kovacs, L., Han, W., Rafikov, R., Bagi, Z., Offermanns, S., Saido, T. C., Black, S. M., & Su, Y. (2015). Activation of Calpain-2 by Mediators in Pulmonary Vascular Remodeling of Pulmonary Arterial Hypertension. American journal of respiratory cell and molecular biology.
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  Calpain mediates the collagen synthesis and cell proliferation and plays an important role in pulmonary vascular remodeling in pulmonary arterial hypertension (PAH). In the present study, we investigate whether and how calpain is activated by PAH mediators in pulmonary artery smooth muscle cells (PASMCs). These data showed that smooth muscle-specific knockout of calpain attenuated and knockout of calpastatin potentiated pulmonary vascular remodeling and pulmonary hypertension. Treatment of PASMCs with the PAH mediators platelet-derived growth factor (PDGF), serotonin, H2O2, endothelin-1 and interleukin-6 caused significant increases in calpain activity, cell proliferation, and collagen-I protein level without changes in protein levels of calpain-1 and -2. The calcium chelator BAPTA/AM did not affect calpain activation but the extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor PD98059 and knocking down of calpain-2 prevented calpain activation in PAH mediator-treated PASMCs. Mass spectrometry data showed that the phosphorylation of calpain-2 at serine 50 (Ser50) was increased and the phosphorylation of calpain-2 at serine 369 (Ser369) was decreased in PDGF-treated PASMCs. The PDGF-induced increase in Ser50 phosphorylation of calpain-2 was prevented by the ERK1/2 inhibitor PD98059, while dephosphorylation of calpain-2 at Ser369 was blocked by the protein phosphatase 2A (PP2A) inhibitor fostriecin. Furthermore, smooth muscle of pulmonary arteries in PAH animal model and PAH patients showed higher levels of P-Ser50-calpain-2 and lower levels of P-Ser369-calpain-2. These data support that calpain modulates pulmonary vascular remodeling in PAH. PAH mediator-induced activation of calpain is caused by ERK1/2-dependent phosphorylation of calpain-2 at Ser50 and PP2A-dependent dephosphorylation of calpain-2 at Ser369 in pulmonary vascular remodeling of PAH.
• Lu, Q., Harris, V. A., Rafikov, R., Sun, X., Kumar, S., & Black, S. M. (2015). Nitric oxide induces hypoxia ischemic injury in the neonatal brain via the disruption of neuronal iron metabolism. Redox biology, 6, 112-21.
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  We have recently shown that increased hydrogen peroxide (H2O2) generation is involved in hypoxia-ischemia (HI)-mediated neonatal brain injury. H2O2 can react with free iron to form the hydroxyl radical, through Fenton Chemistry. Thus, the objective of this study was to determine if there was a role for the hydroxyl radical in neonatal HI brain injury and to elucidate the underlying mechanisms. Our data demonstrate that HI increases the deposition of free iron and hydroxyl radical formation, in both P7 hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD), and the neonatal rat exposed to HI. Both these processes were found to be nitric oxide (NO) dependent. Further analysis demonstrated that the NO-dependent increase in iron deposition was mediated through increased transferrin receptor expression and a decrease in ferritin expression. This was correlated with a reduction in aconitase activity. Both NO inhibition and iron scavenging, using deferoxamine administration, reduced hydroxyl radical levels and neuronal cell death. In conclusion, our results suggest that increased NO generation leads to neuronal cell death during neonatal HI, at least in part, by altering iron homeostasis and hydroxyl radical generation.
• Nadtochiy, S. M., Zhu, Q. M., Urciuoli, W., Rafikov, R., Black, S. M., & Brookes, P. S. (2015). Nitroalkenes confer acute cardioprotection via adenine nucleotide translocase 1. The Journal of biological chemistry, 290(51), 30267.
• Rafikov, R., Sun, X., Rafikova, O., Louise Meadows, M., Desai, A. A., Khalpey, Z., Yuan, J. X., Fineman, J. R., & Black, S. M. (2015). Complex I dysfunction underlies the glycolytic switch in pulmonary hypertensive smooth muscle cells. Redox biology, 6, 278-86.
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  ATP is essential for cellular function and is usually produced through oxidative phosphorylation. However, mitochondrial dysfunction is now being recognized as an important contributing factor in the development cardiovascular diseases, such as pulmonary hypertension (PH). In PH there is a metabolic change from oxidative phosphorylation to mainly glycolysis for energy production. However, the mechanisms underlying this glycolytic switch are only poorly understood. In particular the role of the respiratory Complexes in the mitochondrial dysfunction associated with PH is unresolved and was the focus of our investigations. We report that smooth muscle cells isolated from the pulmonary vessels of rats with PH (PH-PASMC), induced by a single injection of monocrotaline, have attenuated mitochondrial function and enhanced glycolysis. Further, utilizing a novel live cell assay, we were able to demonstrate that the mitochondrial dysfunction in PH-PASMC correlates with deficiencies in the activities of Complexes I-III. Further, we observed that there was an increase in mitochondrial reactive oxygen species generation and mitochondrial membrane potential in the PASMC isolated from rats with PH. We further found that the defect in Complex I activity was due to a loss of Complex I assembly, although the assembly of Complexes II and III were both maintained. Thus, we conclude that loss of Complex I assembly may be involved in the switch of energy metabolism in smooth muscle cells to glycolysis and that maintaining Complex I activity may be a potential therapeutic target for the treatment of PH.
• Rafikova, O., Rafikov, R., Meadows, M. L., Kangath, A., Jonigk, D., & Black, S. M. (2015). The sexual dimorphism associated with pulmonary hypertension corresponds to a fibrotic phenotype. Pulmonary circulation, 5(1), 184-97.
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  Although female predominance in the development of all types of pulmonary hypertension (PH) is well established, many clinical studies have confirmed that females have better prognosis and higher survival rate than males. There is no clear explanation of why sex influences the pathogenesis and progression of PH. Using a rat angioproliferative model of PH, which closely resembles the primary pathological changes observed in humans, we evaluated the role of sex in the development and progression of PH. Female rats had a more pronounced increase in medial thickness in the small pulmonary arteries. However, the infiltration of small pulmonary arteries by inflammatory cells was found only in male rats, and this corresponded to increased myeloperoxidase activity and abundant adventitial and medial fibrosis that were not present in female rats. Although the level of right ventricle (RV) peak systolic pressure was similar in both groups, the survival rate in male rats was significantly lower. Moreover, male rats presented with a more pronounced increase in RV thickness that correlated with diffuse RV fibrosis and significantly impaired right cardiac function. The reduction in fibrosis in female rats correlated with increased expression of caveolin-1 and reduced endothelial nitric oxide synthase-derived superoxide. We conclude that, in the pathogenesis of PH, female sex is associated with greater remodeling of the pulmonary arteries but greater survival. Conversely, in males, the development of pulmonary and cardiac fibrosis leads to early and severe RV failure, and this may be an important reason for the lower survival rate among males.

Presentations

• Rafikov, R., Rafikova, O., Niihori, M., Zemskova, M., Eccles, C., & Kurdyukov, S. (2018, Nov). Pulmonary hypertension in rats with human mutation of NFU1 is oxygen sensitive. Society for redox biology and medicine. Chicago, IL.

Poster Presentations

• Rafikov, R., Rafikova, O., Langlais, P. R., Zemskova, M., Niihori, M., Varghese, M. V., & James, J. (2020, May/Summer). The NFU1 G206C mutation metabolically reprograms pulmonary artery smooth muscle cells, promotes proliferation and apoptosis resistance. American Thoracic Society.
• Rafikov, R., Rafikova, O., Langlais, P. R., Barker, N. K., Niihori, M., Varghese, M. V., Eccles, C. A., Zemskova, M., & James, J. (2019, Novermber/Fall). A single mutation in NFU1 metabolically reprograms pulmonary artery smooth muscle cells and drives proliferation with apoptosis resistance. The Society for Redox Biology and Medicine's 26th Annual Conference.
• Rafikova, O., Eccles, C. A., Niihiri, M., & Rafikov, R. (2019, June). Altered pulmonary vasculature development, reticulocyte maturation and increased hemolysis in PH model of severe mitochondrial dysfunction. The Grover Conference - American Thoracic Society.
• Rafikova, O., James, J., Zemskova, M., McClain, N., & Rafikov, R. (2019, Nov). Distinct HMGB1 signaling induced by endothelial or smooth muscle pulmonary vascular cells. 9th International DAMPs and Alarmins Symposium.
• Rafikova, O., McClain, N., Kurdyukov, S., Zemskova, M., & Rafikov, R. (2019, Nov). Sex difference in the type of cell death predispose males to increased HMGB1 release and signaling. 9th International DAMPs and Alarmins Symposium.
• Desai, A., Rafikov, R., & Rafikova, O. (2018, July). Male gender predisposes PAH patients to reductive stress and disease progression. Pulmonary Hypertension Association's 2018 International PH Conference and Scientific Sessions.
• Eccles, C., Niihori, M., Rafikov, R., & Rafikova, O. (2018, November). Rats with Human Mutation of Fe-S Cluster Scaffold Protein NFU1 Develop Pulmonary Hypertension. American Heart Association - Scientific Sessions.
• Kurdyukov, S., Zemskov, M., Rafikov, R., & Rafikova, O. (2018, July). Gender difference on the cellular level: distinct stress responses in male and female endothelial cells isolated from mouse lungs. Pulmonary Hypertension Association's 2018 International PH Conference and Scientific Sessions.
• Niihori, M., Kurdyukov, S., Eccles, C., Varghese, M., Rafikova, O., & Rafikov, R. (2018, May). Antioxidant conjugated peptide attenuated metabolic reprogramming in pulmonary hypertension. American Thoracic Society meeting. Dallas, TX, USA.
• Rafikov, R., Rafikov, R., Rafikova, O., Rafikova, O., Langlais, P. R., Langlais, P. R., Vasilyev, M., & Vasilyev, M. (2018, Novermber/Fall). Inhibition of respiratory chain Complex III irreversibly changes mitochondria proteomic landscape. The Society for Redox Biology and Medicine's 25th Annual Conference.
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  The role of mitochondria dysfunction in the pathogenesis of pulmonary hypertension (PH) is currently well-recognized. However, the particular mechanisms and the type of mitochondrial dysfunction are still being debated. We have recently shown that chronic inhibition of oxidative phosphorylation by Antimycin A (AA) results in increased pulmonary pressure and remodeled pulmonary arteries. AA (0.35mg/kg) was given to rats three times for six days and lungs were analyzed 30 minutes (acute effect), 12 and 24 days (chronic effect) after the first AA injection. Mitochondria isolated from lungs were subjected to mass spectrometry and quantitative proteomic analysis to estimate changes in the isolated mitochondrial proteome over the course of disease development. Using a 4-fold change cutoff value for effects on protein expression, 48 mitochondrial proteins were discovered to be altered upon AA treatment, with 13 proteins exhibiting a significant difference (ANOVA). Our data indicate that using a 4-fold cutoff w distinguished between the control and 24 day groups by principal component analysis (PCA). Functional analysis of the proteome data revealed major downregulation of enzymes involved in fatty acids oxidation (ACDSB, HADH) and fatty acids transport (CPT1, ACSM5). Electron transport chain proteins showed mixed results with downregulation of Complex IV (COX1/2), altered Complex I subunits (NU4/5M) and assembly protein expression (NDUF4), and upregulation of ubiquinone biosynthesis (COQ3/7). Activation of ubiquinone biosynthesis could be explained as compensation for inhibition of mitochondrial respiration by AA. Mitochondrial machinery for importing proteins into the matrix (TIM9/10/13) was upregulated, but peptidases (LON, MPPA) that control the quality of matrix proteins were downregulated. Finally, proteins that regulate mitochondria morphology (MIRO2, PGAM5, TMM11) and degradation pathways (MIEAP, BCL2) were altered. Collectively, our data indicate that chronic inhibition of the respiratory chain for six days leads to irreversible changes in the proteomic landscape of mitochondria. These changes reprogram mitochondria to a different metabolic state and altered morphology that have been found in various models of PH and PH patients.
• Rafikova, O., Rafikova, O., Rafikova, O., Mandarino, L. J., Mandarino, L. J., Mandarino, L. J., Zemskov, E., Zemskov, E., Zemskov, E., Langlais, P. R., Langlais, P. R., Langlais, P. R., Desai, A., Desai, A., Desai, A., Srivastava, A., Srivastava, A., Srivastava, A., Rafikov, R., , Rafikov, R., et al. (2018, May). Free heme-mediated endothelial barrier dysfunction contributes to the development of pulmonary hypertension. American Thoracic Society.
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  Our data indicate that PH patients have increased levels of free Hb in plasma that correlate with disease severity and progression. There is also a significant accumulation of free Hb and depletion of haptoglobin in the sugen/hypoxia rat model. In rats, perivascular edema was observed during first two weeks of PH concomitant with increased infiltration of inflammatory cells. In the cell culture model of HLMVECs, we found that not hemoglobin but free heme-induced endothelial permeability via activation of the p38/HSP27 signaling pathway. Indeed, the rat model also exhibited an increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling; there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF1a during the first two weeks of PAH regardless of hypoxic conditions. We found that heme-mediated effects on endothelium, at least in part, depend on Heme Carrier Protein 1 (HCP-1) and pharmacological inhibition of HCP-1 by sulfasalazine reduced barrier disruptive potential of the heme. Sulfasalazine administration to sugen/hypoxia rats results in attenuation of PH by a reduction in vascular remodeling in the lungs as well as decreasing right heart hypertrophy.
• Rafikova, O., Rafikova, O., Rafikova, O., Rafikov, R., Rafikov, R., Rafikov, R., Langlais, P. R., Langlais, P. R., Langlais, P. R., McBride, M. L., McBride, M. L., & McBride, M. L. (2018, May). Receptor for Advanced Glycation Endproducts (RAGE) regulates metabolic reprogramming induced over-proliferation in pulmonary hypertension. American Thoracic Society.
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  Our data indicate that PH patients have increased levels of free Hb in plasma that correlate with disease severity and progression. There is also a significant accumulation of free Hb and depletion of haptoglobin in the sugen/hypoxia rat model. In rats, perivascular edema was observed during first two weeks of PH concomitant with increased infiltration of inflammatory cells. In the cell culture model of HLMVECs, we found that not hemoglobin but free heme-induced endothelial permeability via activation of the p38/HSP27 signaling pathway. Indeed, the rat model also exhibited an increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling; there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF1a during the first two weeks of PAH regardless of hypoxic conditions. We found that heme-mediated effects on endothelium, at least in part, depend on Heme Carrier Protein 1 (HCP-1) and pharmacological inhibition of HCP-1 by sulfasalazine reduced barrier disruptive potential of the heme. Sulfasalazine administration to sugen/hypoxia rats results in attenuation of PH by a reduction in vascular remodeling in the lungs as well as decreasing right heart hypertrophy.
• Sergey Kurdyukov, S., Zemskova, M., Rafikov, R., & Rafikova, O. (2018, November). Isolated Pulmonary Endothelial Cells Preserve Gender Specific Sensitivity to Stress. American Heart Association - Scientific Sessions.
• Vasilyev, M., Eccles, C., Niihori, M., Rafikov, R., & Rafikova, O. (2018, May). Pulmonary Hypertension and Metabolic Disease in Rats with Human Mutation in Fe-S Cluster Scaffold Protein NFU1. American Thoracic Society meeting.
• McBride, M. L., Rafikova, O., Williams, E. R., Rafikov, R., Mandarino, L. J., Langlais, P. R., Langlais, P. R., Mandarino, L. J., Williams, E. R., Rafikov, R., McBride, M. L., & Rafikova, O. (2017, June). Inositol Monophosphate 1 (IMPA1) And Rage Interaction: The Role Of Novel Proliferative Pathway In Pulmonary Hypertension. American Heart Association.
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  This is a very first evidence that damage induced RAGE activation is involved in protection from glycolysis-mediated osmotic stress and activation of Akt pathway in PAH by the formation of RAGE/IMPA1 complex.
• Rafikov, R., Srivastava, A., Desai, A., Langlais, P. R., Zemskov, E., Mandarino, L. J., & Rafikova, O. (2017, June). Hemolysis-mediated vascular permeability in lungs contributes to the development of pulmonary hypertension. American Heart Association.
  More info

  Our data indicate that PH patients have increased levels of free Hb in plasma that correlate with disease severity and progression. There is also a significant accumulation of free Hb and depletion of haptoglobin in the sugen/hypoxia rat model. In rats, perivascular edema was observed during first two weeks of PH concomitant with increased infiltration of inflammatory cells. In the cell culture model of HLMVECs, we found that not hemoglobin but free heme-induced endothelial permeability via activation of the p38/HSP27 signaling pathway. Indeed, the rat model also exhibited an increased activation of p38/HSP27 during the initial phase of PH. Surprisingly, despite the increased levels of hemolysis and heme-mediated signaling; there was no heme oxygenase-1 activation. This can be explained by observed destabilization of HIF1a during the first two weeks of PAH regardless of hypoxic conditions. We found that heme-mediated effects on endothelium, at least in part, depend on Heme Carrier Protein 1 (HCP-1) and pharmacological inhibition of HCP-1 by sulfasalazine reduced barrier disruptive potential of the heme. Sulfasalazine administration to sugen/hypoxia rats results in attenuation of PH by a reduction in vascular remodeling in the lungs as well as decreasing right heart hypertrophy.