Pulmonary arterial hypertension (PAH) is a chronic cardiopulmonary disorder instigated by pulmona... more 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.
Pulmonary arterial hypertension (PAH) is an incurable, progressive disorder, and the early diagno... more Pulmonary arterial hypertension (PAH) is an incurable, progressive disorder, and the early diagnosis and treatment of PAH are associated with increased survival [1,2]. However, because of the asymptomatic nature of PAH development, there are severe delays in PAH diagnosis. In most cases, therapy only starts at an advanced stage of the disease. Thus, there is a critical need to improve the diagnostic approaches, for example, by using markers of PAH that can be detected in easily obtained specimens like plasma or urine [3]. Our previous study showed that in the monocrotaline-treated rat model, metabolic reprogramming occurs early in the development of the disease and foreshadows pathophysiological changes [4]. Thus, assessing the profile of circulating metabolites could allow the identification of PAH patients earlier, before the condition has progressed sufficiently to enter an advanced irreversible stage. Several previous reports have already confirmed a significant difference in the metabolic profile of healthy controls and PAH animals and patients [4-7]. However, given that the average age of PAH patients has increased [8], there is a strong need to diagnose PAH in older people that are not entirely "healthy." In this study, we compared the plasma metabolic profiles of well-phenotyped patients with idiopathic PAH (IPAH) obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI) with two other patient cohorts-patients with type 2 diabetes mellitus (DM; University of Arizona (UA) Center for Disparities in Diabetes, Obesity, and Metabolism) and patients with left heart diseases (Heart; UA biobank). The incidence of non-PAH cardiovascular diseases and type 2 diabetes significantly increases with age, especially in people over 40-45 years old [9,10]. Furthermore, these conditions are associated with severe alterations in metabolism that could mask the changes associated with metabolic reprogramming in PAH [11,12]. Therefore, the goal of the study was to evaluate whether the metabolic profile of PAH patients is distinct compared to these patients and could be used to identify PAH when compared to people who have one of these diseases. The patients' ages were (mean ± standard deviation (SD)) 41.1 ± 15.7, 41.8 ± 13.7, and 50.2 ± 10.9, and the sex ratios of female/male (%) were 81.8/18.1, 83.3/16.7, and 72.7/27.3 for PAH (n = 11), DM (n = 12), and heart (n = 11), respectively. All of the PAH patients were receiving PAH therapy. No patients from the PAH cohort had DM; one patient had valvular left ventricular (LV) heart disease. The targeted metabolomics approach was used to quantify the primary plasma metabolites, including carbohydrates, amino acids, and nucleotides, by gas-chromatography time of flight (GC-TOF) mass spectrometry (UC Davis NIH West Coast Metabolomics). Two runs of profiling were acquired separately, PAH versus DM and PAH versus heart. In the DM/PAH run, 84 metabolites (out of 172 analyzed) were significantly altered (p < 0.05). A comparison of the PAH and heart groups showed 76 significantly changed metabolites among the 158 that were identified. Heat maps (Figure 1A,B) show
monary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vas... more monary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vascular remodeling. The receptor for advanced glycation end products (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 1, 2, or 5 wk [right ventricle systolic pressure (RVSP) 56.5 3.2, 63.6 1.6, and 111.1 4.5 mmHg, respectively , vs. 22.9 1.1 mmHg 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 threefold increase in G6P (P 0.0005) levels in lungs of PAH rats starting from week 1 that correlated with accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3), membrane translocation of PI3K, and a threefold 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. glycolysis; inositol pathway; receptor for advanced glycation end products; proliferation; pulmonary hypertension
Significance: Pulmonary arterial hypertension (PAH) is a progressive disease of the lung vasculat... more Significance: 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. Recent Advances: 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. Critical Issues: 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 asymp-tomatically, making it difficult to identify and treat early. Future Directions: 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. Antioxid. Redox Signal. 00, 000-000.
We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation o... more We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation of endothelial nitric-oxide synthase (eNOS) to the mitochondrion via a mechanism that requires protein nitration. Thus, the goal of this study was elucidate how eNOS redistributes to mitochondria and to identify the nitrated protein responsible for this event. Our data indicate that exposure of pulmonary arterial endothelial cells to ADMA enhanced eNOS phosphorylation at the Akt1-dependent phosphorylation sites Ser(617) and Ser(1179). Mutation of these serine residues to alanine (S617A and S1179A) inhibited nitration-mediated eNOS translocation to the mitochondria, whereas the phosphormimic mutations (S617D and S1179D) exhibited increased mitochondrial redistribution in the absence of ADMA. The overexpression of a dominant-negative Akt1 also attenuated ADMA-mediated eNOS mitochondrial translocation. Furthermore, ADMA enhanced Akt1 nitration and increased its activity. Mass spectrometry identified a single nitration site in Akt1 located at the tyrosine residue (Tyr(350)) located within the client-binding domain. Replacement of Tyr(350) with phenylalanine abolished peroxynitrite-mediated eNOS translocation to mitochondria. We also found that in the absence of ADMA, eNOS translocation decreased mitochondrial oxygen consumption and superoxide production without altering cellular ATP level. This suggests that under physiologic conditions, eNOS translocation enhances mitochondria coupling. In conclusion, we have identified a new mechanism by which eNOS translocation to mitochondria is regulated by the phosphorylation of eNOS at Ser(617) and Ser(1179) by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr(350).
Circulating angiogenic cells (CACs) are peripheral blood cells whose functional capacity inversel... more 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. We recruited 40 volunteers varying by sex, age (< or ≥45 years), and CAD and subjected their CACs to well-established functional tests. Age and CAD were associated with reduced CAC intrinsic migration (but not specific response to vascular endothelial growth factor, adherence of CACs to endothelial tubes, eNOS mRNA and protein levels, and NO production. To determine how CAC function influences therapeutic potential, w...
ATP is essential for cellular function and is usually produced through oxidative phosphorylation.... more 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.
American journal of respiratory cell and molecular biology, Jan 6, 2015
Calpain mediates the collagen synthesis and cell proliferation and plays an important role in pul... more 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...
B69. THE ENDOTHELIAL AND EPITHELIAL BARRIERS IN INJURED LUNGS, 2010
Medical College of Georgia, Augusta, GA, United States of America 1 ... Gram-negative bacteria in... more Medical College of Georgia, Augusta, GA, United States of America 1 ... Gram-negative bacteria infection is a major cause of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), characterized by endothelial hyper-permeability and activation of the small ...
Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothel... more Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H 2 O 2 ) levels. These changes correlated with an increase in serine-phosphorylated catalase.
Although female predominance in the development of all types of pulmonary hypertension (PH) is we... more 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.
The zinc tetrathiolate (ZnS 4 ) cluster is an important structural feature of endothelial nitric ... more The zinc tetrathiolate (ZnS 4 ) cluster is an important structural feature of endothelial nitric oxide synthase (eNOS). The cluster is located on the dimeric interface and four cysteine residues (C94 and C99 from two adjacent subunits) form a cluster with a Zn ion in the center of a tetrahedral configuration. Due to its high sensitivity to oxidants this cluster is responsible for eNOS dimer destabilization during periods of redox stress. In this work we utilized site directed mutagenesis to replace the redox sensitive cysteine residues in the ZnS 4 cluster with redox stable tetra-arginines. Our data indicate that this C94R/C99R eNOS mutant is active. In addition, this mutant protein is insensitive to dimer disruption and inhibition when challenged with hydrogen peroxide (H 2 O 2 ). Further, the overexpression of the C94R/C99R mutant preserved the angiogenic response in endothelial cells challenged with H 2 O 2 . The over-expression of the C94R/C99R mutant preserved the ability of endothelial cells to migrate towards vascular endothelial growth factor (VEGF) and preserved the endothelial monolayer in a scratch wound assay. We propose that this dimer stable eNOS mutant could be utilized in the treatment of diseases in which there is eNOS dysfunction due to high levels of oxidative stress.
Pulmonary arterial hypertension (PH) is a fatal disease marked by excessive pulmonary vascular ce... more Pulmonary arterial hypertension (PH) is a fatal disease marked by excessive pulmonary vascular cell proliferation. Patients with idiopathic PH express endothelin-1 (ET-1) at high levels in their lungs. As the activation of both types of ET-1 receptor (ETA and ETB) leads to increased generation of superoxide and hydrogen peroxide, this may contribute to the severe oxidative stress found in PH patients. As a number of pathways may induce oxidative stress, the particular role of ET-1 remains unclear. The aim of this study was to determine whether inhibition of ET-1 signaling could reduce pulmonary oxidative stress and attenuate the progression of disease in rats with occlusiveangioproliferative PH induced by a single dose of SU5416 (200 mg/kg) and subsequent exposure to hypoxia for 21 days. Using this regimen, animals developed severe PH as evidenced by a progressive increase in right-ventricle (RV) peak systolic pressure (RVPSP), severe RV hypertrophy, and pulmonary endothelial and smooth muscle cell proliferation, resulting in plexiform vasculopathy. PH rats also had increased oxidative stress, correlating with endothelial nitric oxide synthase uncoupling and NADPH oxidase activation, leading to enhanced protein nitration and increases in markers of vascular remodeling. Treatment with the combined ET receptor antagonist bosentan (250 mg/kg/day; day 10 to 21) prevented further increase in RVPSP and RV hypertrophy, decreased ETA/ETB protein levels, reduced oxidative stress and protein nitration, and resulted in marked attenuation of pulmonary vascular cell proliferation. We conclude that inhibition of ET-1 signaling significantly attenuates the oxidative and nitrosative stress associated with PH and prevents its progression.
Pulmonary arterial hypertension (PAH) is a chronic cardiopulmonary disorder instigated by pulmona... more 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.
Pulmonary arterial hypertension (PAH) is an incurable, progressive disorder, and the early diagno... more Pulmonary arterial hypertension (PAH) is an incurable, progressive disorder, and the early diagnosis and treatment of PAH are associated with increased survival [1,2]. However, because of the asymptomatic nature of PAH development, there are severe delays in PAH diagnosis. In most cases, therapy only starts at an advanced stage of the disease. Thus, there is a critical need to improve the diagnostic approaches, for example, by using markers of PAH that can be detected in easily obtained specimens like plasma or urine [3]. Our previous study showed that in the monocrotaline-treated rat model, metabolic reprogramming occurs early in the development of the disease and foreshadows pathophysiological changes [4]. Thus, assessing the profile of circulating metabolites could allow the identification of PAH patients earlier, before the condition has progressed sufficiently to enter an advanced irreversible stage. Several previous reports have already confirmed a significant difference in the metabolic profile of healthy controls and PAH animals and patients [4-7]. However, given that the average age of PAH patients has increased [8], there is a strong need to diagnose PAH in older people that are not entirely "healthy." In this study, we compared the plasma metabolic profiles of well-phenotyped patients with idiopathic PAH (IPAH) obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI) with two other patient cohorts-patients with type 2 diabetes mellitus (DM; University of Arizona (UA) Center for Disparities in Diabetes, Obesity, and Metabolism) and patients with left heart diseases (Heart; UA biobank). The incidence of non-PAH cardiovascular diseases and type 2 diabetes significantly increases with age, especially in people over 40-45 years old [9,10]. Furthermore, these conditions are associated with severe alterations in metabolism that could mask the changes associated with metabolic reprogramming in PAH [11,12]. Therefore, the goal of the study was to evaluate whether the metabolic profile of PAH patients is distinct compared to these patients and could be used to identify PAH when compared to people who have one of these diseases. The patients' ages were (mean ± standard deviation (SD)) 41.1 ± 15.7, 41.8 ± 13.7, and 50.2 ± 10.9, and the sex ratios of female/male (%) were 81.8/18.1, 83.3/16.7, and 72.7/27.3 for PAH (n = 11), DM (n = 12), and heart (n = 11), respectively. All of the PAH patients were receiving PAH therapy. No patients from the PAH cohort had DM; one patient had valvular left ventricular (LV) heart disease. The targeted metabolomics approach was used to quantify the primary plasma metabolites, including carbohydrates, amino acids, and nucleotides, by gas-chromatography time of flight (GC-TOF) mass spectrometry (UC Davis NIH West Coast Metabolomics). Two runs of profiling were acquired separately, PAH versus DM and PAH versus heart. In the DM/PAH run, 84 metabolites (out of 172 analyzed) were significantly altered (p < 0.05). A comparison of the PAH and heart groups showed 76 significantly changed metabolites among the 158 that were identified. Heat maps (Figure 1A,B) show
monary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vas... more monary arterial hypertension (PAH) is a lethal disease characterized by progressive pulmonary vascular remodeling. The receptor for advanced glycation end products (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 1, 2, or 5 wk [right ventricle systolic pressure (RVSP) 56.5 3.2, 63.6 1.6, and 111.1 4.5 mmHg, respectively , vs. 22.9 1.1 mmHg 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 threefold increase in G6P (P 0.0005) levels in lungs of PAH rats starting from week 1 that correlated with accumulation of phosphatidylinositol (3,4,5)-trisphosphate (PIP 3), membrane translocation of PI3K, and a threefold 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. glycolysis; inositol pathway; receptor for advanced glycation end products; proliferation; pulmonary hypertension
Significance: Pulmonary arterial hypertension (PAH) is a progressive disease of the lung vasculat... more Significance: 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. Recent Advances: 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. Critical Issues: 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 asymp-tomatically, making it difficult to identify and treat early. Future Directions: 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. Antioxid. Redox Signal. 00, 000-000.
We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation o... more We have recently demonstrated that asymmetric dimethylarginine (ADMA) induces the translocation of endothelial nitric-oxide synthase (eNOS) to the mitochondrion via a mechanism that requires protein nitration. Thus, the goal of this study was elucidate how eNOS redistributes to mitochondria and to identify the nitrated protein responsible for this event. Our data indicate that exposure of pulmonary arterial endothelial cells to ADMA enhanced eNOS phosphorylation at the Akt1-dependent phosphorylation sites Ser(617) and Ser(1179). Mutation of these serine residues to alanine (S617A and S1179A) inhibited nitration-mediated eNOS translocation to the mitochondria, whereas the phosphormimic mutations (S617D and S1179D) exhibited increased mitochondrial redistribution in the absence of ADMA. The overexpression of a dominant-negative Akt1 also attenuated ADMA-mediated eNOS mitochondrial translocation. Furthermore, ADMA enhanced Akt1 nitration and increased its activity. Mass spectrometry identified a single nitration site in Akt1 located at the tyrosine residue (Tyr(350)) located within the client-binding domain. Replacement of Tyr(350) with phenylalanine abolished peroxynitrite-mediated eNOS translocation to mitochondria. We also found that in the absence of ADMA, eNOS translocation decreased mitochondrial oxygen consumption and superoxide production without altering cellular ATP level. This suggests that under physiologic conditions, eNOS translocation enhances mitochondria coupling. In conclusion, we have identified a new mechanism by which eNOS translocation to mitochondria is regulated by the phosphorylation of eNOS at Ser(617) and Ser(1179) by Akt1 and that this is enhanced when Akt1 becomes nitrated at Tyr(350).
Circulating angiogenic cells (CACs) are peripheral blood cells whose functional capacity inversel... more 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. We recruited 40 volunteers varying by sex, age (< or ≥45 years), and CAD and subjected their CACs to well-established functional tests. Age and CAD were associated with reduced CAC intrinsic migration (but not specific response to vascular endothelial growth factor, adherence of CACs to endothelial tubes, eNOS mRNA and protein levels, and NO production. To determine how CAC function influences therapeutic potential, w...
ATP is essential for cellular function and is usually produced through oxidative phosphorylation.... more 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.
American journal of respiratory cell and molecular biology, Jan 6, 2015
Calpain mediates the collagen synthesis and cell proliferation and plays an important role in pul... more 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...
B69. THE ENDOTHELIAL AND EPITHELIAL BARRIERS IN INJURED LUNGS, 2010
Medical College of Georgia, Augusta, GA, United States of America 1 ... Gram-negative bacteria in... more Medical College of Georgia, Augusta, GA, United States of America 1 ... Gram-negative bacteria infection is a major cause of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), characterized by endothelial hyper-permeability and activation of the small ...
Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothel... more Our previous studies have shown that endothelin-1 (ET-1) stimulates catalase activity in endothelial cells and in lambs with acute increases in pulmonary blood flow (PBF), without altering gene expression. The purpose of this study was to investigate the molecular mechanism by which this occurs. Exposing pulmonary arterial endothelial cells to ET-1 increased catalase activity and decreased cellular hydrogen peroxide (H 2 O 2 ) levels. These changes correlated with an increase in serine-phosphorylated catalase.
Although female predominance in the development of all types of pulmonary hypertension (PH) is we... more 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.
The zinc tetrathiolate (ZnS 4 ) cluster is an important structural feature of endothelial nitric ... more The zinc tetrathiolate (ZnS 4 ) cluster is an important structural feature of endothelial nitric oxide synthase (eNOS). The cluster is located on the dimeric interface and four cysteine residues (C94 and C99 from two adjacent subunits) form a cluster with a Zn ion in the center of a tetrahedral configuration. Due to its high sensitivity to oxidants this cluster is responsible for eNOS dimer destabilization during periods of redox stress. In this work we utilized site directed mutagenesis to replace the redox sensitive cysteine residues in the ZnS 4 cluster with redox stable tetra-arginines. Our data indicate that this C94R/C99R eNOS mutant is active. In addition, this mutant protein is insensitive to dimer disruption and inhibition when challenged with hydrogen peroxide (H 2 O 2 ). Further, the overexpression of the C94R/C99R mutant preserved the angiogenic response in endothelial cells challenged with H 2 O 2 . The over-expression of the C94R/C99R mutant preserved the ability of endothelial cells to migrate towards vascular endothelial growth factor (VEGF) and preserved the endothelial monolayer in a scratch wound assay. We propose that this dimer stable eNOS mutant could be utilized in the treatment of diseases in which there is eNOS dysfunction due to high levels of oxidative stress.
Pulmonary arterial hypertension (PH) is a fatal disease marked by excessive pulmonary vascular ce... more Pulmonary arterial hypertension (PH) is a fatal disease marked by excessive pulmonary vascular cell proliferation. Patients with idiopathic PH express endothelin-1 (ET-1) at high levels in their lungs. As the activation of both types of ET-1 receptor (ETA and ETB) leads to increased generation of superoxide and hydrogen peroxide, this may contribute to the severe oxidative stress found in PH patients. As a number of pathways may induce oxidative stress, the particular role of ET-1 remains unclear. The aim of this study was to determine whether inhibition of ET-1 signaling could reduce pulmonary oxidative stress and attenuate the progression of disease in rats with occlusiveangioproliferative PH induced by a single dose of SU5416 (200 mg/kg) and subsequent exposure to hypoxia for 21 days. Using this regimen, animals developed severe PH as evidenced by a progressive increase in right-ventricle (RV) peak systolic pressure (RVPSP), severe RV hypertrophy, and pulmonary endothelial and smooth muscle cell proliferation, resulting in plexiform vasculopathy. PH rats also had increased oxidative stress, correlating with endothelial nitric oxide synthase uncoupling and NADPH oxidase activation, leading to enhanced protein nitration and increases in markers of vascular remodeling. Treatment with the combined ET receptor antagonist bosentan (250 mg/kg/day; day 10 to 21) prevented further increase in RVPSP and RV hypertrophy, decreased ETA/ETB protein levels, reduced oxidative stress and protein nitration, and resulted in marked attenuation of pulmonary vascular cell proliferation. We conclude that inhibition of ET-1 signaling significantly attenuates the oxidative and nitrosative stress associated with PH and prevents its progression.
Uploads
Papers by Ruslan Rafikov