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Caveolin-1 is a Critical Determinant of Autophagy and Oxidative Stress
such as Sildenafil or Tadalafil (which prevent degradation of cGMP) or cGMP analogues protect the heart from DOX-induced cardiomyopathy, reducing cardiomyocyte apoptosis and preventing the loss of ventricular function. cGMP-dependant protein kinase ID (PKGID) is activated by cGMP binding to it or alternatively by oxidant-induced interprotein disulfide formation. However, pre-activation of PKGID with cGMP abrogates PKGID oxidation. We speculated that oxidative activation of PKGID may mediate DOX-induced cardiotoxicity. Thus increased cGMP levels may be protective due to the prevention of PKGID disulfideactivation. to test this hypothesis we used PKGID knock-in (KI) mice that express a mutant Cys42Ser kinase that cannot be oxidant-activated. Male wild-type (WT) or KI age- and weightmatched littermate mice were randomly assigned to intraperitoneal saline (0.2 ml) or DOX (15 mg/kg) injection (n=6 per group). Left ventricular mass (measured by echocardiography) decreased by 19.1% (112.5±2.8mg to 91.1±4.8mg p<0.0005) in WT mice, but only 3.5% in KI (113.0±2.1mg to 109.0±3.9mg) 5 days after the treatment. Ejection fraction, a measure of cardiac contractile function, decreased from 68.7±0.8% to 57.5±0.8% (p<0.0001) in WT mice, but was preserved in KI (68.2±0.7% vs. 69.0±0.6%). These data suggest that oxidant-dependant PKGID disulfide formation plays an important role in DOX-induced cardiomyopathy. Interventions that more effectively limit DOX-induced PKGID oxidation may provide enhanced cardioprotection.
doi: 10.1016/j.freeradbiomed.2013.10.586
doi: 10.1016/j.freeradbiomed.2013.10.587
Protective Effects of Nitro-Fatty Acids in a HypoxiaInduced Murine Model of Pulmonary Hypertension Anna Klinke1, Annika Möller2, Thorben Ravekes1, Michaela Pekarova3, Kai Friedrichs1, Ralph T. Schermuly4, Lukas Kubala3, Hanna Kolarova3, Steven R. Woodcock5, Bruce A. Freeman5, Stephan Baldus1, Volker Rudolph1, and Tanja Rudolph1 1 Heart Center, University Hospital Cologne, Germany, 2University Heart Center, Hamburg, Germany, 3Institute of Biophysics, Czech Academy of Sciences, Czech Republic, 4Lung Center, University of Giessen and Marburg, Germany, 5Department of Pharmacology & Chemical Biology, University of Pittsburg, United States Rationale: Pulmonary arterial hypertension (PAH) is characterized by adverse remodeling of pulmonary arteries. While the origin of the disease and its underlying pathophysiology remains incompletely understood, inflammation has been identified as a central mediator of disease progression. Oxidative inflammatory conditions support the formation of electrophilic fatty acid nitroalkene derivatives, which exert potent anti-inflammatory effects. Objectives: the current study investigated the role of nitrated fatty acids in modulating the pathophysiology of PAH in mice.
Nitrite Attenuates Normoxic Cell Proliferation through the Modulation of Mitochondrial Dynamics Kelly Quesnelle1, Li Mo1, Yinna Wang1, Catherine Corey1, Donna BeerStolz1, Brian Zuckerbraun1, and Sruti Shiva1 1 University of Pittsburgh, United States We have previously shown that nitrite inhibits vascular smooth muscle cell proliferation after vascular injury in a murine model of restenosis. Now, a number of studies demonstrate that physiological levels of nitrite inhibit cell proliferation in vitro. While the cell cycle regulator p21 has been implicated in this effect, the exact mechanism of nitrite-dependent p21 activation is unknown. Mitochondrial dynamics, the rapid and transient formation and breakage of mitochondrial networks within the cell, is tightly linked to cell cycle progression. for example, mitochondrial fusion occurs during S-phase to increase ATP production, while mitochondrial fission (the division of fused mitochondria) occurs primarily during G2 to promote homogenous mitochondrial distribution during mitosis. Here, we hypothesized that nitrite regulates p21 expression through the modulation of mitochondrial dynamics. We show that nitrite concentrationdependently inhibits PDGF-stimulated growth of normoxic rat aortic smooth muscle cells (RASMCs). This growth inhibition is associated with increased protein expression of p21 and prolonged mitochondrial fusion. Mechanistically, this increased fusion was due to an upregulation of the pro-fusion protein mitofusin-1 (mfn-1) and prolonged by the inhibition of the profission protein dynamin related protein 1. Consistent with this mechanism, silencing of Mfn-1 prevented the nitrite-dependent increase in p21 expression and abrogated nitrite-induced cell proliferation. These studies provide novel insight into the mechanism by which nitrite regulates p21 and cell cycle progression and have important physiological and therapeutic implications for vascular restenosis as well as other proliferative diseases.
Methods and Results: Mice were kept for 28 days under either normoxic or hypoxic conditions and 10-nitro-oleic acid (OA-NO2) was infused subcutaneously. Right ventricular systolic pressure was determined and right ventricular and lung tissue was analyzed. the effect of OA-NO2 on cultured pulmonary artery smooth muscle cells (PASMCs) was also investigated. Right ventricular systolic pressure changes revealed increased pulmonary hypertension in mice upon hypoxic exposure, which was significantly decreased by OA-NO2 administration. Right ventricular hypertrophy, fibrosis and right ventricular failure were also attenuated by OA-NO2 treatment. the infiltration of macrophages and increased generation of reactive oxygen species were elevated in lung tissue of mice upon hypoxia and were both diminished by OA-NO2 treatment. Vascular structural remodeling was also limited by OA-NO2. in support of these findings, growth factor-induced proliferation and activation of extracellular signal-regulated kinases 1/2 in cultured PASMCs was less pronounced upon application of OA-NO2. Conclusions: the oleic acid nitroalkene derivative OA-NO2 attenuates hypoxia-induced pulmonary hypertension in mice. Thus, OA-NO2 represents a potential therapeutic agent for the treatment of PAH.
doi: 10.1016/j.freeradbiomed.2013.10.588
Caveolin-1 is a Critical Determinant of Autophagy and Oxidative Stress Natalia Romero1, Takashi Shiroto1, Hermann Kalwa1, Juliano Sartoretto1, Toru Sugiyama1, and Thomas Michel1 1 Brigham and Women's Hospital, Harvard Medical School, United States
6)5%0
6
Caveolin-1 (cav-1) is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Cav-1null mice have marked cardiovascular abnormalities, yet the molecular mechanisms are incompletely understood. We found that plasma 8-isoprostanes were elevated in cav-1null mice. siRNA-mediated cav-1 knockdown in endothelial cells (EC) promoted significant increases in intracellular H2O2 levels (measured using HyPer2 biosensor and Amplex Red), associated with a decrease in GSH/GSSG ratio. Mitochondrial ROS production was increased in EC after cav-1 knockdown and incubation of cells with 2-deoxy-D-glucose attenuated this increase, implicating cav-1 in control of glycolytic pathway. Unbiased metabolomic analysis of EC lysates following cav-1 knockdown show a decrease in glycolytic intermediates and an increase in fatty acids, suggesting a metabolic switch. However, the most striking difference observed in metabolomics studies was the increased levels (up to 30-fold) of cellular dipeptides. This result was consistent with autophagy activation, confirmed by increased cytosolic vacuole formation and accumulation of the autophagy markers LC3II and p62. These results establish that cav-1 plays a central role in regulation of oxidative stress, metabolic switching, and autophagy in the endothelium, and may represent a critical target in cardiovascular diseases.
doi: 10.1016/j.freeradbiomed.2013.10.589
Redox-Dead Protein Kinase a 5,ĮNQRFN-in Mouse Is Not Hypertensive But Has Increased Vascular Reactivity to Oxidants Olena Rudyk1, Oleksandra Prysyazhna1, and Philip Eaton1 1 King's College London, United Kingdom
We initially found that the regulatory subunits of Type I Protein Kinase a 3.$ 5,Į IRUP LQWHU-protein disulfide dimers when oxidant levels are elevated, which may directly activate the kinase. PKA orchestrates combined inotropic, chronotropic and lusitropic effects on the heart and can also regulate blood pressure (BP). to answer the question whether PKA 5,Į redox state impacts on basal BP or hypotensive responses to oxidants such as hydrogen peroxide (H2O2), we developed a QRYHO³redox± GHDG´ &\V6HU 3.$ 5,Į knock-in (KI) mouse that cannot form inter-protein disulfide bonds and compared them to wild-type (WT) littermates. There were no differences in basal systolic (DAY KI: 112±1mmHg, NIGHT KI: 124±1mmHg vs. DAY WT: 112±2mmHg, NIGHT WT: 125±2mmHg) or diastolic (DAY KI: 83±1mmHg, NIGHT KI: 94±2mmHg vs. DAY WT: 84±2mmHg, NIGHT WT: 95±2mmHg) BP, measured by radiotelemetry in vivo; heart rate and locomotor activity were also similar. However, short-term heart rate variability analysis revealed a higher R wave interval (KI: 144±6ms vs. WT: 120±7ms) and ~50% lower LF-toHF ratio (KI: 0.45±0.06 vs. WT: 0.94±0.17) in the KI. This may indicate the KI has decreased sympathetic activity or compensatory autonomic regulation of BP in the absence of the PKA 5,Į oxidation pathway. We went on to characterise the ex vivo vascular responses to exogenous H2O2 or agonists such as phenylephrine (PE) and angiotensin II (AngII) which stimulate endogenous oxidant production. PE is metabolised by monoamine oxidase to yield H2O2, whereas AngII activates NADPH oxidase which generates superoxide and H2O2. in isolated thoracic aortic rings, the maximal force of constriction in response to vasopressor PE (100μM) was higher in the KI compared to WT (KI: 2.6±0.2mN vs. WT: 1.4±0.3mN). in contrast, the vasodilator H2O2 dose-response curve in the KI was shifted to the right (KI EC50: 198±13μM vs. WT EC50:154±13μM). the response to a bolus dose of AngII (1μM) was higher in the KI
6
mesenteric vessels (KI: 2.9±0.5mN vs. WT: 1.1±0.4mN) and abdominal aortic rings (KI: 3.0±0.6mN vs. WT: 1.3±0.7mN). We conclude that PKA R,Į PD\ VHUYH as a pressor-limiting mechanism that prevents hypertension in the setting of elevated levels of endogenous vasoconstrictors.
doi: 10.1016/j.freeradbiomed.2013.10.590
Regulation of the Hyperproliferative Vascular Smooth Muscle Phenotype by Mitochondrial Fission
Joshua K Salabei1, Andrew a Gibb1, Steven P Jones1, Aruni Bhatnagar1, and Bradford G Hill1 1 Diabetes and Obesity Center, University of Louisville, Louisville, KY, United States Introduction: Vascular smooth muscle cells (VSMCs) develop a highly proliferative and synthetic phenotype in arterial diseases such as restenosis and atherosclerosis. Because such phenotypic changes are likely integrated with the energetic state of the cell, we hypothesized that changes in cellular metabolism regulate VSMC plasticity. Methods and Results: VSMCs were exposed to platelet-derived growth factor-BB (PDGF-BB) and changes in mitochondrial morphology, substrate utilization, and contractile protein expression were examined. PDGF-%% GHFUHDVHG Į-smooth muscle actin and calponin and promoted hyperproliferation. Using confocal microscopy, we found that 97% of mitochondria in contractile VSMCs (i.e., non-PDGF-BB exposed) were filamentous; however, synthetic VSMCs showed a mitochondrial pool with >70% fragmented mitochondria (p<0.05). Compared with contractile VSMCs, synthetic VSMCs showed a 50% decrease in the abundance of mitofusin 2 (p<0.05). Synthetic VSMCs demonstrated a 20% decrease in glucose oxidation, which was accompanied by an increase in fatty acid oxidation (p<0.05). Using a comprehensive permeabilized cell protocol for examining mitochondrial function by extracellular flux analysis, we found that complex II-mediated respiration was decreased by 20% in synthetic VSMCs (p<0.05). an inhibitor of mitochondrial fragmentation, mdivi1, decreased mitochondrial fragmentation by ~50% (p<0.05), abolished the hyperproliferative response to PDGF-BB (p<0.05) and reversed all changes in mitochondrial respiration associated with the synthetic phenotype, yet it did not prevent PDGF-induced losses of contractile proteins. Conclusions: These results indicate that changes in mitochondrial morphology and bioenergetics underlie the hyperproliferative features of the synthetic VSMC phenotype, but do not affect the degradation of contractile proteins. We propose that mitochondrial fragmentation occurring during the transition to the synthetic phenotype regulates mitochondrial substrate selection and bioenergetics and is a therapeutic target for hyperproliferative vascular disorders.
doi: 10.1016/j.freeradbiomed.2013.10.591
6)5%0
doi: 10.1016/j.freeradbiomed.2013.10.586
doi: 10.1016/j.freeradbiomed.2013.10.587
Protective Effects of Nitro-Fatty Acids in a HypoxiaInduced Murine Model of Pulmonary Hypertension Anna Klinke1, Annika Möller2, Thorben Ravekes1, Michaela Pekarova3, Kai Friedrichs1, Ralph T. Schermuly4, Lukas Kubala3, Hanna Kolarova3, Steven R. Woodcock5, Bruce A. Freeman5, Stephan Baldus1, Volker Rudolph1, and Tanja Rudolph1 1 Heart Center, University Hospital Cologne, Germany, 2University Heart Center, Hamburg, Germany, 3Institute of Biophysics, Czech Academy of Sciences, Czech Republic, 4Lung Center, University of Giessen and Marburg, Germany, 5Department of Pharmacology & Chemical Biology, University of Pittsburg, United States Rationale: Pulmonary arterial hypertension (PAH) is characterized by adverse remodeling of pulmonary arteries. While the origin of the disease and its underlying pathophysiology remains incompletely understood, inflammation has been identified as a central mediator of disease progression. Oxidative inflammatory conditions support the formation of electrophilic fatty acid nitroalkene derivatives, which exert potent anti-inflammatory effects. Objectives: the current study investigated the role of nitrated fatty acids in modulating the pathophysiology of PAH in mice.
Nitrite Attenuates Normoxic Cell Proliferation through the Modulation of Mitochondrial Dynamics Kelly Quesnelle1, Li Mo1, Yinna Wang1, Catherine Corey1, Donna BeerStolz1, Brian Zuckerbraun1, and Sruti Shiva1 1 University of Pittsburgh, United States We have previously shown that nitrite inhibits vascular smooth muscle cell proliferation after vascular injury in a murine model of restenosis. Now, a number of studies demonstrate that physiological levels of nitrite inhibit cell proliferation in vitro. While the cell cycle regulator p21 has been implicated in this effect, the exact mechanism of nitrite-dependent p21 activation is unknown. Mitochondrial dynamics, the rapid and transient formation and breakage of mitochondrial networks within the cell, is tightly linked to cell cycle progression. for example, mitochondrial fusion occurs during S-phase to increase ATP production, while mitochondrial fission (the division of fused mitochondria) occurs primarily during G2 to promote homogenous mitochondrial distribution during mitosis. Here, we hypothesized that nitrite regulates p21 expression through the modulation of mitochondrial dynamics. We show that nitrite concentrationdependently inhibits PDGF-stimulated growth of normoxic rat aortic smooth muscle cells (RASMCs). This growth inhibition is associated with increased protein expression of p21 and prolonged mitochondrial fusion. Mechanistically, this increased fusion was due to an upregulation of the pro-fusion protein mitofusin-1 (mfn-1) and prolonged by the inhibition of the profission protein dynamin related protein 1. Consistent with this mechanism, silencing of Mfn-1 prevented the nitrite-dependent increase in p21 expression and abrogated nitrite-induced cell proliferation. These studies provide novel insight into the mechanism by which nitrite regulates p21 and cell cycle progression and have important physiological and therapeutic implications for vascular restenosis as well as other proliferative diseases.
Methods and Results: Mice were kept for 28 days under either normoxic or hypoxic conditions and 10-nitro-oleic acid (OA-NO2) was infused subcutaneously. Right ventricular systolic pressure was determined and right ventricular and lung tissue was analyzed. the effect of OA-NO2 on cultured pulmonary artery smooth muscle cells (PASMCs) was also investigated. Right ventricular systolic pressure changes revealed increased pulmonary hypertension in mice upon hypoxic exposure, which was significantly decreased by OA-NO2 administration. Right ventricular hypertrophy, fibrosis and right ventricular failure were also attenuated by OA-NO2 treatment. the infiltration of macrophages and increased generation of reactive oxygen species were elevated in lung tissue of mice upon hypoxia and were both diminished by OA-NO2 treatment. Vascular structural remodeling was also limited by OA-NO2. in support of these findings, growth factor-induced proliferation and activation of extracellular signal-regulated kinases 1/2 in cultured PASMCs was less pronounced upon application of OA-NO2. Conclusions: the oleic acid nitroalkene derivative OA-NO2 attenuates hypoxia-induced pulmonary hypertension in mice. Thus, OA-NO2 represents a potential therapeutic agent for the treatment of PAH.
doi: 10.1016/j.freeradbiomed.2013.10.588
Caveolin-1 is a Critical Determinant of Autophagy and Oxidative Stress Natalia Romero1, Takashi Shiroto1, Hermann Kalwa1, Juliano Sartoretto1, Toru Sugiyama1, and Thomas Michel1 1 Brigham and Women's Hospital, Harvard Medical School, United States
6)5%0
6
Caveolin-1 (cav-1) is a scaffolding/regulatory protein that interacts with diverse signaling molecules. Cav-1null mice have marked cardiovascular abnormalities, yet the molecular mechanisms are incompletely understood. We found that plasma 8-isoprostanes were elevated in cav-1null mice. siRNA-mediated cav-1 knockdown in endothelial cells (EC) promoted significant increases in intracellular H2O2 levels (measured using HyPer2 biosensor and Amplex Red), associated with a decrease in GSH/GSSG ratio. Mitochondrial ROS production was increased in EC after cav-1 knockdown and incubation of cells with 2-deoxy-D-glucose attenuated this increase, implicating cav-1 in control of glycolytic pathway. Unbiased metabolomic analysis of EC lysates following cav-1 knockdown show a decrease in glycolytic intermediates and an increase in fatty acids, suggesting a metabolic switch. However, the most striking difference observed in metabolomics studies was the increased levels (up to 30-fold) of cellular dipeptides. This result was consistent with autophagy activation, confirmed by increased cytosolic vacuole formation and accumulation of the autophagy markers LC3II and p62. These results establish that cav-1 plays a central role in regulation of oxidative stress, metabolic switching, and autophagy in the endothelium, and may represent a critical target in cardiovascular diseases.
doi: 10.1016/j.freeradbiomed.2013.10.589
Redox-Dead Protein Kinase a 5,ĮNQRFN-in Mouse Is Not Hypertensive But Has Increased Vascular Reactivity to Oxidants Olena Rudyk1, Oleksandra Prysyazhna1, and Philip Eaton1 1 King's College London, United Kingdom
We initially found that the regulatory subunits of Type I Protein Kinase a 3.$ 5,Į IRUP LQWHU-protein disulfide dimers when oxidant levels are elevated, which may directly activate the kinase. PKA orchestrates combined inotropic, chronotropic and lusitropic effects on the heart and can also regulate blood pressure (BP). to answer the question whether PKA 5,Į redox state impacts on basal BP or hypotensive responses to oxidants such as hydrogen peroxide (H2O2), we developed a QRYHO³redox± GHDG´ &\V6HU 3.$ 5,Į knock-in (KI) mouse that cannot form inter-protein disulfide bonds and compared them to wild-type (WT) littermates. There were no differences in basal systolic (DAY KI: 112±1mmHg, NIGHT KI: 124±1mmHg vs. DAY WT: 112±2mmHg, NIGHT WT: 125±2mmHg) or diastolic (DAY KI: 83±1mmHg, NIGHT KI: 94±2mmHg vs. DAY WT: 84±2mmHg, NIGHT WT: 95±2mmHg) BP, measured by radiotelemetry in vivo; heart rate and locomotor activity were also similar. However, short-term heart rate variability analysis revealed a higher R wave interval (KI: 144±6ms vs. WT: 120±7ms) and ~50% lower LF-toHF ratio (KI: 0.45±0.06 vs. WT: 0.94±0.17) in the KI. This may indicate the KI has decreased sympathetic activity or compensatory autonomic regulation of BP in the absence of the PKA 5,Į oxidation pathway. We went on to characterise the ex vivo vascular responses to exogenous H2O2 or agonists such as phenylephrine (PE) and angiotensin II (AngII) which stimulate endogenous oxidant production. PE is metabolised by monoamine oxidase to yield H2O2, whereas AngII activates NADPH oxidase which generates superoxide and H2O2. in isolated thoracic aortic rings, the maximal force of constriction in response to vasopressor PE (100μM) was higher in the KI compared to WT (KI: 2.6±0.2mN vs. WT: 1.4±0.3mN). in contrast, the vasodilator H2O2 dose-response curve in the KI was shifted to the right (KI EC50: 198±13μM vs. WT EC50:154±13μM). the response to a bolus dose of AngII (1μM) was higher in the KI
6
mesenteric vessels (KI: 2.9±0.5mN vs. WT: 1.1±0.4mN) and abdominal aortic rings (KI: 3.0±0.6mN vs. WT: 1.3±0.7mN). We conclude that PKA R,Į PD\ VHUYH as a pressor-limiting mechanism that prevents hypertension in the setting of elevated levels of endogenous vasoconstrictors.
doi: 10.1016/j.freeradbiomed.2013.10.590
Regulation of the Hyperproliferative Vascular Smooth Muscle Phenotype by Mitochondrial Fission
Joshua K Salabei1, Andrew a Gibb1, Steven P Jones1, Aruni Bhatnagar1, and Bradford G Hill1 1 Diabetes and Obesity Center, University of Louisville, Louisville, KY, United States Introduction: Vascular smooth muscle cells (VSMCs) develop a highly proliferative and synthetic phenotype in arterial diseases such as restenosis and atherosclerosis. Because such phenotypic changes are likely integrated with the energetic state of the cell, we hypothesized that changes in cellular metabolism regulate VSMC plasticity. Methods and Results: VSMCs were exposed to platelet-derived growth factor-BB (PDGF-BB) and changes in mitochondrial morphology, substrate utilization, and contractile protein expression were examined. PDGF-%% GHFUHDVHG Į-smooth muscle actin and calponin and promoted hyperproliferation. Using confocal microscopy, we found that 97% of mitochondria in contractile VSMCs (i.e., non-PDGF-BB exposed) were filamentous; however, synthetic VSMCs showed a mitochondrial pool with >70% fragmented mitochondria (p<0.05). Compared with contractile VSMCs, synthetic VSMCs showed a 50% decrease in the abundance of mitofusin 2 (p<0.05). Synthetic VSMCs demonstrated a 20% decrease in glucose oxidation, which was accompanied by an increase in fatty acid oxidation (p<0.05). Using a comprehensive permeabilized cell protocol for examining mitochondrial function by extracellular flux analysis, we found that complex II-mediated respiration was decreased by 20% in synthetic VSMCs (p<0.05). an inhibitor of mitochondrial fragmentation, mdivi1, decreased mitochondrial fragmentation by ~50% (p<0.05), abolished the hyperproliferative response to PDGF-BB (p<0.05) and reversed all changes in mitochondrial respiration associated with the synthetic phenotype, yet it did not prevent PDGF-induced losses of contractile proteins. Conclusions: These results indicate that changes in mitochondrial morphology and bioenergetics underlie the hyperproliferative features of the synthetic VSMC phenotype, but do not affect the degradation of contractile proteins. We propose that mitochondrial fragmentation occurring during the transition to the synthetic phenotype regulates mitochondrial substrate selection and bioenergetics and is a therapeutic target for hyperproliferative vascular disorders.
doi: 10.1016/j.freeradbiomed.2013.10.591
6)5%0