- Email: [email protected]
Role of Nox2-induced Reverse Electron Transfer in Production of Mitochondrial Reactive Oxygen Species, Endothelial Dysfunction and Hypertension
oxidation in non-smokers and smokers; the latter have elevated – SCN and may therefore be exposed to different levels and types of oxidants. Studies on plasma from non-smokers indicate that as – SCN levels increase there is an increase in Cys oxidation, consistent with HOSCN being a major oxidant. Similar data has been obtained with isolated BSA. These studies have been extended to a cohort of smokers and non-smokers (n=16 each, matched for age and gender). Smokers had markedly elevated SCN (131±31 vs 40±23 μM; p<0.0001), but not other MPO – – substrates (Br , NO2 p>0.05) or MPO levels (p>0.05). Addition of MPO and H2O2 at levels present at inflammatory foci, resulted in significantly greater thiol oxidation in smokers compared to controls (26±8 vs 13±7 μM; p<0.01). A highly significant correlation (r 0.80; p<0.0001) was observed between the extent of – this thiol oxidation and plasma SCN levels. Computational studies of MPO oxidant formation in plasma predicts a switch – from HOCl to HOSCN with increasing SCN , consistent with the enhanced plasma thiol oxidation, as a result of the selectivity of – HOSCN for Cys residues. These data indicate that plasma SCN levels play a key role in determining the nature and extent of MPO-mediated oxidation, and implicate HOSCN as an important mediator of inflammation-induced oxidative damage in smokers. doi: 10.1016/j.freeradbiomed.2010.10.020
20 Role of Nox2-induced Reverse Electron Transfer in Production of Mitochondrial Reactive Oxygen Species, Endothelial Dysfunction and Hypertension Sergey Dikalov1, Anna Dikalova1, Rafal Nazarewicz1, Alfiya Bikineyeva1, Vladimir Mayorov2, Lula Hilenski1, Bernard Lassègue1, David Harrison1, and Kathy Griendling1 1 2 Emory University Mercer University Hypertension affects more than 50 million individuals in the United States and represents a serious health challenge for Western societies, however, many patients’ blood pressure remains poorly controlled despite treatment with multiple drugs. We have previously shown that mitochondria-targeted antioxidants may represent a new class of anti-hypertensive agent (Circ Res. 2010;107:106-116). In this work we have investigated specific molecular mechanisms responsible for stimulation of mitochondrial ROS and its downstream targets using cultured human aortic endothelial cells (HAEC) and angiotensin II (AngII)induced hypertension. It was found that treatment with the mitochondria-targeted antioxidant mitoTEMPO increased dismutation of superoxide in endothelial and kidney mitochondria, and reduced blood pressure in mice with AngII and DOCA-salt induced hypertension. mitoTEMPO and SOD2 decreased production of mitochondrial ROS, inhibited cellular NADPH oxidase and restored vascular NO. Depletion of the Nox2 isoform of NADPH oxidase with small interfering RNA prevented stimulation of mitochondrial ROS as measured by the fluorescent probe mitoSOX. Supplementation of AngII-stimulated HAEC with mitoTEMPO reduced the activity of redox-dependent c-Src and inhibited superoxide production by Nox2. These data suggest that Nox2 is an activator and a target of mitochondrial ROS. Treatment of isolated mitochondria with specific PKCε peptide antagonist (EAVSLKPT) blocked AngII-induced H2O2 production similar to inhibitor of ATP-sensitive potassium channels 5hydroxydecanoate. Interestingly, treatment with inhibitors of mitochondrial complex II malonate or malate inhibited both endothelial and mitochondrial ROS. Treatment of AngII-infused mice with malate reduced blood pressure and enhanced antihypertensive effect of mitoTEMPO. These studies show that Nox2 is likely to play an important role in stimulation of mitochondrial ROS by activating reverse electron transfer (RET), which can be an important target for the development of antioxidant strategies.
S20
O2•AngII
O2•H2O2
NOX2
RET
SOD2 mT
K+ATP
c‐Src
PKCε
Cytoplasm
Mitochondria
doi:10.1016/j.freeradbiomed.2010.10.021
21 Glucagonlike Peptide1 Enhances Nitric Oxide Production by Coronary Endothelial Cells and improves Endotheliumdependent Coronary Microvascular Function after Cardiac Arrest and Resuscitation Betsy B. Dokken1,2, Charles V Piermarini1, Mary K Teachey1, Michael Gura2, Christian J Dameff2, Ronald Hilwig2, Cynthia J Meininger3, and Karl Kern1,2 1 2 University of Arizona Department of Medicine, University of 3 Arizona Sarver Heart Center, Texas A & M Health Science Center, Division of Vascular Biology Post-resuscitation complications lead to death in 66% of survivors of cardiac arrest. Dysfunction of the coronary microvasculature is a major component of these complications. The aims of this study were to determine: 1) if glucagon-like peptide-1 (GLP-1) improves endothelium-dependent microvascular dysfunction after resuscitation, and 2) the role of nitric oxide in these effects. Methods: Ventricular fibrillation (VF) was induced electrically in 20 anesthetized domestic swine (30-35 kg). Following 8 min of untreated VF, animals were resuscitated and blindly randomized to receive a continuous infusion of either GLP-1 (10 pM/kg/min) or equal volume saline as placebo (PBO) for 4 hr, beginning 1 min after return of spontaneous circulation. Coronary flow reserve (CFR, using intracoronary Doppler flow) was measured before and after intracoronary administration of substance-P (0.2 nG/2 min) pre-arrest and 1 and 4 hr post-resuscitation. In addition, coronary endothelial cells (ECs) were isolated from SpragueDawley rats (250 g) using magnetic beads conjugated to a specific rat EC marker (GSL I-B4 isolectin). At passages 2-4, ECs were incubated in phenol red and nitrite-free media with Lglutamine (0.2 mM), L-arginine (0.4 mM), D-glucose (20 mM), and sodium pyruvate (1 mM), with and without GLP-1 (0.3 nM) and DPPIV inhibition (10 µl/sample). After 48 hr, conditioned media was collected and nitrite concentrations determined using a commercially-prepared fluorometric assay. Results: When normalized to pre-arrest values for each animal, CFR was increased in those treated with GLP-1 after resuscitation (1 Hr: PBO group 0.8 ± 0.1, GLP-1 group 1.3 ± 0.1; 4 Hr: PBO group 0.8 ± 0.1, GLP-1 group 1.1 ± 0.1, p< 0.05). Nitric oxide production was significantly enhanced in rat coronary endothelial cells treated with GLP-1 (un-treated group 1.09 µmol nitrite/µl ± 0.02, GLP-1 group 4.72 µmol nitrite/µl ± 0.14, p< 0.01). Conclusions: GLP-1 attenuates endothelium-dependent postresuscitation coronary microvascular dysfunction in swine. In addition, GLP-1 enhances nitric oxide production in rat coronary ECs in vitro, suggesting one mechanism of improved postresuscitation coronary microvascular function. doi: 10.1016/j.freeradbiomed.2010.10.022
SFRBM/SFRRI 2010
20 Role of Nox2-induced Reverse Electron Transfer in Production of Mitochondrial Reactive Oxygen Species, Endothelial Dysfunction and Hypertension Sergey Dikalov1, Anna Dikalova1, Rafal Nazarewicz1, Alfiya Bikineyeva1, Vladimir Mayorov2, Lula Hilenski1, Bernard Lassègue1, David Harrison1, and Kathy Griendling1 1 2 Emory University Mercer University Hypertension affects more than 50 million individuals in the United States and represents a serious health challenge for Western societies, however, many patients’ blood pressure remains poorly controlled despite treatment with multiple drugs. We have previously shown that mitochondria-targeted antioxidants may represent a new class of anti-hypertensive agent (Circ Res. 2010;107:106-116). In this work we have investigated specific molecular mechanisms responsible for stimulation of mitochondrial ROS and its downstream targets using cultured human aortic endothelial cells (HAEC) and angiotensin II (AngII)induced hypertension. It was found that treatment with the mitochondria-targeted antioxidant mitoTEMPO increased dismutation of superoxide in endothelial and kidney mitochondria, and reduced blood pressure in mice with AngII and DOCA-salt induced hypertension. mitoTEMPO and SOD2 decreased production of mitochondrial ROS, inhibited cellular NADPH oxidase and restored vascular NO. Depletion of the Nox2 isoform of NADPH oxidase with small interfering RNA prevented stimulation of mitochondrial ROS as measured by the fluorescent probe mitoSOX. Supplementation of AngII-stimulated HAEC with mitoTEMPO reduced the activity of redox-dependent c-Src and inhibited superoxide production by Nox2. These data suggest that Nox2 is an activator and a target of mitochondrial ROS. Treatment of isolated mitochondria with specific PKCε peptide antagonist (EAVSLKPT) blocked AngII-induced H2O2 production similar to inhibitor of ATP-sensitive potassium channels 5hydroxydecanoate. Interestingly, treatment with inhibitors of mitochondrial complex II malonate or malate inhibited both endothelial and mitochondrial ROS. Treatment of AngII-infused mice with malate reduced blood pressure and enhanced antihypertensive effect of mitoTEMPO. These studies show that Nox2 is likely to play an important role in stimulation of mitochondrial ROS by activating reverse electron transfer (RET), which can be an important target for the development of antioxidant strategies.
S20
O2•AngII
O2•H2O2
NOX2
RET
SOD2 mT
K+ATP
c‐Src
PKCε
Cytoplasm
Mitochondria
doi:10.1016/j.freeradbiomed.2010.10.021
21 Glucagonlike Peptide1 Enhances Nitric Oxide Production by Coronary Endothelial Cells and improves Endotheliumdependent Coronary Microvascular Function after Cardiac Arrest and Resuscitation Betsy B. Dokken1,2, Charles V Piermarini1, Mary K Teachey1, Michael Gura2, Christian J Dameff2, Ronald Hilwig2, Cynthia J Meininger3, and Karl Kern1,2 1 2 University of Arizona Department of Medicine, University of 3 Arizona Sarver Heart Center, Texas A & M Health Science Center, Division of Vascular Biology Post-resuscitation complications lead to death in 66% of survivors of cardiac arrest. Dysfunction of the coronary microvasculature is a major component of these complications. The aims of this study were to determine: 1) if glucagon-like peptide-1 (GLP-1) improves endothelium-dependent microvascular dysfunction after resuscitation, and 2) the role of nitric oxide in these effects. Methods: Ventricular fibrillation (VF) was induced electrically in 20 anesthetized domestic swine (30-35 kg). Following 8 min of untreated VF, animals were resuscitated and blindly randomized to receive a continuous infusion of either GLP-1 (10 pM/kg/min) or equal volume saline as placebo (PBO) for 4 hr, beginning 1 min after return of spontaneous circulation. Coronary flow reserve (CFR, using intracoronary Doppler flow) was measured before and after intracoronary administration of substance-P (0.2 nG/2 min) pre-arrest and 1 and 4 hr post-resuscitation. In addition, coronary endothelial cells (ECs) were isolated from SpragueDawley rats (250 g) using magnetic beads conjugated to a specific rat EC marker (GSL I-B4 isolectin). At passages 2-4, ECs were incubated in phenol red and nitrite-free media with Lglutamine (0.2 mM), L-arginine (0.4 mM), D-glucose (20 mM), and sodium pyruvate (1 mM), with and without GLP-1 (0.3 nM) and DPPIV inhibition (10 µl/sample). After 48 hr, conditioned media was collected and nitrite concentrations determined using a commercially-prepared fluorometric assay. Results: When normalized to pre-arrest values for each animal, CFR was increased in those treated with GLP-1 after resuscitation (1 Hr: PBO group 0.8 ± 0.1, GLP-1 group 1.3 ± 0.1; 4 Hr: PBO group 0.8 ± 0.1, GLP-1 group 1.1 ± 0.1, p< 0.05). Nitric oxide production was significantly enhanced in rat coronary endothelial cells treated with GLP-1 (un-treated group 1.09 µmol nitrite/µl ± 0.02, GLP-1 group 4.72 µmol nitrite/µl ± 0.14, p< 0.01). Conclusions: GLP-1 attenuates endothelium-dependent postresuscitation coronary microvascular dysfunction in swine. In addition, GLP-1 enhances nitric oxide production in rat coronary ECs in vitro, suggesting one mechanism of improved postresuscitation coronary microvascular function. doi: 10.1016/j.freeradbiomed.2010.10.022
SFRBM/SFRRI 2010