Sachin A. Gupte

Superoxide in the Vascular System

Oxidant production and regulation is becoming increasingly important in the study of vascular signaling mechanisms, and recent reviews have characterized some of the possible roles for known downstream products of superoxide formation. In this review, we will examine current research in the field, with a special emphasis on the role of the superoxide molecule itself and its place amongst the slightly better understood roles of peroxide and peroxynitrite. The regulatory roles of oxidant species are wide-ranging, and their involvement in processes ranging from intracellular and receptor signaling mechanisms that regulate endothelial mediator release and vascular contractile function to processes that control cellular growth and apoptosis has been implied. Cellular sources of superoxide production and metabolism and the chemical interaction of oxidant species with specific components of cellular signaling mechanisms are considered important factors which determine physiological responses that control vascular function.

Dysregulation of mitochondrial biogenesis in vascular endothelial and smooth muscle cells of aged rats

Mitochondrial biogenesis is involved in the control of cell metabolism, signal transduction, and regulation of mitochondrial reactive oxygen species (ROS) production. Despite the central role of mitochondria in cellular aging and endothelial physiology, there are no studies extant investigating age-related alterations in mitochondrial biogenesis in blood vessels. Electronmicroscopy and confocal microscopy (en face Mitotracker staining) revealed that in aortas of F344 rats, a decline in mitochondrial biogenesis occurs with aging. In aged vessels, the expression of the mitochondrial biogenesis factors (including mitochondrial transcription factor A and peroxisome proliferator-activated receptor-gamma coactivator-1) was decreased. The vascular expression of complex I, III, and IV significantly declined with age, whereas aging did not alter the expression of complex II and V. Cytochrome c oxidase (COX) expression/activity exhibited the greatest age-related decline, which was associated with increased mitochondrial ROS production in the aged vessels. In cultured coronary arterial endothelial cells, a partial knockdown of COX significantly increased mitochondrial ROS production. In conclusion, vascular aging is characterized by a decline in mitochondrial mass in the endothelial cells and an altered expression of components of the mitochondrial electron transport chain likely due to a dysregulation of mitochondrial biogenesis factors. We posit that impaired mitochondrial biogenesis and downregulation of COX may contribute to the increased mitochondrial oxidative stress in aged endothelial cells.

Inhibition of guanylate cyclase stimulation by NO and bovine arterial relaxation to peroxynitrite and H2O2

The inhibitor of soluble guanylate cyclase (sGC) stimulation by nitric oxide (NO), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), was examined for its effects on the prolonged relaxation of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to peroxynitrite (ONOO-) and on H2O2-elicited relaxation and sGC stimulation. Our previous studies suggest that ONOO- causes a prolonged relaxation of BPA by regenerating NO and that a 2-min exposure of BCA or BPA to 50 nM NO causes an ONOO--elicited relaxation. The relaxation of K+-precontracted BCA to 50 nM NO or 100 microM ONOO- was essentially eliminated by 10 microM ODQ. ODQ also eliminated relaxation to 0.1 nM-10 microM of NO donor S-nitroso-N-acetyl-penicillamine (SNAP), but it did not alter relaxation to 1-300 microM H2O2. Similar responses were also observed in BPA. ODQ did not increase lucigenin-detectable superoxide production in BCA, and it did not alter luminol-detectable endogenous ONOO- formation observed during a 2-min exposure of BCA to 50 nM NO. In addition, ODQ did not affect tissue release of NO after 2 min exposure of BCA to 50 nM NO. The activity of sGC in BPA homogenate that is stimulated by endogenous H2O2 was not altered by ODQ, whereas sGC activity in the presence of 10 microM SNAP (+fungal catalase) was reduced by ODQ. Thus relaxation of K+-precontracted BCA and BPA to ONOO- appears to be completely mediated by NO stimulation of sGC, whereas the actions of ODQ suggest that NO is not involved in H2O2-elicited relaxation and sGC stimulation. This study did not detect evidence for the participation of additional mechanisms potentially activated by ONOO- in the responses studied.

NADPH and heme redox modulate pulmonary artery relaxation and guanylate cyclase activation by NO

The hemoprotein oxidant ferricyanide (FeCN) converts the iron of the heme on soluble guanylate cyclase (sGC) from Fe2+ to Fe3+, which prevents nitric oxide (NO) from binding the heme and stimulating sGC activity. This study uses FeCN to examine whether modulation of the redox status of the heme on sGC influences the relaxation of endothelium-removed bovine pulmonary arteries (BPA) to NO. Pretreatment of the homogenate of BPA with 50 μM FeCN resulted in a loss of stimulation of sGC activity by the NO donor 10 μM S-nitroso- N-acetylpenicillamine (SNAP). In the FeCN-treated homogenate reconcentrated to the enzyme levels in BPA, 100 μM NADPH restored NO stimulation of sGC, and this effect of NADPH was prevented by an inhibitor of flavoprotein electron transport, 1 μM diphenyliodonium (DPI). In BPA the relaxation to SNAP was not altered by FeCN, inhibitors of NADPH generation by the pentose phosphate pathway [250 μM 6-aminonicotinamide (6-AN) and 100 μM epiandrosterone (Epi)], or 1 μM DPI. However, the combination of FeCN with 6-AN, Epi, or DPI inhibited ( P < 0.05) relaxation to SNAP without significantly altering the relaxation of BPA to forskolin. The inhibitory effects of 1 μM 1 H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one (a probe that appears to convert NO-heme of sGC to its Fe3+-heme form) on relaxation to SNAP were also enhanced by DPI. These observations suggest that a flavoprotein containing NADPH oxidoreductase may influence cGMP-mediated relaxation of BPA to NO by maintaining the heme of sGC in its Fe2+ oxidation state.

Reverse changes in cardiac substrate oxidation in dogs recovering from heart failure.

When recovering from heart failure (HF), the myocardium displays a marked plasticity and can regain normal gene expression and function; however, recovery of substrate oxidation capacity has not been explored. We tested whether cardiac functional recovery is matched by normalization of energy substrate utilization during post-HF recovery. HF was induced in dogs by pacing the left ventricle (LV) at 210-240 beats/min for 4 wk. Tachycardia was discontinued, and the heart was allowed to recover. An additional group was studied in HF, and healthy dogs served as controls (n = 8/group). Cardiac free fatty acids (FFAs) and glucose oxidation were measured with [3H]oleate and [14C]glucose. At 10 days of recovery, hemodynamic parameters returned to control values; however, the contractile response to dobutamine remained depressed, LV end-diastolic volume was 28% higher than control, and the heart mass-to-body mass ratio was increased (9.8 +/- 0.4 vs. 7.5 +/- 0.2 g/kg, P < 0.05). HF increased glucose oxidation (76.8 +/- 19.7 nmol.min(-1).g(-1)) and decreased FFA oxidation (20.7 +/- 6.4 nmol.min(-1).g(-1)), compared with normal dogs (24.5 +/- 6.3 and 51.7 +/- 9.6 nmol.min(-1).g(-1), respectively), and reversed to normal values at 10 days of recovery (25.4 +/- 6.0 and 46.6 +/- 6.7 nmol.min(-1).g(-1), respectively). However, similar to HF, the recovered dogs failed to increase glucose and fatty acid uptake in response to pacing stress. The activity of myocardial citrate synthase and aconitase was significantly decreased during recovery compared with that in control dogs (58 and 27% lower, respectively, P < 0.05), indicating a persistent reduction in mitochondrial oxidative capacity. In conclusion, cardiac energy substrate utilization is normalized in the early stage of post-HF recovery at baseline, but not under stress conditions.

Regulation of NO-elicited pulmonary artery relaxation and guanylate cyclase activation by NADH oxidase and SOD.

We have previously reported that inhibition of Cu/Zn superoxide dismutase (SOD) in endothelium-removed bovine pulmonary arteries (BPA) attenuates nitrovasodilator-elicited relaxation and that a NADH oxidase linked to the redox status of cytosolic NADH is the major detectable source of superoxide ([Formula: see text]) production in this tissue. In the present study, we investigated whether NADH oxidase-derived[Formula: see text] participated in inhibition of nitrovasodilator-elicited relaxation and soluble guanylate cyclase (sGC) stimulation. Lactate (10 mM) and pyruvate (10 mM) were employed to increase and decrease, respectively, NADH-dependent[Formula: see text] production in the BPA presumably by modulating cytosolic NAD(H) through the lactate dehydrogenase reaction. A 30-min pretreatment with 10 mM diethyldithiocarbamate (DETCA) was used to inhibit Cu/Zn SOD, and S-nitroso- N-acetylpenicillamine (SNAP) was employed as a source of nitric oxide (NO). Lactate or pyruvate did not alter relaxation to NO. However, when the response to NO was inhibited by DETCA, lactate potentiated and pyruvate reduced the inhibitory effects of DETCA. SOD attenuated the inhibitory effects of DETCA plus lactate. In the presence of 10 μM SNAP, the activity of sGC in a BPA homogenate preparation (which was reconcentrated to approximate tissue conditions) was not altered by SOD. However, NADH (0.1 mM) decreased sGC activity by 70%, and this effect of NADH was attenuated in the presence of SOD. Thus cytosolic NADH redox and Cu/Zn SOD activity have important roles in controlling the inhibitory effects of [Formula: see text] derived from NADH oxidase on sGC activity and cGMP-mediated relaxation to nitrovasodilators in BPA.We have previously reported that inhibition of Cu/Zn superoxide dismutase (SOD) in endothelium-removed bovine pulmonary arteries (BPA) attenuates nitrovasodilator-elicited relaxation and that a NADH oxidase linked to the redox status of cytosolic NADH is the major detectable source of superoxide (O-2) production in this tissue. In the present study, we investigated whether NADH oxidase-derived O-2 participated in inhibition of nitrovasodilator-elicited relaxation and soluble guanylate cyclase (sGC) stimulation. Lactate (10 mM) and pyruvate (10 mM) were employed to increase and decrease, respectively, NADH-dependent O-2 production in the BPA presumably by modulating cytosolic NAD(H) through the lactate dehydrogenase reaction. A 30-min pretreatment with 10 mM diethyldithiocarbamate (DETCA) was used to inhibit Cu/Zn SOD, and S-nitroso-N-acetylpenicillamine (SNAP) was employed as a source of nitric oxide (NO). Lactate or pyruvate did not alter relaxation to NO. However, when the response to NO was inhibited by DETCA, lactate potentiated and pyruvate reduced the inhibitory effects of DETCA. SOD attenuated the inhibitory effects of DETCA plus lactate. In the presence of 10 microM SNAP, the activity of sGC in a BPA homogenate preparation (which was reconcentrated to approximate tissue conditions) was not altered by SOD. However, NADH (0.1 mM) decreased sGC activity by 70%, and this effect of NADH was attenuated in the presence of SOD. Thus cytosolic NADH redox and Cu/Zn SOD activity have important roles in controlling the inhibitory effects of O-2 derived from NADH oxidase on sGC activity and cGMP-mediated relaxation to nitrovasodilators in BPA.

A Flavoprotein Mechanism Appears to Prevent an Oxygen-Dependent Inhibition of cGMP-Associated Nitric Oxide–Elicited Relaxation of Bovine Coronary Arteries

The redox state of the heme of soluble guanylate cyclase (sGC) may regulate the sensitivity of vascular tissue to nitric oxide (NO). In this study, diphenyliodonium (DPI) is used as an inhibitor of flavoprotein oxidoreductases to examine their potential role in the expression of NO-elicited cGMP-associated arterial relaxation and sGC stimulation. The relaxation of endothelium-removed bovine coronary arteries (BCAs) precontracted with 30 mmol/L KCl to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or to NO is markedly suppressed by 10 micromol/L DPI under an atmosphere of 21% O(2) (5% CO(2)). In contrast, DPI has minimal effects on the relaxation to SNAP under 95% N(2) (5% CO(2)). If BCAs are treated with DPI under 21% O(2) and then exposed to the hemoprotein reductant sodium dithionite (1 mmol/L) under N(2), there is a partial reversal of the inhibitory effects of DPI compared with BCAs that were not treated with dithionite. DPI did not inhibit relaxation elicited by 8-bromo-cGMP or forskolin. Increases in tissue cGMP levels stimulated by SNAP were eliminated by pretreatment of BCAs with DPI under 21% O(2) but not under N(2). Activation of sGC by SNAP in BCA homogenate was also eliminated when vessels were pretreated with 10 micromol/L DPI under 21% O(2), but DPI did not have an inhibitory effect when directly added to the assay of sGC activity. These observations are consistent with a flavoprotein-dependent oxidoreductase functioning to prevent the expression of a novel O(2)-dependent process from oxidizing the heme on sGC and inhibiting NO-elicited cGMP-mediated BCA relaxation.

MicroRNA-140 is elevated and mitofusin-1 is downregulated in the right ventricle of the Sugen5416/hypoxia/normoxia model of pulmonary arterial hypertension.

Heart failure, a major cause of morbidity and mortality in patients with pulmonary arterial hypertension (PAH), is an outcome of complex biochemical processes. In this study, we determined changes in microRNAs (miRs) in the right and left ventricles of normal and PAH rats. Using an unbiased quantitative miR microarray analysis, we found 1) miR-21-5p, miR-31-5 and 3p, miR-140-5 and 3p, miR-208b-3p, miR-221-3p, miR-222-3p, miR-702-3p, and miR-1298 were upregulated (>2-fold; P < 0.05) in the right ventricle (RV) of PAH compared with normal rats; 2) miR-31-5 and 3p, and miR-208b-3p were upregulated (>2-fold; P < 0.05) in the left ventricle plus septum (LV+S) of PAH compared with normal rats; 3) miR-187-5p, miR-208a-3p, and miR-877 were downregulated (>2-fold; P < 0.05) in the RV of PAH compared with normal rats; and 4) no miRs were up- or downregulated with >2-fold in LV+S compared with RV of PAH and normal. Upregulation of miR-140 and miR-31 in the hypertrophic RV was further confirmed by quantitative PCR. Interestingly, compared with control rats, expression of mitofusin-1 (MFN1), a mitochondrial fusion protein that regulates apoptosis, and which is a direct target of miR-140, was reduced in the RV relative to LV+S of PAH rats. We found a correlation between increased miR-140 and decreased MFN1 expression in the hypertrophic RV. Our results also demonstrated that upregulation of miR-140 and downregulation of MFN1 correlated with increased RV systolic pressure and hypertrophy. These results suggest that miR-140 and MFN1 play a role in the pathogenesis of PAH-associated RV dysfunction.

20-HETE-induced mitochondrial superoxide production and inflammatory phenotype in vascular smooth muscle is prevented by glucose-6-phosphate dehydrogenase inhibition

20-Hydroxyeicosatetraeonic acid (20-HETE) produced by cytochrome P-450 monooxygenases in NADPH-dependent manner is proinflammatory, and it contributes to the pathogenesis of systemic and pulmonary hypertension. In this study, we tested the hypothesis that inhibition of glucose-6-phosphate dehydrogenase (G6PD), a major source of NADPH in the cell, prevents 20-HETE synthesis and 20-HETE-induced proinflammatory signaling that promotes secretory phenotype of vascular smooth muscle cells. Lipidomic analysis indicated that G6PD inhibition and knockdown decreased 20-HETE levels in pulmonary arteries as well as 20-HETE-induced 1) mitochondrial superoxide production, 2) activation of mitogen-activated protein kinase 1 and 3, 3) phosphorylation of ETS domain-containing protein Elk-1 that activate transcription of tumor necrosis factor-α gene (Tnfa), and 4) expression of tumor necrosis factor-α (TNF-α). Moreover, inhibition of G6PD increased protein kinase G1α activity, which, at least partially, mitigated superoxide production and Elk-1 and TNF-α expression. Additionally, we report here for the first time that 20-HETE repressed miR-143, which suppresses Elk-1 expression, and miR-133a, which is known to suppress synthetic/secretory phenotype of vascular smooth muscle cells. In summary, our findings indicate that 20-HETE elicited mitochondrial superoxide production and promoted secretory phenotype of vascular smooth muscle cells by activating MAPK1-Elk-1, all of which are blocked by inhibition of G6PD.

Novel Roles for Nox Oxidases in Cardiac Arrhythmia and Oxidized Glutathione Export in Endothelial Function

See related articles, pages 629–636 and pages 637–644 This issue of Circulation Research contains two articles in the area of oxidant regulation that have major implications for their novel roles in mechanisms that contribute to cardiovascular disease processes in humans. An article by Kim et al1 reports data showing that human cardiac myocytes isolated from right atrial appendages express Nox-2, and that these myocytes and homogenates of the atria from patients with atrial fibrillation (AF) have increased levels of Nox-derived superoxide generation that appear to originate from increased Nox oxidase activation. Homogenates of atria from the AF patients also show evidence of nitric oxide synthase (NOS) becoming a source of superoxide generation, which is thought to originate from an uncoupling of the ability of this enzyme to efficiently synthesize NO. The article by Mueller et al2 reports a new important role for multidrug resistance protein-1 (MRP-1) in controlling oxidant regulation in human and animal endothelium by exporting oxidized glutathione (GSSG). In their study, this group demonstrates how the expression of this protein functions to remove increased GSSG in hypertensive rats and endothelial cells exposed to oscillatory shear stress. Interestingly, the inhibition of MRP-1 appears to restore endothelial function in the hypertensive rats and prevent shear-induced apoptosis in these models apparently through preserving endothelial cell glutathione levels. Both of these articles have major new implications for the understanding and therapeutic targeting of human disease processes. There is already much evidence that oxidant processes have a major influence on the expression of AF. As discussed in the article by Kim et al,1 reactive oxygen species (ROS) are known to cause AF, and antioxidant and statin therapies associated with the modulation of ROS, redox and improved nitric oxide regulation modulate the expression of AF. The data in this …