Jinah Hwang

Role of p47 phox in Vascular Oxidative Stress and Hypertension Caused by Angiotensin II

Abstract—Hypertension caused by angiotensin II is dependent on vascular superoxide (O2·−) production. The nicotinamide adenine dinucleotide phosphate (NAD[P]H) oxidase is a major source of vascular O2·− and is activated by angiotensin II in vitro. However, its role in angiotensin II-induced hypertension in vivo is less clear. In the present studies, we used mice deficient in p47phox, a cytosolic subunit of the NADPH oxidase, to study the role of this enzyme system in vivo. In vivo, angiotensin II infusion (0.7 mg/kg per day for 7 days) increased systolic blood pressure from 105±2 to 151±6 mm Hg and increased vascular O2·− formation 2- to 3-fold in wild-type (WT) mice. In contrast, in p47phox-/- mice the hypertensive response to angiotensin II infusion (122±4 mm Hg;P <0.05) was markedly blunted, and there was no increase of vascular O2·− production. In situ staining for O2·− using dihydroethidium revealed a marked increase of O2·−production in both endothelial and vascular smooth muscle cells of angiotensin II-treated WT mice, but not in those of p47phox-/- mice. To directly examine the role of the NAD(P)H oxidase in endothelial production of O2·−, endothelial cells from WT and p47phox-/- mice were cultured. Western blotting confirmed the absence of p47phox in p47phox-/- mice. Angiotensin II increased O2·− production in endothelial cells from WT mice, but not in those from p47phox-/- mice, as determined by electron spin resonance spectroscopy. These results suggest a pivotal role of the NAD(P)H oxidase and its subunit p47phox in the vascular oxidant stress and the blood pressure response to angiotensin II in vivo.

Oscillatory Shear Stress Stimulates Endothelial Production of from p47phox-dependent NAD(P)H Oxidases, Leading to Monocyte Adhesion

Arterial regions exposed to oscillatory shear (OS) in branched arteries are lesion-prone sites of atherosclerosis, whereas those of laminar shear (LS) are relatively well protected. Here, we examined the hypothesis that OS and LS differentially regulate production of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} from the endothelial NAD(P)H oxidase, which, in turn, is responsible for their opposite effects on a critical atherogenic event, monocyte adhesion. We used aortic endothelial cells obtained from C57BL/6 (MAE-C57) and p47phox-/- (MAE-p47-/-) mice, which lack a component of NAD(P)H oxidase. \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} production was determined by dihydroethidium staining and an electron spin resonance using an electron spin trap methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine. Chronic exposure (18 h) to an arterial level of OS (± 5 dynes/cm2) increased \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} (2-fold) and monocyte adhesion (3-fold) in MAE-C57 cells, whereas chronic LS (15 dynes/cm2, 18 h) significantly decreased both monocyte adhesion and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} compared with static conditions. In contrast, neither LS nor OS were able to induce \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} production and monocyte adhesion to MAE-p47-/-. Treating MAE-C57 with a cell-permeable superoxide dismutase compound, polyethylene glycol-superoxide dismutase, also inhibited OS-induced monocyte adhesion. In addition, over-expressing p47phox in MAE-p47-/- restored OS-induced \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} production and monocyte adhesion. These results suggest that chronic exposure of endothelial cells to OS stimulates \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{-}\) \end{document} and/or its derivatives produced from p47phox-dependent NAD(P)H oxidase, which, in turn, leads to monocyte adhesion, an early and critical atherogenic event.

NAD(P)H Oxidase-derived Hydrogen Peroxide Mediates Endothelial Nitric Oxide Production in Response to Angiotensin II*

Recently, it has been shown that the exogenous addition of hydrogen peroxide (H2O2) increases endothelial nitric oxide (NO⋅) production. The current study is designed to determine whether endogenous levels of H2O2 are ever sufficient to stimulate NO⋅ production in intact endothelial cells. NO⋅ production was detected by a NO⋅-specific microelectrode or by an electron spin resonance spectroscopy using Fe2+-(DETC)2 as a NO⋅-specific spin trap. The addition of H2O2 to bovine aortic endothelial cells caused a potent and dose-dependent increase in NO⋅ release. Incubation with angiotensin II (10−7 mol) elevated intracellular H2O2 levels, which were attenuated with PEG-catalase. Angiotensin II increased NO⋅ production by 2-fold, and this was prevented by Losartan and by PEG-catalase, suggesting a critical role of AT1 receptor and H2O2 in this response. In contrast, NO⋅ production evoked by either bradykinin or calcium ionophore A23187 was unaffected by PEG-catalase. As in bovine aortic endothelial cells, angiotensin II doubled NO⋅ production in aortic endothelial cells from C57BL/6 mice but had no effect on NO⋅ production in endothelial cells from p47 phox−/− mice. In contrast, A23187stimulated NO⋅ production to a similar extent in endothelial cells from wild-type and p47 phox−/− mice. In summary, the present study provides direct evidence that endogenous H2O2, derived from the NAD(P)H oxidase, mediates endothelial NO⋅ production in response to angiotensin II. Under disease conditions associated with elevated levels of angiotensin II, this response may represent a compensatory mechanism. Because angiotensin II also stimulates O 2 ⨪ production from the NAD(P)H oxidase, the H2O2 stimulation of NO⋅ may facilitate peroxynitrite formation in response to this octapeptide.

2 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA LIGAND, ROSIGLITAZONE, ATTENUATES VASCULAR OXIDATIVE STRESS IN A MOUSE MODEL OF TYPE 2 DIABETES.

Purpose We have previously shown that peroxisome proliferator-activated gamma (PPARg) ligands reduce superoxide anion (O2 2 ×) generation in vascular endothelial cells in vitro by suppressing expression of selected subunits of NADPH oxidase and by increasing the expression and activity of Cu/Zn superoxide dismutase (SOD). The current study was designed to determine if PPARg ligands modulate vascular endothelial O2 2 × generation in vivo through these same mechanisms. Methods Lean control (db +/db 2) and obese, leptin receptor-deficient (db 2 /db 2) mice were treated with either vehicle or rosiglitazone (3 mg/kg/day) by gavage for 7 days. Aortas were prepared for analysis of O2 2 × production using ESR spectroscopy and for RNA analysis, and serum was collected for analysis of metabolic parameters. Results Compared to db +/db 2 mice, obese, db 2 /db 2 mice had higher serum glucose, insulin, leptin, triglyceride, and fatty acid levels and lower adiponectin levels. Rosiglitazone had no effect on these metabolic derangements. Aortic O2 2 × generation measured with ESR spectroscopy was significantly increased in db 2 /db 2 mice. Aortic tissue from these mice also demonstrated higher relative mRNA levels of the NADPH oxidase subunits, Nox-1 and Nox-4, as measured by real-time PCR analysis and lower mRNA levels of PPARg. Rosiglitazone treatment decreased O2 2 × generation and mRNA levels of Nox homologues in db 2 /db 2 mice. Conclusions These data indicate that short-term treatment with the PPARg agonist rosiglitazone suppressed vascular NADPH oxidase expression and O2 2 × production in an animal model of vascular oxidative stress. Because these findings occurred in the absence of significant metabolic effects, these results indicate that rosiglitazone and other PPARg ligands may exhibit direct vascular protective effects.

161 PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR GAMMA LIGAND 15d-PGJ2 REPRESSES PROINFLAMMATORY RESPONSES IN VASCULAR ENDOTHELIAL CELLS: THE ROLE OF NITRIC OXIDE.

Purpose Peroxisome proliferator-activated receptor gamma (PPARg) activation prevents atherosclerotic vascular disease and reduces vascular dysfunction and inflammation in diabetic and nondiabetic subjects. The precise molecular mechanisms for this vascular protection remain to be defined. We have previously shown that PPARg ligands enhance endothelial nitric oxide (NO) bioavailability, in part, by reducing superoxide anion (O2 2.) generation and suppressing expression of selected subunits of NADPH oxidase and by enhancing eNOS activity at the level of post-translational regulation. We hypothesized that PPARg-induced increases in endothelial NO production contribute to previously described vascular anti-inflammatory effects following PPARg activation. Methods Human umbilical vein endothelial cells (HUVECs) were treated with 10 μM 15d-PGJ2 with or without human recombinant TNFa (100 U for 2 hours) followed by analysis of cytokine production, adhesion molecule expression, and monocyte adhesion. In separate studies, HUVECs were treated with the NOS inhibitor L-NAME prior to treatment with PPARg ligands. Results 15d-PGJ2 attenuated TNFa-mediated induction of IL-6, IL-8, MCP-1, and IP-10, endothelial-monocyte adhesion, and ICAM, VCAM, and E-selectin expression. The NOS inhibitor L-NAME reduced 15d-PGJ2-mediated NO production and attenuated 15d-PGJ2 repression of TNFa-mediated ICAM expression. Conclusions These data indicate that treatment with 15d-PGJ2 suppressed TNFa-stimulated expression of inflammatory genes in vascular endothelial cells. L-NAME-mediated attenuation of the ability of 15d-PGJ2 to suppress ICAM expression suggests that PPARg-stimulated NO production may contribute to the anti-inflammatory effects of PPARg ligands in vascular endothelial cells.

5 CHRONIC LEPTIN STIMULATION DOES NOT MODULATE NITRIC OXIDE RELEASE FROM HUMAN AORTIC ENDOTHELIAL CELLS.

Acute exposure to the adipokine leptin has been shown to stimulate endothelial nitric oxide (NO) production. However, obesity and other states associated with chronic hyperleptinemia are often characterized by endothelial dysfunction and impaired NO bioavailability. Therefore, we hypothesized that chronic exposure to pathophysiologic leptin levels would decrease NO bioavailability. To test this hypothesis, human aortic endothelial cells (HAECs) were treated with graded concentrations of leptin (5-60 ng/mL) for 72 hours. Endothelial NO production, detected by chemiluminescence analysis of NO and its oxidation products in culture media, cyclic guanosine monophosphate (cGMP) activity, and endothelial nitric oxide synthase (eNOS) activity, was similar to control for all concentrations of leptin studied. Leptin had no effect on eNOS expression assessed with Western blotting and calculated relative to actin expression (n = 7). Because superoxide reacts at diffusion limited rates with NO and reduces its bioavailability, we examined NADPH oxidase and xanthine oxidase, two major sources of superoxide generation in vascular endothelial cells. Chronic leptin exposure did not alter the expression of Nox-4, gp91phox, p22phox, or p67phox, all important components of the NADPH oxidase electron transfer complex in human endothelial cells (n = 5). Similarly, the expression of xanthine oxidase was unchanged when all concentrations of leptin exposures were compared (n = 6). In conclusion, chronic leptin exposure even at pathophysiologic levels does not modulate endothelial NO bioavailability and is not associated with increased superoxide anion production via NADPH oxidase activity.