Patrick J. Pagano

Expression of a Functionally Active gp91phox-Containing Neutrophil-Type NAD(P)H Oxidase in Smooth Muscle Cells From Human Resistance Arteries Regulation by Angiotensin II

A major source of vascular smooth muscle cell (VSMC) superoxide is NAD(P)H oxidase. However, the molecular characteristics and regulation of this enzyme are unclear. We investigated whether VSMCs from human resistance arteries (HVSMCs) possess a functionally active, angiotensin II (Ang II)–regulated NAD(P)H oxidase that contains neutrophil oxidase subunits, including p22phox, gp91phox, p40phox, p47phox, and p67phox. mRNA expression of gp91phox homologues, nox1 and nox4, was also assessed in HVSMCs, human aortic smooth muscle cells, and rat VSMCs. HVSMCs were obtained from resistance arteries from gluteal biopsies of healthy subjects. gp91phox and nox4, but not nox1, were detected in HVSMCs. Nox1 and nox4, but not gp91phox, were expressed in human aortic smooth muscle cells and rat VSMCs. All NAD(P)H oxidase subunits were present in HVSMCs as detected by reverse transcriptase–polymerase chain reaction and immunoblotting. Ang II increased NAD(P)H oxidase subunit abundance. These effects were inhibited by cycloheximide. Acute Ang II stimulation (10 to 15 minutes) increased p47phox serine phosphorylation and induced p47phox and p67phox translocation. This was associated with NAD(P)H oxidase activation. In cells transfected with gp91phox antisense oligonucleotides, Ang II–mediated actions were abrogated. NADPH-induced superoxide generation was reduced by gp91ds-tat and apocynin, inhibitors of p47phox-gp91phox interactions. Our results suggest that HVSMCs possess a functionally active gp91phox-containing neutrophil-like NAD(P)H oxidase. Ang II regulates the enzyme by inducing phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. These novel findings provide insight into the molecular regulation of NAD(P)H oxidase by Ang II in HVSMCs. Furthermore, we identify differences in gp91phox homologue expression in VSMCs from rats and human small and large arteries.

Novel Role of gp91phox-Containing NAD(P)H Oxidase in Vascular Endothelial Growth Factor–Induced Signaling and Angiogenesis

Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell proliferation and migration, primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). Reactive oxygen species (ROS) derived from NAD(P)H oxidase are critically important in many aspects of vascular cell regulation, and both the small GTPase Rac1 and gp91(phox) are critical components of the endothelial NAD(P)H oxidase complex. A role of NAD(P)H oxidase in VEGF-induced angiogenesis, however, has not been defined. In the present study, electron spin resonance spectroscopy is utilized to demonstrate that VEGF stimulates O2*- production, which is inhibited by the NAD(P)H oxidase inhibitor, diphenylene iodonium, as well as by overexpression of dominant-negative Rac1 (N17Rac1) and transfection of gp91(phox) antisense oligonucleotides in human umbilical vein endothelial cells (ECs). Antioxidants, including N-acetylcysteine (NAC), various NAD(P)H oxidase inhibitors, and N17Rac1 significantly attenuate not only VEGF-induced KDR tyrosine phosphorylation but also proliferation and migration of ECs. Importantly, these effects of VEGF are dramatically inhibited in cells transfected with gp91(phox) antisense oligonucleotides. By contrast, ROS are not involved in mediating these effects of sphingosine 1-phosphate (S1P) on ECs. Sponge implant assays demonstrate that VEGF-, but not S1P-, induced angiogenesis is significantly reduced in wild-type mice treated with NAC and in gp91(phox-/-) mice, suggesting that ROS derived from gp91(phox)-containing NAD(P)H oxidase play an important role in angiogenesis in vivo. These studies indicate that VEGF-induced endothelial cell signaling and angiogenesis is tightly controlled by the reduction/oxidation environment at the level of VEGF receptor and provide novel insights into the NAD(P)H oxidase as a potential therapeutic target for angiogenesis-dependent diseases.

Endothelin-1 increases vascular superoxide via endothelin (A)-NADPH oxidase pathway in low-renin hypertension

Background—Angiotensin II–induced hypertension is associated with NAD(P)H oxidase–dependent superoxide production in the vessel wall. Vascular superoxide level is also increased in deoxycorticosterone acetate (DOCA)–salt hypertension, which is associated with a markedly depressed plasma renin activity because of sodium retention. However, the mechanisms underlying superoxide production in low-renin hypertension are undefined. Methods and Results—This study investigated (1) whether and how endothelin-1 (ET-1), which is increased in DOCA-salt hypertensive rats, contributes to arterial superoxide generation and (2) the effect of gene transfer of manganese superoxide dismutase and endothelial nitric oxide synthase. Both superoxide and ET-1 levels were significantly elevated in carotid arteries of DOCA-salt rats compared with that of the sham-operated controls. ET-1 concentration-dependently stimulated superoxide production in vitro in carotid arteries of normotensive rats. The increase in arterial superoxide in both ET-1–treated normotensive and DOCA-salt rats was reversed by a selective ETA receptor antagonist, ABT-627, the flavoprotein inhibitor diphenyleneiodonium, and the NADPH oxidase inhibitor apocynin but not by the nitric oxide synthase inhibitor N&ohgr;-l-arginine methyl ester or the xanthine oxidase inhibitor allopurinol. Furthermore, in vivo blockade of ETA receptors significantly reduced arterial superoxide levels, with a concomitant decrease of systolic blood pressure in DOCA-salt rats. Ex vivo gene transfer of manganese superoxide dismutase or endothelial nitric oxide synthase also suppressed superoxide levels in carotid arteries of DOCA-salt rats. Conclusions—These findings suggest that ET-1 augments vascular superoxide production at least in part via an ETA/NADPH oxidase pathway in low-renin mineralocorticoid hypertension.

Angiotensin II Induces p67phox mRNA Expression and NADPH Oxidase Superoxide Generation in Rabbit Aortic Adventitial Fibroblasts

Superoxide radical (O2-) is ubiquitously critical to the bioactivity of endothelial nitric oxide. In angiotensin-dependent hypertension, vascular O2- levels rise and impede endothelium/nitric oxide-dependent vascular relaxation. We have reported that the major O2- source in the rabbit aorta is adventitial fibroblast phagocyte-like NADPH oxidase and shown that angiotensin (Ang) II treatment of adventitial fibroblasts causes a concentration-dependent increase in particulate NADPH-dependent O2-. From cultured rabbit aortic adventitial fibroblasts treated or not treated with Ang II, we prepared particulate fractions and measured lucigenin-enhanced chemiluminescence. Because [Sar1,Thr8]-Ang II, a generalized antagonist of Ang II and plausible inhibitor of the conversion of Ang II, reversed Ang II (10 nmol/L)-induced NADH- and NADPH-dependent O2- to basal levels, we tested the effect of the inhibitor of aminopeptidase N, amastatin (10 micromol/L), and found no effect on Ang II-stimulated O2-. Ang(1-7), Ang III, and Ang IV also were not effective in stimulating O2- levels at concentrations similar to those of Ang II. Kinetic analysis showed a rise in NADPH oxidase O2- production in response to Ang II, which peaks at 3 hours and returns to basal levels by 16 hours. p67phox, a cytosolic factor, appears to be affected at both the level of transcription and protein synthesis because actinomycin and cycloheximide individually inhibited the observed effect. A partial sequence of p67phox was recovered by reverse transcriptase from mRNA harvested from cultured rabbit aortic adventitial fibroblasts. Furthermore, the p67phox mRNA transcript in aortic fibroblasts is induced by Ang II before the peak of NADPH oxidase by Northern analysis and ribonuclease protection assays. These data suggest that Ang II stimulates NAD(P)H oxidase O2- generation in fibroblasts of aortic adventitia via transcriptional activation of p67phox. These data also provide preliminary evidence for the regulation of factors of the NADPH oxidase and potentially provide a novel means by which to abrogate the development of O2(-)-dependent hypertension.

Hydrogen Peroxide as a Paracrine Vascular Mediator: Regulation and Signaling Leading to Dysfunction

Numerous studies have demonstrated the ability of a variety of vascular cells, including endothelial cells, smooth muscle cells, and fibroblasts, to produce reactive oxygen species (ROS). Until recently, major emphasis was placed on the production of superoxide anion (O2–) in the vasculature as a result of its ability to directly attenuate the biological activity of endothelium-derived nitric oxide (NO). The short half-life and radius of diffusion of O2– drastically limit the role of this ROS as an important paracrine hormone in vascular biology. On the contrary, in recent years, the O2– metabolite hydrogen peroxide (H2O2) has increasingly been viewed as an important cellular signaling agent in its own right, capable of modulating both contractile and growth-promoting pathways with more far-reaching effects. In this review, we will assess the vascular production of H2O2, its regulation by endogenous scavenger systems, and its ability to activate a variety of vascular signaling pathways, thereby leading to vascular contraction and growth. This discussion will include the ability of H2O2 to (i) Initiate calcium flux as well as (ii) stimulate pathways leading to sensitization of contractile elements to calcium. The latter involves a variety of protein kinases that have also been strongly implicated in vascular hypertrophy. Previous Intensive study has emphasized the ability of NADPH oxidase-derived O2– and H2O2 to activate these pathways in cultured smooth muscle cells. However, growing evidence indicates a considerably more complex array of unique oxidase systems in the endothelium, media, and adventitia that appear to participate in these deleterious effects in a sequential and temporal manner. Taken together, these findings seem consistent with a paracrine effect of H2O2 across the vascular wall.

Oxidases and Peroxidases in Cardiovascular and Lung Disease: New Concepts in Reactive Oxygen Species Signaling

Reactive oxygen species (ROS) are involved in numerous physiological and pathophysiological responses. Increasing evidence implicates ROS as signaling molecules involved in the propagation of cellular pathways. The NADPH oxidase (Nox) family of enzymes is a major source of ROS in the cell and has been related to the progression of many diseases and even environmental toxicity. The complexity of this familys effects on cellular processes stems from the fact that there are seven members, each with unique tissue distribution, cellular localization, and expression. Nox proteins also differ in activation mechanisms and the major ROS detected as their product. To add to this complexity, mounting evidence suggests that other cellular oxidases or their products may be involved in Nox regulation. The overall redox and metabolic status of the cell, specifically the mitochondria, also has implications on ROS signaling. Signaling of such molecules as electrophilic fatty acids has an impact on many redox-sensitive pathologies and thus, as anti-inflammatory molecules, contributes to the complexity of ROS regulation. This review is based on the proceedings of a recent international Oxidase Signaling Symposium at the University of Pittsburghs Vascular Medicine Institute and Department of Pharmacology and Chemical Biology and encompasses further interaction and discussion among the presenters.

The Reactive Adventitia Fibroblast Oxidase in Vascular Function

Abstract—The vascular adventitia is activated in a variety of cardiovascular disease states and has recently been shown to be a barrier to nitric oxide bioactivity. Vascular fibroblasts produce substantial amounts of NAD(P)H oxidase–derived reactive oxygen species (ROS) that appear to be involved in fibroblast proliferation, connective tissue deposition, and perhaps vascular tone. However, the physiological and pathophysiological roles of the adventitia have not been extensively studied, possibly because of its location in large blood vessels remote from the vascular endothelium. In recent years, substantial information has been gathered on pathways leading to oxidase activation in smooth muscle cells and fibroblasts and the downstream signaling pathways leading to hypertrophy and proliferation. A clearer understanding of the molecular mechanisms involved will likely lead to therapeutic strategies aimed at preventing vascular dysfunction in diseases such as atherosclerosis, in which these pathways are activated.

Vascular effects following homozygous disruption of p47(phox) : An essential component of NADPH oxidase.

BACKGROUND Evidence suggests that the vessel wall contains an oxidase similar, if not identical, to phagocytic NADPH oxidase. We tested the contribution of this specific oxidase to the progression of atherosclerosis and the regulation of blood pressure. METHODS AND RESULTS An examination of aortic rings from wild-type mice and mice with homozygous targeted disruptions in p47(phox) revealed that p47(phox) knockout mice had a reduction in vascular superoxide production. However, analyses of apoE -/- p47(phox)+/+ and apoE -/- p47(phox) -/- strains of mice demonstrated no significant differences in atherosclerotic lesion sizes. Similarly, analyses of wild-type and p47(phox) knockout mice revealed no differences in either basal blood pressure or the rise in blood pressure seen after the pharmacological inhibition of nitric oxide synthase. CONCLUSIONS NADPH oxidase contributes to basal vascular superoxide production. However, the absence of a functional oxidase does not significantly affect the progression of atherosclerosis in the standard mouse apoE -/- model, nor does it significantly influence basal blood pressure.

NAD(P)H Oxidase Mediates Angiotensin II–Induced Vascular Macrophage Infiltration and Medial Hypertrophy

Objective—Our preliminary data suggested that angiotensin II (Ang II)–induced reactive oxygen species are involved in intercellular adhesion molecule-1 (ICAM-1) expression and leukocyte infiltration in the rat thoracic aorta. Other reports demonstrating reactive oxygen species–induced cell growth suggested a potential role of NAD(P)H oxidase in vascular hypertrophy. In the present study, we postulate that NAD(P)H oxidase is functionally involved in Ang II–induced ICAM-1 expression, macrophage infiltration, and vascular growth, and that oxidase inhibition attenuates these processes independently of a reduction in blood pressure. Methods and Results—Rats were infused subcutaneously with vehicle or Ang II (750 &mgr;g/kg per day) for 1 week in the presence or absence of gp91 docking sequence (gp91ds)-tat peptide (1 mg/kg per day), a cell-permeant inhibitor of NAD(P)H oxidase. Immunohistochemical staining for ICAM-1 and ED1, a marker of monocytes and macrophages, showed that both were markedly increased with Ang II compared with vehicle and were reduced in rats receiving Ang II plus gp91ds-tat. This effect was accompanied by an Ang II–induced increase in medial hypertrophy that was attenuated by coinfusion of gp91ds-tat; however, gp91ds-tat had no effect on blood pressure. Conclusions—Ang II–enhanced NAD(P)H oxidase plays a role in the induction of ICAM-1 expression, leukocyte infiltration, and vascular hypertrophy, acting independently of changes in blood pressure.

Novel NAD(P)H Oxidase Inhibitor Suppresses Angioplasty-Induced Superoxide and Neointimal Hyperplasia of Rat Carotid Artery

Abstract— Neointimal proliferation occurring after vascular or endovascular procedures is a major complication leading to end-organ or limb ischemia. In experimental models, balloon injury has been shown to induce NAD(P)H oxidase to produce vascular superoxide anion (O2·−) production, which has been implicated in cell proliferation, but a direct link is still unclear. We postulated that inhibition of arterial NAD(P)H oxidase, resulting in decreased O2·−, would lessen the neointimal hyperplasia caused by balloon injury to the common carotid artery (CCA). Sprague-Dawley rats were implanted with osmotic minipumps containing either vehicle, a cell-permeant peptide that inhibits NAD(P)H oxidase (gp91ds-tat, 10 mg/kg per day), or a scrambled peptide control (scrmb-tat). Two days after pump implantation, the left CCA was injured using an intravascular balloon embolectomy catheter (2F Fogarty). Systolic blood pressure was monitored by tail cuff. Fourteen days after injury, CCAs were harvested and analyzed by digital morphometry. Rats in both groups remained normotensive, with no significant differences in systolic blood pressure. Reactive oxygen species measurements after injury indicated a significant reduction in vascular O2·− in rats infused with gp91ds-tat, and the neointima/media area and thickness ratios were significantly lower in their arteries compared with control. On the contrary, no significant change in overall CCA diameter was observed in any group. Our data indicate that in response to balloon injury of the rat carotid artery, NAD(P)H oxidase activity contributes to neointimal hyperplasia and is involved in vascular cell proliferation and migration during restenosis.