Insulin and insulin-like growth factor-I promotes angiotensinogen production and growth in vascular smooth muscle cells

BACKGROUND: Circulating insulin and insulin-like growth factor-I (IGF-I) levels are increased in patients with hypertension and insulin resistance. Since both hormones are known to have cell growth-promoting effects, they may contribute to the progression of vascular hypertrophy in patients with insulin resistance. Insulin-mediated activation of the vascular renin-angiotensin system (RAS) stimulates growth in cultured rat vascular smooth muscle cells (VSMC). OBJECTIVE: In order to evaluate the role of IGF-I-mediated activation of components of the tissue RAS, we examined the effect of IGF-I receptor stimulation on cell proliferation, and production of angiotensinogen in cultured VSMC. STUDY DESIGN: Aortic VSMC were derived from male Sprague-Dawley rats. IGF-I and insulin-mediated DNA synthesis were estimated by 3H-thymidine uptake (3H-TdR) with or without the angiotensin I converting enzyme inhibitor, captopril. Moreover, angiotensinogen released by the cells to the culture medium was determined by radioimmunoassay with or without the anti-IGF-I receptor antibody alphaIR3 or captopril. RESULTS: Both IGF-I and insulin increased 3H-TdR uptake by cultured rat VSMC (P < 0.05). Captopril blocked IGF-I and insulin-mediated 3H-TdR uptake (-34.4 +/- 1.9% and -32.7 +/- 1.8%, P < 0.05, respectively). IGF-I increased the angiotensinogen level in the medium by 30.6 +/- 2.9% (P < 0.01). Insulin also stimulated angiotensinogen synthesis by 26.3 +/- 2.2% (P < 0.01). Captopril and alphaIR3 significantly suppressed angiotensinogen production stimulated by both IGF-I and insulin. CONCLUSIONS: These results indicate that IGF-I as well as insulin stimulates angiotensinogen production and growth in VSMC. Thus, both hormones may independently play a role in progression of the vascular hypertrophy and atherosclerosis in patients with hypertension and insulin resistance through activation of the tissue RAS.     

Uric acid stimulates vascular smooth muscle cell proliferation and oxidative stress via the vascular renin-angiotensin system

BACKGROUND: Plasma uric acid has been associated with hypertension in a variety of disorders, and has been shown to be predictive of hypertension. The mechanistic role of uric acid in the development of hypertension is not known however. METHOD: We tested the hypothesis that uric acid stimulates vascular smooth muscle cell (VSMC) proliferation and oxidative stress by stimulating the vascular renin-angiotensin system (RAS). Rat VSMC were exposed to 0-300 micromol uric acid for 48 h. RESULTS: Uric acid (200 and 300 micromol) stimulated the proliferation of VSMC as measured by thymidine uptake. This effect was prevented by 10(-6) mol losartan or by 10(-6) mol captopril. Incubation of VSMC with uric acid for 48 h also increased angiotensinogen messenger RNA expression and intracellular concentrations of angiotensin II. These responses were also inhibited by losartan and captopril. Increased expression of angiotensinogen mRNA was also inhibited by co-incubation with PD 98059, a mitogen-activated protein (MAP) kinase inhibitor. Uric acid stimulated the production of hydrogen peroxide and 8-isoprostane in VSMC. These increases in oxidative stress indicators were significantly reduced by co-incubating the cells with captopril or losartan. Uric acid also decreased nitrite and nitrate concentrations in the culture medium, an effect that was prevented by losartan and captopril. CONCLUSION: These results demonstrate that uric acid stimulates proliferation, angiotensin II production, and oxidative stress in VSMC through tissue RAS. This suggests that uric acid causes cardiovascular disorders by stimulating the vascular RAS, and this stimulation may be mediated by the MAP kinase pathway.     

Regulation of discoidin domain receptor 2 by cyclic mechanical stretch in cultured rat vascular smooth muscle cells

Discoidin domain receptor 2 (DDR2) plays potential roles in the regulation of collagen turnover mediated by smooth muscle cells in atherosclerosis. How mechanical stretch affects the regulation of DDR2 in smooth muscle cells is not fully understood. We sought to investigate the cellular and molecular mechanisms of regulation of DDR2 by cyclic stretch in smooth muscle cells. Rat vascular smooth muscle cells grown on a flexible membrane base were stretched by vacuum to 20% of maximum elongation, at 60 cycles/min. Cyclic stretch significantly increased DDR2 protein and mRNA expression after stretch. Cyclic stretch also significantly increased DNA-protein binding activity of Myc-Max. Addition of SB203580, transforming growth factor-beta1 (TGF-beta1) monoclonal antibody, p38 small interfering RNA (siRNA), and c-myc siRNA 30 minutes before stretch inhibited the induction of DDR2 protein and abolished the DNA-protein binding activity induced by cyclic stretch. Cyclic stretch increased, whereas SB203580 abolished the phosphorylated p38 protein. Conditioned medium from stretched smooth muscle cells and exogenous administration of angiotensin II and TGF-beta1 recombinant proteins to the nonstretched cells increased DDR2 protein expression similar to that seen after stretch. In conclusion, cyclic mechanical stretch enhances DDR2 expression in cultured rat smooth muscle cells. The stretch-induced DDR2 is mediated by angiotensin II and TGF-beta1, at least in part, through p38 mitogen-activated protein kinase and Myc pathway.     

Inhibition of cardiovascular cell proliferation by angiotensin receptor blockers: are all molecules the same?

BACKGROUND: Few studies have directly compared the effects of different angiotensin receptor blockers (ARBs) on mechanisms involved in the pathogenesis of cardiovascular disease. METHODS: We studied the ability of different ARBs to inhibit the proliferation of vascular smooth muscle cells (VSMCs) and cardiac fibroblasts (CFs) in continuous culture and in quiescent cells stimulated to proliferate by platelet-derived growth factor (PDGF) and insulin. We also investigated whether the antiproliferative effects of ARBs depended on their ability to block angiotensin II receptors or activate the peroxisome proliferator-activated receptor gamma (PPAR gamma). RESULTS: Dose-response studies showed that candesartan, eprosartan, and irbesartan had little or no effect on the proliferation of VSMC or CF in continuous culture even when tested at concentrations as high as 10 mumol/l or when tested in cells stimulated with PDGF/insulin. In contrast, telmisartan inhibited VSMC and CF proliferation by 50-70% (P < 0.05) in a dose-dependent and reversible fashion and significantly inhibited the increases in cyclin D1 levels and cell proliferation induced by PDGF/insulin. Antiproliferative effects of telmisartan were also observed in Chinese hamster ovary (CHO-K1) cells that lack functional angiotensin II receptors and in human VSMCs treated with the PPAR gamma antagonist GW9662. CONCLUSION: Telmisartan, but not candesartan, irbesartan, or eprosartan, can significantly inhibit the proliferation of VSMC and CF in culture when tested at concentrations near those that can be achieved in plasma with usual oral dosing. Telmisartan can also inhibit the proliferation of cells that lack angiotensin II receptors and cells treated with a PPAR gamma antagonist suggesting that the antiproliferative effects of telmisartan may involve more than just angiotensin II receptor blockade or activation of PPAR gamma.     

多巴胺D_1类受体对胰岛素受体介导的血管平滑肌细胞增殖的影响

目的观察多巴胺D1类受体对胰岛素受体介导的血管平滑肌细胞增殖的影响。方法本研究以A10细胞为研究对象,观察刺激D1类受体对胰岛素促增殖作用的影响,并用免疫印迹研究D1类受体影响胰岛素作用的机制。细胞增殖作用采用[3H]胸腺嘧啶核苷(3H-TdR)掺入量表示。结果胰岛素可促进A10细胞的增殖,该作用呈现浓度依赖性的。D1类受体激动剂Fenoldopam本身对A10细胞无增殖影响,但Fenoldopam通过D1类受体可完全阻断胰岛素介导的血管平滑肌细胞增殖作用。免疫印迹显示刺激D1类受体可降低胰岛素受体的蛋白表达,提示该机制可能参与了D1类受体对胰岛素受体作用的过程。结论D1类受体对胰岛素受体介导的血管平滑肌细胞增殖具有抑制作用,该作用可能在高血压的发生发展中发挥一定作用。     

Butylated hydroxyanisole stimulates heme oxygenase-1 gene expression and inhibits neointima formation in rat arteries

OBJECTIVE: Butylated hydroxyanisole (BHA) is a synthetic phenolic compound that is a potent inducer of phase II genes. Since heme oxygenase-1 (HO-1) is a vasoprotective protein that is upregulated by phase II inducers, the present study examined the effects of BHA on HO-1 gene expression and vascular smooth muscle cell proliferation. METHODS: The regulation of HO-1 gene expression and vascular cell growth by BHA was studied in cultured rat aortic smooth muscle cells and in balloon injured rat carotid arteries. RESULTS: Treatment of cultured smooth muscle cells with BHA stimulated the expression of HO-1 protein, mRNA and promoter activity in a time- and concentration-dependent manner. BHA-mediated HO-1 expression was dependent on the activation of NF-E2-related factor-2 by p38 mitogen-activated protein kinase. BHA also inhibited cell cycle progression and DNA synthesis in an HO-1-dependent manner. In addition, the local perivascular delivery of BHA immediately after arterial injury of rat carotid arteries induced HO-1 protein expression and markedly attenuated neointima formation. CONCLUSIONS: These studies demonstrate that BHA stimulates HO-1 gene expression in vascular smooth muscle cells, and that the induction of HO-1 contributes to the antiproliferative actions of this phenolic antioxidant. BHA represents a potentially novel therapeutic agent in treating or preventing vasculoproliferative disease.     

多巴胺D1类受体对胰岛素受体介导的血管平滑肌细胞增殖的影响

目的 观察多巴胺D1类受体对胰岛素受体介导的血管平滑肌细胞增殖的影响.方法 本研究以A10细胞为研究对象,观察刺激D1类受体对胰岛素促增殖作用的影响,并用免疫印迹研究D1类受体影响胰岛素作用的机制.细胞增殖作用采用[3H]胸腺嘧啶核苷(3H-TdR)掺入量表示.结果 胰岛素可促进A10细胞的增殖,该作用呈现浓度依赖性的.D1类受体激动剂Fenoldopam本身对A10细胞无增殖影响,但Fenoldopam通过D1类受体可完全阻断胰岛素介导的血管平滑肌细胞增殖作用.免疫印迹显示刺激D1类受体可降低胰岛素受体的蛋白表达,提示该机制可能参与了D1类受体对胰岛素受体作用的过程.结论 D1类受体对胰岛素受体介导的血管平滑肌细胞增殖具有抑制作用,该作用可能在高血压的发生发展中发挥一定作用.     

Angiotensin II increases proto-oncogene expression and phosphoinositide turnover in vascular smooth muscle cells via the angiotensin II AT1 receptor.

OBJECTIVES: The aim of this study was to determine which angiotensin II receptor (AT receptor) mediates proto-oncogene expression and phosphoinositide metabolism in vascular smooth muscle cells in vitro. DESIGN: The AT receptor antagonists DuP753 (losartan), an AT1 antagonist, and PD 123319, an AT2 antagonist, were used to characterize AT receptors on cultured vascular smooth muscle cells derived from the rat mesenteric artery and to identify which receptor subtype mediates the angiotensin II-induced increase in proto-oncogene expression and phosphoinositide metabolism. METHODS: Rat mesenteric artery vascular smooth muscle cells were grown using standard cell culture methods. Proto-oncogene induction was measured using Northern blotting. Phosphoinositide breakdown was assessed by measuring [3H]-inositol phosphates released from prelabelled cells. RESULTS: Receptor-binding studies revealed that the AT1 receptor predominated on vascular smooth muscle cells. Incubation of quiescent cells with 0.1 mumol/l angiotensin II resulted in a 65% increase in total [3H]-inositol phosphates released compared with unstimulated cells and in a rapid accumulation of c-fos messenger RNA (mRNA). Pre-incubation of the cells with 10(-5) mol/l PD 123319 had no effect on either total inositol phosphates release or c-fos mRNA induction. Both responses, however, were totally abolished by pre-incubation of the cells with 10(-5) mol/l losartan or saralasin. CONCLUSIONS: Angiotensin II acts through the AT1 receptor to increase c-fos expression and phosphoinositide turnover in vascular smooth muscle cells. These mechanisms may be important in angiotensin II-induced smooth muscle hypertrophy.     

Mitogen-activated protein kinase-activated protein kinase 2 in angiotensin II-induced inflammation and hypertension: regulation of oxidative stress

Vascular oxidative stress and inflammation play an important role in angiotensin II-induced hypertension, and mitogen-activated protein kinases participate in these processes. We questioned whether mitogen-activated protein kinase-activated protein kinase 2 (MK2), a downstream target of p38 mitogen-activated protein kinase, is involved in angiotensin II-induced vascular responses. In vivo experiments were performed in wild-type and Mk2 knockout mice infused intravenously with angiotensin II. Angiotensin II induced a 30 mm Hg increase in mean blood pressure in wild-type that was delayed in Mk2 knockout mice. Angiotensin II increased superoxide production and vascular cell adhesion molecule-1 in blood vessels of wild-type but not in Mk2 knockout mice. Mk2 knockdown by small interfering RNA in mouse mesenteric vascular smooth muscle cells caused a 42% reduction in MK2 protein and blunted the angiotensin II-induced 40% increase of MK2 expression. Mk2 knockdown blunted angiotensin II-induced doubling of intracellular adhesion molecule-1 expression, 2.4-fold increase of nuclear p65, and 1.4-fold increase in Ets-1. Mk2 knockdown abrogated the angiotensin II-induced 4.7-fold and 1.3-fold increase of monocyte chemoattractant protein-1 mRNA and protein. Angiotensin II enhanced reactive oxygen species levels (by 29%) and nicotinamide adenine dinucleotide phosphate oxidase activity (by 48%), both abolished by Mk2 knockdown. Reduction of MK2 blocked angiotensin II-induced p47phox translocation to the membrane, associated with a 53% enhanced catalase expression. Angiotensin II-induced increase of MK2 was prevented by the nicotinamide adenine dinucleotide phosphate oxidase inhibitor Nox2ds-tat. Mk2 small interfering RNA prevented the angiotensin II-induced 30% increase of proliferation. In conclusion, MK2 plays a critical role in angiotensin II signaling, leading to hypertension, oxidative stress via activation of p47phox and inhibition of antioxidants, and vascular inflammation and proliferation.     

Angiotensin II increases expression of IP-10 and the renin-angiotensin system in endothelial cells

Angiotensin II promotes vascular inflammation, which plays important roles in vascular injury. In this study, we found that angiotensin II-stimulated human endothelial cells increased the release of a CXC chemokine, IP-10, according to an antibody array. IP-10 expression was higher in the endothelium of coronary blood vessels in mice infused with angiotensin II than in control. Quantitative real-time PCR analysis revealed that angiotensin II significantly increased IP-10 mRNA expression compared to control. Pretreatment with valsartan, but not with PD123319, blocked angiotensin II-induced IP-10 mRNA expression. IP-10 levels in conditioned media detected by ELISA increased in response to angiotensin II compared to control, which was blocked by the pretreatment with valsartan. These data indicate that angiotensin II stimulates IP-10 production from endothelial cells via angiotensin II type 1 receptors. In endothelial cells, IP-10 significantly increased mRNA expression of renin, angiotensin-converting enzyme, and angiotensinogen. IP-10 also increased angiotensin II levels in conditioned media compared to control. Angiotensin II significantly increased mRNA expression of renin, angiotensin converting enzyme and angiotensinogen, which was blocked by neutralization of IP-10 with antibody in endothelial cells. IP-10 neutralization with antibody blocked angiotensin II-induced apoptosis and cell senescence in endothelial cells. These data indicate that IP-10 is involved not only in leukocyte-endothelial interaction but also in the circuit of endothelial renin-angiotensin system activation that potentially promotes atherosclerosis.     

Enhanced expression of TLR4 in smooth muscle cells in human atherosclerotic coronary arteries

Toll-like receptors (TLRs) play an essential role in innate immunity as components of the primary defense system against microbial infections. It has become evident that TLRs are also involved in the pathogenesis of various cardiovascular diseases. However, the expression patterns of TLRs in the human coronary arteries of coronary artery disease (CAD) patients and the regulatory mechanisms of their expression remain unknown. The TLR4 expression patterns were invstigated by immunohistochemical analysis of coronary specimens obtained from autopsy cases or CAD patients by using directional coronary atherectomy. In atherosclerotic coronary arteries (n = 8), TLR4 immunoreactivity was colocalized with infiltrating inflammatory cells. Interestingly, vascular smooth muscle cells of atherosclerotic coronary arteries intensely expressed TLR4 even in the regions that had few inflammatory cells. In contrast, TLR4 expression was barely detected in the vascular smooth muscle cells of nonatherosclerotic coronary arteries (n = 4). Furthermore, intense expression of smooth muscle TLR4 was observed in the coronary arteries of CAD patients (n = 52). Stimulation with tumor necrosis factor α and angiotensin II increased the expression of TLR4 mRNA in cultured human vascular smooth muscle cells. Candesartan, an antagonist of the angiotensin II type 1 receptor (AT1), and N-acetylcystine inhibited angiotensin II-induced TLR4 mRNA expression in these cells. These findings suggest that the vascular smooth muscle cells of atherosclerotic coronary arteries may be activated to express TLR4. Furthermore, proinflammatory cytokines and oxidative stress in the inflammatory lesions might contribute to the enhanced expression of TLR4 in vascular smooth muscle cells of atherosclerotic arteries.     

COX-2 up-regulation and vascular smooth muscle contractile hyperreactivity in spontaneous diabetic db/db mice

OBJECTIVE: Abnormalities in vascular constriction and dilatation are associated with early diabetes and contribute to diabetic vascular complications. However, mechanisms underlying such vascular dysfunction remain to be fully elucidated. The current study tests the role of cyclooxygenase-2 (COX-2) in diabetic vascular smooth muscle dysfunction. METHODS: Small mesenteric artery and aorta are isolated from type 2 diabetic db/db and control mice. Isometric contractions in response to serotonin, angiotensin II, phenylephrine and high potassium are determined in small spiral mesenteric arterial or aortic strips. COX-2 mRNA and protein levels are analyzed by using DNA microarray, real-time PCR and immunoblot. RESULTS: Contractions induced by serotonin, angiotensin II, phenylephrine and high potassium are significantly higher in endothelium-denuded smooth muscle strips isolated from db/db mice than in those isolated from control mice. The contractile hyperreactivity is observed in aortic and third-order branch small mesenteric arterial smooth muscle strips. DNA microarray, real-time PCR and immunoblot analysis show that compared with control mice, COX-2 mRNA and protein are significantly increased in db/db mice aortic smooth muscle. The COX-2 up-regulation is temporally associated with the development of diabetes mellitus and vascular smooth muscle contractile hyperreactivity. Inhibition of COX-2 with NS-398 or SC-58125 partially--but significantly--alleviates agonist-induced but not potassium-induced contractile hyperreactivity. In addition, serum isolated from db/db mice induces COX-2 expression and increases thromboxane A2 production in primary cultured vascular smooth muscle cells (VSMC). SQ-29548, a TP receptor antagonist, diminished the db/db mice vascular smooth muscle contractile hyperreactivity. CONCLUSIONS: COX-2 is up-regulated and contributes at least in part to the vascular smooth muscle contractile hyperreactivity in db/db mice.     

Endothelin-mediated remodeling in aortas of diabetic rats

BACKGROUND: Smooth muscle cells proliferation and extracellular matrix (ECM) protein deposition are key features of diabetic macroangiopathy. In the present study, we have studied the role of endothelin(A) (ET(A)) receptor, the predominant receptor on smooth muscle cells, in diabetes-induced vascular hypertrophy and remodeling. METHODS: Streptozotocin-induced diabetic rats were administrated a selective ET(A) receptor antagonist, TBC3214, for 26 weeks. Following treatment, aortas were harvested and subjected to gene expression and morphometric analyses. We quantified fibronectin (FN) and plasminogen activator inhibitor-1 (PAI-1) expression as indicators of increased ECM protein synthesis. ET-1, ET-3, transforming growth factor-beta1 (TGF-beta1) and angiotensinogen mRNA levels were measured to elucidate genes involved in FN expression. We have investigated an embryonic splice variant of FN, oncofetal FN, and nonmuscle myosin heavy chain (SMemb) as vascular remodeling indicators. RESULTS: Our results show that diabetes leads to upregulation of FN, PAI-1, ET-1, ET-3, TGF-beta1 and angiotensinogen mRNA levels in association with increased medial thickness. Immunohistochemical analyses revealed concurrent protein level changes. Diabetes also upregulated oncofetal FN and SMemb mRNA levels. Treatment with TBC3214 attenuated the mRNA levels of several genes and prevented increased medial thickness. CONCLUSIONS: These results indicate that diabetes-induced vascular hypertrophy and remodeling is associated with reexpression of embryonic forms of FN and myosin heavy chain. Such changes are ET-dependent and may be mediated via TGF-beta1 and angiotensin.     

阿托伐他汀对血管紧张素Ⅱ诱导的人肠系膜动脉平滑肌细胞增殖和细胞内NADPH氧化酶-活性氧通路的影响

目的观察阿托伐他汀钙对血管紧张素Ⅱ的促人肠系膜动脉平滑肌细胞增殖和其诱导的平滑肌细胞内NADPH氧化酶-活性氧通路的干预作用。方法用组织贴块法培养的第2～4代人肠系膜动脉平滑肌细胞作为实验标本;以四甲基偶氮唑盐(MTF)法比色测定各组平滑肌细胞增殖率;流式细胞仪检测被双氢乙酰乙酸-二氯荧光黄(DCFH-DA)标记的细胞内活性氧;反转录聚合酶链式反应(RT-PCR)方法检测细胞内p22phox蛋白mRNA的表达变化。结果 (1)与空白对照组比较,加入血管紧张素Ⅱ药物组的细胞增殖率、细胞内活性氧物质生成量、细胞内p22phox mRNA的表达均明显增高(P0.01)。(2)与浓度为10~(-6)mol/L血管紧张素Ⅱ孵育组比较,血管紧张素Ⅱ和阿托伐他汀钙共孵育组的细胞增殖率[阿托伐他汀组(0.242±0.018)比血管紧张素Ⅱ组(0.359±0.024),P0.01]、细胞内活性氧[阿托伐他汀组(130±29)比血管紧张素Ⅱ组(180±34),P0.01]、细胞内p22phox蛋白mRNA的表达[阿托伐他汀组(0.47±0.07)比血管紧张素Ⅱ组(0.71±0.05),P0.01]均降低。结论阿托伐他汀钙可以部分抑制血管紧张素Ⅱ的促人肠系膜动脉平滑肌细胞增殖和其诱导的细胞内NADPH氧化酶-活性氧通路的作用。     

The angiotensin II type 2 (AT2) receptor antagonizes the growth effects of the AT1 receptor: gain-of-function study using gene transfer.

The type 1 angiotensin II (AT1) receptor is well characterized but the type 2 (AT2) receptor remains an enigma. We tested the hypothesis that the AT2 receptor can modulate the growth of vascular smooth muscle cells by transfecting an AT2 receptor expression vector into the balloon-injured rat carotid artery and observed that overexpression of the AT2 receptor attenuated neointimal formation. In cultured smooth muscle cells, AT2 receptor transfection reduced proliferation and inhibited mitogen-activated protein kinase activity. Furthermore, we demonstrated that the AT2 receptor mediated the developmentally regulated decrease in aortic DNA synthesis at the latter stages of gestation. These results suggest that the AT2 receptor exerts an antiproliferative effect, counteracting the growth action of AT1 receptor.     

Insulin-stimulated NAD(P)H oxidase activity increases migration of cultured vascular smooth muscle cells

BACKGROUND: We reported that insulin stimulates NAD(P)H oxidase activity but not migration of cultured rat vascular smooth muscle cells (VSMCs). Because angiotensin II (Ang II) increases NAD(P)H oxidase activity in these cells, we wished to determine whether insulin stimulates migration of Ang II-treated VSMCs by synergistically stimulating enzyme activity. METHODS: Cultured rat VSMC superoxide anion (O2-) production, cyclic GMP production, and migration were measured by lucigenin luminescence, immunoassay, and wound closure rate, respectively. Nitric oxide (NO) scavenging was measured by inhibition of NO-induced fluorescence of 4-5-diaminofluorescin. RESULTS: Insulin (1 nmol/L) did not affect and Ang II (100 nmol/L) stimulated VSMC migration by 65% (P < .05), but together stimulated it by 150% (P < .05 versus Ang II) by a mechanism inhibited by the NAD(P)H oxidase inhibitors, diphenyleneiodonium (DPI) or gp91ds-tat. Insulin and Ang II stimulated O2- production by 34% and 35%, respectively (both P < .05), but together synergistically stimulated it by 143% (P < .05 versus insulin or Ang II) in a DPI or gp91ds-tat-sensitive manner. Neither insulin nor Ang II measurably affected NO scavenging, but together reduced NO availability by 46% in a DPI-sensitive manner (P < .05) and significantly inhibited NO-stimulated cyclic GMP production. CONCLUSIONS: Insulin synergestically stimulates NAD(P)H oxidase activity in Ang II-treated cultured rat VSMCs causing increased migration.