3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may exert direct effects on vascular cells and beneficially influence endothelial dysfunction. Because reactive oxygen species (ROS) may lead to vascular damage and dysfunction, we investigated the effect of atorvastatin on ROS production and the underlying mechanisms in vitro and in vivo. Cultured rat aortic vascular smooth muscle cells were incubated with 10 micromol/L atorvastatin. Angiotensin II-induced and epidermal growth factor-induced ROS production were significantly reduced by atorvastatin (dichlorofluorescein fluorescence laser microscopy). Atorvastatin downregulated mRNA expression of the NAD(P)H oxidase subunit nox1, whereas p22phox mRNA expression was not significantly altered (reverse transcription-polymerase chain reaction, Northern analysis). Membrane translocation of rac1 GTPase, which is required for the activation of NAD(P)H oxidase, was inhibited by atorvastatin (Western blot). mRNA expression of superoxide dismutase isoforms and glutathione peroxidase was not modified by atorvastatin, whereas catalase expression was upregulated at mRNA and protein levels, resulting in an increased enzymatic activity. Effects of atorvastatin on ROS production and nox1, rac1, and catalase expression were inhibited by L-mevalonate but not by 25-hydroxycholesterol. In addition, spontaneously hypertensive rats were treated with atorvastatin for 30 days. ROS production in aortic segments was significantly reduced in statin-treated rats (lucigenin chemiluminescence). Treatment with atorvastatin reduced vascular mRNA expression of p22phox and nox1 and increased aortic catalase expression. mRNA expression of superoxide dismutases, glutathione peroxidase, and NAD(P)H oxidase subunits gp91phox, p40phox, p47phox, and p67phox remained unchanged. Translocation of rac1 from the cytosol to the cell membrane was also reduced in vivo. Thus, atorvastatin exerts cellular antioxidant effects in cultured rat vascular smooth muscle cells and in the vasculature of spontaneously hypertensive rats mediated by decreased expression of essential NAD(P)H oxidase subunits and by upregulation of catalase expression. These effects of atorvastatin may contribute to the vasoprotective effects of statins.

Abstract-3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) significantly reduce cardiovascular mortality associated with hypercholesterolemia. There is evidence that statins exert beneficial effects in part through direct effects on vascular cells independent of lowering plasma cholesterol. We characterized the effect of a 30-day treatment with atorvastatin in normocholesterolemic, spontaneously hypertensive rats (SHR). Systolic blood pressure was significantly decreased in atorvastatin-treated rats (184Ϯ5 versus 204Ϯ6 mm Hg for control). Statin therapy improved endothelial dysfunction, as assessed by carbachol-induced vasorelaxation in aortic segments, and profoundly reduced angiotensin II-induced vasoconstriction. Angiotensin type 1 (AT 1 ) receptor, endothelial cell NO synthase (ecNOS), and p22phox mRNA expression were determined with quantitative reverse transcription-polymerase chain reaction. Atorvastatin treatment downregulated aortic AT 1 receptor mRNA expression to 44Ϯ12% of control and reduced mRNA expression of the essential NAD(P)H oxidase subunit p22phox to 63Ϯ7% of control. Aortic AT 1 receptor protein expression was consistently decreased. Vascular production of reactive oxygen species was reduced to 62Ϯ12% of control in statin-treated SHR, as measured with lucigenin chemiluminescence assays. Accordingly, treatment of SHR with the AT 1 receptor antagonist fonsartan improved endothelial dysfunction and reduced vascular free-radical release. Moreover, atorvastatin caused an upregulation of ecNOS mRNA expression (138Ϯ7% of control) and an enhanced ecNOS activity in the vessel wall (209Ϯ46% of control). Treatment of SHR with atorvastatin causes a significant reduction of systolic blood pressure and a profound improvement of endothelial dysfunction mediated by a reduction of free radical release in the vasculature. The underlying mechanism could in part be based on the statin-induced downregulation of AT 1 receptor expression and decreased expression of the NAD(P)H oxidase subunit p22phox, because AT 1 receptor activation plays a pivotal role for the induction of this redox system in the vessel wall.

Background-The AT 1 receptor has been implicated in the pathogenesis of hypertension and atherosclerosis. Estrogen deficiency is also associated with cardiovascular diseases. Therefore, we examined the AT 1 receptor gene expression in ovariectomized rats with and without estrogen replacement therapy and the influence of estrogen on AT 1 receptor expression in cultured vascular smooth muscle cells. Methods and Results-Rat aortic tissue was examined 5 weeks after ovariectomy. In one group, estrogen (1.7 mg estradiol) was administered during the 5-week period. Functional experiments assessed angiotensin II-induced contraction of aortic rings. AT 1 receptor mRNA levels were measured by quantitative polymerase chain reaction and Northern blotting. AT 1 receptor density was assessed by radioligand binding assays. These techniques were also applied in cultured vascular smooth muscle cells. The efficacy of angiotensin II on vasoconstriction was significantly increased in aortas from ovariectomized rats. As assessed by radioligand binding assays, AT 1 receptor density was increased to 160% without changes in receptor affinity during estrogen deficiency. AT 1 receptor mRNA levels were consistently increased to 187% in ovariectomized rats compared with sham-operated animals. Estrogen substitution therapy in ovariectomized rats reversed this AT 1 receptor overexpression. To explore the underlying mechanisms, the direct influence of estradiol on AT 1 receptor expression was investigated in VSMCs. Estradiol (1 mol/L) led to a time-dependent downregulation of AT 1 receptor mRNA, with a maximum of 33.3% at 12 hours. There was a correlative decrease in AT 1 receptor density. Conclusions-This novel observation of estrogen-induced downregulation of AT 1 receptor expression could explain the association of estrogen deficiency with hypertension and atherosclerosis, because activation of the AT 1 receptor plays a key role in the regulation of blood pressure, fluid homeostasis, and vascular cell growth. (Circulation. 1998;97:2197-2201.)Key Words: angiotensin Ⅲ hypertension Ⅲ hormones Ⅲ genes Ⅲ muscle, smooth Ⅲ atherosclerosis T he low incidence of vascular diseases in premenopausal women and the rapid increase of the risk of cardiovascular events after menopause as well as the beneficial effects of estrogen replacement therapy on cardiac and vascular morbidity have suggested a important role of estrogens in the pathogenesis of atherosclerosis. [1][2][3] In addition to its effects on classic cardiovascular risk factors, eg, in the sense of a decrease of cholesterol plasma levels, 4,5 estrogen has been recognized to directly influence vascular as well as myocardial cells. Indeed, VSMCs, myocytes, and cardiac fibroblasts have been shown to contain functional estrogen receptors. [6][7][8] Moreover, there is increasing evidence that estrogen interferes with the RAS. The production of angiotensinogen is enhanced, whereas ACE levels are decreased, by estrogens. According to a recent report, plasma renin levels are also reduced during estroge...

3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) may exert pleiotropic effects on vascular cells independent of lowering plasma cholesterol. To elucidate the molecular mechanisms involved in these effects, we investigated the impact of statins on production of reactive oxygen species (ROS) in rat aortic vascular smooth muscle cells (VSMC). Exposure of VSMC to angiotensin II caused production of ROS via angiotensin AT1 receptor activation. Pretreatment with atorvastatin inhibited angiotensin II-induced ROS production. Atorvastatin decreased AT1 receptor mRNA levels in a time- and concentration-dependent manner and consistently reduced AT1 receptor density. L-Mevalonate but not hydroxy-cholesterol reversed the inhibitory effect of atorvastatin on AT1 receptor transcript levels. Inhibition of geranylgeranyl-transferase but not of farnesyl-transferase mimicked the effect of atorvastatin on AT1 receptor gene expression. Atorvastatin did not decrease AT1 receptor gene transcription but did reduce the half-life of the AT1 receptor mRNA. AT1 receptor activation by angiotensin II increased the expression of the GTPase rac1, enhanced rac1 GTP-binding activity, and increased the geranylgeranyl-dependent translocation of rac1 to the cell membrane. In contrast, statins inhibited rac1 activity and membrane translocation. Consequently, specific inhibition of rac1 with Clostridium sordellii lethal toxin blocked angiotensin II-induced production of free radicals. Finally, treatment of rats with atorvastatin caused down-regulation of aortic AT1 receptor mRNA expression and reduced aortic superoxide production in vivo. Cholesterol-independent down-regulation of AT1 receptor gene expression and inhibition of rac1, leading to decreased ROS production, demonstrates a novel regulatory mechanism of statins that may contribute to the beneficial effects of these drugs beyond lowering of plasma cholesterol.