Superoxide anion formation is vital to the microbicidal activity of phagocytes. Recently, however, there is accumulating evidence that it is also involved in cell growth in vascular smooth muscle cells (VSMCs). We have shown that the hypertrophic agent angiotensin II stimulates superoxide production by activating the membrane-bound NADH/NADPH oxidase and that inhibition of this oxidase attenuates vascular hypertrophy. However, the molecular identity of this oxidase in VSMCs is unknown. We have recently cloned the cytochrome b558 alpha-subunit, p22(phox) (one of the key electron transfer elements of the NADPH oxidase in phagocytes), from a rat VSMC cDNA library, but its role in VSMC oxidase activity remains unclarified. Here we report that the complete inhibition of p22(phox) mRNA expression by stable transfection of antisense p22(phox) cDNA into VSMCs results in a decrease in cytochrome b content, which is accompanied by a significant inhibition of angiotensin II-stimulated NADH/NADPH-dependent superoxide production, subsequent hydrogen peroxide production, and [3H]leucine incorporation. We provide the first evidence that p22(phox) is a critical component of superoxide-generating vascular NADH/NADPH oxidase and suggest a central role for this oxidase system in vascular hypertrophy.

Atherosclerotic lesions are found opposite vascular flow dividers at sites of low shear stress and oscillatory flow. Since endothelial proinflammatory genes prominent in lesions are regulated by oxidation-sensitive transcriptional control mechanisms, we examined the redox state of cultured human umbilical vein endothelial cells after either oscillatory or steady laminar fluid shear stress. Endothelial oxidative stress was assessed by measuring activity of the superoxide (O2.- )-producing NADH oxidase (a major source of reactive oxygen species in vascular cells), intracellular O2.- levels, induction of the redox-sensitive gene heme oxygenase-1 (HO-1), and abundance of Cu/Zn superoxide dismutase (Cu/Zn SOD), an antioxidant defense enzyme whose level of expression adapts to changes in oxidative stress. When cells were exposed to oscillatory shear (+/-5 dyne/cm2, 1 Hz) for 1, 5, and 24 hours, NADH oxidase activity and the amount of HO-1 progressively increased up to 174+/-16% (P<0.05) and 505+/-111% (P<0.05) versus static conditions, respectively, whereas levels of Cu/Zn SOD remained unchanged. This upregulation of HO-1 was completely blocked by the antioxidant N-acetylcysteine (NAC, 20 mmol/L). In contrast, steady laminar shear (5 dyne/cm2) induced NADH oxidase activity and NAC-sensitive HO-1 mRNA expression only at 1 and 5 hours, a transient response that returned toward baseline at 24 hours. Levels of Cu/Zn SOD mRNA and protein were increased after 24 hours of steady laminar shear. Furthermore, intracellular O2.-, as measured by dihydroethidium fluorescence, was higher in cells exposed to oscillatory than to laminar shear. These data are consistent with the hypothesis that continuous oscillatory shear causes a sustained activation of pro-oxidant processes resulting in redox-sensitive gene expression in human endothelial cells. Steady laminar shear stress initially activates these processes but appears to induce compensatory antioxidant defenses. We speculate that differences in endothelial redox state, orchestrated by different regimens of shear stress, may contribute to the focal nature of atherosclerosis.

Recent studies suggest that superoxide production by the NADPH/NADH oxidase may be involved in smooth muscle cell growth and the pathogenesis of hypertension. We previously showed that angiotensin II (Ang II) activates a p22phoxbased NADPH/NADH oxidase in cultured rat vascular smooth muscle cells and in animals made hypertensive by infusion of Ang II. To investigate the mechanism responsible for this increased oxidase activity, we examined p22phox mRNA expression in rats made hypertensive by implanting an osmotic minipump that delivered Ang II (0.7 mg/kg per day). Blood pressure began to increase 3 days after the start of Ang II infusion and remained elevated for up to 14 days. Expression of p22phox mRNA in aorta was also increased after 3 days and reached a maximum increase of 338 +/- 41% by 5 days after pump implantation compared with the value after sham operation. This increase in mRNA expression was accompanied by an increase in the content of the corresponding cytochrome (twofold) and NADPH oxidase activity (179 +/- 11% of that in sham-operated rats 5 days after pump implantation). Treatment with the antihypertensive agents losartan (25 mg/kg per day) or hydralazine (15 mg/kg per day) inhibited this upregulation of mRNA levels and activity. Furthermore, infusion of recombinant heparin-binding superoxide dismutase decreased both blood pressure and p22phox mRNA expression. In situ hybridization of aortic tissue showed that p22phox mRNA was expressed in medial smooth muscle as well as in the adventitia. These findings suggest that Ang II-induced hypertension activates the NADPH/NADH oxidase system by upregulating mRNA levels of one or several components of this oxidase system, including the p22phox, and that the NADPH/NADH oxidase system is associated with the pathology of hypertension in vivo.

ACE angiotensin converting enzyme BAL bronchoalveolar lavage CRT cardiac resynchronization therapy CT computed tomography 18 F-FDG fluorine-18 fluorodeoxyglucose 67 Ga gallium-67 HRCT high resolution computed tomography ICD implantable cardioverter defibrillator MRI magnetic resonance imaging PET positron emission tomography sIL-2R soluble interleukin 2 recepter SPECT single photon emission computed tomography JCS GUIDELINES