Increased Generation of Vascular Reactive Oxygen Species by Ang II Is Mediated Via Src-Dependent Pathways in Essential Hypertension.
We tested the hypothesis that augmented Ang II-induced vascular smooth muscle cell (VSMC) growth in human hypertension is mediated via Src-dependent pathways that generate reactive oxygen species (ROS). VSMCs from arteries of normotensive and hypertensive subjects were studied. Production of ROS was measured by fluorescence digital imaging using dichlorofluorescin diacetate (6 μM). The roles of Src and NADH/NADPH oxidase were assessed with the specific inhibitors, PP2 (10 μM) and diphenylene iodinium (DPI) (10 μM) respectively. c-Src phosphorylation was determined by western blot and kinase activity was assessed by measuring enolase phosphorylation. Ang II increased DCFDA fluorescence. This effect was inhibited by catalase, indicating that the signal was derived predominantly from H2O2. Ang II increased H2O2production within 40 minutes. Responses were greater (p<0.05) in cells from hypertensive patients (Emax=82±nM) than normotensive subjects (Emax= 67±nM). DPI and PP2, but not PP3 (inactive analogue) attenuated (p<0.05) Ang II-induced H2O2 production. PP2 effects were greater in cells from hypertensive patients (delta H2O2, 28±5nM) vs controls (delta H2O2, 16±2nM). Ang II increased c-Src phosphorylation and activity, with responses 3-4 fold higher in hypertensives. DPI and PP2 (p<0.01) attenuated Ang II-induced DNA and protein synthesis, as measured by 3H-thymidine and 3H-leucine incorporation respectively. Growth responses in hypertensive patients were normalized by PP2. In VSMCs from hypertensive patients, Ang II-induced generation of ROS and growth are augmented. These effects are mediated, in part, by Src-dependent, NADH/NADPH oxidase-dependent cascades. Thus increased Src activity may be an upstream modulator of redox-sensitive pathways that regulate vascular growth and remodeling in essential hypertension.