Role of NADH/NADPH Oxidase–Derived H ₂ O ₂ in Angiotensin II–Induced Vascular Hypertrophy

Abstract

Abstract —Recent evidence suggests that oxidative mechanisms may be involved in vascular smooth muscle cell (VSMC) hypertrophy. We previously showed that angiotensin II (Ang II) increases superoxide production by activating an NADH/NADPH oxidase, which contributes to hypertrophy. In this study, we determined whether Ang II stimulation of this oxidase results in H ₂ O ₂ production by studying the effects of Ang II on intracellular H ₂ O ₂ generation, intracellular superoxide dismutase and catalase activity, and hypertrophy. Ang II (100 nmol/L) significantly increased intracellular H ₂ O ₂ levels at 4 hours. Neither superoxide dismutase activity nor catalase activity was affected by Ang II; the SOD present in VSMCs is sufficient to metabolize Ang II–stimulated superoxide to H ₂ O ₂ , which accumulates more rapidly than it is degraded by catalase. This increase in H ₂ O ₂ was inhibited by extracellular catalase, diphenylene iodonium, an inhibitor of the NADH/NADPH oxidase, and the AT ₁ receptor blocker losartan. In VSMCs stably transfected with antisense p22phox, a critical component of the NADH/NADPH oxidase in which oxidase activity was markedly reduced, Ang II–induced production of H ₂ O ₂ was almost completely inhibited, confirming that the source of Ang II–induced H ₂ O ₂ was the NADH/NADPH oxidase. Using a novel cell line that stably overexpresses catalase, we showed that this increased H ₂ O ₂ is a critical step in VSMC hypertrophy, a hallmark of many vascular diseases. Inhibition of intracellular superoxide dismutase by diethylthiocarbamate (1 mmol/L) also resulted in attenuation of Ang II–induced hypertrophy (62±2% inhibition). These data indicate that AT ₁ receptor–mediated production of superoxide generated by the NADH/NADPH oxidase is followed by an increase in intracellular H ₂ O ₂ , suggesting a specific role for these oxygen species and scavenging systems in modifying the intracellular redox state in vascular growth.