Role of NADH/NADPH oxidase-derived H₂O₂ in angiotensin II-induced vascular hypertrophy

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 H₂O₂ production by studying the effects of Ang II on intracellular H₂O₂ generation, intracellular superoxide dismutase and catalase activity, and hypertrophy. Ang II (100 nmol/L) significantly increased intracellular H₂O₂ 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 H₂O₂, which accumulates more rapidly than it is degraded by catalase. This increase in H₂O₂ was inhibited by extracellular catalase, diphenylene iodonium, an inhibitor of the NADH/NADPH oxidase, and the AT₁ 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 H₂O₂ was almost completely inhibited, confirming that the source of Ang II-induced H₂O₂ was the NADH/NADPH oxidase. Using a novel cell line that stably overexpresses catalase, we showed that this increased H₂O₂ 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 AT₁ receptor-mediated production of superoxide generated by the NADH/NADPH oxidase is followed by an increase in intracellular H₂O₂, suggesting a specific role for these oxygen species and scavenging systems in modifying the intracellular redox state in vascular growth.