Nox2-Induced Production of Mitochondrial Superoxide in Angiotensin II-Mediated Endothelial Oxidative Stress and Hypertension

Aims: Angiotensin II (AngII)-induced superoxide (O-2(center dot-)) production by the NADPH oxidases and mitochondria has been implicated in the pathogenesis of endothelial dysfunction and hypertension. In this work, we investigated the specific molecular mechanisms responsible for the stimulation of mitochondrial O-2(center dot-) and its downstream targets using cultured human aortic endothelial cells and a mouse model of AngII-induced hypertension. Results: Western blot analysis showed that Nox2 and Nox4 were present in the cytoplasm but not in the mitochondria. Depletion of Nox2, but not Nox1, Nox4, or Nox5, using siRNA inhibits AngII-induced O-2(center dot-) production in both mitochondria and cytoplasm. Nox2 depletion in gp91phox knockout mice inhibited AngII-induced cellular and mitochondrial O-2(center dot-) and attenuated hypertension. Inhibition of mitochondrial reverse electron transfer with malonate, malate, or rotenone attenuated AngII-induced cytoplasmic and mitochondrial O-2(center dot-) production. Inhibition of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)(+)) with 5-hydroxydecanoic acid or specific PKC peptide antagonist (EAVSLKPT) reduced AngII-induced H2O2 in isolated mitochondria and diminished cytoplasmic O-2(center dot-). The mitoK(ATP)(+) agonist diazoxide increased mitochondrial O-2(center dot-), cytoplasmic c-Src phosphorylation and cytoplasmic O-2(center dot-) suggesting feed-forward regulation of cellular O-2(center dot-) by mitochondrial reactive oxygen species (ROS). Treatment of AngII-infused mice with malate reduced blood pressure and enhanced the antihypertensive effect of mitoTEMPO. Mitochondria-targeted H2O2 scavenger mitoEbselen attenuated redox-dependent c-Src and inhibited AngII-induced cellular O-2(center dot-), diminished aortic H2O2, and reduced blood pressure in hypertensive mice. Innovation and Conclusions: These studies show that Nox2 stimulates mitochondrial ROS by activating reverse electron transfer and both mitochondrial O-2(center dot-) and reverse electron transfer may represent new pharmacological targets for the treatment of hypertension. Antioxid. Redox Signal. 20, 281-294.