Nox2-induced production of mitochondrial superoxide in angiotensin II-mediated endothelial oxidative stress and hypertension.
OBJECTIVE
Angiotensin II (AngII)-induced superoxide (O2(•-)) 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 O2(•-) 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 O2(•-) production in both mitochondria and cytoplasm. Nox2 depletion in gp91phox knockout mice inhibited AngII-induced cellular and mitochondrial O2(•-) and attenuated hypertension. Inhibition of mitochondrial reverse electron transfer with malonate, malate, or rotenone attenuated AngII-induced cytoplasmic and mitochondrial O2(•-) production. Inhibition of the mitochondrial ATP-sensitive potassium channel (mitoK(+)ATP) with 5-hydroxydecanoic acid or specific PKCɛ peptide antagonist (EAVSLKPT) reduced AngII-induced H2O2 in isolated mitochondria and diminished cytoplasmic O2(•-). The mitoK(+)ATP agonist diazoxide increased mitochondrial O2(•-), cytoplasmic c-Src phosphorylation and cytoplasmic O2(•-) suggesting feed-forward regulation of cellular O2(•-) 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 O2(•-), diminished aortic H2O2, and reduced blood pressure in hypertensive mice.


CONCLUSIONS
These studies show that Nox2 stimulates mitochondrial ROS by activating reverse electron transfer and both mitochondrial O2(•-) and reverse electron transfer may represent new pharmacological targets for the treatment of hypertension.
1 Division of Clinical Pharmacology, Vanderbilt University Medical Center , Nashville, Tennessee.
Sergey I
SI
Dikalov
Rafal R
RR
Nazarewicz
Alfiya
A
Bikineyeva
Lula
L
Hilenski
Bernard
B
Lassègue
Kathy K
KK
Griendling
David G
DG
Harrison
Anna E
AE
Dikalova
D000804:Angiotensin II / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D003497:Cyclic N-Oxides / Q000378:metabolism / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004195:Disease Models, Animal; D004579:Electron Transport; D042783:Endothelial Cells / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006973:Hypertension / Q000378:metabolism* / Q000503:physiopathology; D008293:Malates / Q000378:metabolism / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008929:Mitochondria, Heart / Q000235:genetics / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D020033:Protein Isoforms; D021381:Protein Transport; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism
D003497:Cyclic N-Oxides; D008293:Malates; D008562:Membrane Glycoproteins; D020033:Protein Isoforms; D017382:Reactive Oxygen Species; D013481:Superoxides; D000804:Angiotensin II; C030298:malic acid; D006861:Hydrogen Peroxide; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; C003959:TEMPO