Genetic Demonstration of p47phox-Dependent Superoxide Anion Production in Murine Vascular Smooth Muscle Cells

Abstract

Background —Previous investigations provide evidence that an enzyme related to the phagocyte NADPH oxidase produces superoxide in the blood vessel wall. These data, however, are confounded by observations that both NADPH and NADH serve as substrates for superoxide production in vascular cells. To clarify this issue, we compared the superoxide-generating capabilities of vascular smooth muscle cells (VSMCs) derived from wild-type (p47phox +/+ ; ph agocyte ox idase) mice with those from mice that lack p47phox (p47phox −/− ; “knockout”), an essential component of the phagocyte NADPH oxidase. Methods and Results —VSMCs were derived from aortic explants harvested from p47phox +/+ or p47phox −/− mice. VSMCs from p47phox +/+ but not those from p47phox −/− mice produced superoxide after stimulation by phorbol myristate acetate. Consistent with this, p47phox was detected only in p47phox +/+ VSMCs. p47phox-transduced p47phox −/− but not enhanced green fluorescent protein–transduced p47phox −/− VSMCs generated significant levels of superoxide after stimulation by angiotensin II or platelet-derived growth factor-BB (PDGF-BB). Enhanced expression of recombinant p47phox in p47phox-transduced p47phox −/− cells correlated with superoxide production in these cells. Conclusions —These data provide direct functional proof that an oxidase requiring the p47phox component mediates superoxide release from VSMCs in the blood vessel wall in response to angiotensin II or PDGF-BB.