Genetic demonstration of p47phox-dependent superoxide anion production in murine vascular smooth muscle cells

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 N ADH serve as substrates for superoxide production in vascular cells. To cla rify this issue, we compared the superoxide-generating capabilities of vasc ular smooth muscle cells (VSMCs) derived from wild-type (p47phox(+/+); phag ocyte oxidase) mice with those from mice that lack p47phox (p47phox(-/-); " knockout"), an essential component of the phagocyte NADPH oxidase, Methods nod Results-VSMCs were derived from aortic explants harvested from p47phox(+/+) or p47phox(-/-) mice. VSMCs from p47phox(+/+) but not those fr om p47phox(-/-) mice produced superoxide after stimulation by phorbol myris tate acetate. Consistent with this, p47phox was detected only in p47phox(+/ +) VSMCs. p47phox-transduced p47phox(-/-) but not enhanced green fluorescen t protein-transduced p47phox(-/-) VSMCs generated significant levels of sup eroxide after stimulation by angiotensin II or platelet-derived growth fact or-BE (PDGF-BB). Enhanced expression of recombinant p47phox in p47phox-tran sduced p47phox(-/-) cells correlated with superoxide production in these ce lls. Conclusions-These data provide direct functional proof that an oxidase requ iring the p47phox component mediates superoxide release from VSMCs in the b lood vessel wall in response to angiotensin II or PDGF-BB.