Augmented Superoxide Production By Nox2-Containing NADPH Oxidase Causes Cerebral Artery Dysfunction During Hypercholesterolemia

Background and Purpose— We tested the hypothesis that elevated superoxide production by Nox2-NADPH oxidase occurs in cerebral arteries during hypercholesterolemia and causes decreased nitric oxide function. Methods— Wild-type (WT), apolipoprotein E-deficient (ApoE−/−) and Nox2−/−/ApoE−/− mice were fed a high-fat diet for 7 to 14 weeks. Basal superoxide production by cerebral arteries was measured using L-012 (100 &mgr;mol/L)-enhanced chemiluminescence. Nitric oxide function was assessed in isolated middle cerebral arteries through the constrictor response to N&ohgr;-nitro-l-arginine methyl ester (l-NAME; 100 &mgr;mol/L). Western blotting was used to measure protein expression of Nox2, p47phox, endothelial nitric oxide synthase, and superoxide dismutases (1–3). Results— Morphology of cerebral arteries was similar in WT and ApoE−/− mice. In ApoE−/−, but not Nox2−/−/ApoE−/− mice, superoxide production by cerebral arteries was approximately 50% greater than in WT mice (P<0.05). Moreover, the magnitude of l-NAME-induced contractions of isolated middle cerebral arteries from ApoE−/− mice was <50% of that in WT mice (P<0.05), whereas in Nox2−/−/ApoE−/− mice, the contractile response was comparable to WT responses. In the presence of the superoxide scavenger, tempol (1 mmol/L), l-NAME-induced contractions of middle cerebral arteries were similar between WT and ApoE−/− mice. Expression of p47phox was approximately 2-fold higher in ApoE−/− versus WT mice, whereas Nox2, endothelial nitric oxide synthase, and superoxide dismutase isoforms were unchanged. Conclusions— Elevated superoxide production and reduced basal nitric oxide-mediated relaxation occur in cerebral arteries of hypercholesterolemic mice even in the absence of lesions. These changes appear to be exclusively due to increased activity of Nox2-NADPH oxidase, possibly through increased expression of its regulatory subunit p47phox.