Abstract 5328: Excessive Superoxide Production and Endothelial Dysfunction in Cerebral Arteries of Atherosclerotic Mice Are Due to Enhanced Activity of Nox2-containing NADPH-oxidase

Abstract

We tested if enhanced superoxide (O2−) production by Nox2-containing NADPH oxidase leads to impaired endothelial function in cerebral arteries of high fat-fed apolipoprotein E-deficient (ApoE−/−) mice. All mice were fed a high fat diet from 5 wks of age for 7–14 wks. Intracranial cerebral arteries were isolated from C57Bl6/J wild-type (n=13), ApoE−/− (n=26), Nox2−/− (n=10) and novel Nox2−/−/ApoE−/− (n=23) mice. Basal O2− production by cerebral arteries was measured using L012 (100 umol/L)-enhanced chemiluminescence. Endothelial function was assessed in isolated cannulated middle cerebral arteries using a perfusion myograph via the vasoconstrictor response to the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester (L-NAME; 100 umol/L). Plasma total cholesterol levels of wild-type (n=6), ApoE−/− (n=7) and Nox2−/−/ApoE−/− (n=8) mice were 6.1±0.2, 39.5±5.3, and 41.93±1.6 mg/L, respectively. En face oil red O staining showed that there were no atherosclerotic lesions in aortae of wild-type mice (n=13), whereas aortic lesions were prominent in ApoE−/− mice (~9% of the total aortic surface, n=13). By contrast, no lesions were observed in cerebral arteries from ApoE−/− mice. O2− production was elevated in cerebral arteries from ApoE−/− (35±5 × 103 counts/mg; n=14, P<0.05) versus wild-type (16±2 × 103 counts/mg; n=11) and Nox2−/− (10±2 ×103 counts/mg; n=6) mice. However, in Nox2−/−/ApoE−/− mice, cerebral artery O2− production was not elevated (11±1 ×103 counts/mg; n=17; P< 0.05). The magnitude of L-NAME-induced contractions of isolated middle cerebral arteries from ApoE−/− mice (Δ diameter =−13±4 %; n=6) was <50% of that in wild-type (−34±2 %; n=6) mice, whereas in Nox2−/−/ApoE−/− (−33±10 %; n=6) mice endothelial function was comparable to that in wild-types. By contrast, similar constrictor responses to high K+ (124 mM) were observed in wild-type (−59±4 %), ApoE−/− (−60±5 %), and Nox2−/−/ApoE−/− (−65±3 %) mice. In summary, excessive O2− production and endothelial dysfunction occur in cerebral arteries of atherosclerotic mice even in the absence of lesions. Furthermore, we report for the first time that these changes appear to be exclusively due to increased activity of Nox2-containing NADPH oxidase.