Oxysterol-induced activation of macrophage NADPH-oxidase enhances cell-mediated oxidation of LDL in the atherosclerotic apolipoprotein E deficient mouse: inhibitory role for vitamin E.

In the present study we provide evidence, both direct and circumstantial, that macrophage oxysterols induce translocation of p47phox from the cytosol to the cell's plasma membrane, forming an active NADPH-oxidase complex which produces superoxide anion and facilitates cell-mediated oxidation of LDL. The study was performed on macrophages from atherosclerotic apolipoprotein E deficient (E(0)) mice, which are under oxidative stress. The oxysterol content in peritoneal macrophages (MPM) from E(0) mice was significantly higher (by 50-80%) than that observed in MPM from control (C57BL6) mice. E(0) MPM release 2-fold more superoxide anions and oxidize LDL by 2.5-fold more than control MPM. Furthermore, macrophage protein kinase C (PKC) activity and arachidonic acid (AA) release (which are both involved in NADPH-oxidase activation) were elevated by 60 and 70%, respectively, in E(0) MPM compared with control MPM. Dietary supplementation of vitamin E (40 mg/kg per day for 2 months) to E(0) mice resulted in a reduction in MPM total oxysterols content (-27%) and this effect was associated with a reduction in PKC activity (-36%), AA release (-39%), cytosolic p47phox translocation to the plasma membrane (-30%), superoxide anion release (-25%) and MPM-mediated LDL oxidation (-28%), compared with unsupplemented E(0) mice. Enrichment of MPM from control mice with the major oxysterols found in E(0) MPM (7-ketocholesterol, beta-epoxycholesterol and 7beta-hydroxycholesterol) resulted in a dose-dependent increase (60-80%) in PKC activity, AA release, p47phox translocation, superoxide anion release and cell-mediated oxidation of LDL. These data clearly demonstrate for the first time that under oxidative stress, cellular lipids are oxidized, and that macrophage enrichment with oxysterols (as exists in E(0) mice) activates the NADPH-oxidase system and enhances cell-mediated oxidation of LDL, a key event during early atherogenesis.