In vitro knockout of human p47phox blocks superoxide anion production and LDL oxidation by activated human monocytes

Abstract

AbstractWe previously reported that superoxide dismutase (SOD) blocked human monocyte oxidation of LDL and therefore concluded that superoxide anion ( O2.‒ ) was required for oxidation. Others, however, have suggested that SOD may inhibit by mechanisms alternative to the dismutation of O2.‒ . This study definitively addresses the involvement of O2.‒ in monocyte oxidation of LDL. Using an antisense ODN designed to target p47phox mRNA, we found that treatment of monocytes with antisense ODN caused a substantial and selective decrease in expression of p47phox protein, whereas sense ODN was without effect. Corresponding functional assays demonstrated that antisense ODN inhibited production of O2.‒ . As sense ODN caused no inhibition of O2.‒ production, these results suggested that inhibition of p47phox expression caused reduction in O2.‒ production. Evaluation of the contribution of O2.‒ production to monocyte-mediated oxidation of LDL lipids confirmed that O2.‒ production is required for LDL lipid oxidation as antisense ODN treatment significantly inhibited LDL oxidation whereas sense ODN treatment caused no inhibition. This is the first report of the reduction of NADPH oxidase activity in intact human monocytes by directly targeting the mRNA of a significant member of this enzyme complex. Our results provide convincing data that O2.‒ is indeed required for monocyte-mediated LDL oxidation. —Bey, E. A., and M. K. Cathcart. In vitro knockout of human p47phox blocks superoxide anion production and LDL oxidation by activated human monocytes. J. Lipid Res. 2000. 41: 489–495.