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

Correspondence to: Martha K. Cathcart

We 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.

Supplementary key words: lipid oxidation, LDL oxidation, atherosclerosis, monocyte-mediated LDL oxidation, superoxide anion, NADPH oxidase, inflammation, antisense oligonucleotides, monocyte activation, oxygen-free radicals, respiratory burst oxidase

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