In vitro knockout of human p47phox blocks superoxide anion production and LDL oxidation by activated human monocytes.
We previously reported that superoxide dismutase (SOD) blocked human monocyte oxidation of LDL and therefore concluded that superoxide anion (O(2)(.-)) was required for oxidation. Others, however, have suggested that SOD may inhibit by mechanisms alternative to the dismutation of O(2)(.-). This study definitively addresses the involvement of O(2)(.-) 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 O(2)(.-). As sense ODN caused no inhibition of O(2)(.-) production, these results suggested that inhibition of p47phox expression caused reduction in O(2)(.-) production. Evaluation of the contribution of O(2)(.-) production to monocyte-mediated oxidation of LDL lipids confirmed that O(2)(.-) 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 O(2)(.-) is indeed required for monocyte-mediated LDL oxidation.
Bey
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EA
Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA.
Cathcart
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MK
D001483:Base Sequence; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000378:metabolism*; D019255:NADPH Oxidases; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage; D010084:Oxidation-Reduction; D010750:Phosphoproteins / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D008077:Lipoproteins, LDL; D016376:Oligonucleotides, Antisense; D010750:Phosphoproteins; D012333:RNA, Messenger; D013481:Superoxides; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1