title: Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells :a potential atherogenic source of reactive oxygen species.
abstract: Cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein levels have increased reactive oxygen species (ROS) generation. The enzyme responsible for this ROS production elevation is unknown. We have examined for the presence of a functional leukocyte-type NADPH oxidase in EC to elucidate whether this enzyme could be the ROS source. The plasma membrane fraction of disrupted EC showed a reduced-minus-oxidized difference spectra with absorption peaks identical to those observed in the spectra of the leukocyte NADPH oxidase component, cytochrome b558. Western-blot analysis, using anti-gp91 -phox. anti -p22-phox. anti -p47-phox. and anti -p67-phox antibodies, demonstrated the protein expression of NADPH oxidase subunits in EC. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed the mRNA expression of gp91-phox, p22-phox, p47-phox, and p67-phox in EC. Sonicates from unstimulated EC produced no measurable superoxide; whereas, exogenously applied arachidonic acid activated superoxide generation in a manner that was dependent upon the presence of NADPH and both membrane and cytosolic fractions combined. Apocynin, a specific leukocyte NADPH oxidase inhibitor, was shown by Western-blot analysis of membrane and cytoplasmic fractions to inhibit the translocation of p47-phox to the membrane of stimulated EC. These findings support the presence of a functionally active leukocyte-type NADPH oxidase in EC. NADPH oxidase could be the major cellular ROS source in EC perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.
authors.affiliation: Department of Anatomy & Cell Biology, State University of New York Health Science Center Syracuse, 13210, USA.
authors.forename: J W
authors.initials: JW
authors.lastname: Meyer
authors.forename: J A
authors.initials: JA
authors.lastname: Holland
authors.forename: L M
authors.initials: LM
authors.lastname: Ziegler
authors.forename: M M
authors.initials: MM
authors.lastname: Chang
authors.forename: G
authors.initials: G
authors.lastname: Beebe
authors.forename: M E
authors.initials: ME
authors.lastname: Schmitt
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology; D001161:Arteriosclerosis / Q000201:enzymology; D001692:Biological Transport; D002462:Cell Membrane / Q000737:chemistry; D002474:Cell-Free System / Q000201:enzymology; D002478:Cells, Cultured; D003573:Cytochrome b Group / Q000032:analysis; D004730:Endothelium, Vascular / Q000166:cytology* / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007962:Leukocytes / Q000201:enzymology; D008562:Membrane Glycoproteins / Q000235:genetics; D026901:Membrane Transport Proteins; D009252:NADPH Dehydrogenase / Q000235:genetics; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000235:genetics; D010750:Phosphoproteins / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D014178:Translocation, Genetic / Q000187:drug effects; D014471:Umbilical Veins / Q000166:cytology / Q000201:enzymology
chemical_list: D000098:Acetophenones; D000975:Antioxidants; D003573:Cytochrome b Group; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; C036888:cytochrome b558; C056165:acetovanillone; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D009252:NADPH Dehydrogenase