Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells: A potential atherogenic source of reactive oxygen species

Cultured human endothelial cells (EC) exposed to atherogenic low-density li poprotein levels have increased reactive oxygen species (ROS) generation. T he 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 mem brane fraction of disrupted EC showed a reduced-minus-oxidized difference s pectra with absorption peaks identical to those observed in the spectra of the leukocyte NADPH oxidase component, cytochrome b(558). Western-blot anal ysis, using anti-gp91-phox. anti -p22-phox. anti -p47-phox. and anti -p67-p hox antibodies, demonstrated the protein expression of NADPH oxidase subuni ts in EC. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed t he mRNA expression of gp91-phox, p22-phox, p47-phox, and p67-phox in EC. So nicates from unstimulated EC produced no measurable superoxide; whereas, ex ogenously applied arachidonic acid activated superoxide generation in a man ner that was dependent upon the presence of NADPH and both membrane and cyt osolic fractions combined. Apocynin, a specific leukocyte NADPH oxidase inh ibitor, was shown by Western-blot analysis of membrane and cytoplasmic frac tions to inhibit the translocation of p47-phox to the membrane of stimulate d EC. These findings support the presence of a functionally active leukocyt e-type NADPH oxidase in EC. NADPH oxidase could be the major cellular ROS s ource in EC perturbation, which has been hypothesized to be a major contrib uting factor in the pathogenesis of atherosclerosis.