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.

Antioxidative Potential of Fluvastatin <i>via</i> the Inhibition of Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase Activity

“…These findings also support the presence of a functionally active leukocyte type NADPH oxidase in EC. 27) From these facts, it is likely that fluvastatin inhibits the progression of atherosclerosis via the inhibition of NADPH oxidase in not only neutrophils but also in EC. Rueckschloss et al reported that oxidized LDL induces proatherosclerotic NADPH oxidase expression and superoxide anion formation in human endothelial cells, and an antioxidative potential of HMG-CoA reductase inhibition via the reduction of vascular NADPH oxidase expression.…”

Apocynin attenuates ventilator-induced lung injury in an isolated and perfused rat lung model

“…Apocynin is a strong oxidative inhibitor that has been demonstrated to block NADPH oxidase in neutrophils [21,22], macrophages [22,23], and endothelium [24] through inhibition of p47phox translocation [25], without interfering with other immune biological functions of cell systems. This is an important therapeutic mechanism of apocynin, as NADPH plays a crucial role in the complex cell-cell interaction during oxidative status [26].…”

Mechanisms of H2O2-induced oxidative stress in endothelial cells

“…These include: xanthine oxidase (Miyachi et al 1986;Satoh et al 1998;Fleming et al 2001), NADPH oxidase (Meyer et al 1999;Hohler et al 2000;Barry-Lane et al 2001;Beswick et al 2001;Seno et al 2001;Brandes et al 2002;Li et al 2002;Lassegue et al 2003;Parinandi et al 2003), and uncoupled NOS (Beretta et al 2003;Landmesser et al 2003). NADPH oxidase and uncoupled NOS are thought to be major…”