Expression of phagocyte NADPH oxidase components in human endothelial cells

Abstract

Low-level generation of reactive oxygen species (ROS) by endothelial cells in response to a variety of stimuli has been observed; however, the enzyme system responsible is unknown. Using a variety of techniques, we examined for components of the phagocyte superoxide-generating NADPH oxidase to elucidate whether this enzyme could be a source of endothelial-derived ROS. Superoxide generation on addition of 100 microM NAD(P)H to human umbilical vein endothelial cell (HUVEC) sonicates (using lucigenin-enhanced chemiluminescence) was partially inhibited on addition of the flavoenzyme inhibitor diphenyliodonium (IDP). Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated expression of gp91phox, p22phox, p67phox, and p47phox in four independent HUVEC isolates. Expression of p22phox was also confirmed by Northern blotting. RT-PCR for tumor necrosis factor-alpha was negative, indicating an absence of mononuclear cell contamination (a potential source of NADPH oxidase). Immunoperoxidase staining, using anti-p47phox (JW-1)- and anti-p67phox (JW-2)-specific antibodies, showed protein expression of these cytosolic components. However, heme spectroscopy failed to indicate the presence of the low-potential cytochrome b558. These data indicate that cultured human endothelial cells express both mRNA and protein for cytosolic components of the phagocyte superoxide-generating NADPH oxidase. However, because the cytochrome b558 heme could not be conclusively demonstrated, a contribution of the phagocyte NADPH oxidase to endothelial oxidant generation may be unlikely.

Elevated superoxide production by active H-ras enhances human lung WI-38VA-13 cell proliferation, migration and resistance to TNF-α

“…In the presence of cofactors and regulators, the NADPH oxidase catalyzes one electron reduction of oxygen to superoxide (Irani and Goldchmidt-Clermont, 1998). It has been shown by several groups that the NADPH oxidase, or NADPH oxidase-like activity exists in non-phagocytic cells, including ®broblasts (Irani et al, 1997;Jones et al, 1994), endothelial cells (Jones et al, 1996) and keratinocyte cells (Yang et al, 1999). The elevated superoxide production in WI-38 VA-13 cells expressing active H-ras was primarily derived from the NADPH oxidase activity, because superoxide production can be inhibited by DPI or apocynin, but not rotenone, allopurinol or oxypurinol (Figure 4).…”

Redox Control of Renal Function and Hypertension

“…It is now established that NAD(P)H oxidase expressed in endothelial cells is an important source of free oxygen radical generation in the arterial wall (147). In contrast, smooth muscle cells either lack Nox2 or have several orders of magnitude lower expression of this subunit when compared with Nox2 homologues such as Nox1 and Nox4 (174).…”

Decreased neointimal formation in Nox2-deficient mice reveals a direct role for NADPH oxidase in the response to arterial injury

“…10). Although early studies of oxidant generation were typically limited to phagocytic cells that contain the prototypical NADPH oxidase, it is now well accepted that nonphagocytic cells, such as endothelial cells (11), vascular SMC (12), and fibroblasts (13), also express NADPH oxidase isoforms that participate in the generation of both ROS and RNS.…”