gp91phox Contributes to NADPH oxidase activity in aortic fibroblasts but not smooth muscle cells

Abstract

Reactive oxygen species (ROS) derived from vascular NADPH oxidase are important in normal and pathological regulation of vessel growth and function. Cell-specific differences in expression and function of the catalytic subunit of NADPH oxidase may contribute to differences in vascular cell response to NADPH oxidase activation. We examined the functional expression of gp91 phox on NADPH oxidase activity in vascular smooth muscle cells (SMC) and fibroblasts (FB). As measured by dihydroethidium fluorescence in situ, superoxide [Formula: see text] levels were greater in adventitial cells compared with medial SMC in wild-type aorta. In contrast, there was no difference in [Formula: see text] levels between adventitial cells and medial SMC in aorta from gp91 phox-deficient (gp91 phox KO) mice. Adventitial-derived FB and medial SMC were isolated from the aorta of wild-type and gp91 phox KO mice and grown in culture. Consistent with the observations in situ, basal and stimulated ROS levels were reduced in FB isolated from aorta of gp91 phox KO compared with FB from wild-type aorta, whereas ROS levels were similar in SMC derived from gp91 phox KO and wild-type aorta. There were no differences in expression of superoxide dismutase between gp91 phox KO and wild-type FB to account for these observations. Because gp91 phox is associated with membranes, we examined NADPH-stimulated [Formula: see text] production in membrane-enriched fractions of cell lysate. As measured by chemiluminescence, NADPH oxidase activity was markedly greater in wild-type FB compared with gp91 phox KO FB but did not differ among the SMCs. Confirming functional expression of gp91 phox in FB, antisense to gp91 phox decreased ROS levels in wild-type FB. Finally, deficiency of gp91 phox did not alter expression of the gp91 phox homolog NOX4 in isolated FB. We conclude that the neutrophil subunit gp91 phox contributes to NADPH oxidase function in vascular FB, but not SMC.