Abstract 3388: Calcium Dependent Nox5 NADPH Oxidase Contributes To Vascular Oxidative Stress In Human Coronary Artery Disease
The Nox-based NADPH oxidases are major sources of ROS in human vessels. Several Nox homologs have been identified but their relative contribution to vascular ROS production in coronary artery disease (CAD) is unclear. Nox5, a calcium dependent homolog, has been previously identified in immature lymphatic tissues, cancer cells and in endothelial cells but has not yet been studied in human vessels. Moreover, the role of calcium-dependent superoxide production in human coronary artery disease remains unknown. We hypothesized that Nox5 is present in human coronary arteries and that CAD increases its expression and activity. Western blotting using a novel anti-Nox5 antibody yielded a single 70 kDa band, consistent with the known Nox5 molecular weight, in both human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC). Small interfering RNA against Nox5 reduced this band and inhibited Ca++-dependent NADPH oxidase activity as measured by electron paramagnetic spin resonance. Studies of human coronary arteries indicated that calcium-dependent NADPH oxidase activity measured by ESR in vascular membranes was markedly greater in athero-sclerotic vs. normal human coronary vessels (1390±170 vs. 195±100 AU/mg protein; p<0.001). In keeping with these findings, Nox 5 mRNA, measured by real time PCR, markedly increased in atherosclerotic vessels compared to normals (9e5±5e4 vs. 2.1e2±1e2 AU/18S mRNA; p<0.01). Nox5 protein demonstrated by Western blotting was also dramatically increased in human coronary arteries obtained from patients with CAD. Immunofluorescence demonstrated that as atherosclerosis progresses, Nox5 expression extends beyond the endothelium to the intima and vascular smooth muscle. Moreover levels of Nox5 mRNA were significantly correlated to the Ca++ dependent NADPH oxidase activity measured from artery homogenates (p<0.01). These studies identify Nox5 as a novel calcium dependent source of reactive oxygen species in atherosclerosis. It also raises the possibility that calcium channel antagonists could reduce ROS production in CAD.