Calcium-dependent NOX5 nicotinamide adenine dinucleotide phosphate oxidase contributes to vascular oxidative stress in human coronary artery disease

Abstract

Objectives: This study sought to examine the expression and activity of the calcium-dependent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) in human atherosclerotic coronary arteries.

Background: The NOX-based NADPH oxidases are major sources of reactive oxygen species (ROS) in human vessels. Several NOX homologues have been identified, but their relative contribution to vascular ROS production in coronary artery disease (CAD) is unclear; NOX5 is a unique homolog in that it is calcium dependent and thus could be activated by vasoconstrictor hormones. Its presence has not yet been studied in human vessels.

Methods: Coronary arteries from patients undergoing cardiac transplantation with CAD or without CAD were studied; NOX5 was quantified and visualized using Western blotting, immunofluorescence, and quantitative real-time polymerase chain reaction. Calcium-dependent NADPH oxidase activity, corresponding greatly to NOX5 activity, was measured by electron paramagnetic resonance.

Results: Both Western blotting and quantitative real-time polymerase chain reaction indicated a marked increase in NOX5 protein and messenger ribonucleic acid (mRNA) in CAD versus non-CAD vessels. Calcium-dependent NADPH-driven production of ROS in vascular membranes, reflecting NOX5 activity, was increased 7-fold in CAD and correlated significantly with NOX5 mRNA levels among subjects. Immunofluorescence showed that NOX5 was expressed in the endothelium in the early lesions and in vascular smooth muscle cells in the advanced coronary lesions.

Conclusions: These studies identify NOX5 as a novel, calcium-dependent source of ROS in atherosclerosis.

Figure 1. Contribution of Nox5 to calcium-dependent NADPH oxidase activity in human endothelial cells

Panel A. Example ESR spectra of nitroxide adduct formation by membranes prepared from human aortic endothelial cells (HAEC) in Ca⁺⁺ free media and in the presence of 1 mM Ca⁺⁺ (left) and the effects of small interfering RNA (siRNA) against Nox5 on calcium dependent signal (right). Panel B. Effects of siRNA Nox5 on Nox5 protein in HAECs; n=4 experiments; Panel C. Average Ca++ dependent (top) and Ca⁺⁺ independent (bottom) NADPH oxidase activity in the presence of control siRNA (black bars) and Nox5 siRNA (open bars); n=4; values are presented as mean +/−SEM; *-p<0.01 vs control siRNA.

Figure 2. Calcium dependent NADPH oxidase activity and Nox5 expression in coronary artery disease

Panel A. Calcium independent (left; panel A) and calcium dependent (right; panel A) NADPH oxidase activity in human coronary arteries in relation to the presence of coronary artery disease (CAD). NADPH oxidase activity was measured by ESR as described in methods in membranes isolated from coronary arteries of subjects with (n=8) and without (n=8) CAD. Panel B. Nox5 mRNA expression in coronary arteries from patients with (n=13) and without (n=11) CAD. TaqMan real time PCR analysis was performed using commercially available Gene expression assays. Panel C. Relationship between Ca++ dependent NADPH oxidase activity and Nox5 mRNA expression in studied coronary arteries. Data were expressed as mean+/−SEM. *-p<0.05 vs non CAD; **-p<0.01 vs non CAD.

Figure 3. Nox5 expression in human coronary arteries in relation to coronary artery disease

Example Western blots (Panel A.) average data showing Nox5 protein expression in non-CAD (n=7) and CAD (n=7) coronary arteries (Panel B). Lysates of DU 145 prostate cancer cells were used as positive control. Bars represent mean+/− SEM. *-p<0.01.

Figure 4. Nox5 localization in human coronary artery disease at different stages of atherosclerosis

Immunofluorecent localization of Nox5 in human coronary arteries. Nox5 (red) was studied in control, non CAD coronary arteries (panel A); in coronary artery segments from CAD patient showing no atherosclerosis (panels B and C), in coronary artery segments showing neointimal hyperplasia (panel D) and in coronary artery segment with severe complex lesion (panel E). Panel C presents a magnification of panel B showing presence of Nox5 in endothelium (double staining; arrows). Green staining represents endothelial cell marker CD31 in panels A–D and smooth muscle cell alpha actin in panel E. Micrographs show representative staining of at least 5 independent experiments.