Objectives To examine the expression and activity of the calcium dependent NADPH oxidase in human atherosclerotic coronary arteries. Background The Nox based NADPH oxidases are major sources of reactive oxygen species (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 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 transplant with CAD or without CAD were studied. Nox5 was quantified and visualized using Western blotting, immunofluorescence and quantitative real-time PCR. Calcium dependent NADPH oxidase activity, corresponding greatly to Nox5 activity was measured by electron paramagnetic resonance. Results Both western blotting and quantitative real time PCR indicated a marked increase in Nox5 protein and mRNA in CAD vs non CAD vessels. Calcium dependent NADPH driven production of reactive oxygen species in vascular membranes, reflecting Nox5 activity was increased 7 fold in CAD and correlated significantly with Nox5 mRNA levels among subjects. Immunofluorescence demonstrated that Nox5 was expressed in the endothelium in the early lesions and in vascular smooth muscle cells in the advanced in coronary lesions. Conclusions These studies identify Nox5 as a novel, calcium dependent source of reactive oxygen species in atherosclerosis.

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Abstract-We previously found that angiotensin II-induced hypertension increases vascular extracellular superoxide dismutase (ecSOD), and proposed that this is a compensatory mechanism that blunts the hypertensive response and preserves endothelium-dependent vasodilatation. To test this hypothesis, we studied ecSOD-deficient mice. ecSODand C57Blk/6 mice had similar blood pressure at baseline; however, the hypertension caused by angiotensin II was greater in ecSOD Ϫ/Ϫ compared with wild-type mice (168 versus 147 mm Hg, respectively; PϽ0.01). In keeping with this, angiotensin II increased superoxide and reduced endothelium-dependent vasodilatation in small mesenteric arterioles to a greater extent in ecSOD Ϫ/Ϫ than in wild-type mice. In contrast to these findings in resistance vessels, angiotensin II paradoxically improved endothelium-dependent vasodilatation, reduced intracellular and extracellular superoxide, and increased NO production in aortas of ecSOD Ϫ/Ϫ mice. Whereas aortic expression of endothelial NO synthase, Cu/ZnSOD, and MnSOD were not altered in ecSOD Ϫ/Ϫ mice, the activity of Cu/ZnSOD was increased by 80% after angiotensin II infusion. This was associated with a concomitant increase in expression of the copper chaperone for Cu/ZnSOD in the aorta but not in the mesenteric arteries. Moreover, the angiotensin II-induced increase in aortic reduced nicotinamide-adenine dinucleotide phosphate oxidase activity was diminished in ecSOD Ϫ/Ϫ mice as compared with controls. Thus, during angiotensin II infusion, ecSOD reduces hypertension, minimizes vascular superoxide production, and preserves endothelial function in resistance arterioles. We also identified novel compensatory mechanisms involving upregulation of copper chaperone for Cu/ZnSOD, increased Cu/ZnSOD activity, and decreased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity in larger vessels. These compensatory mechanisms preserve large vessel function when ecSOD is absent in hypertension. , which, in turn, leads to inactivation of NO-altering endothelium-dependent vasodilatation. 1 In some models, such as angiotensin II-induced hypertension and in the spontaneously hypertensive rat, membrane-targeted forms of superoxide dismutase (SOD) and SOD mimetics lower blood pressure suggesting that the oxidative inactivation of NO contributes to blood pressure elevation. 2-4 Increased production of O 2 ⅐Ϫ in kidneys and circumventricular organs of the central nervous system also contributes to the hypertension caused by angiotensin II. 5,6 A major antioxidant mechanism against O 2 ⅐Ϫ is the SODs, including the cytosolic Cu/ZnSOD (SOD1), the mitochondrial MnSOD (SOD2), and the extracellular superoxide dismutase (ecSOD or SOD3). 7 In most tissues, ecSOD is present in small amounts, however, in vascular tissues ecSOD represents 30% to 50% of total SOD. 8 We have shown previously that angiotensin II increases ecSOD expression in mice and human vascular smooth muscle cells. 9 One consequence of this increase in ecSOD might be a blunting of hypert...