Calcium-Dependent NOX5 Nicotinamide Adenine Dinucleotide Phosphate Oxidase Contributes to Vascular Oxidative Stress in Human Coronary Artery Disease
Tomasz J. Guzik, MD, PhD*, ,
Wei Chen, MD, PhD*,
Maria C. Gongora, MD*,
Bartlomiej Guzik, MD, PhD,
Heinrich E. Lob, PhD*,
Deepa Mangalat, MD*,
Nyssa Hoch, PhD*,
Sergey Dikalov, PhD*,
Pawel Rudzinski, MD, PhD,
Boguslaw Kapelak, MD, PhD,
Jerzy Sadowski, MD, PhD and
David G. Harrison, MD, FACC*,*
* Division of Cardiology, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia
Department of Pharmacology and Internal Medicine, Jagiellonian University School of Medicine, Cracow, Poland
Department of Cardiovascular Surgery and Transplantation, Jagiellonian University School of Medicine, Cracow, Poland
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Figure 1 Contribution of NOX5 to Calcium-Dependent NADPH Oxidase Activity in Human Endothelial Cells
(A) Example ESR spectra of nitroxide adduct formation by membranes prepared from human aortic endothelial cells (HAECs) in Ca++-free media and in the presence of 1 mM Ca++
(left) and the effects of small interfering ribonucleic acid (siRNA) against NOX5 on calcium-dependent signal (right). (B) Effects of siRNA NOX5 on NOX5 protein in HAECs; n = 4 experiments. (C) Average Ca++-dependent (top) and Ca++-independent (bottom) NADPH oxidase activity in the presence of control siRNA (solid bars) and NOX5 siRNA (open bars). n = 4. Values are presented as mean ± SEM. *p < 0.01 versus control siRNA. ESR = electron spin resonance; NADPH = nicotinamide adenine dinucleotide phosphate.
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Figure 2 Calcium-Dependent NADPH Oxidase Activity and NOX5 Expression in CAD
(A) Calcium-independent (left) and -dependent (right) NADPH oxidase activity in human coronary arteries in relation to the presence of coronary artery disease (CAD). The NADPH oxidase activity was measured by ESR as described in the Methods section in membranes isolated from coronary arteries of subjects with (n = 8) and without (n = 8) CAD. (B) The NOX5 messenger ribonucleic acid (mRNA) expression in coronary arteries from patients with (n = 13) and without (n = 11) CAD. TaqMan real-time polymerase chain reaction analysis was performed using commercially available gene expression assays. (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 versus non-CAD. **p < 0.01 versus non-CAD. Abbreviations as in Figure 1.
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Figure 3 NOX5 Expression in Human Coronary Arteries in Relation to CAD
Example Western blots (A), average data showing NOX5 protein expression in non–coronary artery disease (CAD) (n = 7) and CAD (n = 7) coronary arteries (B). Lysates of DU 145 prostate cancer cells were used as positive control. Bars represent mean ± SEM. *p < 0.01. Abbreviations as in Figure 1.
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Figure 4 NOX5 Localization in Human CAD at Different Stages of Atherosclerosis
Immunofluorescent localization of NOX5 in human coronary arteries. The NOX5 (red) was studied in control, non-coronary artery disease (CAD) coronary arteries (A), in coronary artery segments from CAD patients showing no atherosclerosis (B,C), in coronary artery segments showing neointimal hyperplasia (D), and in coronary artery segments with severe complex lesions (E). (C) A magnification of (B) showing the presence of NOX5 in endothelium (double staining; arrows). Green staining represents endothelial cell marker CD31 in (A to D) and smooth muscle cell alpha actin (E). Micrographs show representative staining of at least 5 independent experiments. Abbreviations as in Figure 1.
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