Increased superoxide anion production is associated with early atherosclerosis and cardiovascular dysfunctions in a rabbit model Bertrand Collin, 1 David Busseuil, 1 Marianne Zeller, 1 Caroline Perrin, 1 Olivier Barthez, 1 Laurence Duvillard, 2 Catherine Vergely, 1 Marc Bardou, 1 Monique Dumas, 1 Yves Cottin 3 and Luc Rochette 1 1 Laboratory of Cardiovascular and Experimental Physiopathology and Pharmacology, Faculty of Medicine, IFR n°100, University of Burgundy, Dijon, France; 2 INSERM U498, CHU Bocage , IFR n°100, 21034 Dijon, France; 3 Cardiology Department, CHU Bocage, 21034 Dijon, France Received 13 January 2006; accepted 26 June 2006 Abstract Objective: Hypercholesterolemia (HC) has been associated with impairment of vascular and myocardial functions. As HC could generate an alteration in the oxidative status, we studied the effects of a 1-month cholesterol diet on cardiovascular oxidative stress. Methods and Results: New Zealand rabbits received cholesterol (1%) or normal chow for 1 month. At 30 days, superoxide anion levels, assessed by ESR spectroscopy, NAD(P)H oxidase (NOX) activity, and dihydroethidium (DHE) staining of aortas were higher in the cholesterol-fed (CF) group compared with control (respectively, 4.0 ± 0.6 Arbitrary Units/mg (AU/mg) vs. 2.6 ± 0.3, p < 0.05; 4231 ± 433 vs. 2931 ± 373 AU/mg, p < 0.05; 21.4 ± 1.2 vs. 12.9 ± 1.7% fluorescence/mm 2 , p < 0.001). NOX gp91phox and p67phox expression in the aortas were higher in the CF group vs. control (1.5 ± 0.2 vs. 0.5 ± 0.2, p < 0.001; 0.9 ± 0.2 vs. 0.3 ± 0.2, p < 0.05). The endothelium-dependent relaxation evaluated on the iliac arteries was higher in control than in the CF group (64.8 ± 10.1 vs. 13.1 ± 3.70%, p < 0.001). The cardiac diastolic pressure estimated on isolated hearts was higher in the CF group than in control (21.1 ± 4.1 vs. 10.3 ± 1.4 mmHg, p < 0.05) after 60 min of ischemia. Conclusions: Hypercholesterolemia induced increased levels of superoxide in the aortas and a higher expression of NOX subunits, associated with altered vasorelaxation. The increased diastolic pressure observed in hearts, consistent with a post- ischemic contractile dysfunction might be mediated by the production of superoxide. Key words: endothelial dysfunction, hypercholesterolemia, ischemia-reperfusion, NAD(P)H oxidase, superoxide Introduction Reactive oxygen species (ROS) and in particular superoxide anion (O 2 Æ) ) have been shown to initiate several processes involved in atherogenesis, including expression of adhesion molecules, stimulation of proliferation and migration of vascular smooth muscle cells (VSMC), endothelial apop- tosis and alteration of vasomotor activity. Vascular O 2 Æ) originates from various enzymatic sources of which the most important are NAD(P)H oxidases [1]. Vascular Address for offprints: Bertrand Collin, Laboratory of Cardiovascular and Experimental Physiopathology and Pharmacology, Faculty of Medicine, IFR n°100, University of Burgundy, 7, Boulevard Jeanne dÕArc, 21079 Dijon Cedex, BP 87900, France (E-mail: bertrand.collin@voila.fr) 1 Molecular and Cellular Biochemistry 294: 225–235, 2007. Ó Springer 2006 DOI 10.1007/s11010-006-9263-y