Clin Res Cardiol 98, Suppl 1, April 2009

V234 - Effects of Vascular Oxidative Stress Induced by Endothelium-Specific Overexpression of Uncoupled eNOS in Mice
T. Suvorava1, M. Weber2, S. Valcaccia3, V. T. Dao1, G. Kojda3
1Institut für Pharmakologie, Heinrich-Heine-Universität, Düsseldorf; 2Division of Cardiology, Emory University School of Medicine, Atlanta, USA; 3Institut für Pharmakologie und Klinische Pharmakologie, Universitätsklinikum Düsseldorf, Düsseldorf;
Purpose: Numerous pathological conditions are associated with increased vascular oxidative stress. Whether elevated vascular levels of reactive oxygen species promote cardiovascular pathology or are the consequences of the disease state is an important but ananswered question.
Methods: We have generated a mutant of bovine eNOS in which cysteine 101 was replaced by alanine (C101A) resulting in uncoupling of eNOS. The effects of C101A mutation have been characterized in cultured human embryonic kidney cells (HEK) transfected with either mutant eNOS (eNOSmu) or wild type eNOS (eNOSwt). Transgenic mice carrying eNOSmu have been generated on a C57BL/6 background using the endothelium-specific Tie-2 promotor. By breeding these mice with eNOS knockouts (eNOS-/-), mice that only express eNOSmu (eNOS-/-/eNOSmu+) or no eNOS at all (eNOS-/-/eNOSmu-) were obtained.
Results: Studies in HEK cells transfected with eNOSmu revealed decreased eNOS activity, increased superoxide generation inhibited by L-NAME treatment, less NO production (45.8±6.2 %), strongly elevated protein oxidation level and decreased eNOS dimer stability in native SDS-gels as compared to eNOSwt suggesting that eNOSmu has the typical features of uncoupled eNOS. Organ bath experiments showed increased aortic sensitivity of eNOS-/-/eNOSmu+ to NO-donor S-nitroso-N-acetyl-penicillamine. In striking contrast, there was a significant rightward shift in acetylcholine dose-response curve in eNOS-/-/eNOSmu+ (p=0.0062 vs C57Bl/6, n=11-13). eNOS-/-/eNOSmu+ mice have elevated systolic blood pressure as compared to C57Bl/6 (138±2.3 vs 118.4±3.1 mmHg in C57BL/6, n=6-8, p<0.05), but not to eNOS-/-/eNOSmu- (135.5±0.8, n=8). Western blot analysis confirmed the expression of eNOS in eNOS-/-/eNOSmu+ and revealed an increased phosphorylation of eNOS at Ser1179 in aorta, heart and muscle of eNOS-/-/eNOSmu+ as compared to C57Bl/6 (p<0.05, n=4-8). Since the number of circulting endothelial progenitor cells may serve as a marker of ongoing endothelial dysfunction, EPCs were counted using Fluorescent Activated Cell Sorter. Amount of circulating EPCs defined as CD34/Flk-1 or as Sca-1/Flk-1 double positive cells was significantly lower in peripheral blood of eNOS-/-/eNOSmu+ as compared to eNOS-/-/eNOSmu- (n=7-8, p<0.05) strongly suggesting a role of uncoupled eNOS and increased vascular oxidative stress in regulation of circulating EPCs.
Conclusions: Endothelial specific overexpression of uncoupled eNOS and increased vascular oxidative stress decrease the number of circulating stem cells with endothelial progenitor capacity.