P302	Endothelial nitric oxide synthase is essential for vasodilation during blood flow recovery , but not for collateral artery growth.
1B.Mees, 2S.Wagner, 2E.Ninci, 2S.Tribulova, 2S.Martin, 1R.van Haperen, 2S.Kostin, 2M.Heil, 1R.de Crom, 2W.Schaper
1Erasmus University Medical Center, Rotterdam, NL; 2Max-Planck-Institute for Heart & Lung Research, Bad Nauheim, DE.

Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS), is a powerful vasodilator and angiogenic mediator having several anti-atherogenic effects. Therefore, patients with cardiovascular disease could benefit from a therapy stimulating vascular growth by eNOS upregulation. Since blood flow deficits caused by occlusion or stenosis of a major artery can only be efficiently compensated for by arteriogenesis (i.e. collateral growth), we investigated the role of NO specifically in arteriogenesis. Hind limb ischemia was induced in eNOS knockout (KO) mice (n=26), mice overexpressing eNOS (n=21) (TG) and wild type (WT) controls (n=23) and mice were monitored for 3 weeks. Limb function of eNOS TG and WT mice recovered to normal within 1 week, as assessed with a clinical score. In contrast, limb function of eNOS KO mice did not even recover fully in 3 weeks. Laser Doppler and Oxygen saturation measurements in the mouse feet demonstrated decreased tissue perfusion in eNOS KO mice until endpoint (21 day: 37±4% vs. 85±4%, P<0.001; eNOS KO vs. WT). Only immediately following ligation tissue perfusion was significantly increased in eNOS TG mice (33±4% vs. 18±4%, P<0.01; eNOS TG vs. WT) suggesting higher acute maximal vasodilation in eNOS TG mice. Magnetic resonance imaging (MRI) measurements demonstrated that collateral dependent blood flow in the calf was significantly higher immediately after ligation in eNOS TG mice. However, MRI measured diminished collateral dependent blood flow in eNOS KO mice only in the 1st week after ligation (3 day: 40±7% vs. 12±2%, P<0.001; 14 day: 89±13% vs. 83±8%, P=NS; WT vs. eNOS KO). Histology demonstrated that collateral diameters and wall areas did not differ between the 3 groups (day 21, diameter(µm): 58.2±5.5, 52.6±5.4 and 61.3±5.1 for WT, eNOS TG and eNOS KO P=NS). Treatment with the NO-donor SNAP at day 7 resulted in equal collateral dependent blood flow in eNOS KO and WT mice confirming normal collateral growth in eNOS KO mice. Our findings stress the essential role of vasodilation in blood flow recovery following ischemia. Overexpression of eNOS only has a beneficial effect in the acute recovery phase but no further beneficial effects on arteriogenesis. Furthermore, deficient blood flow recovery and excessive tissue damage in eNOS KO mice is caused by lack of vasodilation, not by lack of collateral vessel growth.

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