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Abstract-Hypercholesterolemia is associated with impaired neovascularization in response to ischemia. Potential mechanisms include defective NO bioactivity and a reduction in the number/function of endothelial progenitor cells (EPCs). Here we tested the hypothesis that sildenafil, a phosphodiesterase 5 inhibitor that increases NO-driven cGMP levels, could stimulate EPC function and improve ischemia-induced neovascularization in hypercholesterolemic conditions. Apolipoprotein E-deficient (ApoE Ϫ/Ϫ ) mice were treated (or not treated) with sildenafil (40 mg/kg per day in water), and hindlimb ischemia was surgically induced by femoral artery removal. Sildenafil treatment led to an improved blood flow recovery, an increased capillary density, and a reduction of oxidative stress levels in ischemic muscles at day 7 after surgery. Sildenafil therapy is associated with an increased activation of angiogenic transduction pathways, including Akt, p44/42 mitogen-activated protein kinase, and p38. In vitro, sildenafil increases cellular migration and tubule formation of mature endothelial cells (human umbilical vascular endothelial cells) in a cGMP-dependent manner. In vivo, ApoE Ϫ/Ϫ mice treated with sildenafil exhibit a significant increase in the number of bone marrow-derived EPCs. Moreover, the angiogenic activities of EPCs (migration and adhesion) are significantly improved in ApoE Ϫ/Ϫ mice treated with sildenafil. In summary, this study demonstrates that sildenafil treatment is associated with improved ischemia-induced neovascularization in hypercholesterolemic ApoE Ϫ/Ϫ mice. The mechanisms involve beneficial effects on angiogenic transduction pathways together with an increase in the number and the functional activity of EPCs. Sildenafil could constitute a novel therapeutic strategy to reduce tissue ischemia in atherosclerotic diseases. Key Words: sildenafil Ⅲ endothelial Ⅲ progenitor Ⅲ cells Ⅲ angiogenesis hypercholesterolemia T he ability of the organism to develop new blood vessels (neovascularization) constitutes an important adaptive response to vascular occlusive diseases. 1 Postnatal neovascularization necessitates the activation, migration, and proliferation of mature endothelial cells (angiogenesis). 2 In response to ischemia, hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF) have been shown to be critical limiting factors for the induction of angiogenesis. 3,4 The importance of NO for endothelial cell migration and VEGF-induced angiogenesis was also demonstrated recently. 5,6 However, increasing evidence suggests that postnatal neovascularization relies not exclusively on the sprouting of preexisting vessels, but also the contribution of bone marrowderived circulating endothelial progenitor cells (EPCs). 7 In young patients and in animal models with young and healthy animals, the neovascularization process is very effective so that blood flow restoration after ischemia is almost complete. Neovascularization, however, is impaired in several clinical situations, which leads to i...

Moderate consumption of red wine is associated with a decreased incidence of cardiovascular diseases in populations with relatively high amount of fat in the diet. However, the mechanisms involved in this protective effect are not completely understood. Here we show that moderate consumption of red wine (equivalent to 2 glasses/day in humans) but not ethanol only, improves blood flow recovery by 32% after hindlimb ischemia in hypercholesterolemic ApoE-deficient mice. In ischemic tissues, red wine consumption reduces oxidative stress and increases capillary density by 46%. Endothelial progenitor cells (EPCs) have been shown to have an important role in postnatal neovascularization. We found that the number of EPCs is increased by 60% in ApoE mice exposed to red wine. Moreover, the migratory capacity of EPCs is significantly improved in red wine-drinking mice. The wine used in our study is a cabernet sauvignon from Languedoc-Roussillon, France, which contains a relatively high concentration (4-6 mg/L) of the polyphenolic antioxidant resveratrol. We demonstrate that resveratrol can rescue oxidized low-density lipoprotein (oxLDL)-induced impairment of in vitro angiogenic activities in human umbilical vein endothelial cells (HUVECs). Resveratrol exposure is also associated with increased activation of Akt/eNOS together with a restoration of nitric oxide production in HUVECs exposed to oxLDL. Our study suggests that moderate consumption of red wine improves ischemia-induced neovascularization in high-cholesterol conditions by increasing the number and the functional activities of EPCs and by restoring the Akt-eNOS-NO pathway.

Objective-Because Nox2-containing NADPH oxidase is a major source of ROS in the vasculature, we investigated its potential role for the modulation of ischemia-induced neovascularization in conditions of increased oxidative stress. Methods and Results-To mimic a clinical situation of increased oxidative stress, mice were exposed to cigarette smoke before and after the surgical induction of hindlimb ischemia. Nox2 expression and oxidative stress in ischemic tissues were significantly increased in wild-type mice, but not in mice deficient for the Nox2-containing NADPH oxidase (Nox2 Ϫ/Ϫ ). Nox2 Ϫ/Ϫ mice demonstrated faster blood flow recovery, increased capillary density in ischemic muscles, and improved endothelial progenitor cell functional activities compared to Nox2 ϩ/ϩ mice. In addition, Nox2 deficiency was associated with increased antioxidant and nitrite concentrations in plasma, together with a preserved expression of eNOS in ischemic tissues. In vitro, Nox2Ϫ/Ϫ endothelial cells exhibit resistance against superoxide induction and improved VEGF-dependent angiogenic activities compared to Nox2 ϩ/ϩ endothelial cells. Importantly, the beneficial effects of Nox2 deficiency on neovascularization in vitro and in vivo were lost after treatment with the NO inhibitor L-NAME. Conclusions-Nox2-containing NADPH oxidase deficiency protects against ischemia in conditions of increased oxidative stress. The mechanism involves improved neovascularization through a reduction of ROS formation, preserved activation of the VEGF/NO angiogenic pathway, and improved functional activities of endothelial progenitor cells.

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