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Background: Exposure to cigarette smoke is associated with impaired formation of new blood vessels (neovascularization) in response to ischemia. Here we investigated the potential role of miRs in that physiopathology. Methods and Results: Using Affimetrix GeneChip miRNA array analysis, we identified let-7f as a pro-angiogenic miR which expression is reduced following cigarette smoke exposure. qRT-PCR analyses confirmed that the expression of let-7f is significantly reduced in HUVECs treated with cigarette smoke extracts (CSE), and in the ischemic muscles of mice that are exposed to cigarette smoke (MES). In a mouse model of hindlimb ischemia, intramuscular injection of let-7f mimic restored ischemia-induced neovascularisation in MES. At day 21 after ischemia, Doppler flow ratios and capillary density in ischemic muscles were significantly improved in MES treated with let-7f mimic compared to those treated with a mimic control. Clinically, mice treated with let-7f mimic also exhibited reduced ambulatory impairment and hindlimb ischemic damages. Interestingly, we found that treatment with let-7f mimic can rescue endothelial progenitor cell (EPC) number and function (migration, integration into tubules) in MES. Let-7f has previously been shown to target ALK5 (TGF-βRI), an important modulator of angiogenesis. We found that ALK5 is significantly increased in HUVECs exposed to CSE and in the ischemic muscles of MES. This is associated with a downstream activation of anti-angiogenic factors such as SMAD2 and PAI-1. Importantly, treatment with let-7f mimic reduces the expression of ALK5, SMAD2 and PAI-1 both in vitro and in vivo. Moreover, let-7f mimic can also rescue angiogenesis in HUVECs exposed to CSE. Conclusion: Cigarette smoke exposure is associated with reduced expression of let-7f, which leads to impaired neovascularization following ischemia. Our results suggest that the mechanism involves increased activation of ALK5, together with a downstream stimulation of the anti-angiogenic SMAD2/PAI-1 pathway. Overexpression of let-7f using a miR mimic could constitute a novel therapeutic strategy to improve ischemia-induced neovascularization in pathological conditions.

Background: The activation of the renin-angiotensin system is associated with impaired formation of new blood vessels (neovascularization) in response to ischemia. Aliskiren is the only direct renin inhibitor that is clinically used as an orally active antihypertensive drug. Here we tested the hypothesis that aliskiren might improve neovascularization following ischemia. Methods and Results: C57BL/6 mice were treated with a high dose of aliskiren (50 mg/kg), a low dose of aliskiren (10 mg/kg), or drinking water only. After two weeks of treatment, hindlimb ischemia was surgically induced by femoral artery removal. Treatment with aliskiren led to a significantly faster rate of blood flow recovery after hindlimb ischemia (Laser Doppler). Interestingly the lower dose of aliskiren, which did not reduce blood pressure, provided similar improvement of blood flow recuperation compared to the higher dose of aliskiren. At day 21 after surgery, Doppler flow ratios were significantly improved in mice treated with aliskiren (0.69+/-0.07 vs. 0.52+/-0.03; p Conclusions: Direct renin inhibition with aliskiren leads to improved ischemia-induced neovascularization that is not dependant on blood pressure lowering. The mechanisms involve beneficial effects of aliskiren on NO and angiogenic pathways in ischemic tissues, together with an increase in the number and the functional activity of EPCs.

Background: Sildenafil is widely used to treat erectile dysfunction. Sildenafil inhibits phospho-diesterase 5 (PDE5), thereby increasing cGMP levels in target tissues. The NO-cGMP pathway has been shown to be essential for angiogenesis, vasculogenesis and postnatal neovascularization. Therefore, here we tested the hypothesis that sildenafil might improve neovascularization following ischemia. Methods and results: Hypercholesterolemic ApoE−/− mice, which are characterised by reduced neovascularization in response to ischemia, were treated or not with sildenafil (40 mg/kg/day in water) for the whole duration of the study. Sildenafil was well tolerated by ApoE−/− mice and led to a significant increase of plasmatic cGMP levels compared to controls (13.4±1.9 vs. 4.6±2.6 pmol/mL, p Conclusions: Sildenafil treatment is associated with improved ischemia-induced neovascularization in hypercholesterolemic conditions. The mechanisms involve beneficial effects of sildenafil on angiogenic pathways in ischemic tissues together with an increase in the number and the functional activity of EPCs.

Background: Several risk factors for cardiovascular diseases are associated with reduced neovascularization in response to ischemia. A common feature of all cardiovascular risk factors is increased production of reactive oxygen species (ROS) such as superoxide. We have recently demonstrated that mice that are deficient for CuZnSOD present impaired neovascularization in response to ischemia. Here we tested the hypothesis that endothelial progenitor cell (EPC) dysfunction might be involved in this neovascularization defect. Methods and results: Seven days after surgically induced hindlimb ischemia, EPCs were isolated from CuZnSOD deficient mice (CuZnSOD −/ − , n=8 –10) or control mice (CuZnSOD +/+ , n=8 –10). We found that the number of EPCs was significantly reduced in the bone marrow (40.6±3.0 vs 70.5±5.0, p−/ − mice compared to controls. Moreover, EPC functional activities were significantly impaired in CuZnSOD −/ − mice. More specifically, we found that EPC adhesion was reduced by 41%, migration by 88%, proliferation by 46% and integration into tubules by 77% in CuZnSOD −/ − mice. To confirm the role of EPCs in vivo, we isolated CD117+ cells (also acLDL and lectin positive when cultured for 4 days in vitro) from the bone marrow of CuZnSOD +/+ mice and performed an intravenous injection (5X10 5 cells) into CuZnSOD −/ − mice at 24 hours after surgically-induced hindlimb ischemia. Interestingly, EPC supplementation in CuZnSOD −/ − mice led to a complete rescue of ischemia-induced neovascularization, as assessed by blood flow recuperation in the ischemic hindlimb (Laser-Doppler analysis). Moreover, EPC therapy in CuZnSOD −/ − mice was also associated with a significant reduction of oxidative stress in ischemic tissues (nitrotyrosine and DHE staining). Conclusions: CuZnSOD deficiency is associated with a significant reduction in the number and the functional activities of EPCs. Cellular therapy with EPC can rescue the impaired neovascularization in CuZnSOD −/ − mice. Our results suggest that increased oxidative stress could contribute to explain EPC dysfunction and impaired neovascularization associated with different cardiovascular risk factors.