Abstract 14445: Reduced Expression of Let-7f Leads to Impaired Ischemia-Induced Neovascularization Following Cigarette Smoke Exposure: Role of ALK5/SMAD2/PAI-1 Pathway

Abstract

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.