Anti-angiogenic Effect Of Platelet P2Y₁₂ Inhibitor In Ischemia-induced Angiogenesis In Mice Hindlimb And Possible Mechanisms

Objective:Postischemic inammation induces angiogenesis,while platelet P2Y₁₂ inhibitors can alleviate this inammation.Therefore,we studied the potential effects of P2Y₁₂inhibitor,ticagrelor,on angiogenesis in a mouse model of hindlimb ischemia and possible mechanisms.Methods:Mouse hindlimb ischemia model was established,and then wild-type mice(IL-10^+/+)were randomized to sham（sham group）,control(IL-10^+/+)and ticagrelor treatment group(IL-10^+/++tica);knockout IL-10 mice(IL-10^-/-)to control(IL-10^-/-group)and drug treatment group(IL-10^-/-+tica group).Laser Doppler perfusion imaging and capillary density measurements were used to quantify vasculogenesis.CD31 was detected by immunofluorescence staining.IL-10activity in mice was measured by enzyme-linked immunosorbent assay（ELISA）and Western blot analysis of vascular endothelial growth factor（VEGF）levels.To understand the effect of ticagrelor on angiogenesis in vitro,we isolated and cultured SMCs from mice and treated them with b FGF.This experiment was divided into five groups:Control group,b FGF group,b FGF+tica group,IL-10 depleted group and tica group,CCK-8 and Edu;tube-forming ability assay for tube-forming capacity;Western blot for intracellular AKT/VEGFR2/VEGF/e IF4E1 protein expression.To analyze the effect of ticagrelor inflammatory response in SMCs in vitro,we induced the inflammatory response in SMCs by LPS（bacterial lipopolysaccharide）stimulation.ELISA detected the inflammatory factors IL-1β,IL-2,IL-6,IL-8,IL-13,IFY-γ,and TNF-α.Results:Ischemic hindlimb angiogenesis was sharply decreased in IL-10^+/+mice than IL-10^-/-mice.In the IL-10^+/+mice,ticagrelor inhibited angiogenesis and blood reperfusion recovery significantly elevated the levels of IL-10 and decreased the expression of VEGF in the IL-10^+/+mouse ischemic hindlimb,which were abolished in IL-10-deficient(IL-10^–/–)C57BL/6J mice.In cell experiments,the proliferation,tubular formation and migration of b-FGF-induced rat smooth muscle cells were decreased by ticagrelor,which recovered after IL-10 depletion.The expressions of IL-1β,IL-2,IL-6,IL-8,IL-13,IFY-γand TNF-αin rat aortic smooth muscle cells were increased in LPS group,and all inflammatory factors were decreased after treatment with ticagrelor,while IL-10 was depleted after treatment with IL-10 neutralizing antibody,and all inflammatory factors were increased.The protein expressions of AKT/p-AKT,VEGFR2,VEGF and e IF4E1 in rat aortic smooth muscle cells were increased in the b-FGF group,while the expression of all proteins decreased after treatment with ticagrelor,while the expression of all detected proteins increased after treatment with IL-10 neutralizing antibody.Conclusions:The study underscore the effect of ticagrelor antiangiogenic function is related with the higher IL-10 expression.The mechanism may involve the inhibition of inflammatory response and migration of SMCs through IL-10 pathway.