Effect Of Integrin ?3 On Autophagy Of Vascular Endothelial Cells

Native Arteriovenous Fistulae(AVF)is a common method for hemodialysis in patients with end-stage renal disease.However,lumen stenosis often leads to the AVF failure due to intimal hyperplasia.One candidate for triggering neointima hyperplasia is the dysfunction of vascular endothelial cells.To prevent the intimal hyperplasia and further restenosis is remained to be resolved.As an adhesive molecule located on the surface of cell membrane,Integrin ?3 can mediate the cell-cell interaction and cell-extracellular matrix intereaction,it also participate in the physiological processes such as autophagy,cell proliferation and migration.Our previous work has found that Integrin ?3 induces neointimal hyperplasia through promoting the endothelial-to-mesenchymal transition.Autophagy is a self-renewal mechanism to maintain homeostasis,which plays an important role in vascular repair after injury.It has been reported that abnormal autophagy in endothelial cells may induce intimal hyperplasia.However,whether Integrin ?3 is involved in this process remains unknown.To analysis the effects of Integrin ?3 on autophagy and its regulatorymechanism,we collected clinical AVF specimens and explored the relationship between intimal hyperplasia and autophagy in endothelial cells.Then,Integrin ?3 was induced to express or knocked down in Human Umbilical Vein Endothelial Cells(HUVEC),and the effects of integrin ?3on the autophagy of endothelial cells and its underlying mechanism was detected.Finally,the effect of metformin in autophagy of endothelial cells and its regulatory mechanism was also detected.The results are as follows:1.The autophagy-related molecules were highly expressed in endothelial cells of AVF with severe intimal hyperplasia.HE staining results showed that the there has obviously intima hyperplasia near the anastomosis.Immunofluorescence assay showed that LC3 positve cells located in the endothelium of the thickened intima.2.Integrin ?3 promotes autophagy of endothelial cells.The results of real-time PCR and Western Blot showed that the expression of Integrin ?3in endothelial cells was induced by TGF?1.The expression of LC3 II and Beclin-1,two autophagy markers,was also increased.However,the mRNA and protein levels of LC3 II and Beclin-1 was significantly decreased in Integrin ?3 knocked down HUVECs,.3.Integrin ?3 regulates endothelial cells autophagy through YAP signal.Western Blot results showed that the expression of NICD(Notch signal transcription factors),AKT,pAKT and pJNK remained unchange in Integrin ?3 highly expressed HUVECs or knock down HUVECs.However,Integrin ?3 knockdown could down-regulate the expression of YAP(Yes-Associated Protein),CTGF(the downstream transcription factors of YAP)and LC3 II,suggesting that Integrin ?3 may prevent endothelial cells autophagy through the inhibition of YAP activation.4.Metformin reduces autophagy in endothelial cells by inhibiting the expression of YAP and promoting the inactivation of YAP.The results of CCK-8 showed that treated with 10,20 and 40mmol/L metformin for 24 h could reduce the cell survival rate.The results of transmission electronmicroscope,immunofluorescence and Western Blot showed that metformin could prevent the formation of autophagosome,decrease the expression of LC3 II,Belin-1 and CTGF,and promote the phosphorylation level of YAP in endothelial cells.Our study showed that Integrin ?3 may induce endothelial cell autophagy through activating YAP,the transcription factor of the Hippo signal pathway.To knock down or block the expression of integrin ?3 can inhibit autophagy,which may have a protective effect on endothelial cells in the process of neointimal hyperplasia,but these needs to be further verified in animal models.This study not only deepens our understanding of the molecular mechanism of endothelial cell autophagy,but also provides a new target for the clinical prevention of vascular intimal hyperplasia in AVFs.