Deptor Deficiency-mediated MTORC1 Hyperactivation In Vascular Endothelial Cells Promotes Angiogenesis

BackgroundAngiogenesis is the physiological process through which new blood vessels sprouts from pre-existing vessels.VEGFs plays an import role in promoting angiogenesis and maintain the integrity of endothelium.It has been reported that DEPTOR regulates vascular endothelial cell activation and plays an importment role in proinflammatory and angiogenic response in vitro.However,the roles of DEPTOR in angiogenesis in vivo remain poorly understood.Therefore,our studies expect to illustrate the relationship of Deptor in angiogenesis in vivo at first.Methods1.Using the Cre-loxp system,we have successfully created mice with vascular endothelial cell specific deletion of DEPTOR.2.Verify knock out effect of the vascular endothelial cell specific DEPTOR knockout mice.3.HE staining,immunohistochemistry and western blot methods were used to detect the number of vessels in knockout mice.Observe the changes after rapamycin treatment.4.The expression of VEGF and HIF-la were detected in KO mice.Observe the changes after rapamycin treatment.5.HUVECs was transfected into DEPTOR siRNA to observe the changes of CD31,angiogenesis factor and hypoxia inducible factor.Observe the changes after rapamycin treatment.The control group,DEPTOR siRNA group and DEPTOR siRNA+ rapamycin group were set up to observe the formation of HUVEC tubules.Statistical analysisIn this experiment,all of the data and results were expressed as the mean±SEM.Each experiment was done by three individual experiments or more.The data in each group were analyzed using t test and two-way ANOVA.When the P values<0.05 were considered statistically significant.Result1.Generation Deptor-conditional knockout mice in the ECs.Immnuofluoscent staining indicated that DEPTOR protein expression was decreased in vascular ECs of KO mice.Furthermore,western blotting analysis demonstrated that DEPTOR was dramatically decreased in Deptor KO mice tissues.2.ECs-specific deletion of Deptor activated mTOR signaling.The phosphorylation of S6 was elevated in the KO mice tissues compared with WT littermates.3.EC-specific deletion of Deptor increases the tissue angiogenesis,and rapamycin can reverse the phenotype.CD31 histochemical staining was significantly increased in KO mice tissue compared with WT mice.After treated with rapamycin to KO mice,the CD31 histochemical staining was decreased.Western blot analysis confirmed this result.4.Increased expression of VEGF and HIF-1α in Deptor KO mice,and rapamycin can reverse the phenotype.Immunohistochemical staining of VEGF and HIF-1α was significantly increased in KO mice compared with the WT mice.However,following treatment with rapamycin,there was a trend of decreasing staining of VEGF in KO+RAPA mice,as compared with KO mice.Western blot analysis confirmed this result.5.HUVECs were transfected with siRNA to knockdown DEPTOR,Knockdown of DEPTOR in ECs led to a marked increase in phosphorylation of pS6K1(T389)and pS6(S235/236).Similarly,the expression of CD31,VEGF and HIF-la were also significantly increased after DEPTOR knockdown.The cells were treated with rapamycin,the increase in pS6K,pS6 was inhibited,and the expression of CD31,VEGF and HIF-1α were markedly reduced.DEPTOR knockdown by siRNA increase the angiogenesis compared with control group in endothelial tube formation assay,whereas,treatment with rapamycin can reverse the increase.Conclusion1.Vascular endothelial cell knockout Deptor,mTOR signaling pathway to promote the formation of tissue blood vessels.2.Activation of mTOR signaling pathway in endothelial cells to promote the formation of hypoxia-inducible factor and VEGF to promote the release of angiogenic factors to promote angiogenesis,plus rapamycin can reduce the formation of hypoxia-inducible factor.