Roles Of Rictor In The Angiogenesis And Maturation

Objective:Ischemic diseases including cerebral ischemia,limb ischemia,myocardial ischemia have become one of the killers of human health.It has been a hot topic in the field of human health in promoting angiogenesis after ischemia and improving collateral circulation to treat ischemic disease.Vascular endothelial cells(ECs)play a critical role in the process of angiogenesis and maturation.Studies have shown that m TOR-Rictor plays an important role in the regulation of endothelial cell function,but its mechanism in the process of angiogenesis to maturation is unclear.Previous studies have shown that neovascularization in the ischemic area of mice after Rictor deletion was improved,but the arrangement of normal vascular endothelial cells was irregular.In order to verify whether Rictor plays a significant part in vascular maturation and to explore its mechanism,this paper indirectly demonstrates the mechanism of m TORC2 in vascular maturation by using m TOR blocker KU0063794 and m TORC1 blocker Rapamycin at the condition of hypoxic,And the role of m TOR-Rictor in vascular maturation was verified by sh RNA interference in vivo and in vitro.Methods:1.Mouse aortic endothelial cells(MAECs)were primary cultured and identificated by immunofluorescence staining method.i CELLigence real-time cell analysis system and CCK8 test method were used to examine the effects of KU0063794 and Rapamycin on the survival rate of MAECs.2.Cell invasion assay,cell migration assay and cell tube formation assay were used to examine the effect of m TOR-Rictor on the invasion,migration and loop formation of MAECs.3.Under the condition of hypoxia,VEGF and ANG1 in the supernatant of culture medium added KU0063794 and Rapamycin was detected by ELISA method.4.Under the condition of hypoxia,Western Blotting method was used to investigate the expression of Akt,p-Akt,e NOs,p-e NOs,Rictor,CX43,PDGF,TGF?1 and Smad3 protein in MAECs after treated by KU0063794 and Rapamycin.5.sh RNA down-regulated the expression of Rictor in MAECs.Western blotting was used to detected the expression of CX43,PDGF,TGF?1 and Smad3 protein.3The changes of Rictor,CX43 and TGF?1 beta 1 in gene level were detected by QPCR.And ANG1 was detected by ELISA method.6.Established the model of lower limb ischemia.Rictor was interferenced in vivo,after 7 days,western blotting method was used to examine the Rictorprotein expression in gastrocnemius muscle.Then immunofluorescence staining method was used to observe the level of neovascularization Results:1.Immunofluorescence was used to identify MAECs.KU0063794 and Rapamycin can Inhibit the proliferation and survival of MAECs with high concentration.2.Compared with control group,KU0063794 and Rapamycin can significantly inhibit MAECs migration,invasion and tube formation(P<0.05).While compared with KU0063794 group,the Inhibition of Rapamycin was weaker,and there were statistically significant(P<0.05).These results suggested that m TOR-Rictor can promote migration,invasion and loop formation of endothelial cells.3.Under the condition of hypoxic.The release of VEGF and ANG1 in KU0063794 group was significantly decreased than control group(P<0.05).While in Rapamycin group,the release of VEGF decreased significantly(P<0.05),but ANG1 not changed.These results suggested that m TOR-Rictor can promote the release of VEGF and ANG1.4.Under the condition of hypoxic,the expression of p-Akt,p-e NOs,TGF?1,Smad3,CX43 and PDGF proteins were significantly decreased in KU0063794 groupcompared with Rapamycin group(P<0.01).These results suggested that m TOR-Rictor can promote the expression of p-Akt,p-e NOs,TGF?1,Smad3,CX43 and PDGF proteins.5.Down-regulation of Rictor expression by sh RNA interference in MAECs was relatively successful,Rictor knockdown can significantly decreased the expression of TGF?1,CX43,PDGF and Smad3 proteins(P<0.05).After detection of sh RNA interference by QPCR,the gene levels of Rictor,CX43 and TGF?1 beta 1 were down regulated.The release of ANG1 was decreased.The results of the experiment were further verified the outcomes with blocking agents.6.The model of lower limb ischemia in mouse was established.After Rictor knockdown,the protein expression of Rictor was decreased.Immunofluorescence staining showed that the number of new vessels was increased,while the MAECs arranged disorderly.These results suggested that Rictor can promote the stability of blood vessels.Conclusion:In low-oxygen environment,m TORC2 can increase the release of VEGF and other angiogenic-related factors to promoted the migration,invasion and angiogenesis of vascular endothelial cells,and increased the release of ANG1 to promoted the stability of new vessels.m TOR-Rictor regulated the angiogenesis and stabilization through PI3K-Akt-e NOS,PDGFR-PI3K-Akt,and TGF?1-ALK-Smad cell signaling pathway.