Rap1GAP Overexpression Inhibits Proliferation And Migration Of Endothelial Cellsvia ERK And Akt Pathways

Background&Objective:Small GTPase Rap1 has been extensively studied in vitro and shown to regulate multiple basic cellular processes. Until recently, the best studied aspect of Rapl function in endothelial cells involved its role in regulation of cell-cell junction formation and remodeling. Several recent reports provide emerging evidence that Rapl function in endothelial cells is not limited to promoting barrier but that it also regulates basic endothelial responses to angiogenic stimulation and that Rapl may act as a positive regulator of angiogenesis in vivo.Rap1GAP, a Rapl GTPase-activating protein, inhibits the RAS superfamily protein Rapl by facilitating hydrolysis of GTP to GDP.Some researcher found Rap1GAP could block Rapl activation and prevent angiogenesis.But others demonstrated Rap1GAP could not inhibit angiogenesis.So,more works are needed to illustrate the role of Rap1GAP in angiogenesis.To reveal the role of Rap1GAP in angiogenesis will further the understanding of the mechanism of angiogenesis and provide more specific, novel therapeutic targets for diseases where either inhibition of angiogenesis (i.e. tumors) or induction of angiogenesis.Angiogenesis, sprouting of new capillaries from existing vascular beds,,which is complex and involves several discrete steps. Many growth factors have been shown to regulate angiogenesis,including the activation of PI3K/Akt and ERK signaling pathway.We hypothesized that Rap1GAP may exert affects angiogenesis via ERK and AKT pathways.Therefore,the purpose of this study was to determine the precise mechanisms Rap1GAP inhibition of endothelial processes related to angiogenesisMethods and results:Primary HUVECs were isolated from fresh human umbilical veins and cultured,the HUVECs were indentified by eletroscope observation and Factor VIII immuocytochemical staining.HUVECs proliferation assay was evaluated using BrdU-ELISA method,Cell migration assay was examined by transwelland tube formation test was performed on Matrigel when human umbilical vein endothelial cells (HUVECs) were transfected with pcDNA3.1,pcDNA-Rap1GAP-Flagged and Myc-tagged-Rap1N17.we demonstrate a significant reduction of proliferation, migration and tube formation compared with empty vector-transfected controls.These changes were coincident with increased Rap1GAP expression and decreased activated Rap1, ERK and Akt phosphorylation. These results were confirmed in dominant negative Rap1N1transfected HUVECs with decreased Rapl activity. We then treated Rap1GAP with 8CPT-2'OMe-cAMP,the Possible signaling Pathways include ERK and PI3K/AKT were detected by Westernblot analysis.Pretreatment with PD98059 or LY294002 prior to stimulation not only blocked 8CPT-2'OMe-cAMP-induced ERK and Akt phosphorylation, but also significantly reduced cell proliferation and migration, implicating ERK and Akt pathways in these processes.Finally, we examined the effect of vascular endothelial growth factor (VEGF) on Rap1GAP overexpressing HUVECs. VEGF-stimulated Rapl activity, ERK and Akt phosphorylation, cyclin D1 expression and cell proliferation were repressed in Rap1GAP overexpressing cells compared to vector-transfected controls.Conclusions:Taken together, our findings demonstrate that Rap1GAP/Rapl and their downstream effectors regulate endothelial cell proliferation and migration via ERK and AKT pathways.