The Research About The Effect Of Overexpression Of Annexin A2 Receptor On Neovascularization

Purpose:To explore the effect of overexpression of annexin A2 receptor(AX2R)on neovascularization.Methods:The PCR primers for AX2 R were designed according to the coding sequence of Gene Bank by using the primer premier 5 software.The preparation method of eukaryotic recombinant plasmid Lenti-AX2 R comprised the following steps: synthesizing the human AX2 R gene fragment by PCR;the nucleotide sequence fragment obtained from former step was connected with the eukaryotic plasmid;the cleavage sites were Xbar I and Sal I respectively;the eukaryotic recombinant plasmid was transformed into competent bacterial;coating plate,picking clones,colonies amplification and the use of alkaline lysis method to extract eukaryotic recombinant plasmid;screening correctly inserted recombinant plasmid clone by DNA sequencing.The recombinant plasmids were transfected into human embryonic kidney cells(293T).Western blotting and RT-PCR were used to detect the protein and m RNA expression levels of eukaryotic expression plasmid.Lenti-AX2 R was packaged as lentivirus to infect human retinal endothelial cells(HRECs)and umbilical vein endothelial cells(HUVECs)in order to achieve the purpose of overexpressing AX2 R gene in endothelial cells(ECs).The effects of Lenti-AX2 R on the function of HRECs and HUVECs were detected by cell proliferation assay,cell migration assay and tube formation assay.The effect of Lenti-AX2 R on neovascularization was examined by mouse aortic ring assay.The effect of overexpression of AX2 R on cell cycle of HRECs and HUVECs was detected by flow cytometry.The expression of Cyclin A1,Cyclin B1,Cyclin D1,Cyclin E1,CDK1 and p-CDC2 which were a series of cell cycle related proteins was detected by Western blotting.Fluorescence microscope was used to observe the localization of Lenti-EGFP-AX2 R fusion protein in HRECs and 293 T cells.The protein and gene expression levels of KLF2,VEGF and VEGFR2 were detected by Western blotting and RT-PCR.Results:By comparing the sequencing results of the constructed overexpressed AX2 R plasmid with BALST,it found that the sequences were consistent with the one published in Gene Bank.The results of Western blotting and RT-PCR showed that compared with blank control group and negative control group,the protein and gene expression levels of Lenti-AX2 R were significantly increasing.The numbers of cells proliferation,cells migration to the distant and tubular structures were significantly decreasing compared with control group after infection with Lenti-AX2 R lentivirus in HRECs and HUVECs for a certain period of time.The mouse aortic rings which had been infected Lenti-AX2 R lentivirus were cultured in vitro.The initial time of initiation of neovascularization in the experimental group significantly delayed.Regardless of the length and the number of the new blood vessels,or the vascular morphology and integrity,they were obvious weaker than the other two groups.Flow cytometry results were analyzed by Modfitsoftware software.The number of cells in G1 phase was invarient in the overexpression of AX2 R group,but the number of cells in S phase was increasing,in G2 phase decreasing.The expression level of cell cycle-associated proteins CDK1 and p-CDC2 was increasing and the expression level of cyclin A1 and cyclin E1 was decreasing.It found that Lenti-EGFP-AX2 R protein in HRECs and 293 T cells was agglomerated in the intracellular using fluorescence microscope.The results of Western blotting and RT-PCR showed that the protein and gene expression level of KLF2 was significantly increasing after overexpressing AX2 R gene.The expression levels of VEGF and VEGFR2 were decreasing.Conclusions : The overexpression AX2 R plasmid(Lenti-AX2R)was successfully constructed.Overexpression of AX2 R co significantly inhibit the function of HRECs and HUVECs including migration,proliferation,tube formation and inhibit the vessel sprouting of aortic rings in mice,which might inhibit cell cycle and the pathways of protein degradation of KLF2 to influence the formation of neovascularization.