The Role And Mechanism Of CKAP2 In Proliferative Diabetic Retinopathy

Objective Through proliferative diabetic retinopathy(PDR)tissue samples and by the establishment of human retinal capillary endothelial cells(HRCECs)in vitro with high glucose,high glucose or/with VEGF conduction,gradually exploring the expression of CKAP2 in the PDR model and its effect on the biological behavior of HRCECs,the interaction relationship between CKAP2 and VEGF,and the interaction relationship between CKAP2 and p53,with a view to preliminarily clarifying the molecular mechanism of CKAP2 participating in PDR,which will provide the new ideas and scientific basis for PDR.Methods 1.A total of 36 blood samples were collected from patients with DM without retinopathy(DM group),non-proliferative diabetic retinopathy(NPDR group),proliferative retinopathy(PDR group),or age-,sex-,ethnically-matched healthy persons(CON group).Peripheral bold mononuclear cell(PBMC)and monocytes were isolated from blood samples by lymphocyte separation medium to be subjected to the following experiments: Through Western blot and q PCR to clarify the protein and m RNA expression of CKAP2 and p53.Ocular samples were collected from patients with PDR(PDR group)and patients with idiopathic macular epiretinal membrane(IMEM group as control)undergoing pars plana vitrectomy.Nine samples of undiluted vitreous fluid in each group were obtained immediately when performing the surgery.Three samples of proliferating membranes or preretinal membranes were collected from PDR patients or IMEM patients during the surgery,for the purpose of CKAP2 and p53 m RNA and protein detection by the techniques of q PCR and immunofluorescence.2.For HG experiment,cells were cultured in the medium with human endothelial medium plus HG(30 m M D-glucose,HG group),or human endothelial medium plus HG and VEGF(30 m M D-glucose+30 ng /ml VEGF,VEGF+HG group).Cells were cultured with normal human endothelial medium((8.3 m M D-glucose,control group),or human endothelial medium plus mannitol(8.3 m M D-glucose+21.7?m M D-mannitol,HM group),or human endothelial medium plus VEGF(8.3 m M D-glucose+30 ng /ml VEGF,VEGF group)served as controls.MTT,cell scratches and tube formation experiments were used to detect the biological behavior of cells.The protein and m RNA levels of CKAP2 were examined respectively through Western blot and q PCR.CKAP2 RNAi was added in the cells cultured with the medium plus HG or/and VEGF to block CKAP2.3.In order to block VEGF,Lucentis at concentration of 106μg/ml was added in the cells cultured with the medium plus HG or/and VEGF.The expression level of CKAP2 protein was detected by Western blot.Furthermore,the effects of blocking CKAP2 and VEGF on the biological characteristics of HRCECs were detected through MTT,cell scratches and tube formation experiments.4.CKAP2 and p53 were blocked in vitro to clarify the interaction between p53 and CKAP2,and clarify the mechanism of CKAP2 participating in PDR.Results 1.The expression of CKAP2 protein and m RNA in the PDR group of peripheral blood and vitreous were significantly higher than the other groups.Cell proliferation,migration and angiogenesis were enhanced in the cells cultured with the medium plus HG or/and VEGF.After the block of CKAP2,the proliferation,migration and angiogenesis ability of HRCECs were significantly weakened(P <0.05).2.The blocking of VEGF by Lucentis results in the downregulation of CKAP2.Blocking CKAP2 and VEGF can both cause HRCECs to decrease the ability of proliferation,migration and angiogenesis,but the cell proliferation and migration decreased more significantly after blocking CKAP2.3.The expression of p53 m RNA in the PDR group of peripheral blood and vitreous were significantly higher than the other groups.The expression of p53 protein was enhanced in the cells cultured with the medium plus HG or/and VEGF.Compared with the retinal premembrane of IMEM patients,the co-localization staining of p53 and CKAP2 showed obvious strong fluorescence co-localization on the retinal proliferation membrane of PDR patients.The blocking of p53 results in the downregulation of CKAP2 protein in VEGF or HG+ VEGF group.However,no down-regulation of p53 protein was observed after blocking CKAP2.Conclusion 1.High expression of CKAP2 may be involved in the PDR by affecting the proliferation,migration and tube formation ability of retinal vascular endothelial cells;2.CKAP2 may be incompletely regulated by VEGF ayin the process of PDR;3.CKAP2 may have a stronger ability to promote the proliferation and migration of retinal vascular endothelial cells than that of VEGF in the process of PDR;4.CKAP2 may also be regulated by p53 signaling pathway during the PDR.