Study On The Function And Mechanism Of Annexin A2 In Regulating PI3K/AKT Signaling Pathway In Retinal Neovascularization

ObjectiveTo explore the role and mechanism of AnxA2 in the formation of RNV and seek for new potential targets for the prevention and treatment of diseases related to RNV.MethodsWestern Blot,RT-PCR was used to detect whether AnxA2 was successfully interfered with and overexpressed in HRECs,and changes in phosphorylation degree of major proteins in PI3K/AKT signaling pathway in each group of HRECs were detected by Western Blot in order to explore the relationship between AnxA2 and PI3K/AKT signaling pathway in HRECs.By experiment in vitro we investigated whether AnxA2 plays a role in the formation of new blood vessel function of HRECs by regulating the PI3K/AKT signaling pathway.Lentivirus containing plasmids which can interfer AnxA2 and overexpress AnxA2 were packaged and infected HRECs,dividing HRECs into four groups: shNC group,shA2 group,lenti-EGFP group,lentiA2 group.Moreover,1?l AKT activation agent SC79 solution was added in shA2 group,1?l PI3 K inhibitor LY294002 solution was added into lentiA2 group,so that SC79 treatment group and LY294002 treatment group were built respectively.96 healthy C57BL/6J mice were equally distributed into control group,OIR group and LY294002 treatment group.Classic model of OIR mice was constructed.Mice of OIR treatment group were injected intraperitoneally with 0.05 ml PBS every day from 7th to 11 th day after birth.Mice of LY294002 treatment group were injected intraperitoneally with 0.05 ml LY294002 solution,an effective PI3 K inhibitor,every day from 7th to 11 th day after birth,while no treatment in the control group.Technique of lectin GS-IB4 fluorescence staining was used to observe RNV in mice in all groups.The expression of AnxA2 in mouse retinas was detected by Western Blot and RT-PCR to verify the role of AnxA2 in retinal neovascularization in vivo.ResultsCompared with HRECs in shNC group,we found AnxA2 expression level was much lower in cells shA2 group.Western blot showed that when AnxA2 in HRECs was interfered,the activity of PI3K/AKT signaling pathway was significantly decreased.Experiment in vitro showed that the number of HRECs cells in the replication and proliferation stage,cells in migration and the number of tube structures were also significantly reduced in shA2 group(P=0.006;P=0.001;P=0.001).When AKT activator SC79 was added,however,the activity of PI3K/AKT signaling pathway was significantly enhanced,the number of HRECs cells in the replication and proliferation stage,cells in migration and the number of tube structures were also obviously increased(P=0.014;P=0.02;P<0.001).Compared with HRECs in lenti-EGFP group,we found AnxA2 expression level was much higher in cells in lentiA2 group,Western blot showed that when AnxA2 in HRECs was overpressed,the activity of PI3K/AKT signaling pathway was significantly increased.Experiment in vitro showed that the number of HRECs cells in the replication and proliferation stage,cells in migration and the number of tube structures were also significantly increased in lentiA2 group(P<0.001;P<0.001;P<0.001).When PI3 K inhibitor activation agent LY294002 was added,however,the activity of PI3K/AKT signaling pathway significantly was reduced,the number of HRECs cells in the replication and proliferation stage,cells in migration and the number of tube structures were also obviously decreased(P=0.002;P=0.002;P=0.006).Compared with mice of normal group in this experiment,retina of mice in OIR group contained much more disorganized neovascularization and the layer of blood vessels was obscure,success ratio of model reached 87.5%.Compared with mice in the OIR group,new vascular plexus in the retina of mice in the LY294002 treatment group was significantly reduced and the severity of abnormal branches was much lower,non-perfused area was significantly diminished.We found that the expression level of AnxA2 in OIR group and LY294002 treatment group was significantly increased(P<0.001).ConclusionAnnexin A2 can promote the formation of retinal neovascularization and the promotion may be carried out through the process that AnxA2 ehhance the proliferation,migration and tubular formation of HRECs by activating the PI3K/AKT signaling pathway in HRECs.The promotion may ultimately lead to retinal neovascularization.