Anxa2 Expression In Retinal Neovascularization Process And Mechanism Of Action

Purpose:Assess the production of ANXA2 in Human umbilical vein endothelial cells (HUVEC) cultured with high glucose in vitro and in oxygen-induced retinal neovascularization in C57BL/6J mouse model. To evaluate the effect of ANXA2 on the proliferation, migration, and tube formation in HUVEC, the development of oxygen-induced retinal neovascularization, and the regulation of proangiogenic factors' expression. Study the role of ANXA2 in the process of retinal neovascularization.Methods:(1)HUVEC were cultured and classified into four groups:Mock group, SiANXA2 group (transfected with SiRNA target ANXA2), SiControl group (transfected with irrelevant SiRNA as control) (these three groups were cultured with DMEM containing 50 mmol/1 glucose),and Normal group (cultured with DMEM containing 25 mmol/1 glucose). The proliferation, migration capacity and tube fonnation of HUVEC in the four groups were estimated by MTT, migration assays, and tube formation assay. And the production of ANXA2, VEGFA, MMP2, MMP9, and TIMP2 were assessed by real-time PCR and western blot. The results of all factors between the Mock group and the Normal group were analyzed, as well as those among the SiANXA2 group, the SiControl group and the Mock group.(2) C57BL/6J mouse were classified into four groups. Normal group, Mock group, SiANXA2 group (transfected with SiRNA target ANXA2),and SiANXA2_M group. On P12, P13,P14, P15,P16, P17, P21, and P30. stretched preparation of retina after angiography was used to observe the morphology change of retinal neovascularization in Normal group and OIR Mock group, and real-time PCR was used to test the expression of ANXA2 in these days. On P17, the mRNA production of Vegfa, Mmp2, Mmp9, and Timp2 in four groups were assessed by real-time PCR. The results of all factors between four groups were analyzed by SPSS software. A value of P<0.05 was considered statistically significant. Results:(1) The expression of ANXA2 in the Mock group was significantly higher than that in Normal group. The cell proliferation, and the number of migration cells (102.3±4.9 VS 50.3±2.5) and tubes (17.3±1.5 VS 10±1.4) in the Mock group was significantly higher than that in Normal group (P<0.05), and the expression of VEGFA, MMP2, MMP9 and TIMP2 was significantly increased in the Mock group (P<0.05). The cell proliferation, and the number of migration cells (41.1±1.5 VS 102.3±4.9) and tubes (7.6±1.2 VS 17.3±1.5) was significantly decreased in the SiANXA2 group than that in the Mock group. The production of ANXA2, VEGFA, MMP2, MMP9 and TIMP2 was significantly decreased in the SiANXA2 group compared with the Mock group (P<0.05).(2) The production of ANXA2 in mouse retina was associated with the level of retinal neovascularization. The retinal neovascularization of SiANXA2 group in P17 was more health than that in Mock group and SiANXA2_M group. ANXA2 was successfully silenced by SiANXA2(P<0.05),the Vegfa, Mmp2, and Mmp9 was upregulated by overproduction of ANXA2 and downregulated when ANXA2 was silenced(P<0.05).Conclusion:ANXA2 is overexpressed in HUVEC cultured with high glucose in vitro, and in oxygen-induced retinal neovascularization in a mouse model.the overexpression of ANXA2 may affect the proliferation, migration, tube formation of endothelial cells and regulate the expression of proangiogenic factors. ANXA2 may involved in the development of the retinal neovascularization. ANXA2 maybe a new target for inhibition of retinal neovascularization.