Restrain of hypoxia on the expression of annexin A2 in mouse RGC-5 cells

Hypoxia is the main factor of retinal neovascularization and is closely associated with retinal ganglial cells (RGCs) degeneration.However, the study of retinal neural tissue lesions is rare.

This study was to investigate the influence of hypoxia environment on the expression of annexin A2 (ANXA2) in mouse RGC-5 cells and explore the mechanism of RGCs damage induced by hypoxia.

Immortalized mouse RGC-5 cells were cultured in high glucose DMEM with 10% fetal bovine serum.The cells were identified by detecting the expression of Thy-1, a specific biomarker of RGCs.CoCl₂ was added into the medium at the final concentrations of 50, 100, 200 and 300 μmol/L, and the cells without CoCl₂ served as the control group.Cell viability (absorbance) was assayed by cell counting kit-8 (CCK-8) method in 12, 24 and 48 hours after addition of CoCl₂.The hypoxic cell models were established in DMEM with 100 μmol/L CoCl₂ and divided into the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group, with the normal cultured cells as the normal control group.Apoptotic cells were determined by using hoechst 33342 stain.The expression levels of ANXA2 mRNA and protein in the cells were detected by real-time quantification PCR and Western blot, respectively.The expression and location of ANXA2 in the cells were examined by using immunofluorescence technique.

The cultured cells grew well and showed the fusiform and polygonal shape, with positive expression of Thy-1 protein.Compared with the normal control group, the viabilities of the cells were insignificantly changed in the 50 μmol/L CoCl₂ group and 100 μmol/L CoCl₂ group (all at P>0.05), but the cell viabilities were significantly reduced in the 200 μmol/L CoCl₂ group and 300 μmol/L CoCl₂ group in various time points (all at P<0.05). Hoechst 33342 staining showed that the apoptotic cells with nuclear condensation and high green fluorescence intensity were obtained in the hypoxia groups.The relative expression levels of ANXA2 mRNA were significantly lower in the hypoxic groups than those in the normal control group (all at P<0.05). The relative expression levels of ANXA2 protein were significantly lower in the hypoxia 3-, 6-, 12- and 24-hour group than those in the normal control group (all at P<0.05). Apoptotic cells were seen in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group compared with the normal control group, showing the bright blue fluorescence in cellular nucleus for hoechst 33342.The relative expressing levels of ANXA2 mRNA in the cells were 0.80±0.14, 0.67±0.33, 0.49±0.17 and 0.39±0.02 in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group, which were significantly declined in comparison with the normal control group, with a statistically difference among the groups (F=434.354, P=0.000). The relative expression values of ANXA2 protein were 0.552 6±0.012 3, 0.425 9±0.033 4, 0.344 9±0.017 8 and 0.382 7±0.022 1 in the hypoxic 3-hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group, which were remarkably lower than 0.602 1±0.001 4 in the normal control group, showing considerably difference among the groups (F=3.057, P=0.000). ANXA2 proteins were highly expressed in the cellular nucleus and less expressed in the cell membrane and cytoplasm in the normal cells.Compared with the normal control group, the ANXA2 protein showed weak expression in the hypoxia group and primarily in the cytoplasm.

The expression of ANXA2 down-regulates in hypoxic mouse RGC-5 cells, which may participate in the apoptosis process of RGCs in high glucose environment.