5α-HA inhibits VEGF164-induced proliferation, migration and angiogenesis of choroidal vascular endothelial cells

Objective To investigate the role and underlying mechanism of 5α-hydroxycostic acid (5α-HA) in inhibiting choroidal neovascularization (CNV) model in vitro. Methods Rat choroidal vascular endothelial cells (CECs) were randomly grouped into blank control group, model group (30 ng/mL VEGF164), 5α-HA treatment group (30 ng/mL VEGF164+100 μmol/L 5α-HA) and inhibition group (30 ng/mL VEGF164+5 μmol/L semaxanib) (n=3). CCK-8 assay was used to detect the proliferation of CECs in each group. Cell scratch assay, Transwell assay and Matrigel tube formation assay were used to observe the proliferation, migration and tube formation of the CECs. RT-qPCR was employed to detect the mRNA expression of leakage-related factors angiopoietin 2 (Ang 2), VE-Cadherin and ZO-1. Western blotting was performed to measure the expression of p-VEGFR2, p-Tie2, p-FAK, VE-Cadherin, ZO-1 and occudin. Cell immunofluorescence assay was conducted to observe the expression of Ang 2 protein in the cells. Results CCK-8 assay showed that 5α-HA treatment inhibited the abnormal proliferation of CECs in the model group (P < 0.01). Cell scratch assay, Transwell assay and Matrigel tube formation assay displayed that 5α-HA treatment also reversed the migration and tube formation of CECs induced by VEGF164 in the model group (P < 0.01). RT-qPCR indicated that the treatment also inhibited the up-regulation of Ang 2 mRNA and the down-regulation of VE-Cadherin and ZO-1 in the model group (P < 0.01). Western blotting results showed that 5α-HA significantly reduced the protein levels of p-VEGFR2, p-Tie2 and p-FAK in the model group (P < 0.01), and up-regulated the levels of VE-Cadherin, ZO-1 and occudin (P < 0.05). The results of cell immunofluorescence showed 5α-HA reduced the expression of Ang 2 protein in the model group (P < 0.01). Conclusion 5α-HA inhibits VEGF164-induced proliferation, migration and tube formation in CECs, possibly by inhibiting the phosphorylation of VEGFR2 and Tie2 and VEGFR2/FAK signaling pathway