Role of Autophagy in Hypoxia-Induced Angiogenesis of RF/6A Cells in vitro

Abstract

ABSTRACTPurpose: Autophagy plays a role in the pathogenesis of tumor angiogenesis and cardiovascular diseases. The autophagy level in mammalian cells was found to increase in the state of hypoxia or ischemia reperfusion. However, the role of autophagy in ocular angiogenesis has not yet been elucidated. The aim of this study was to investigate the effects of autophagy on hypoxia-induced choroidal and retinal angiogenesis in vitro using a rhesus macaque choroid-retinal endothelial (RF/6A) cell line.Materials and Methods: RF/6A cells were cultured and randomly divided into three groups according to the different culture media: control, hypoxia model[adding 125μMcobalt chloride (CoCl2) to the culture medium], and hypoxia with an autophagy inhibition group [pretreatment with 5 mmol/L 3-methyladenine (3-MA) for 1.5 h and then adding125μmol/LCoCl2 to the culture medium]. The impact of 3-MA’s effect on the level of autophagy proteins (Beclin-1 and LC3) was tested by Western blot analysis. The cell proliferation was assessed using the chromogenicmethylthiazol tetrazolium bromide (MTT) dye after 24 and 48 hours. Cell migration was investigated by wound assay. The tube formation was measured on Matrigel.Results: Under chemical hypoxia conditions, Beclin-1 and LC3 levels increased and this change can be inhibited by 3-MA. Cell viability was decreased in cells treated with CoCl2 for 24 and 48 h compared with the control, and pretreatment with 3-MA slightly promoted CoCl2-inhibited cell proliferation. Cell migration and tube formation were increased in cells treated with CoCl2 for 24 and 48 h compared with the control. Pretreatment with 3-MA significantly inhibited CoCl2-induced cell migration and tube formation.Conclusions: Hypoxia-induced autophagy decreased the cell viability and increased the cell migration and tube formation of RF/6A cells. 3-MA can inhibit hypoxia-induced angiogenesis of RF/6A cells in vitro. The present study suggests that autophagy plays a role in retinal and choroidal angiogenesis and the autophagy inhibitor can be a potential candidate for the treatment of choroidal or retinal neovascularization.