Role of autophagy in angiogenesis of retinal vascular endothelial cells under hypoxia condition

Objective

To study the role of autophagy in proliferation, migration and tube formation of retinal vascular endothelial cells (RVECs) under the condition of hypoxia in vitro.

Methods

Mouse RVECs were isolated from 50 C57BL/6J mice primarily cultured using explant culture method, and the cells were identified by immunofluorescence of CD34.Well cultured RVECs were divided into normal control group, hypoxia control group and hypoxia+ 3-methyladenine (3-MA) group.The cells in the normal control group were cultured in the normal environment for 24 hours, and the cells in the hypoxia control group were cultured 1% O₂ environment for 24 hours, and the cells in the hypoxia+ 3-MA group were pretreated with 5 mmol/L 3-MA for 4 hours and then exposed to 1% O₂ environment for 24 hours.Microtubule-related protein 1 light chain 3 (LC3-Ⅱ/LC3-Ⅰ) and Beclin-1 protein contents in the cells were detected by Western blot analysis; the ultrastructure of autophagosome was examined under the transmission electron microscope.The proliferation, migration and tube formation of the cells were detected by Click-iTEdU kit, scratch assay and matrigel, respectively.

Results

Primarily culture cells grew well with the cobblestone-like appearance 5-7 days after culture and showed positive response for CD34.The autophagosome number was increased in the hypoxia control group compared with the hypoxia+ 3-MA group and normal control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.243±0.030, 0.658±0.032 and 0.405±0.095; the relative expression of Beclin-1 protein in the cells was 0.260±0.040, 0.650±0.071 and 0.461±0.089; the proliferation rate of the cells was (45.93±6.39)%, (22.74±2.35)% and (24.12±3.59)%; the scratch healing rate of the cells was (36.02±5.84)%, (57.26±11.98)% and (18.16±9.73)%; the number of tube formation was 29.20±6.10, 41.40±4.04 and 22.00±2.92 in the normal control group, hypoxia control group and hypoxia+ 3-MA group, respectively, with significant differences among the groups (F=35.86, 23.53, 34.28, 21.12, 23.27; all at P<0.01). Compared with the normal control group, the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein, scratch healing rate and the number of tube formation were significantly increased, and the proliferation rate was significantly reduced in the hypoxia control group (all at P<0.05). Compared with the hypoxia control group, the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein, scratch healing rate and the number of tube formation were significantly decreased in the hypoxia+ 3-MA group.

Conclusions

Hypoxia environment activates autophagy of mouse RVECs, which enhances the migration and tube formation abilities of the cells.3-MA inhibits the angiogenesis abilities of mouse RVECs.