Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia

• Main Authors: Rong Li, Li-Zhao Wang, Jun-Hui Du, Lei Zhao, Yang Yao
• Format: Article
• Language: English
• Published: Press of International Journal of Ophthalmology (IJO PRESS) 2018-04-01
• Series: International Journal of Ophthalmology
• Subjects: 574; autophagy; retinal microvascular endothelial cells; hypoxia
• Online Access: http://www.ijo.cn/en_publish/2018/4/20180405.pdf
• ISSN: 2222-3959; 2227-4898

AIM: To explore the state of autophagy and related mechanisms in the murine retinal microvascular endothelial cells (RMECs) under hypoxia stimulation. METHODS: The murine RMECs were primarily cultured and randomly divided into three groups: hypoxia group (cultured in 1% O2 environment), hypoxia+autophagy inhibition group [pretreated with 5 mmol/L 3-methyladenine (3-MA) for 4h followed by incubation in 1% O2] and control group (cultured under normoxic condition). The state of autophagy in RMECs was examined by assaying the turnover of light chain 3B (LC3BB) and expression of Beclin-1, Atg3 and Atg5 proteins with Western blotting, by detecting formation of autophagosomes with transmission electron microscopy (TEM) and by counting the number of GFP+ puncta in RMECs. The protein levels of AMPK, P-AMPK, Akt, P-Akt, m-TOR and P-mTOR were also assayed by Western blotting. RESULTS: Primary murine RMECs were successfully cultured. Under hypoxic conditions, the ratio of LC3BB-II/I and the expression of Beclin-1, Atg3 and Atg5 proteins were increased when compared with the control group. In addition, the numbers of autophagosome and the GFP+ puncta were also increased under hypoxia. However, pre-treatment with 3-MA obviously attenuated these changes in autophagy in RMECs under hypoxia. Protein expression of P-Akt and P-AMPK was increased but P-mTOR level was decreased in cells exposed to hypoxia. CONCLUSION: In murine RMECs autophagy is activated under hypoxia possibly through activation of the AMPK/mTOR signaling pathway.