Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia
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Rong Li. Department of Ophthalmology, the First Affiliated Hospital of Xi’an Medical University, Xi’an 710077, Shaanxi Province, China. rechelrong198222@163.com; Jun-Hui Du. Department of Ophthalmology, Xi’an Ninth Hospital Affiliated to Medical College of Xi’an Jiaotong University, Xi’an 710054, Shaanxi Province, China. djh79918@163.com

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Supported by the National Natural Science Foundation of China (No.81500726).

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    Abstract:

    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.

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Rong Li, Li-Zhao Wang, Jun-Hui Du, et al. Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia. Int J Ophthalmol, 2018,11(4):567-574

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Publication History
  • Received:August 04,2017
  • Revised:February 10,2018
  • Adopted:
  • Online: April 10,2018
  • Published: