Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia
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+aut...
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doaj-555906eac5c84f52a8dfc3e235585c942020-11-25T02:03:11ZengPress of International Journal of Ophthalmology (IJO PRESS)International Journal of Ophthalmology2222-39592227-48982018-04-0111456757410.18240/ijo.2018.04.05Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxiaRong Li0Li-Zhao Wang1Jun-Hui Du2Lei Zhao3Yang Yao4Department of Ophthalmology, the First Affiliated Hospital of Xi’an Medical University, Xi’an 710077, Shaanxi Province, ChinaDepartment of Cataract, Xi’an aier eye hospital, Xi’an 710061, Shaanxi Province, ChinaDepartment of Ophthalmology, Xi’an Ninth Hospital Affiliated to Medical College of Xi’an Jiaotong University, Xi’an 710054, Shaanxi Province, ChinaDepartment of Molecular Physiology and Biophysics, Holden Comprehensive Cancer Center, University of Iowa Carver College of Medicine, Iowa City, IA 52242, USADepartment of Central laboratory, the First Affiliated Hospital of Xi’an Medical University, Xi’an 710077, Shaanxi Province, ChinaAIM: 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.http://www.ijo.cn/en_publish/2018/4/20180405.pdf574autophagyretinal microvascular endothelial cellshypoxia |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Rong Li Li-Zhao Wang Jun-Hui Du Lei Zhao Yang Yao |
spellingShingle |
Rong Li Li-Zhao Wang Jun-Hui Du Lei Zhao Yang Yao Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia International Journal of Ophthalmology 574 autophagy retinal microvascular endothelial cells hypoxia |
author_facet |
Rong Li Li-Zhao Wang Jun-Hui Du Lei Zhao Yang Yao |
author_sort |
Rong Li |
title |
Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia |
title_short |
Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia |
title_full |
Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia |
title_fullStr |
Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia |
title_full_unstemmed |
Autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia |
title_sort |
autophagy activation and the mechanism of retinal microvascular endothelial cells in hypoxia |
publisher |
Press of International Journal of Ophthalmology (IJO PRESS) |
series |
International Journal of Ophthalmology |
issn |
2222-3959 2227-4898 |
publishDate |
2018-04-01 |
description |
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. |
topic |
574 autophagy retinal microvascular endothelial cells hypoxia |
url |
http://www.ijo.cn/en_publish/2018/4/20180405.pdf |
work_keys_str_mv |
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1724948944449961984 |