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Yujing Bai, Jingjing Zhang, Yalin Wu, Xiaoxin Li; Autophagy inhibitor attenuated A2E induced inflammatory factors in ARPE-19 cells: implications for age-related macular degeneration. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2393.
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Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. A2E, a major component in lipofuscin, is deposited in the RPE with age. We aim to explore the role of autophagy in the inflammatory response induced by A2E in RPE.
(1) ARPE-19 cells were stimulated with different concentrations of A2E at different time points. Cell morphology changeswere observed. Cell viability was assessed by CCK-8 assays.<br /> (2) Transmission electron microscopy was performed to examine the autophagosomes formation treated by A2E. The p62 and LC3 protein levels and localization were analyzed by immunofluorescence staining. The expression of mTOR, p-mTOR, P62, LC3, TMEM166 were detected by Western blot. 14 different human inflammatory chemokines and cytokines in the RPE cell culture supernatants were detected simultaneously by ProcartaPlex Human 14-plex kit.<br /> (3) Autophagy specific inhibitor 3-methyladenine (3-MA) or the mTOR inhibitor rapamycin were used to observe the changes induced by A2E in RPE cells. The expression of mTOR, p-mTOR, P62, LC3, TMEM166 were detected by Western blot. The changes of inflammatory chemokines and cytokines were determined by Procarta.
(1) The cell proliferation was inhibited by A2E with dose-dependent manner.<br /> (2) Autophagosomes and LC3 were found in RPE treated with 25μmol/L A2E from 30min.<br /> (3) The expression of LC3 II, p62 and TMEM166 in RPE cells were increased, while p-mTOR was down-regulated by A2E treatment. ICAM, IL1β, IL2, IL6, IL8, IL17A, IL22, MCP-1, SDF-1 and VEGFA in the RPE cell culture supernatants were increased by A2E treatment.<br /> (4) After the treatment with 3-MA, LC3 puncta and the transformation of LC3 I to LC3II significantly decreased, and blocked the activation and degradation of p62. The expression of autophagy-associated protein also had varying degrees of decline. ICAM, IL1β, IL2, IL6, IL8, IL17A, IL22, SDF-1 and VEGFA in the RPE cell culture supernatants were increased.<br /> (5) mTOR inhibitor rapamycin atenuated RPE death by CCK-8 assay. Both the number of autophagosomes and the LC3 puncta increased. The expression of inflammatory chemokines and cytokines in the RPE cell culture supernatants had different degree of inhibition.
A2E could induce autophagy in RPE cells and increase the expression of autophagy-associated protein through the mTOR pathway.
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