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Agnes Boltz, Andreas Pollreisz, Maria Laggner, Leopold Eckhart, Ying-Ting Chen, Ursula Schmidt-Erfurth; Autophagy is essential for Retinal Pigmented Epithelial cells’ redox response to oxidized lipid. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2405.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidized phospholipids (OxPL) are pleiotropic lipid mediators inducing proinflammatory and angiogenic cellular effects and have been shown to accumulate in retinal pigmented epithelial (RPE) cells and photoreceptors in healthy eyes with increasing levels in ageing eyes or eyes with age-related macular degeneration (AMD). The aim of this study was to determine the role of autophagy in RPE cells’ stress response to OxPL ex vivo.
The essential autophagy gene 7 (Atg7) was specifically deleted in RPE cells using tyrosinase-driven Cre-loxP system in C57/BL6 mice (Atg7ΔRPE) with Cre-negative littermate controls (Atg7f/f). Synthetic 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Dry lipids were oxidized by exposure of a thin lipid film to air until approximately 80% of the lipid was oxidized. Oxidation was monitored by thin-layer chromatography and electrospray ionization mass spectrometry. RPE-choroid explants were surgically dissected from Atg7ΔRPE and Atg7f/f mouse eyes and incubated with oxidized PAPC (OxPAPC) resuspended in RPE cell medium for six hours. RPE cells were isolated by dispase-assisted microdissection for cytospin and gene expression assay.
The purity of isolated RPE in cytospin samples was validated by immunostaining showing approx. 96% of RPE65 immunoreactivity. RT-qPCR revealed 85% decrease of Atg7 gene transcription in Atg7ΔRPE vs. Atg7f/f (p<0.001). An array of downstream genes of redox master regulator Nrf2 was studied in OxPAPC treated Atg7ΔRPE and Atg7f/f RPE including Gclc, GST-1, HO-1, and Nqo-1. Comparison of autophagy sufficient and deficient RPE cells revealed a significant upregulation of Gclc in Atg7ΔRPE (p=0.005) suggesting an association between Nrf2-Gclc pathway and autophagy. Induction of intracellular oxidative stress by administration of OxPAPC, Nqo-1 and HO-1 were significantly upregulated in Atg7ΔRPE RPE compared to Atg7f/f (p=0.03 and 0.05, respectively) indicating a role of Nrf2-HO-1 and -Nqo-1 in maintaining cellular redox balance.
These data suggest that Atg7-dependent autophagy is implicated in oxidative stress response of RPE. In autophagy-deficient RPE cells, Nrf2/Nqo-1/HO-1 may serve as a compensatory antioxidant mechanism.
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