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Shwu-Jiuan Sheu, Yi-An Chen, Chih-Wen Shu; Differential autophagic effects of vital dyes in retinal pigment epithelial and photoreceptor cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):290.
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© ARVO (1962-2015); The Authors (2016-present)
Indocyanine green (ICG) and Brilliant blue G (BBG) are commonly used vital dyes for removal of internal limiting membrane (ILM). Recent reports showed the dyes impair mitochondria and induce autophagy in ocular cells in vitro and in vivo. Autophagy is an evolutionarily conserved process, of which cells catabolize damaged proteins and organelles in lysosome dependent manner during nutrient deprivation and stress. Autophagy is highly associated with development and degeneration of eye. However, the role of autophagy in ocular cells in response to the vital dyes is completely unknown. The purpose of this study is to investigate the differential effects of vital dyes in retinal pigment epithelial and photoreceptor cells.
Human retinal pigment epithelial ARPE-19 cells and mouse photoreceptor 661W cells were treated with ICG or BBG (0.05 mg/ml) for 30 mins and recovered for 24h to examine cell viability with CellTiter-Glo® Luminescent Cell Viability Assay. The plasmid expressing GFP-LC3 was transfected into cells and treated with ICG or BBG to examine the GFP-LC3 puncta and fluorescence intensity with fluorescence microscopy and flow cytometry, respectively. Autophagic flux in the treated cells was further determined with immunoblotting using antibody against LC3.
We found that ICG and BBG reduced cell viability in both ARPE19 and 661W cells. Moreover, the conversion of LC3-1 to LC3-II and GFP-LC3 puncta were increased in the vital dyes treated-ARPE19 and 661W cells, indicating the vital dyes modulate autophagy in ocular cells. We further combine the treatment with autophagy inhibitor chloroquine (CQ) to inspect the role ICG and BBG on autophagic flux in ocular cells. Interestingly, ICG and BBG inhibited autophagic flux in ARPE-19 cells, whereas the vital dyes induced autophagic flux in 661W cells. Ablation of autophagy with inhibitor CQ or shRNA against ATG7 diminished cell viability in ARPE-19, but elevated cell viability in 661W cell, suggesting autophagy play protective and detrimental role in vital dyes treated-ARPE-19 and 661W cells, respectively.
Our results imply autophagy modulation could prevent the damage caused by vital dyes during ocular therapy.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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