Differential autophagic effects of vital dyes in retinal pigment epithelial ARPE-19 and photoreceptor 661W cells

Abstract

Indocyanine green (ICG) and brilliant blue G (BBG) are commonly used vital dyes to remove internal limiting membrane (ILM) in vitreoretinal surgery. The vital dyes have shown cytotoxic effects in ocular cells. Autophagy is a stress responsive pathway for either protecting cells or promoting cell death. However, the role of autophagy in ocular cells in response to the vital dyes remains unknown. In this study, we found that ICG and BBG reduced cell viability in both human retinal pigment epithelial ARPE-19 and mouse photoreceptor 661W cells. ICG and BBG induced lipidated GFP-LC3-II and LC3-II in ARPE-19 and 661W cells. Combination treatment with the autophagy inhibitor chloroquine indicated that ICG and BBG reduced autophagic flux in ARPE-19 cells, whereas the vital dyes induced autophagic flux in 661W cells. Moreover, genetic and pharmacological ablation of autophagy enhanced vital dyes-induced cytotoxicity in ocular cells. Dietary supplements, including resveratrol, lutein, and CoQ10, induced autophagy and diminished the cytotoxic effects of ICG and BBG in ocular cells. These results suggest that autophagy may protect ARPE-19 and 661W cells from vital dyes-induced damage.

Fig 1. The effects of vital dyes on cell viability in ocular cells.

(A and B) Human RPE cells ARPE-19 or (C and D) mouse photoreceptor cell 661W cells were treated with various concentration of ICG or BBG for 5 or 30 mins. The cells were then rinsed with PBS to remove the vital dyes and recover for 24 h. The cell viability of recovered cells was measured with Cell-titer Glo. The data were analyzed with Prism 5, and the results are shown as the means ± SEM from three independent experiments.

Fig 2. The effects of vital dyes on GFP-LC3 puncta and retention in treated-ARPE19 and 661W cells.

ARPE19 and 661W cells harboring GFP-LC3 expression plasmid were treated with ICG or BBG (0.05 mg/ml) for 30 mins (A and B). Bar: 20 μm. The treated cells were recovered for 6 h and fixed to examine the GFP-LC3 puncta with fluorescence microscopy. ARPE19 (C and D) and 661W (E and F) cells expressing GFP-LC3 were treated with ICG or BBG (0.05 mg/ml) for 30 mins. The treated cells were recovered for 6 hr or 24 hr and fixed to examine the GFP-LC3 fluorescence intensity with flow cytometry. The quantitative results are shown as the means ± SEM from three independent experiments.

Fig 3. The effects of ICG and BBG on autophagy markers in ocular cells.

Human RPE ARPE-19 cells were treated with (A and B) ICG (0.05 mg/ml) or (C and D) BBG (0.05 mg/ml) for 30 mins and then recovered with fresh media for different time period as indicated. The cells were harvested to determine the protein levels of autophagy markers SQSTM1 and LC3 with immunoblotting. Mouse photoreceptor cells 661W were treated with (E) ICG (0.05 mg/ml) or (F) BBG (0.05 mg/ml) for 30 mins and then recovered with fresh media for different time period as indicated. The cells were harvested to determine the protein levels of autophagy markers LC3 with immunoblotting. The quantitative results are shown as the means ± SEM from three independent experiments.

Fig 4. The effects of vital dyes on autophagic flux in ocular cells.

Human RPE ARPE-19 cells were treated with ICG or BBG (0.05 mg/ml) for 30 mins in the presence or absence of autophagy inhibitor chloroquine (CQ, 20 μM) to determine the protein levels of autophagy markers with immunoblotting. The quantitative results for protein level of (B) SQSTM1、LC3-II/LC3-I conversion and (C) autophagic flux in treated cells were shown. (D) Photoreceptor cells 661W were treated with ICG or BBG (0.05 mg/ml) for 30 mins in the presence or absence of autophagy inhibitor chloroquine (CQ, 20 μM) to determine the protein levels of autophagy markers with immunoblotting. The quantitative results for LC3-II/LC3-I conversion and autophagic flux (E) in treated cells were shown. The quantitative results are shown as the means ± SEM from three independent experiments.

Fig 5. The effects of autophagy inhibition on cytotoxicity in ocular cells treated with vital dyes.

(A) ARPE19 and (B) 661W cells were treated with autophagy inhibitor chloroquine (CQ, 20 μM) for 1 h. The cells were treated with ICG or BBG (0.05 mg/ml) for 30 mins and then recovered with CQ for 24 h to determine cell viability with CellTiter Glo. (C) ARPE-19 cells were infected with shRNA against ATG5 or ATG7 or (E) transfected with siRNA against ATG5, ATG7 or BECN1. The knockdown efficiency was verified with immunoblotting. (D and F) The knockdowned cells were exposed to ICG or BBG as panel A to evaluate cytotoxicity. The quantitative results are shown as the means ± SEM from three independent experiments.

Fig 6. The effects of ocular supplements on autophagy and cell viability in vital dye-treated ocular cells.

(A)Human RPE ARPE-19 cells were treated with resveratrol (RSV, 10 μM), lutein (10 μM) or CoQ10 (10 μM) for 4 h in the presence or absence of autophagy inhibitor CQ (20 μM) to determine the protein level of LC3-II protein level with immunoblotting. (B) The net LC3-II between cells with and without CQ was used to quantitate LC3 flux. (C) ARPE-19 cells were exposed to ICG (0.05 mg/ml) or BBG (0.05 mg/ml) for 30 mins in the presence or absence of ocular supplements. The ICG and BBG were then removed from cells to recover for 24 h and examine the cell viability with CellTiter Glo. (D) Photoreceptor cells 661W were treated with resveratrol (RSV, 10 μM), lutein (10 μM) or CoQ10 (10 μM) for 4 h in the presence or absence of CQ (20 μM) to determine the protein levels of LC3-II and (E) quantitate autophagic flux. (F) 661W cells treated as panel C were used to examine the cell viability with CellTiter Glo. The quantitative results are shown as the means ± SEM from three independent experiments.