Abstract
Purpose :
Serum deprivation in cultured human retinal pigment epithelial (RPE) cells has been shown to result in cell responses similar to those seen in age-related macular degeneration (AMD). In this study, we investigated the impact of serum deprivation on mitochondrial respiration of cultured ARPE-19 cells. Furthermore, influence of an uncoupler under serum deprivation conditions was also examined.
Methods :
ARPE-19 cells were cultured in Dulbecco’s modified Eagle’s Medium (DMEM, 4.5 g/L glucose) with either normal (10%) or deprived (1%) fetal calf serum (FCS). Under both conditions, some cells were treated with different concentrations of the uncoupling agent carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) for 24 hours. Cell viability was assessed by cytotoxicity assay with WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate). ARPE-19 cells were pre-treated with sublethal doses of FCCP (0.5 µM and 1 µM) for 24 hours before their mitochondrial respiratory function was analyzed by Mitostress Assay with the Seahorse XFe24 Analyzer.
Results :
WST-1 assay results showed FCCP at concentrations up to 1 µM did not affect the viability of ARPE-19 cells after 24 hours of exposure under both normal and serum-deprived conditions. Serum deprivation reduced basal and maximal respiration of ARPE-19 cells by 23% and 21%, respectively, compared to normal serum conditions (p<0.01). Pre-treatment with 0.5 µM and 1 µM FCCP for 24 hours significantly reduced basal respiration of RPE cells’ under serum deprived condition, while maximum respiration and spare capacity were significantly increased after pre-treatment with 1 μM FCCP. Under normal serum condition, pre-treatment of FCCP showed no significant effect on mitochondrial respiratory functions of ARPE-19 cells.
Conclusions :
Results indicate that low doses of mitochondrial uncoupling may enhance potential mitochondrial respiratory function of RPE cells under serum deprived conditions, mimicking the phenotypes of RPE cells in AMD. This suggests that mitochondrial uncoupling of RPE cells may be a potential therapeutic target for degenerative retinal disorders, and further studies are needed to clarify this.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.