Abstract
Purpose :
Age-related changes of melanosomes in the human RPE reduce melanin content of the pigment granules, increase their photochemical reactivity and decrease their antioxidant properties. Here we asked whether photic stress, mediated by melanosomes (MS) or melanolipofuscin granules (MLFG) isolated from donors of different age, induces measurable phototoxicity, affects specific phagocytosis of ARPE-19 cells and causes peroxidation of cellular proteins. We also examined the modulatory effect of selected antioxidants on photoreactivity and phototoxicity of the pigment granules.
Methods :
ARPE-19 cells pre-loaded with MS or MLFG, isolated from human RPEs from donors of different age or MS and MLFG enriched with a combination of antioxidants (MS-A and MLFG-A) were irradiated with blue light for selected time intervals and the cell survival was determined by MTT assay. Phagocytosis of FITC-labeled photoreceptor outer segments (POS) isolated from bovine retinas was analyzed by flow cytometry 5.5 hr after cell irradiation. Cells were also analyzed for the presence of protein hydroperoxides using the fluorogenic coumarin boronic acid (CBA) indicator.
Results :
Irradiation or ARPE-19 cells containing phagocytized MS or MLFG with sub-threshold doses of blue light induced significant inhibition of the specific phagocytosis of POS with the effect being substantially stronger for granules obtained from older donors (age:50-59) compared to younger donors (age:18-29). Photic stress mediated by the MS or MLFG induced peroxidation of cellular proteins with the effect being more pronounced for granules isolated from older donors. Enrichment of MS and MLFG with antioxidants reduced the inhibitory effect of MS or MLFG mediated stress on POS phagocytosis and decreased photoperoxidation of cellular proteins.
Conclusions :
Our study has demonstrated for the first time that one of the key functions of the RPE, its specific phagocytosis, can be impaired by photic stress mediated by the aging pigment granules and that the effect can partially be reversed by natural antioxidants. The observed inhibition of the phagocytic activity of ARPE-19 cell is accompanied by oxidation of cellular proteins, which is also reduced by the antioxidants.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.