Abstract
Retinal pigment epithelium (RPE) provides important metabolic support for the entire retina and is involved in biological renewal of photoreceptor outer segments. It is believed that key changes involved in the pathology of age-related macular degeneration, the major cause of blindness in people over 60, occur in the RPE. Being exposed to high oxygen tension and intense visible light from focused irradiation, human RPR is at risk of oxidative stress, which is aggravated by the accumulating age pigment lipofuscin. In this work, we examined the effect of photic stress, induced by blue light irradiation of ARPE-19 cells containing phagocytized RPE lipofuscin granules isolated from human donors of different age, on cell survival, oxidation of cellular proteins, morphology of the cells and their mechanical properties, employing standard cell culture techniques, coumarin boronic acid (CBA) assay, immunofluorescence analysis with laser scanning confocal microscopy (LSCM), and atomic force microscopy (AFM) and spectroscopy. Photic stress mediated by lipofuscin from older donors reduced survival of ARPE-19 cells more efficiently than lipofuscin from younger donors. Sub-lethal stress induced oxidation of cellular proteins in a dose-dependent manner with the effect being stronger for lipofuscin from older donors. These biochemical changes correlated with modifications of the cell morphology, which indicated substantial decrease in the formation of actin stress fibers in lipofuscin-containing cells subjected to sub-lethal photic stress. In addition, nanomechanical analysis revealed that such cells were significantly softer than control non-irradiated cells, or irradiated cells without lipofuscin. Our study demonstrates that AFM analysis can be used for sensitive detection of early changes of cultured RPE cells subjected to sub-lethal oxidative stress. Supported by Poland National Science Centre (grant Maestro 2013/08/A/NZ1/00194).
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