FORMATION OF LIPOFUSCIN IN CULTURED RETINAL-PIGMENT EPITHELIAL-CELLS EXPOSED TO PRE-OXIDIZED PHOTORECEPTOR OUTER SEGMENTS

Accumulation of lipofuscin in the retinal pigment epithelium (RPE) wit h increasing age may affect essential supportive functions for the pho toreceptors. Earlier, we described a model system for the study of lip ofuscinogenesis in RPE cell cultures and showed that mild oxidative st ress enhances lipofuscin formation from phagocytized photoreceptor out er segments (POS). In the present study, bovine POS were photo-oxidize d, and turned into a lipofuscin-like material, by irradiation with UV light. Transmission electron microscopy of irradiated POS showed loss of the normal stacks of the disk membranes with conversion into an amo rphous osmiophilic electron-dense mass. The formation of thiobarbituri c acid reactive substances (TEARS), estimated during the irradiation p rocess, indicated lipid peroxidation. Irradiated POS also showed a str ong granular yellow autofluorescence. RPE cell cultures, kept at 21% a mbient oxygen, were fed daily for 3, 5 or 7 days with either (i) UV-pe roxidized POS, (ii) native POS or (iii) culture medium only. RPE cells fed irradiated POS showed significantly higher levels of lipofuscin-s pecific autofluorescence compared to cells exposed to native POS after 3 days (p = 0.0056), 5 days (p = 0.0037) and 7 days (p = 0.0020), and to the non-exposed control cells (3 days: p = 0.005, 5 days: p = 0.00 37, 7 days: p = 0.0094). The lipofuscin content of cells exposed to ir radiated POS increased significantly between days 3 and 7 (p = 0.0335) . Ultrastructural studies showed much more numerous and larger lipofus cin-like inclusions in RPE cells fed irradiated POS compared to cells exposed to native POS. In the control cells, lipofuscin-like granules were small and sparse. It appears that exposing RPE cells to previousl y peroxidized POS, thus artificially converted to lipofuscin and obvio usly not digestible by the lysosomal enzymes, accelerates the formatio n of severely lipofuscin-loaded cells. The results will be useful for further studies of possible harmful effects of lipofuscin in heavily l oaded RPE cells.