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This report reviews our experimental work on cultured retinal pigment epithelial (RPE) cells, fed native or UV-irradiated photoreceptor outer segments (POS). We showed that significantly more lipofuscin (LF) was formed in cells cultured in 40% oxygen than in cells cultured in 8% oxygen, indicating an involvement of oxidative mechanisms in LF formation. The antioxidants alpha-tocopherol, lycopene, zeaxanthin and lutein significantly reduced LF formation. RPE cells high in melanin content exhibited significantly less formation of LF than cells low in or devoid of melanin, suggesting that melanin acts as an effective antioxidant. The phagocytic capacity of LF-loaded RPE cells was significantly reduced compared to that of unloaded control cells, indicating that LF-loaded RPE cells may be unable to serve the photoreceptors sufficiently regarding phagocytosis of shed outer segment tips. Blue light irradiation destabilized lysosomal membranes in LF-loaded RPE cells and significantly reduced the viability of such cells compared to unloaded, irradiated control cells. These results may be of significance in relation to the development of age-related macular degeneration (AMD).

Lipofuscin accumulates in retinal pigment epithelial (RPE) cells with increasing age in response to phagocytotic degradation of worn-out disks of photoreceptor outer segments (POS). This study investigates the ability of iris pigment epithelial (IPE) cells to ingest POS and compares the phagocytotic capacity of RPE and IPE cells by measuring their amounts of lipofuscin-specific autofluorescence.IPE and RPE cells were isolated from the same calf eyes. After growing to confluence, primary cultures from 15 eyes (69 wells) were exposed to bovine POS daily, or kept as controls. After 1, 2 and 3 weeks, respectively, the cellular amounts of lipofuscin were quantified by a computerized static cytofluorometer system (Nikon Microphot SA with a Nikon p102 photometer). Lipofuscin-specific fluorescence (550 nm barrier filter eliminating nonspecific autofluorescence) of 100 randomly selected individual cells from each well was measured. Electron microscopy revealed phagosomes (containing POS disk membranes) and lipofuscin-like inclusions in the cytoplasm of RPE and IPE cells. In both IPE and RPE cultures, the POS-challenged cells had significantly higher (P<0. 01) lipofuscin-specific fluorescence than the control cells. The fluorescence levels were higher in POS-challenged RPE cells as compared to IPE cells after 1 (P< 0.001), 2 (P<0.01) and 3 (P<0.001) weeks, respectively. The fluorescence level of POS-fed IPE cells at 3 weeks was 56% of that of RPE cells.IPE cells have the ability to phagocytose POS, resulting in an accumulation of lipofuscin intracellularly. The amount of lipofuscin was lower in IPE cells compared to RPE cells, probably reflecting the lower phagocytotic capacity of IPE., (Copyright 1999 Academic Press.)

Purpose: Retinal pigment epithelial (RPE) cells slowly accumulate lipofuscin pigment within their acidic vacuolar apparatus as a result of extra- and/or intralysosomal oxidative alterations of phagocytosed photoreceptor outer segments (POS) with consequent imperfect degradation of these structures. In old age, lipofuscin accumulation may become quite substantial. It has been suggested that pronounced accumulation of lipofuscin is related to decreased RPE function and, possibly, to age-related macular degeneration. The aim of the present investigation was to study whether heavy loading with lipofuscin of RPE acidic lysosomes would affect the further phagocytic ability of the cells., Methods: In the first section of the investigation, cultures of rabbit RPE cells were exposed daily to bovine UV-irradiated POS (artificial lipofuscin) for 4 weeks, resulting in a pronounced lipofuscin accumulation of the cells. Fluorescent latex beads (labelled with a red fluorophore) were added to unloaded control cultures at O and 4 weeks after their establishment, and to lipofuscin loaded cells after 4 weeks of feeding with artificial lipofuscin. Cellular amounts of lipofuscin, and their phagocytotic activity, were quantified by static fluorometry measuring lipofuscin-specific and red bead-specific fluorescence, respectively. The intracellular location of the beads was verified by confocal laser scanning microscopy., Results: Unloaded, and thus almost lipofuscin-free, control cells exposed to latex beads showed numerous cytoplasmic particles emitting reddish fluorescence, while few particles were taken up by cells initially loaded with artificial, POS-derived, lipofuscin. Measurement of the latex bead-specific fluorescence showed significantly (p < 0.001) higher levels in unloaded control cells than in lipofuscin-loaded ones. In the second part of the investigation, unloaded control cultures and lipofuscin-loaded cultures were exposed to native bovine Texas Red-X-labelled POS 4 weeks after the establishment of the cultures. Unloaded control cells showed a large number of cytoplasmic POS emitting reddish fluorescence, while fewer POS were phagocytosed by cells loaded with artificial lipofuscin. Measurement of the Texas Red-X-specific fluorescence, thus quantifying the phagocytic ability of the cells, showed significantly (p < 0.001) higher levels in control cells than in lipofuscin-loaded ones., Conclusions: Severe lipofuscin accumulation of RPE cells appears to result in a greatly decreased phagocytic capacity. The resulting reduction in ability to cope with the needs of the overlying photoreceptor cells, in order to eliminate the obsolete tips of their POS, may well be of significance in the development of age-related macular degeneration.

Accumulation of lipofuscin in the retinal pigment epithelium (RPE) with increasing age may affect essential supportive functions for the photoreceptors. Earlier, we described a model system for the study of lipofuscinogenesis in RPE cell cultures and showed that mild oxidative stress enhances lipofuscin formation from phagocytized photoreceptor outer segments (POS). In the present study, bovine POS were photo-oxidized, 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 amorphous osmiophilic electron-dense mass. The formation of thiobarbituric acid reactive substances (TBARS), estimated during the irradiation process, indicated lipid peroxidation. Irradiated POS also showed a strong granular yellow autofluorescence. RPE cell cultures, kept at 21% ambient oxygen, were fed daily for 3, 5 or 7 days with either (i) UV-peroxidized POS, (ii) native POS or (iii) culture medium only. RPE cells fed irradiated POS showed significantly higher levels of lipofuscin-specific 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 = 0.0037, 7 days: p = 0.0094). The lipofuscin content of cells exposed to irradiated POS increased significantly between days 3 and 7 (p = 0.0335). Ultrastructural studies showed much more numerous and larger lipofuscin-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 previously peroxidized POS, thus artificially converted to lipofuscin and obviously not digestible by the lysosomal enzymes, accelerates the formation of severely lipofuscin-loaded cells. The results will be useful for further studies of possible harmful effects of lipofuscin in heavily loaded RPE cells.

Lipofuscin accumulates in the course of time in the acidic vacuolar apparatus of retinal pigment epithelial (RPE) cells and may influence their metabolic functions. In order to study the effect of oxidative stress on lipofuscin accumulation, rabbit RPE cell cultures were kept at an ambient oxygen concentration of either 8% or 40%. To simulate the normal phagocytic function of RPE cells, bovine photoreceptor outer segments (POS) were added daily. The lipofuscin-specific autofluorescence was measured after 1, 2 and 3 weeks. RPE cells cultured under normobaric hyperoxic conditions (40% oxygen) showed significantly higher levels of lipofuscin-like autofluorescence than those kept under normobaric and probably normoxic conditions (8% oxygen) after 1 (p = 0.0050), 2 (p = 0.0001) as well as 3 (p = 0.0077) weeks. At both oxygen concentrations, the lipofuscin accumulation level was increased after 2 weeks of POS exposure (40% p = 0.0001; 8% p = 0.0037) and even further after 3 weeks (40% p = 0.0541; 8% p = 0.0377). The results suggest an involvement of oxidative mechanisms in the formation of lipofuscin from phagocytized POS by RPE cells. The autofluorescence of control cells, not exposed to POS, was significantly (40%: 1 week p = 0.0011, 2 weeks p = < 0.0001, 3 weeks p = 0.0001; 8%: 1 week p = 0.0036, 2 weeks p = 0.0063, 3 weeks p = 0.0066) lower than that of the POS-fed cells. The autofluorescence increased significantly (40% p = 0.0059; 8% p = 0.0034) between week 1 and week 3 in the control cells. This finding may reflect a contribution to lipofuscin formation by autophagocytized intracellular material. The present model seems to be useful for further studies on the mechanisms behind lipofuscinogenesis of RPE cells as well as the possible effects of lipofuscin accumulation on cell functions and viability.