Lipofuscin-formation in retinal pigment epithelial cells is reduced by antioxidants.

The accumulation of lipofuscin by retinal pigment epithelium may be an important feature in the pathogenesis of age-related macular degeneration, suggesting the possibility that this common cause of blindness might be prevented or delayed by antioxidants. In support of this idea, we now report significantly reduced formation of lipofuscin when the antioxidant substances lutein, zeaxanthin, lycopene (carotenoids), or alpha-tocopherol were added to rabbit and bovine (calf) retinal pigment epithelial (RPE) cells exposed to normobaric hyperoxia (40%) and photoreceptor outer segments. Rabbit and calf RPE cells were grown for 2 weeks with addition of one of the test substances every 48 h. The cellular uptake of carotenoids and alpha-tocopherol was assayed by HPLC after 2 weeks. The lipofuscin-content was measured by static fluorometry (rabbit cells) or by image analysis (calf cells). Both rabbit and calf RPE showed similar results with significantly lower amounts of lipofuscin in antioxidant-treated cells. The effect of carotenoids is especially interesting, since the result is not dependent on their protective effect against photo-oxidative reactions. The chain-breaking abilities of these antioxidants in peroxidative reactions of lipid membranes and quenching of free radicals seem to be of importance for inhibition of lipofuscin formation.

Lipofuscin-formation in cultured retinal pigment epithelial cells is related to their melanin content.

Age-related macular degeneration (AMD), the leading cause of blindness in the developed world, is accompanied by degeneration of the retinal pigment epithelial (RPE) cells. There is an inverse correlation between the melanin content of the eye and the incidence of AMD. Lipofuscin (LF)-accumulation in RPE cells accompanies the process of aging, and may also be related to AMD. This study was designed to evaluate the effect of melanin/melanosomes on the rate of LF formation in cultured rabbit and bovine RPE cells subjected to oxidative stress (40% normobaric O(2)) and daily supplementation with photoreceptor outer segments for 4 weeks. The LF content was measured at 0, 2, and 4 weeks in RPE cells from pigmented and albino rabbits, as well as in pigment-rich and pigment-poor bovine cells. Albino rabbit and pigment-poor bovine cells accumulated significantly higher amounts of LF than pigmented rabbit cells and pigment-rich bovine RPE cells after both 2 and 4 weeks of exposure. Autometallography of melanin-containing cells, without previous exposure to ammonium sulfide, showed a positive outcome, indicating either the occurrence of pre-existing iron-sulphur clusters or an extremely high intrinsic reducing capacity. These results suggest that melanin acts as an efficient antioxidant, perhaps by interacting with transition metals.

Lipofuscin accumulation in cultured retinal pigment epithelial cells reduces their phagocytic capacity.

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.