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F Schutt, FG Holz, M Bergmann, J Kopitz; Effect of A2-E on phagocytic capacity of human RPE-cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2560.
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Purpose: Lipofuscin (LF) accumulates in the lysosomal compartment of RPE cells with age and in association with various retinal diseases including age-related macular degeneration (ARMD). Toxic A2-E, a major retinoid component of LF, has been shown to interfere with normal RPE-cell function by various mechanisms including inhibition of catabolic lysosomal pathways and detergent effects. Herein we sought to evaluate the effect of A2-E on the phagocytic capacity of human RPE-cells. Methods: ROS were isolated from pig eyes, radiolabelled by the Iodobead method and included in the culture medium of A2-E-loaded and control human RPE cells. Phagocytosis rates were calculated from the time-dependent disappearance of TCA-insoluble radioactivity from the medium. Intracellular accumulation (storage) of phagocytosed material was determined by measurement of intracellular TCA-insoluble radioactivity. Differentiation between prelysosomal and intralysomal storage was achieved after separation of lysosomes and phagosomes by means of glycyl-phenylalanine-naphtylamide, a lysosome-disrupting cathepsin-C substrate. Proteolysis rates of the labelled ROS were quantitated by determination of intracellular and extracellular TCA-soluble radioactivity. Control experiments with RPE cells where lysosomal function is totally blocked by NH4Cl validated the outlined methodology. Results: Intracellular degradation rates of radiolabeled ROS in A2-E-treated cells were reduced to 30% of the rates in control cells and consequently striking intracellular accumulation of undegraded ROS was observed. Secondary to intracellular storage of ROS phagocytosis was reduced by more than 60% in A2-E treated cells as compared to controls. Conclusions: The results indicate that phagocytic capacity of human RPE cells is impaired by A2-E, a major fluorophore of LF. The underlying mechanism for the inhibitory effect of A2-E remains to be elucidated. We speculate that a reduced phagocytosis rate of photoreceptor outer segments would interfere with normal photoreceptor function, which may explain recent in vivo observations of impaired sensitivity of apposing photoreceptors over areas with markedly elevated fundus autofluorescence (AF) due to excessive LF accumulation when examined with cSLO-AF-imaging and microperimetry.
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