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The formation of autofluorescent granules in cultured human RPE
M Boulton1, N M McKechnie, J Breda
1Department of Clinical Ophthalmology, Institute of Ophthalmology, London, England.
Abstract:
Although the incomplete degradation of phagocytosed outer segment discs is thought to result in the formation of lipofuscin, there has to date been no proof of this concept. We report for the first time that cultured human retinal pigment epithelial (RPE) cells fed daily doses of isolated rod outer segments for periods of up to 3 months were capable of developing intracellular autofluorescent granules whose morphology and fluorescence characteristics were similar to lipofuscin. These autofluorescent granules were observed as early as 2 weeks following daily challenge with rod outer segments and the number of granules increased with the number of challenge doses until the experiment was terminated after 3 months of daily feeding. We also studied the effects of the antioxidant vitamin E and a drug, Centrophenoxine, which has been purported to slow the formation of lipofuscin formation in vivo. Neither of these additives given either with the challenge or subsequently had any effect on the formation of the intracellular granules. In conclusion, the development of these "lipofuscin-like" inclusions in cultured RPE may provide a model with which to study ageing processes and may provide an understanding of the increased lipofuscin accumulation observed in certain retinal pathologies.
Insights
Researchers cultured human retinal pigment epithelial (RPE) cells with outer segments, observing lipofuscin-like granules. This study provides the first evidence for lipofuscin formation from incomplete phagosome degradation in RPE cells.
Area of Science:
- Ophthalmology
- Cell Biology
- Biochemistry
Background:
- Lipofuscin, an age-related autofluorescent pigment, accumulates in retinal pigment epithelial (RPE) cells.
- Incomplete degradation of phagocytosed photoreceptor outer segments is hypothesized to cause lipofuscin formation.
- Direct experimental proof for this hypothesis has been lacking.
Purpose of the Study:
- To provide the first direct experimental evidence for the formation of lipofuscin in human RPE cells.
- To investigate the effect of antioxidants and specific drugs on lipofuscin formation in vitro.
- To establish a cell culture model for studying RPE aging and related pathologies.
Main Methods:
- Cultured human RPE cells were fed daily doses of isolated rod outer segments for up to 3 months.
- Intracellular autofluorescent granules were analyzed for morphology and fluorescence characteristics.
- The effects of Vitamin E and Centrophenoxine on granule formation were assessed.
Main Results:
- Cultured RPE cells developed intracellular autofluorescent granules resembling lipofuscin after 2 weeks of daily outer segment feeding.
- Granule accumulation increased with the duration and dose of outer segment challenge.
- Neither Vitamin E nor Centrophenoxine affected the formation of these lipofuscin-like granules.
Conclusions:
- The study provides the first direct evidence that incomplete degradation of rod outer segments leads to lipofuscin-like inclusions in RPE cells.
- Cultured RPE cells offer a viable model for studying RPE aging and lipofuscin accumulation.
- This model may aid in understanding retinal pathologies associated with increased lipofuscin.