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Florian Schütt, Sallyanne Davies, Jürgen Kopitz, Frank G. Holz, Michael E. Boulton; Photodamage to Human RPE Cells by A2-E, a Retinoid Component of Lipofuscin. Invest. Ophthalmol. Vis. Sci. 2000;41(8):2303-2308.
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purpose. A fluorescent component of lipofuscin, A2-E
(N-retinylidene-N-retinylethanol-amine) has been
shown to impair lysosomal function and to increase the intralysosomal
pH of human retinal pigment epithelial (RPE) cells. In addition to its
lysosomotropic properties A2-E is known to be photoreactive.
The purpose of this study was to determine the phototoxic potential of
A2-E on RPE cells.
methods. A2-E (synthesized by coupling all-trans-retinaldehyde to
ethanolamine) was complexed to low-density lipoprotein (LDL) to allow
for specific loading of the lysosomal compartment. Human RPE cell
cultures were loaded with the A2-E–LDL complex four times within 2
weeks. A2-E accumulation was confirmed by fluorescence microscopy and
flow cytometry analysis. Acridine orange staining allowed assessment of
lysosomal integrity and intralysosomal pH. The phototoxic properties of
A2-E were determined by exposing A2-E–free and A2-E–fed RPE cell
cultures to short wavelength visible light (400–500 nm) and assessing
cell viability and lysosomal integrity.
results. Fluorescence microscopy and flow cytometry analysis demonstrated that
the intralysosomal accumulation of A2-E in cultured RPE cells increased
with the number of feedings. Acridine orange staining confirmed that
the A2-E was located in the lysosomal compartment and induced an
elevation of intralysosomal pH. Exposure of A2-E–fed cells to light
resulted in a significant loss of cell viability by 72 hours, which was
not observed in either RPE cells maintained in the dark or A2-E–free
cultures exposed to light. Toxicity was associated with a loss of
conclusions. A2-E is detrimental to RPE cell function by a variety of mechanisms:
inhibition of lysosomal degradative capacity, loss of membrane
integrity, and phototoxicity. Such mechanisms could contribute to
retinal aging as well as retinal diseases associated with excessive
lipofuscin accumulation—for example, age-related macular degeneration
and Stargardt’s disease.
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