Photodamage to human RPE cells by A2-E, a retinoid component of lipofuscin.
OBJECTIVE
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 lysosomal integrity.


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.
Schütt
F
F
Department of Ophthalmology, University of Heidelberg, Germany.
Davies
S
S
Kopitz
J
J
Holz
F G
FG
Boulton
M E
ME
D000165:Acridine Orange; D002470:Cell Survival / Q000528:radiation effects; D002478:Cells, Cultured; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D008027:Light; D008062:Lipofuscin / Q000737:chemistry / Q000502:physiology* / Q000528:radiation effects; D008247:Lysosomes / Q000528:radiation effects; D008856:Microscopy, Fluorescence; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism / Q000528:radiation effects*; D012168:Retinal Pigments / Q000502:physiology* / Q000528:radiation effects*; D012176:Retinoids / Q000378:metabolism* / Q000528:radiation effects*
C112040:A2-E (N-retinylidene-N-retinylethanolamine); D005456:Fluorescent Dyes; D008062:Lipofuscin; D012168:Retinal Pigments; D012176:Retinoids; D000165:Acridine Orange