Photodamage to human RPE cells by A2-E, a retinoid component of lipofuscin

Invest Ophthalmol Vis Sci. 2000 Jul;41(8):2303-8.

Abstract

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acridine Orange
  • Cell Survival / radiation effects
  • Cells, Cultured
  • Flow Cytometry
  • Fluorescent Dyes
  • Humans
  • Light
  • Lipofuscin / chemistry
  • Lipofuscin / physiology*
  • Lipofuscin / radiation effects
  • Lysosomes / radiation effects
  • Microscopy, Fluorescence
  • Pigment Epithelium of Eye / cytology
  • Pigment Epithelium of Eye / metabolism
  • Pigment Epithelium of Eye / radiation effects*
  • Retinal Pigments / physiology*
  • Retinal Pigments / radiation effects*
  • Retinoids / metabolism*
  • Retinoids / radiation effects*

Substances

  • A2-E (N-retinylidene-N-retinylethanolamine)
  • Fluorescent Dyes
  • Lipofuscin
  • Retinal Pigments
  • Retinoids
  • Acridine Orange