The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells.
OBJECTIVE
To determine whether the lipofuscin fluorophore A2E participates in blue light-induced damage to retinal pigmented epithelial (RPE) cells.


METHODS
Human RPE cells (ARPE-19) accumulated A2E from 10, 50, and 100 microM concentrations in media, the levels of internalized A2E ranging from less than 5 to 64 ng/10(5) cells, as assayed by quantitative high-performance liquid chromatography (HPLC). Restricted zones (0.5-mm diameter spots) of confluent cultures were subsequently exposed to 480 +/- 20-nm (blue) or 545 +/- 1-nm (green) light for 15 to 60 seconds. Phototoxicity was quantified at various periods after exposure by fluorescence staining of the nuclei of membrane-compromised cells, by TdT-dUTP terminal nick-end labeling (TUNEL) of apoptotic cells and by Annexin V labeling for phosphatidylserine exposure.


RESULTS
Nonviable cells were located in blue light- exposed zones of A2E-containing RPE cells, whereas cells situated outside the illuminated areas remained viable. As shown by fluorescence labeling of the nuclei of membrane-damaged cells and by the presence of TUNEL-positive cells, the numbers of nonviable cells increased with exposure duration and as a function of the concentration of A2E used to load the cells before illumination. The numbers of blue light-induced TUNEL-positive cells also increased in advance of the increase in labeling of membrane-compromised cells, a finding that, together with Annexin V labeling, indicates an apoptotic form of cell death. Conversely, blue light- exposed RPE cells that did not contain A2E remained viable. In addition, illumination with green light resulted in the appearance of substantially fewer nonviable cells.


CONCLUSIONS
These studies implicate A2E as an initiator of blue light-induced apoptosis of RPE cells.
Sparrow
J R
JR
Department of Ophthalmology, Columbia University, New York, New York 10032, USA. jrs88@columbia.edu
Nakanishi
K
K
Parish
C A
CA
D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000528:radiation effects*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D006801:Humans; D008027:Light; D008062:Lipofuscin; D008856:Microscopy, Fluorescence; D010857:Pigment Epithelium of Eye / Q000378:metabolism* / Q000473:pathology / Q000528:radiation effects*; D011726:Pyridinium Compounds / Q000378:metabolism*; D012168:Retinal Pigments; D012176:Retinoids / Q000378:metabolism*
D017304:Annexin A5; D008062:Lipofuscin; D011726:Pyridinium Compounds; D012168:Retinal Pigments; D012176:Retinoids; D004247:DNA