Hui Sheng1, Yi Lu, Feng-ling Qing. 1. Department of Ophthalmology, Eye & ENT Hospital of Fudan University, Shanghai 200031, China.
Abstract
OBJECTIVE: To observe the internalization of A2E by human retinal pigmented epithelial (hRPE) cells and study whether the lipofuscin fluorophore A2E (N-retinylidene-N-retinylethanolamine) participates in blue light-induced damage to hRPE cells. METHODS: A mixture of all-trans-retinal and ethanolamine was used to produce A2E in one step. A2E granules were delivered to medium of cultured hRPE cells for internalization. Confluent cultures were subsequently exposed to 450 nm (blue) light for 20 minutes with or without A2E (25, 50, 100 micromol/L). The light intensity was 70 mW/mm(2). Phototoxicity was quantified at 12, 24, 36, and 48 h after exposure by CCK-8 of viable cells. Apoptosis of cells was detected by Hoechst 33342 DNA staining and flow cytometry. RESULTS: The reaction of all-trans-retinal (100 mg) and ethanolamine (9.5 mg) produced 53.8 mg A2E in one step. When A2E was delivered to hRPE cells in culture, it accumulated intracellularly. Internalized A2E presented as autofluorescent granules having a perinuclear distribution. As shown by CCK-8 analysis, the A2E-fed hRPE cell viability reduced with duration after 450 nm light exposure. Conversely, blue light-exposed hRPE cells that did not contain A2E showed less loss of cell viability. The percentage of hRPE cell apoptosis with 25 micromol/L A2E 12, 24, 36 and 48 h after blue light exposure was (12.11 +/- 2.32)%, (31.21 +/- 3.72)%, (64.23 +/- 3.53)% and (58.71 +/- 3.48)% respectively. Conversely, the apoptosis was less than 5% in other hRPE cells. CONCLUSIONS: A2E is essential to blue light-induced hRPE cell damage. Only blue light exposure and without A2E lead to little cell injury. hRPE cells in old people which contain much lipofuscin are sensitive to blue light injury.
OBJECTIVE: To observe the internalization of A2E by humanretinal pigmented epithelial (hRPE) cells and study whether the lipofuscin fluorophore A2E (N-retinylidene-N-retinylethanolamine) participates in blue light-induced damage to hRPE cells. METHODS: A mixture of all-trans-retinal and ethanolamine was used to produce A2E in one step. A2E granules were delivered to medium of cultured hRPE cells for internalization. Confluent cultures were subsequently exposed to 450 nm (blue) light for 20 minutes with or without A2E (25, 50, 100 micromol/L). The light intensity was 70 mW/mm(2). Phototoxicity was quantified at 12, 24, 36, and 48 h after exposure by CCK-8 of viable cells. Apoptosis of cells was detected by Hoechst 33342 DNA staining and flow cytometry. RESULTS: The reaction of all-trans-retinal (100 mg) and ethanolamine (9.5 mg) produced 53.8 mg A2E in one step. When A2E was delivered to hRPE cells in culture, it accumulated intracellularly. Internalized A2E presented as autofluorescent granules having a perinuclear distribution. As shown by CCK-8 analysis, the A2E-fed hRPE cell viability reduced with duration after 450 nm light exposure. Conversely, blue light-exposed hRPE cells that did not contain A2E showed less loss of cell viability. The percentage of hRPE cell apoptosis with 25 micromol/L A2E 12, 24, 36 and 48 h after blue light exposure was (12.11 +/- 2.32)%, (31.21 +/- 3.72)%, (64.23 +/- 3.53)% and (58.71 +/- 3.48)% respectively. Conversely, the apoptosis was less than 5% in other hRPE cells. CONCLUSIONS:A2E is essential to blue light-induced hRPE cell damage. Only blue light exposure and without A2E lead to little cell injury. hRPE cells in old people which contain much lipofuscin are sensitive to blue light injury.