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P. Gupta, T. S. Hsu; Induction of "Lipofuscin-like" Pigment in Cultured Retinal Pigment Epithelial Cells: An in-vitro Model. Invest. Ophthalmol. Vis. Sci. 2009;50(13):713.
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Lipofuscin, a byproduct of cellular metabolism, is believed to accumulate due to oxidative stress and as well as due to decreased lysosomal activity in post mitotic cells. The purpose of this study is to standardize a method to induce lipofuscinogenesis with simultaneous dual effects of oxidative stress and a lysosomal enzyme inhibitor in retinal pigment epithelial cells in culture.
ARPE-19 cells were seeded on glass cover slips and grown to confluence. Oxidative stress was induced by treating cells daily with two different concentrations of hydrogen peroxide (25 µM and 50 µM) in combination with 40µM leupeptin, a thiol protease inhibitor for 7 days. Negative controls were treated with complete cell growth medium. Cells were imaged for lipofuscin analysis under confocal microscope LSM 510 with excitation at 488nm and a 520nm barrier filter on the emission side. Lipofuscin quantitation was performed on Metamorph image analysis. Mito green FM and lyso DND red dyes were used on live cells to visualize the mitochondrial and lysosomal functions. Student’s t-tests were performed to find the statistical significances between samples.
There was a slow progressive increase in the accumulation of "lipofuscin -like" pigment in the stress induced RPE cells, mimicking the normal chronic lipofuscin accumulation with age. The golden bright autofluorescent "lipofuscin-like" bodies increased by 31% (mean fluorescence per cell; P<.001) in the stress (25µM H2O2 and 40µM leupeptin) induced group when compared to the untreated group at the end of 7 days. Along with an increase in autofluorescence, alteration of the cell morphology with 50µM H2O2 and 40µM leupeptin was noted suggesting toxicity due to chronic stress treatment. A positive correlation between increased lysosomal staining and accumulated autoflourescent bodies was seen, confirming the lysosomal role in lipofuscin accumulation. RPE cells exposed to chronic H2O2 and leupeptin treatment also resulted in a 25% decrease in total mitochondrial mass.
We have demonstrated that a combination of oxidative stress (25µM H2O2) and a protease inhibitor (leupeptin 40 µM) induces chronic "lipofuscin-like" accumulation in cultured RPE cells that bear similar autofluorescent properties to normal ageing lipofuscin. This model can be used to study the pathophysiological mechanism of lipofuscin accumulation as well as help discover novel lipofuscinolytic drugs in vitro for future disease therapy.
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