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B.F. Godley, R. Alsaadi, F. Liang; Increased Oxidative Stress in Human Macular RPE Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5288.
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Purpose: Although the etiology of age–related macular degeneration (AMD) is unknown, it is likely that oxidative stress–induced RPE changes play a critical role in the development and/or progression of this disease. The present study tests the hypothesis that freshly isolated RPE cells from the macular region are more oxidatively stressed than RPE from peripheral regions of elderly human donor eyes. This appears to be the first study to examine levels of reactive oxygen species (ROS) and mtDNA damage in freshly isolated RPE cells from human donor eyes. Methods: Elderly human eyes (n=16) without advanced AMD lesions were obtained within 10–40 hrs postmortum. RPE cells were freshly harvested from macular and peripheral regions, respectively. Reactive oxygen species in RPE (n=4) were detected with fluorescent microscopy using a fluorescent probe. Mitochondrial (mt) DNA damage was examined by quantitative PCR. Mitochondrial redox function was measured by MTT assay (n=4). To determine the susceptibility of macular RPE to oxidative stress, these cells were exposed to H2O2 (100 um) for 1 hr, and then tested for mtDNA damage by PCR. Results: ROS levels, as indicated by fluorescence intensity, were strikingly higher in macular RPE compared to peripheral RPE. The PCR showed significantly higher mtDNA lesions in macular RPE than the peripheral RPE (1.05±0.8 vs 0.51±0.44, p<0.01). Mitochondrial redox function significantly decreased (54.1±12.8%) in macular RPE cells relative to peripheral RPE. H2O2 exposure induced more mtDNA lesions in macular RPE (1.83±0.77) than in peripheral RPE cells (0.99±0.61, p<0.01), suggesting diminished antioxidant protection and/or DNA repair capacity. Conclusions: Our data demonstrate that freshly harvested macular RPE from elderly human donors exhibit higher baseline levels of ROS and mtDNA damage than peripheral RPE, which correlates with reduced mitochondrial function in the macular RPE. Moreover, macular RPE are more susceptible to oxidative damage. The results strongly support our hypothesis that oxidatively–mediated mtDNA damage may be an initial step in macular aging and AMD (Liang and Godley, 2003).
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