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H. Cai, L. Geng, T.H. Tezel, L.V. Del Priore; Aging of Bruch’s Membrane (BM) Upregulates Human Retinal Pigment Epithelium (RPE) Apoptosis Genes: Implications for Age-related Macular Degeneration . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4225.
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Purpose: We have determined the effects of age-related changes within BM on RPE gene expression profile. Methods: First passage human RPE (51year old donor) cells were seeded onto human BM (ages: 48, 51, 68 and 80). RPE cells were harvested 72 hours after seeding and total RNA was isolated using a Qiagen RNeasy Mini Kit. First and second strand cDNAs were synthesized with a T7- (dT)24 oligomer for priming and double-stranded cDNA was cleaned with Phase Lock Gels-Phenol/Chloroform extraction and ethanol precipitation. Biotin-labeled antisense cRNA was produced by an in vitro transcription reaction (ENZO BioArray High Yield RNA Transcript Labeling Kit) and incubated with fragmentation buffer (Tris-acetate, KOAc and MgOAcat; 94oC for 35 minutes). Target hybridization, washing, staining and scanning probe arrays were done following an Affymetrix GeneChip Expression Analysis Manual. RPE gene expression profile was analyzed with Affymetrix Miroarray Suite 5.0, GeneCluster and Genesprint software. LightCycler system (Roche Diagnostics Corp.) were used for real time quantitative RT-PCR. Results: The expression of approximately 6,000 genes (out of 12,600 genes on microarray Human 95UA chip) was detected. 74 genes were upregulated and 50 genes were downregulated with increasing age of BM. Aging of BM resulted in up regulation of several apoptosis genes and genes involved in inhibition of cell proliferation, and down regulation of genes related to cell motility and inhibition of apoptosis. A VEGF receptor (Flt-1) was upregulated in RPE seeded on older BM, but there was no change in VEGF mRNA expression. Selected data are being confirmed with real-time quantitative RT-PCR. Conclusions: Aging within BM alters the gene expression profile within RPE attached to this surface, and age-related changes within BM alone may initiate senescence and apoptosis and thus lead to death of overlying RPE. These results suggest that age-related macular degeneration, including the development of geographic atrophy, may be the result of substrate-induced alterations in the behavior of the overlying RPE.
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