Abstract
Abstract: :
Purpose: Principal component analysis (PCA) is a technique used to determine global changes in gene expression in response to changing cellular conditions. We have used PCA to determine the gene expression pattern of the retinal pigment epithelium (RPE) in response to age–related changes within Bruch’s membrane (BM). Methods: Immortalized human ARPE–19 cells were seeded onto human BM (five young samples: donor age = 31– 47 yr and five older samples: donor age = 71 – 81 years) harvested from human eye bank eyes. ARPE–19 were harvested 72 hours after seeding onto human BM and total RNA was isolated using a Qiagen RNeasy Mini Kit. First and second strand cDNAs synthesis, Biotin–labeled antisense cRNA 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, SAM and Genesis 1.3 software. Results:The expression of approximately 6,000 genes (out of 12,600 genes on microarray Human 95UA chip) was detected, and we analyzed the results with a PCA of functional gene clusters. The expression profile of proliferation, apoptosis and cell migration genes was very similar among RPE samples seeded onto young BM group. Aging of human BM induced a significant spread in the gene expression profile for these 3 sets of genes. There was no apparent difference in the gene expression profile or spread of angiogenesis genes as a function of BM aging. Using Bonferroni adjusted statistic analysis we are able to generate a list of genes with altered expression levels. Conclusions:Age–related changes within BM alone induce significant spreading of the gene expression profile of proliferation, apoptosis and cell migration genes, with no change in angiogenesis genes. These observations suggest some of cellular changes that develop within the RPE as a function of age, such as occur in age–related macular degeneration, may be the result of substrate–induced alterations in the behavior of the overlying RPE.
Keywords: Bruch's membrane • aging • retinal pigment epithelium