Abstract
Abstract: :
Purpose: Previous studies shows that vitronectin is a major constituent of ocular drusen and vitronectin mRNA is synthesized in RPE cells. The purpose of this study is to determine if Bruch’s membrane aging alters the level of vitronectin mRNA and its receptor in the overlying RPE. Methods: DNA microarray and semi–quantitative RTPCR method were used for this study. Immortalized human ARPE–19 cells were seeded onto human Bruch’s membrane (five samples from donors age < 50 yr and five samples from donors age > 70 yr) harvested from eye bank eyes. ARPE–19 cells were harvested 72 hours after seeding onto human Bruch’s membrane and total RNA was isolated using a Qiagen RNeasy Mini Kit. First and second strand cDNAs synthesis, Biotin–labeled antisense cRNA target hybridization (on Affymetrix U95A chip), washing, staining and scanning probe arrays were done following an Affymetrix GeneChip Expression Analysis Manual. Real–time quantitative polymerase chain reaction (Roche Lightcycler) using samples generated from different experiments with different donor tissues were used to confirm and further study the genes expression patterns. Oligo primers were determined with LC Primer Design and RTPCR data were analyzed with Lightcycler3 Data Analysis software. Results: Microarray analysis demonstrates that vitronectin mRNA in RPE cells is down regulated by culturing onto older Bruch’s membrane by 1.4–2.5 fold. RTPCR of samples from four other experiments demonstrates consistent downregulation of vitronectin mRNA in RPE seeded onto older versus younger Bruch’s membrane. RTPCR studies of the mRNA expression of vitronectin receptor alphaVbeta5 also demonstrate downregulation upon culturing onto older Bruch’s membrane. Conclusions: Aging of Bruch’s membrane downregulates the expression profile of vitronectin mRNA and its receptor in human RPE. It is known that the vitronectin receptor plays an important role in phagocytosis of photoreceptor outer segments and vitronectin partially mediates RPE attachment to human Bruch’s membrane. These observations suggest some of the changes seen in age–related macular degeneration may be the result of substrate–induced alterations in the behavior of the overlying RPE.
Keywords: age–related macular degeneration • retinal degenerations: cell biology • Bruch's membrane