Abstract
Abstract: :
Purpose: The molecular composition of the substrate is known to affect RPE behavior. Since drusen are rich in vitronectin, we have used DNA microarray technology to determine the effects of vitronectin-rich substrates on gene expression of overlying RPE. Methods: First passage human RPE were seeded onto vitronectin-coated (10ng/ml) or uncoated culture dishes; in some wells RPE were preincubated with a monoclonal anti-αVß5 antibody (1 µg /ml; 60 minutes) against the vitronectin receptor. 24 hours after seeding RPE were harvested and total RNA was isolated using a Qiagen RNeasy Mini Kit. First and second strand cDNA 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 MgOAc at 94C 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 4.0, GeneCluster and Genesprint software. RPE gene expression changes specifically due to vitronectin treatment was defined as 2-fold increase/decrease in density of signals (vitronectin-treated vs controls) if the blocking antibody can reverse the change. Results: The expression of approximately 6,000 genes (out of 12,600 genes on microarray Human 95UA chip) was detected. 22 genes were upregulated and 9 genes were downregulated ≷2x in response to vitronectin and expression of each of these genes was reversed by RPE exposure to anti-αVß5 antibody. Among them, vitronectin-enrichment upregulates interferon-inducible RNA-dependent protein kinase, thus promoting RPE death; simultaneous up regulation of thymosin beta-4 suggests vitronectin may induce release of angiogenic factors that stimulate choroidal neovascularization. Conclusion: Vitronectin upregulates genes related to RPE apoptosis and induction of choroidal angiogenesis. Further work is necessary to determine the role of these changes in the development of apoptosis-dependent geographic atrophy and subfoveal neovascularization in age-related macular degeneration.
Keywords: 567 retinal pigment epithelium • 403 extracellular matrix • 417 gene/expression