While a number of genes have been postulated to be involved in age-related macular degeneration (AMD), the leading cause of blindness among the elderly in the US, a comprehensive mRNA phenotype of the retinal pigment epithelium (RPE) in vivo is at present unknown. Determination of an RPE mRNA phenotype could identify the genes critical to the development of AMD, identify markers of disease, and lead to new preventative and treatment strategies. Basal deposits in Bruch's membrane are an early recognizable change that occur prior to degeneration of the RPE and are a histopathological marker for AMD. Recently, our laboratory identified an age-dependent accumulation of advanced glycation end products (AGEs) in Bruch's membrane and basal deposits. AGEs are structures formed during the series of nonenzymatic reactions between sugars or other precursors, and long-lived proteins that alter the phenotype of a variety of cell types. Our laboratory has also recently shown that AGEs alter the expression of genes involved in matrix regulation, cell polarity, and apoptosis in RPE cells. Our long term goal is to define an extensive mRNA phenotype of the RPE in health and AMD which will in turn, yield insights into the pathogenesis of AMD. Specifically, we want to define the component of that phenotype which is regulated by AGEs. We hypothesize that the presence of AGEs in basal deposits is responsible for a switch in the phenotype of RPE cells that is consistent with alterations in matrix regulation, RPE cell polarity, and apoptosis. To test this hypothesis we ask 3 questions: 1. Are the mRNA phenotypes of macular RPE cells overlying basal deposits and normal Bruch's membrane different? 2. Does AGE modified matrix induce an RPE mRNA phenotype in vitro that is a subset of the phenotype expressed by RPE cells overlying basal deposits? 3. Is a subset of the AGE induced mRNA phenotype of RPE cells mediated by the Receptor for Advanced Glycation End products? To answer these questions we will use laser capture microdissection to obtain pure samples of RPE cells from tissue specimens for mRNA phenotyping by microarray analysis. We will also utilize an in vitro AGE-matrix system to determine what subset of genes is regulated by AGEs. This project hopes to establish an extensive mRNA phenotype of the RPE in both health and AMD, and define a subset of genes induced by AGEs.
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