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Understanding the Pathogenesis of Age-related Macular Degeneration Open Access

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Age related macular degeneration (AMD) is the leading cause of central vision loss in the elderly population. The disease affects the macula and it can occur in "dry" or "wet" form. The dry AMD is the most prevalent form and is characterized by accumulation of extracellular deposits named "drusen" between the retinal pigment epithelium (RPE) and Bruch's membrane while the wet AMD is less common form and is characterized with growth of new blood vessels that breach the RPE monolayer and invade the photoreceptors. AMD is a complex disease in which age, genetic variants, and environmental factors are all considered to play a role. Although aging is known as a major risk factor for AMD, it has been shown that more than 75 % of AMD patients harbor certain genetic variations. Three major genetic risk factors have been identified to date and these include variant in the complement factor H (CFH)gene, in the promoter region of the high temperature requirement protease 1 (HTRA1)gene and in the Age-related maculopathy susceptibility 2 (ARMS2) gene. However it is not clear how these apparently unrelated genes lead to the same pathological features of AMD (e.g. drusen formation and choroidal neovascularization). The goal of this dissertation is to understand the downstream consequences of these polymorphisms on RPE cells and define molecular mechanisms of AMD pathogenesis. We hypothesized that AMD risk genotypes influence homeostasis of RPE secretome leading to progressive drusen formation and macular degeneration. To test this hypothesis: (i) we established primary RPE cell cultures from human autopsy eyes of donors with and without AMD risk genotypes, (ii) compared their secretome profiles using stable isotope labeling by amino acid in cell culture (SILAC) strategy, and (iii) characterized key pathways linking the different risk genotypes to drusen formation and choroidal neovascularization. Our SILAC strategy readily facilitated high throughput screening for both detection and quantitation of RPE secreted proteins. In our first study of the RPE secretome, we found that RPE cells derived from AMD donors secrete elevated amounts of protein components found in drusen and these include complement components, amyloid, clusterin, fibronectin and TIPM-3 (Chapter 2). We also found that TNF-α, a pleiotropic cytokine, modulates secretion of specific proteins of RPE cells leading to dysregulation of the complement pathway and extracellular matrix remodeling (Chapter 3). Moreover, we found that RPE culture with HTRA1/ARMS2 risk genotype secrete elevated amounts of HTRA1 and that HTRA1 cleaves key proteins involved in regulation of the complement pathway (e.g. clusterin, vitronectin and fibromodulin) and in amyloid deposition (clusterin, alpha 2 macroglobulin and ADAM9). We propose models where alteration of RPE extracellular environment due to genetic variants and external stimuli favors amyloid accumulation and complement deposition leading to progressive drusen buildup and ocular cell degeneration.

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