Determining the Influence of AMD Phenotype/Genotype on the RPE Stress Response

Abstract

Purpose: Association of high risk AMD genes with diverse biological pathways suggests different mechanisms lead to AMD. Effective treatments will require incorporating both the ocular phenotype and an individual’s genetic risk factors. However, we must first understand how genotype and AMD phenotype affects specific biological pathways. The aim is to test how genotype/phenotype affects the RPE’s susceptibility to stress or responsiveness to specific drugs.

Methods: RPE cells were obtained by the Minnesota Lions Eye Bank following consent of the donor or family for medical research. Fundus images were taken of donor eyes to evaluate the stage of AMD using the Minnesota Grading System. Genotype analysis was performed for the major AMD risk alleles CFH, ARMS2, APOE, C3, LIPC, and VEGF. Cells were seeded either in polystyrene plastic flasks or fibronectin-coated transwell membranes. Primary cultures of RPE from different donors were evaluated for RPE-specific genes and proteins by RT-PCR, Western immunoblotting and immunohistochemistry. Cells were photographed to record changes in pigmentation and morphology over time. Peroxide-induced (H2O2 or TBHP) decrements in cellular ATP production, susceptibility to cell death, production of reactive oxygen species, and rescue by N-acetylcysteine (NAC) were compared for cells from donors with and without AMD.

Results: Initially, the majority of cultured cells were pigmented. However, cells lost their pigment between 2-3 weeks in culture but regained pigment once confluent. By 3 months, pigment granules were present in 94 ± 1.5% of cells. Confluent cells in long-term cultures (>1 month) have a cobblestone appearance, express ZO-1 at cell margins and the Na-K ATPase on the apical border. Bestrophin, CRALBP, and RPE65 were also present. Following peroxide exposure, a dose-dependent decrease in ATP production was observed for cells from both healthy and AMD donors. However, significantly less ATP (p=0.003) was produced at higher peroxide concentrations in RPE from AMD donors. Pre-treatment with NAC was effective in rescuing ATP production by 53± 1% and in reducing reactive oxygen species by 69± 3% following exposure to peroxide.

Conclusions: Primary cultures of donor RPE genotyped and graded for AMD severity provides a valuable pre-clinical model system for testing whether a specific AMD genotype or phenotype alters RPE cell response to stress and drugs.

Keywords: 412 age-related macular degeneration • 694 retinal culture • 634 oxidation/oxidative or free radical damage