A2E modulates mRNA levels of genes expressed in human retinal pigment epithelial cells in culture

Abstract

Abstract: : Purpose: A2E, the major lipophilic flourophore in lipofuscin, accumulates in the retinal pigment epithelium (RPE) with age and has been suggested to be associated with RPE dysfunction in age–related macular degeneration. In order to gain insight into the molecular pathways by which A2E may lead to RPE damage, we studied alterations in RPE gene expression induced by A2E. Methods: 25–µM or 100–µM A2E was loaded into human RPE cells (ARPE–19). RNA was extracted 3 or 7 days following A2E loading, and gene expression profiles were obtained using a custom human retina and RPE cDNA microarray. Differential expression was determined using the Significance Analysis of Microarrays (SAM) algorithm. Results were validated by quantitative real time RT–PCR (QPCR). Apoptosis and cell death rates were compared across all groups by labeling cells with Hoechst and propidium iodide dyes. Results: Several genes showed altered mRNA levels after A2E loading. Among these, nine of ten that were also tested by QPCR showed results that were consistent with the array data. The fold–difference between the A2E loaded and control samples for these ten genes ranged between 1.3–2.6 and 1.1–1.9 for the array and QPCR experiments, respectively. The apoptosis and cell death rates were similar across all groups. Conclusions: These findings suggest that A2E accumulation in RPE cells may be associated with mRNA level alterations of several RPE expressed genes. These alterations are not limited to an acute phase following A2E loading, and are not explained by RPE cells apoptosis, but rather, may potentially contribute to prolonged RPE cell dysfunction.

Keywords: age–related macular degeneration • retinal pigment epithelium • gene/expression