SRF Exposure Up–Regulates Cytokine and Anti–Apoptotic Factor Expression in Cultured Human RPE Cells

Abstract

Abstract: : Purpose:Following a retinal detachment, retinal pigment epithelial (RPE) cells are exposed to sub–retinal fluid (SRF), a mixture of vitreous, serum proteins, and proteins produced by a variety of cells. After detachment, RPE cells can migrate, proliferate and contribute to membrane formation characteristic of proliferative vitreoretinopathy (PVR), the leading cause of surgical reattachment failure. Herein, we determined if exposure to sub–retinal fluid up–regulates survival factor and cytokine expression in RPE cells, contributing to the development of PVR. Methods:SRF was collected from patients during re–attachment surgery. Cultured human RPE cells were grown to confluency, then incubated for 8 hours in serum–free media (control), or in SRF pooled from 25 patients with detachment durations of 7 days or less. Cells were then harvested, and RNA extracted. Gene expression was determined by microarrays containing cDNAs for 96 genes associated with the human apoptosis pathway. Real–time RT–PCR was perfomed to validate microarray results using optimized cycling parameters and primers to quantitate changes in RPE cell mRNA expression. Significant up or down regulation was considered to be a two–fold or greater change in expression compared to basal (control) levels. Results:Microarray analysis showed up–regulation of 34 apoptosis–related genes, including both pro– and anti–apoptotic factors. Real–time RT–PCR confirmed significant up–regulation of several anti–apoptotic factors including MCL–1, CFLAR (c–FLIP), cIAP–1, cIAP–2, BFL–1, TRAF1 and TRAF2. BCL–2 was down–regulated after SRF exposure, and neither BCL–X nor Survivin showed any significant change from basal levels. The cytokines IL–1B, MCSF, MCP–1 and ICAM–1 were all up–regulated after SRF exposure, whereas RANTES and MGSA were not significantly affected. Conclusions: Survival factors important for apoptosis resistance were significantly up–regulated in human RPE cells after 8 hour SRF exposure. Expression of these anti–apoptotic factors may help to promote survival of migrating and proliferating RPE cells in PVR, thereby contributing to the progression of the disease. Exposure to SRF also caused human RPE cells to up–regulate cytokine expression. These cytokines could serve to attract and activate macrophages and may trigger the inflammatory response associated with PVR.

Keywords: proliferative vitreoretinopathy • retinal pigment epithelium • cytokines/chemokines