Abstract
Abstract: :
Purpose: In proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cells enter and proliferate in the vitreous, and participate in the formation of epiretinal membranes, a process that can ultimately lead to retinal detachment. In PVR, RPE cells undergo morphological changes and become more fibroblast–like. Vitreous–treatment of cultured RPE cells results in similar morphological changes. We are investigating changes in gene expression in cultured RPE cells treated with vitreous. Methods: Oligonucleotide microarrays (representing 21,322 human genes) were used to detect changes in mRNA expression in RPE cells from three donors treated with complete medium or complete medium containing 25% vitreous (isolated from three donors). Microarrays for each RPE donor were done using mRNA extracted from control or vitreous–treated cells after 6, 12, 24 or 48 hours of treatment. Cyanine–3 (control) or cyanine–5 labeled (vitreous–treated) mRNA was prepared by coupling reactive dye esters to amino–allyl mRNA prepared using the procedure of Eberwine et al. (Proc. Natl. Acad. Sci. 89:3010).Control and vitreous probes were mixed and hybridized to the microarrays. Each mRNA control/vitreous pair was also analyzed using cyanine–5 (control) and cyanine–3 (vitreous) labeled mRNAs. The data were analyzed using GeneSpring (Silicon Genetics). Changes in levels of mRNA for certain genes were confirmed by real–time quantitative PCR using different RPE and vitreous donors from those used for the microarray experiments. Results: The microarray data indicated that the mRNA for 202 genes was changed at least two–fold upon vitreous treatment of all three RPE donors. These included genes associated with adhesion, extracellular matrix (ECM) and ECM remodeling, inflammation, stress, and control of transcription. Some of the genes which showed increased mRNA expression code for transcription factors associated with the transition from an epithelial to a mesenchymal phenotype. Conclusions:Vitreous causes a reproducible two–fold or more change in approximately 1% of the 21,322 genes on the microarrays. Many of these changes are consistent with the known biology of RPE cells, their interaction with vitreous and their putative role in PVR.
Keywords: retinal pigment epithelium • proliferative vitreoretinopathy