Purchase this article with an account.
R.C. Hunt, R. Ganti, L. Li, S.K. Parapuram, D.M. Hunt; Changes in Gene Expression in Retinal Pigment Epithelial Cells During Vitreous–Induced Transformation . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5538.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
In proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cells enter the vitreous and proliferate. They become fibroblast–like and participate in the formation of epiretinal membranes, a process that can lead to retinal detachment. Vitreous–treatment of RPE cells in vitro results in a similar morphological change. In this study we examined vitreous–induced modulation of gene expression in RPE cells.
Low passage RPE cell lines derived from three donors were treated with complete medium or complete medium containing 25% vitreous. mRNA was extracted at 6, 12, 24 or 48 hours of treatment. Labeled RNA, prepared using a linear amplification method, was hybridized to oligonucleotide microarrays (21,318 genes) and the data analysed using programs including Genespring, Significance Analysis of Microarrays (SAM) and a variety of methods to analyse the pathways involved. Changes in levels of mRNA for selected genes were confirmed by real–time quantitative PCR (QPCR). Immunoblotting and immunohistochemistry were used to confirm changes in protein levels.
SAM analysis of microarray results indicated that vitreous–treated cells underwent a progressive re–programming of their gene expression; many of these changes were consistent with a transition from an epithelial to a mesenchymal type of cell. QPCR confirmed vitreous modulation of mRNA levels for 14/14 regulated–genes in at least 3/3 additional RPE/vitreous donors with similar kinetics to those found by array analysis. Pathway analysis and experimental data indicated that TGF–beta and BMP–2 may play a role in this process.
Despite the biological variation in vitreous and RPE donors, vitreous reproducibly caused changes in expression of a limited number of mRNAs. Many of these changes are consistent with the more fibroblast–like appearance of vitreous–treated cells and with the pathobiology of PVR. TGF–beta and BMP–2 may be important modulators of RPE cell transformation.
This PDF is available to Subscribers Only