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R.C. Hunt, O. Olasimbo, R. Ganti, D.M. Hunt; Comparison of the Effects of Vitreous and Collagen on mRNA Expression by RPE Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5367.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In proliferative vitreoretinopathy (PVR), retinal pigment epithelial (RPE) cells enter and proliferate in the vitreous, and participate in the formation of epiretinal membranes. They undergo morphological changes and become more fibroblast–like. Treatment of cultured RPE cells with vitreous or collagen results in similar morphological changes. We examined whether these two treatments caused similar changes in gene expression by RPE cells. Methods: Oligonucleotide microarrays (21,318 human genes) were used to detect changes in mRNA expression in RPE cells from three donors treated with medium or medium containing 25% vitreous or 100ug collagen/ml. mRNA was extracted after 6, 12, 24 or 48 hours of treatment. Data were analyzed using GeneSpring (Silicon Genetics), Significance Analysis of Microarrays (SAM) (Tusher et al., Proc. Natl. Acad. Sci. U S A. 2001 98:5116–5121) and other programs. Changes in levels of mRNA for selected genes were validated by real–time quantitative PCR using different donors from those used for the microarray experiments. Results: The mRNA levels for 200 genes were changed at least two–fold upon vitreous treatment of all three RPE donors. SAM detected significant changes in expression of 1333 genes using a false discovery rate (FDR) of 5%, and in 2335 genes using a FDR of 10%. With collagen I treatment, a similar change in morphology was observed, but mRNA expression of only 47 genes was found to alter by two fold or more. SAM analysis found no significant changes at a FDR of 5% and only 27 changes at a FDR of 10%. 10 of the 27 genes up–regulated by collagen were down–regulated by vitreous. Of the four genes up–regulated by both collagen and vitreous (SAM FDR of 10%), we were able to obtain real–time PCR data from two. For both of these genes, the mRNA levels in cells from three additional donors were up–regulated by both collagen and vitreous, with a longer period of up–regulation by vitreous, in agreement with the results from the arrays. Conclusions: Vitreous causes a reproducible two–fold or more change in approximately 1% of the 21,318 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. However, although collagen treatment causes a similar morphological change in RPE cells, it has only minimal effects on the program of mRNA expression.
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