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Heuy-Ching Hetty Wang, Teresa A Burke, Ramesh Kaini, Whitney Greene; Expression of pro-fibrotic genes in an in vitro model of Proliferative Vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5966.
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© ARVO (1962-2015); The Authors (2016-present)
Proliferative vitreoretinopathy (PVR) is the result of aberrant wound healing and fibrosis following a retinal tear or detachment. Expression of genes that promote TGF-β/CTGF signaling, epithelial-mesenchymal transition, extracellular matrix remodeling and cell adhesion, inflammation, and angiogenesis is increased during fibrosis and may lead to the development of PVR. The purpose of this study is to examine the expression of 84 pro-fibrotic genes using an in vitro model of PVR.
Retinal pigment epithelium derived from induced pluripotent stem cells (iPS-RPE) was cultured on matrigel-coated transwells for 60 days until confluent and pigmented. The cell monolayers were then scratched to create a wound. To recapitulate the in vivo conditions that lead to the development of PVR, one set of cells was treated with 5% fresh vitreous immediately after wounding. Unscratched cells and fresh vitreous alone were included as controls. Cells were lysed on Days 0, 3, and 12 post wounding. Total RNA was extracted and reverse transcribed to prepare cDNA. A commercially-available human fibrosis PCR array was used to analyze expression of 84 genes associated with fibrosis. Data was analyzed to identify pro-fibrotic genes that were differentially expressed during wound healing 4-fold or greater compared to controls.
Analysis of eleven sample sets revealed a distinctive pattern of pro-fibrotic gene expression. Wounded cells had increased expression of 10 pro-fibrotic genes (Day 3), and 29 pro-fibrotic genes (Day 12) compared to control cells. The most highly expressed pro-fibrotic genes included TIMP1, TIMP3, STAT1, and COL1A2 (Day 3); and COL1A2, CTGF, TIMP1, TIMP2, TIMP3, and MMP2 (Day 12). Cells treated with vitreous immediately after wounding had increased expression of 20 pro-fibrotic genes (Day 3) and 17 pro-fibrotic genes (Day 12) compared to unscratched cells also treated with vitreous. The most highly expressed genes during wound healing with vitreous treatment included COL1A2, CTGF, and CAV1 in both Day 3 and Day 12 samples.
The results of this study reveal distinct patterns of pro-fibrotic gene expression during wound healing. The most significantly increased genes are involved in tissue remodeling, collagen production, cell proliferation, and caveolar-mediated endocytosis. These results may provide novel therapeutic targets for the treatment of this blinding disorder.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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