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Xiaoqing Q Guo, Sriniwas Sriram, Jennifer A Tran, Audrey E K Hutcheon, James D Zieske; Potential Approach to Prevent Corneal Fibrosis. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
In previous studies, we have found that transforming growth factor beta (TGFβ) is involved in corneal wound healing and that thrombospondin-1 (TSP1) expression is regulated through different TGFβ-signaling pathways in corneal epithelial cells and fibroblasts. We also have observed that upon exposure to TGF-β1, human corneal fibroblasts (HCF) change to a myofibroblastic phenotype; however, with the addition of p38 inhibitor and TGF-β1, HCF maintained their fibroblastic morphology. Due to these observations, we studied the relationship between α-smooth muscle actin (SMA) expression and TGFβ/p38-signaling pathway.
HCF ± Trx-SARA (an inhibitor of the Smad pathway) or -GA (control) were serum starved overnight in EMEM ± 10μM p38 inhibitor (SB202190), and then grown in EMEM ± 2ng/ml TGF-β1 (T1) ± 10μM p38 inhibitor for 24 hours. After 24 hours, cells were collected and processed for indirect-immunofluorescence (IF), western blot (WB), or quantitative real-time polymerase chain reaction (qRT-PCR) to examine for SMA. In addition to SB202190, three other p38 inhibitors—Birb-796, SB239063, and SB203580—were examined for their inhibitory effect on SMA-protein expression in HCF.
SMA-protein expression increased in TGF-β1 stimulated HCF as compared with HCF control (EMEM only). This level of protein expression was maintained, even when the Smad pathway was inhibited (Trx-SARA-HCF); however, when p38 inhibitor was added, SMA-positive cells were decreased to control levels compared with TGF-β1 samples by IF. This data was confirmed by WB and qRT-PCR, which showed that both SMA protein and mRNA levels in HCF treated with p38 inhibitor was significantly decreased with TGF-β1 stimulation. All the p38 inhibitors gave similar results.
TGF-β1 regulates SMA expression through the p38 pathway in HCF. Therefore, blocking the p38 pathway may be a potential approach to prevent corneal haze after wounding.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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