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Xiaoqing Q Guo, Sriniwas Sriram, Jennifer A Tran, Audrey E K Hutcheon, James D Zieske; Potential Approach to Reverse Corneal Myofibroblast Formation. Invest. Ophthalmol. Vis. Sci. 2017;58(8):136.
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© ARVO (1962-2015); The Authors (2016-present)
In previous studies, we found that transforming growth factor beta (TGFb) was involved in corneal wound healing. We also observed that blocking the TGFb/p38-signaling pathway in human corneal fibroblasts (HCF) prevented TGF-b1-stimulated HCFs from changing to a myofibroblastic phenotype. In the current study, we examined if p38 inhibitor (p38inh) could not only reduce, but also reverse levels of αSMA, a marker of myofibroblasts, in TGF-b1-stimulated HCFs, thus converting myofibroblasts back to their fibroblastic phenotype.
HCF were treated with EMEM ± 2ng/ml TGF-β1 (T1) for 3 days. After rinsing with PBS three times, the 3 day T1-treated cells were split into two groups: 1) regular medium (RM: EMEM+10%FBS), and 2) p38inh medium (RM+10μM SB202190, a p38 inhibitor) for an additional 3 days. Media was changed everyday, and cells were harvested on days 1, 2, and 3 (D1, D2, and D3, respectively) for aSMA analysis by western blot (WB) and qRT-PCR. HCF grown in EMEM only (HCF control) or EMEM + T1 (T1 control) served as controls.
The WB results showed that αSMA protein levels in T1 control were significantly higher than HCF control (p<0.001). After splitting T1 cells into RM or p38inh, αSMA levels in both groups continued to increase at D1, compared to T1 control. On D2, the αSMA protein levels in the RM sample leveled out; however, in the p38inh sample, the αSMA levels decreased and were significantly lower than the D2 RM sample (p<0.05). By D3, αSMA protein levels in both medias decreased; however, the p38inh’s αSMA level continued to be lower than that in the RM sample, which returned to T1 control level. The qRT-PCR data showed a similar pattern as WB.
αSMA levels in both RM and p38inh samples abated with time; however, the presence of p38inh significantly accelerated this reverse. Therefore, blocking the p38 pathway may be a potential approach to reversing corneal myofibroblast formation after wounding.
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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