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Sriniwas Sriram, Jennifer A Tran, Xiaoqing Q Guo, Hetian Lei, Andrius Kazlauskas, James D Zieske; Identifying the role of PDGFRα in the fibrotic pathway of TGF-β3. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2355.
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© 2017 Association for Research in Vision and Ophthalmology.
The difference in function between two highly similar transforming growth factor beta (TGF-β) isoforms, TGF-β1 and -β3 (T1 and T3, respectively), still has not been clearly elucidated in the literature. The purpose of this study was to develop a 3-dimensional (3D) cell culture model that aimed to identify this difference in function in vitro. Our hypothesis was that T1 and T3 show varied effects on fibrosis by differentially activating the platelet derived growth factor receptor alpha (PDGFRα).
Human corneal fibroblasts (HCF) were infected with lentivirus containing optimized short-hairpin RNA (shRNA) sequences for PDGFRα, which permanently knocked down the PDGFRα in these cells (HCF-P). 3D constructs of HCF or HCF-P were cultured on polycarbonate inserts for 4 weeks ± 0.1ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for immunofluorescence (IF) and RT-qPCR analysis. RNA from these constructs also was analyzed with a custom mRNA array that looked at 86 key fibrosis genes.
IF and RT-qPCR results showed that when compared to HCF no growth factor control, SMA was not stimulated as much by T3 as T1; however, in HCF-P, SMA expression remarkably increased with the presence of T3. The mRNA array results showed that in HCF, T3 stimulated a majority of the fibrotic markers that T1 stimulated but to a lesser degree—ITGB6, CTGF, PDGF and SERPINA1. There also were some signaling factors that were downregulated by T3 when compared to T1—Integrin’s (ITGAV, ITGA2), MMPs (MMP3, MMP14), STATs (STAT1, 6) and SMADs (SMAD2-7). When we looked at the difference between T3 treatment in HCF and HCF-P to identify the source of T3’s fibrotic response, STAT3, SRC and ITGAV were some of the signaling growth factors that were highly upregulated in HCF-P.
The presence or absence of PDGFRα elicited contrasting fibrotic responses to the same T3 treatment. Identifying the signaling factors that make T3 cause fibrosis may help in understanding how it differs from T1, and may potentially help in developing a treatment that stimulates wounding with reduced levels of scarring.
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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