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Sriniwas Sriram, Xiaoqing Q Guo, Audrey E K Hutcheon, James D Zieske; FAK Signaling Regulates the Difference in T1 and T3’s Ability to Stimulate SMA. Invest. Ophthalmol. Vis. Sci. 2017;58(8):135.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we have shown that transforming growth factor beta 1 and 3 (T1 and T3, respectively) differentially effect α-smooth muscle actin (SMA) in our three-dimensional (3D) cell culture model, which secretes its own extracellular matrix (ECM) due to the addition of Vitamin C (VitC). In this study, we hypothesize that the reason for this difference in function may be due to the presence of ECM, which activates a fibrotic signaling pathway through focal adhesion kinase (FAK).
Human corneal fibroblasts (HCF) were cultured two ways—with ECM (3D model) and without (regular cell culture model). HCF in the 3D model were cultured on poly-transwell membranes for 2 weeks in culture medium (EMEM + 10% FBS + 1% Antibiotic-Antimycotic) + 0.5mM VitC. In the regular cell culture model, the HCF were grown on cell culture plates to confluence in culture medium. Both models were treated for 24 hours with ± 1ng/ml T1 or T3, or T1+ 10mM FAK inhibitor (FAKi). Samples from both experiments were processed for analysis by indirect-immunofluorescence (IF), Western blot, and qRT-PCR. All experiments were conducted in triplicate.
In the regular cell culture model, which lacked the presence of ECM, there was no difference in T1 and T3’s ability to stimulate SMA. Both T1 and T3 treatment resulted in an increase in SMA (1.5 fold in mRNA, 2 fold in protein). In the 3D model, with the self assembled ECM, T1 stimulated SMA (10 fold in mRNA) while in contrast, T3’s stimulation of SMA was significantly less than that of T1 (5 fold in mRNA). Interestingly, T1 stimulated twice the expression of FAK (4 fold in mRNA) when compared to T3 (2 fold in mRNA). In both systems, the FAKi reduced the expression of both FAK and SMA following T1 treatment. All the SMA results were confirmed by IF.
These results show that ECM plays an important role in the ability of T1 and T3 to stimulate SMA by potentially activating the FAK signaling pathway. Using FAKi to block this pathway negates the ability of T1 to stimulate SMA and can be developed as a potential treatment to reduce corneal scarring.
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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