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Sriniwas Sriram, Jennifer A Tran, Xiaoqing Q Guo, Andrius Kazlauskas, James D Zieske; PDGFRα is required for the anti-fibrotic action of TGFβ3. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):719.
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In corneal wound healing, transforming growth factor beta 1 and 2 (T1 and T2, respectively) have been shown to stimulate fibrosis, while interestingly, T3 decreased scarring. The goal of this study was to study the mechanism behind this unique anti-fibrotic action of T3. It was our hypothesis that platelet derived growth factor receptor alpha (PDGFRα) played a key role in determining T3’s response to fibrosis.
PDGFRα expression was permanently knocked out of human corneal fibroblasts (HCF-P) using optimized shRNA sequences delivered by lentiviral particles. A 3D in vitro model was constructed by culturing HCF and HCF-P cells on poly-transwell membranes for 4 weeks in 10% FBS ± 0.1ng/ml T1 or T3. At the end of 4 weeks, the constructs were processed for indirect-immunofluorescence (IF) and qRT-PCR. For the ex vivo model, corneas of freshly excised rabbit globes were injured using a 6-mm biopsy punch. After which, the epithelium and stroma were removed using sterile forceps. The corneas were then excised, segregated into groups treated with T1 or T3 (1ng/ml). At the end of 2 weeks, the corneas were assessed for levels of haze using a Nikon D7000 digital camera. The media in both systems was supplemented with 0.5mM vitamin C, which allowed them to secrete a self-assembled matrix.
In HCF cells, T3 (0.86-fold) treatment resulted in significantly lower mRNA expression of smooth muscle actin (SMA) when compared to T1 (2-fold). IF data from HCF constructs confirmed that T3-treated samples had up to 95% (p<0.05) lower levels of SMA protein expression when compared to T1. Interestingly, in the HCF-P cells, both T1 (1.74 fold) and T3 (1.45 fold) treatment increased the expression of SMA to almost twice that of T3-treated HCF cells. IF data confirmed that T3-treated HCF-P cells resulted in 18-fold higher SMA expression when compared to T3-treated HCF cells. In the ex vivo model, T3 reduced scarring in the corneas when measured by both haze grading (48% scar reduction from T1-treated corneas) and image analysis (55% scar reduction from T1-treated corneas).
These results show that T3 decreases SMA expression in HCF cells, while remarkably increasing SMA expression in HCF-P cells. The presence or absence of PDGFRα elicits contrasting responses to the same T3 treatment. Understanding the role of PDGFRα in T3’s anti-fibrotic response may potentially help in stimulating corneal healing with reduced levels of scarring.
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