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A. E. Kuriyan, A. Khulrani, G. M. Lehmann, S. E. Feldon, P. Sime, K. R. Huxlin, R. P. Phipps; Electrophilic Ppar Ligands Inhibit Corneal Fibroblast to Myofibroblast Differentiation by Modulating Pfak: A Potential Therapy for Corneal Scarring. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2499.
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© ARVO (1962-2015); The Authors (2016-present)
Corneal scarring, the second most frequent cause of blindness worldwide, is due to the differentiation of corneal fibroblasts into myofibroblasts. This is driven by TGFβ-induced Smad signaling and phosphorylation of focal adhesion kinase (FAK). We previously demonstrated that the electrophilic peroxisome proliferator-activated receptor gamma (PPARγ) ligands, cyano-3,12-dioxolean-1,9-dien-28-oic acid (CDDO) and 15-deoxy-Δ-12,14-prostaglandin J2 (15d-PGJ2), potently inhibit TGFβ-induced corneal fibroblast to myofibroblast differentiation in vitro. Here, we studied both the ability of CDDO-methyl ester (Me) and 15d-PGJ2 to inhibit myofibroblast marker mRNA levels and the mechanism of action of these agents.
Primary human corneal fibroblasts were treated with 0-5ng/ml TGFβ with or without CDDO-Me or 15d-PGJ2 in all experiments. Quantitative RT-PCR was used to assess myofibroblast marker mRNA levels after 24 hours. Western blotting for αSMA in genetically and pharmacologically manipulated cells was used to determine if the myofibroblast-inhibiting property of these agents is PPARγ dependent. Western blotting was used to assess the levels of phosphorylated Smad 2/3 (pSmad 2/3) after 15 and 30 minutes, nuclear Smad 4 after 30 minutes, and the phosphorylated FAK (pFAK)/FAK ratio after 12 and 24 hours. One-way ANOVA with post-hoc Tukey HSD test was used for statistical analysis.
TGFβ-induced αSMA and collagen III mRNA levels were inhibited by CDDO-Me (p<0.01 & p<0.01, respectively) and 15d-PGJ2 (p<0.01 & p<0.05, respectively). TGFβ alone resulted in only a 1.2-fold increase in collagen I mRNA at 24 hours. Genetic and pharmacological methods demonstrated that the myofibroblast-inhibiting property of these agents is PPARγ independent. CDDO-Me and 15d-PGJ2 did not have a sustained effect on TGFβ-induced pSmad 2/3, nuclear Smad 4, or pFAK/FAK after 12 hours. After 24 hours, TGFβ-induced pFAK/FAK was significantly decreased by CDDO-Me (p<0.05) and was non-significantly decreased by 15d-PGJ2.
CDDO-Me and 15d-PGJ2 significantly inhibit key TGFβ1-induced myofibroblast marker mRNA levels in primary human corneal fibroblasts. These agents inhibit myofibroblast differentiation in a PPARγ independent manner, likely by decreasing the pFAK/FAK ratio at 24 hours. CDDO-Me, 15d-PGJ2, and other compounds that modulate pFAK are potentially effective therapies to combat corneal scarring in humans.
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