143 Galunisertib Exerts Targeted Anti-Fibrotic Effects in In Vitro Models of Burn Wound Healing

Abstract

Abstract Introduction Inhibition of TGF-β has shown promising in vitro and in vivo results for reduction of hypertrophic scarring after burn injury. However, TGF-β regulates diverse cellular pathways apart from fibrosis, including physiologic wound healing, cell cycle control, and homeostasis. Galunisertib, a novel small molecular tyrosine kinase inhibitor of TGF-beta receptor type 1, specifically targets downstream pro-fibrotic pathways of TGF-β signaling, has an excellent adverse effect profile, and minimal off-target effects. We hypothesized that galunisertib diminishes fibrotic phenotypes in a targeted fashion, making it a promising candidate drug for prevention of hypertrophic burn scar and contracture. Methods Commercially available fibroblasts were treated with TGF-β at concentrations typical of burn wounds to induce fibrotic phenotype fibroblasts (FPF). FPF cells were treated with galunisertib for 0, 1, 2, 3, and 7 days at concentrations ranging from 0.01 μM to 100 μM. FPF viability and proliferation were assessed with MTT assay. Modulation of FPF cell fibrotic gene and was analyzed using quantitative real-time PCR (qRT-PCR) of COL1A1, COL3A1, FN1, αSMA, CTGF, DCN, MMP1, and MMP13. Analysis of phenotype modulation was performed by western blotting of P-Smad2/3, collagen-1, fibronectin, and αSMA. Statistical analysis performed with students t-test and ANOVA. Results TGF-β treated FPF cells had significantly increased proliferation relative to commercially available fibroblasts, which was attenuated by treatment with 2.5–10 μM galunisertib at 2 or 3 days after treatment in a concentration dependent manner (p < 0.05) while also not causing a relative decrease in proliferation. Expression of FPF downstream pro-fibrotic genes underwent significant fold changes (FN1, αSMA, CTGF: p < 0.05). Protein expression showed decrease in both SMAD2/3 phosphorylation and fibrotic protein expression. Scratch assay analysis is ongoing. Conclusions Galunisertib exerts targeted dose-dependent inhibition of fibrotic gene expression and phenotypes in vitro without hindering cellular proliferation.