A. A. Joglar1, J. W. Jay1, J. M. Peterson1, S. E. Wolf1, A. El Ayadi1 1University Of Texas Medical Branch, Surgery, Galveston, TX, USA
Introduction: Hypertrophic scars result from an increased production of extracellular matrix by myofibroblasts, which are regulated through TGF-β signaling during the wound healing process. Current treatments to prevent or treat hypertrophic scars are suboptimal. However, inhibiting the TGF-β signaling pathway may be a viable mechanism for pharmacologic treatment of hypertrophic scars. Therefore, the purpose of our present work was to investigate the effects of Galunisertib, a novel small molecular inhibitor of type 1 TGF-β1 receptor (TGF-βR1), in a wound healing and fibrosis in vitro model.
Methods: Primary neonatal dermal fibroblasts were treated with either recombinant human TGF-β1 (10 ng/mL), Galunisertib (10 μM), TGF-β1 and Galunisertib, or siRNA for TGF-βR1 knockdown. Our control group was defined as cells that did not receive any treatment. Scratch assays were preformed to assess fibroblast migration. Cells were seeded to a six-well plate (2.0 x 105 cells per well) and were grown to 90% confluence. Two parallel scratches (~500 μm width) were created per well. Digital images were acquired at 0, 4, 8, 12, 24 hours and analyzed for scratch gap closure through TScratch software. To examine the fibrotic TGF-β1 signaling cascade, protein expression of phosphorylated SMAD, αSMA, and collagen-1a was assessed at 72 hours by Western blot. Gene expression of αSMA, fibronectin expression, CTGF, collagen-1a, MMP1, decorin, and collagen-3 were assessed through qRT-PCR at 72 hours.
Results: Scratch assay analysis indicated that primary neonatal fibroblasts treated with Galunisertib alone increased wound closure rate compared to fibroblasts treated with TGF-β1 alone (p<0.05). Additionally, there was no significant difference in gap closure between TGF-βR1 knockdown fibroblasts and fibroblasts treated with Galunisertib or TGF-β1. Protein expression of phosphorylated SMAD and αSMA were reduced in fibroblasts treated with Galunisertib compared to fibroblasts treated with TGF-β1. However, there was no significant difference in protein expression of collagen-1a. Galunisertib reduced the transcript level of TGF-β-induced expression of αSMA, fibronectin, CTGF, and collagen-1a. Galunisertib increased gene expression of MMP1 and decorin. There was no significant difference in gene expression of collagen-3 in fibroblasts treated with Galunisertib.
Conclusion: Galunisertib can reduce the fibrotic phenotype of primary neonatal dermal fibroblasts, but also increase wound closure rate in vitro. Therefore, Galunisertib may be a potential option for pharmacologic treatment of hypertrophic scars.