Fig. 1: Effect of nintedanib on fibroblasts from healthy human muscle in vitro.

Fibroblasts proliferation was analyzed after 2, 4, and 6 days of culture (a–d). We cultured fibroblast with increasing concentrations of nintedanib in a culture containing 10% fetal bovine serum (FBS) and we observed decreased fibroblast proliferation after 4 and 6 days in a dose-dependent manner (a). Fibroblasts were cultured in the presence of platelet-derived growth factor AA (PDGF-AA, 10 ng/mL) (b), basic fibroblast growth factor (FGFb, 10 ng/mL),(c) or vascular endothelial growth factor A (VEGF-A, 50 ng/mL) (d) with 1% FBS with or without nintedanib 0.4 μM. Only PDGF-AA increased cell proliferation, an effect that was reversed with the addition of nintedanib to the culture. Fibroblast migration was analyzed using a scratch assay. Nintedanid treatment at 0.4 μM reverted the promigratory effect of PDGF-AA, FGFb, VEGF, and CTGF (e). Representative images of this assay are shown in (f). Nintedanib treatment at 0.4 μM (blue bars) reverted the effect of PDGF-AA in the expression of ADAM metallopeptidase domain 17 (ADAM-17), tissue inhibitor of metalloproteinase 1 (TIMP-1) and tissue inhibitor of metalloproteinase 2 (TIMP-2) (g). Nintedanib treatment at 0.4 μM (blue bars) produced a statistically significant reduction of collagen type I alpha 1 chain (COL1A1), collagen type III alpha 1 chain (COL3A1), fibronectin 1 (FN1), platelet-derived growth factor A (PDGFA), connective tissue growth factor (CTGF), transforming growth factor beta 1 (TGFβ1) expression compared with control samples (black bars) analyzed by qPCR (h). Data are expressed as means ± SD. N = 3 per group. *P < 0.05, **P < 0.01, ***P < 0.005