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. 2014 Dec 12;15(1):157.
doi: 10.1186/s12931-014-0157-3.

Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

Katrin E Hostettler  1   2 , Jun Zhong  3 , Eleni Papakonstantinou  4 , George Karakiulakis  5 , Michael Tamm  6   7 , Petra Seidel  8 , Qingzhu Sun  9 , Jyotshna Mandal  10 , Didier Lardinois  11 , Christopher Lambers  12 , Michael Roth  13
Affiliations

Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

Katrin E Hostettler et al. Respir Res. .

Abstract

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The kinase inhibitor nintedanib specific for vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) significantly reduced the rate of decline of forced vital capacity versus placebo.

Aim: To determine the in vitro effect of nintedanib on primary human lung fibroblasts.

Methods: Fibroblasts were isolated from lungs of IPF patients and from non-fibrotic controls. We assessed the effect of VEGF, PDGF-BB and basic FGF (bFGF) ± nintedanib on: (i) expression/activation of VEGFR, PDGFR, and FGFR, (ii) cell proliferation, secretion of (iii) matrix metalloproteinases (MMP), (iv) tissue inhibitor of metalloproteinase (TIMP), and (v) collagen.

Results: IPF fibroblasts expressed higher levels of PDGFR and FGFR than controls. PDGF-BB, bFGF, and VEGF caused a pro-proliferative effect which was prevented by nintedanib. Nintedanib enhanced the expression of pro-MMP-2, and inhibited the expression of TIMP-2. Transforming growth factor-beta-induced secretion of collagens was inhibited by nintedanib.

Conclusion: Our data demonstrate a significant anti-fibrotic effect of nintedanib in IPF fibroblasts. This effect consists of the drug's anti-proliferative capacity, and on its effect on the extracellular matrix, the degradation of which seems to be enhanced.

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Figures

Figure 1
Figure 1
Effect of nintedanib on receptor expression and phosphorylation. (A) Fibroblasts derived from patients with IPF were treated with increasing concentrations of nintedanib (0.01, 0.1, and 1 μM) for 24 hours and cell viability was analysed by trypan blue exclusion staining. (B) Representative immuno-blots showing the expression of total PDGF-receptor (PDGFR), VEGF-receptor (VEGFR) and FGF-receptor (FGFR), in primary human lung IPF fibroblasts and non-fibrotic control cells. The bar charts summarise the densitometric analysis of total receptor expression normalised to GAPDH in IPF and control cells. Data are presented as mean ± SEM of experiment performed in three different cell lines. *p < 0.05. (C) Representative immuno-blots showing total and phosphorylated PDGFR, VEGFR, and FGFR in primary human lung IPF fibroblasts. Fibroblasts were starved for 24 hours. Before stimulation with PDGF-BB, bFGF, or VEGF (all 10 ng/ml) for 30 minutes, cells were pre-incubated with nintedanib (400 nM) for 30 minutes. The bar charts summarise the densitometric analysis of receptor phosphorylation normalised to total receptor expression in IPF cells. Data are presented as mean ± SEM of experiment performed in three different cell lines. *p < 0.05.
Figure 2
Figure 2
Effect of nintedanib on PDGF-BB- (A, B), bFGF- (C, D), and VEGF- (E, F) induced fibroblast proliferation compared with serum-free RPMI (control) in fibroblasts derived from IPF lungs (A, C, E) and non-fibrotic control lungs (B, D, F). Primary human lung fibroblasts derived from IPF patients and from non-fibrotic lungs were pre-incubated for 30 minutes with increasing concentrations of nintedanib (0.001, 0.01, 0.1, and 1 μM), before adding PDGF-BB (10 ng/ml), bFGF (10 ng/ml), and VEGF (10 ng/ml) for 48 hours. Serum-free RPMI served as control. Cell growth was assessed by manual cell counting, and values are presented as mean ± SEM of triplicate independent experiments performed in 4 different cell lines, expressed as relative proliferation compared to control (serum-free RPMI medium) which was set to 100%. *p < 0.05.
Figure 3
Figure 3
Effect of nintedanib on enzymatic activity (panel A-D) and secretion of matrix metalloproteinase (MMP)-2 (panel E, F) and tissue inhibitor of metalloproteinase (TIMP)-2 (panel G, H) by primary human lung IPF fibroblasts (A, C, E, G) and by non-fibrotic control cells (B, D, F, H). Fibroblasts were treated with increasing concentrations of nintedanib (0.001, 0.01, 0.1, and 1 μM) for 24 hours and conditioned cell medium was collected. (A, B) Representative gelatine-based zymograms of the effect of nintedanib on pro-MMP-2 enzymatic activity by IPF fibroblasts (A) and by control cells (B). Arrows indicate the migration position of purified pro-MMP-2. (C, D) Densitometric analysis of pro-MMP-2 enzymatic activity in primary human lung fibroblasts obtained from IPF lungs (C), and from non-fibrotic controls (D). Values are presented as mean ± SEM of independent experiments performed in 4 different cell lines, expressed as percentage of control (serum-free RPMI medium). Each experiment was performed at least in duplicates. *p < 0.05. The effect of nintedanib on the secretion of MMP-2 and TIMP-2 by IPF fibroblasts (E, G) and control cells (F, H) was assessed by ELISA. Values are presented as mean ± SEM of independent experiments performed in 4 different cell lines, expressed as percentage of control (serum-free RPMI medium). Each experiment was performed at least in duplicates. *p < 0.05.
Figure 4
Figure 4
Effect of nintedanib on TGF-β-induced secretion (panel A, B) and deposition (panel C) of collagens by primary human lung IPF fibroblasts (panel A, C) and by non-fibrotic control cells (B). Primary human lung fibroblasts were pre-incubated for 30 minutes with nintedanib (0.001, 0.01, 0.1, and 1 μM), before adding TGF-β (5 ng/ml) for 48 hours. Collagen secretion and deposition were quantitated by the Sircol™ Assay, and values are presented as mean ± SEM of independent experiments performed in 4 different cell lines, expressed as percentage of control (0.1% FCS) which was set to 100%. Each experiment was performed at least in duplicates. *p < 0.05. (D) Representative immuno-blots showing the expression of total and phosphorylated ERK1/2 and c-Abl in primary human lung IPF fibroblasts. Fibroblasts were starved for 24 hours. Before stimulation with TGF-β1 (5 ng/ml) for 5 minutes, cells were pre-incubated with nintedanib (1 μM) for 30 minutes. The bar charts summarise the densitometric analysis of receptor phosphorylation normalised to α–tubulin and total receptor expression in IPF cells. Data are presented as mean ± SEM of experiment performed in three different cell lines. *p < 0.05.
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