Nintedanib modulates type III collagen turnover in viable precision-cut lung slices from bleomycin-treated rats and patients with pulmonary fibrosis

Abstract

Background: Aberrant extracellular matrix (ECM) deposition and remodelling is important in the disease pathogenesis of pulmonary fibrosis (PF). We characterised neoepitope biomarkers released by ECM turnover in lung tissue from bleomycin-treated rats and patients with PF and analysed the effects of two antifibrotic drugs: nintedanib and pirfenidone.

Methods: Precision-cut lung slices (PCLS) were prepared from bleomycin-treated rats or patients with PF. PCLS were incubated with nintedanib or pirfenidone for 48 h, and levels of neoepitope biomarkers of type I, III and VI collagen formation or degradation (PRO-C1, PRO-C3, PRO-C6 and C3M) as well as fibronectin (FBN-C) were assessed in the culture supernatants.

Results: In rat PCLS, incubation with nintedanib led to a reduction in C3M, reflecting type III collagen degradation. In patient PCLS, incubation with nintedanib reduced the levels of PRO-C3 and C3M, thus showing effects on both formation and degradation of type III collagen. Incubation with pirfenidone had a marginal effect on PRO-C3. There were no other notable effects of either nintedanib or pirfenidone on the other neoepitope biomarkers studied.

Conclusions: This study demonstrated that nintedanib modulates neoepitope biomarkers of type III collagen turnover and indicated that C3M is a promising translational neoepitope biomarker of PF in terms of therapy assessment.

Keywords: Antifibrotic therapy; Collagen; Extracellular matrix; Human lung; Neoepitope biomarkers; Nintedanib; Pirfenidone; Precision-cut lung slices; Pulmonary fibrosis.

Fig. 1

Nintedanib reduced C3M concentrations in the PCLS supernatant from bleomycin in vivo treated rats. C3M concentrations from PCLS from bleomycin-treated rats incubated with nintedanib for 48 h compared with PCLS from vehicle-treated rats. Number of slices indicates the total number from all rats evaluated at that dose. Statistical differences between groups were analysed by Kruskal–Wallis test followed by Dunn’s multiple comparison test for nonparametric data. Data are shown as mean ± SEM. ***P < 0.001. C3M neo-epitope of MMP-9 mediated degradation of type III collagen, LLOD lower limit of detection, PCLS precision-cut lung slices, SEM standard error of the mean

Fig. 2

Nintedanib reduced C3M and PRO-C3 concentrations in the PCLS supernatant from patients with PF. a Absolute changes in C3M and b PRO-C3 concentrations from PCLS incubated with nintedanib and pirfenidone for 48 h compared with PCLS incubated with medium. Samples were generated by pooling supernatant from two wells, with each well containing two PCLS. Statistical differences between groups were analysed by Kruskal–Wallis test followed by Dunn’s multiple comparison test for nonparametric data. Two data points in part A and three in part B lie outside the plotted axes. C3M neo-epitope of MMP-9 mediated degradation of type III collagen, PCLS precision-cut lung slices, PF pulmonary fibrosis, PRO-C3 ADAMTS-2 mediated release of the N-terminal pro-peptide of type III collagen

Fig. 3

Inhibition of C3M and PRO-C3 in the PCLS supernatant from patients with PF. Percentage changes in a C3M and b PRO-C3 concentrations from PCLS incubated with nintedanib compared with PCLS incubated with medium. The EC₅₀ were cal culated with asymmetric (five parameter) least squares fit. Six data points lie outside the plotted axes. C3MC3M neo-epitope of MMP-9 mediated degradation of type III collage n; EC₅₀ half- maxi mal inhibition values; PCLS precision-cut lung slices; PF pulmonary fibrosis; PRO-C3 ADAMTS-2 mediated release of the N-terminal pro-peptid e of type III collagen