Potential novel targets: Protease-activated receptors in idiopathic pulmonary fibrosis

Background Pulmonary fibrosis is a devastating disease of unknown etiology. Protease-activated receptor-1 recently emerged as a critical component in the context of fibrotic lung diseases. In the present study, we aimed to address the potential importance of macrophages in PAR-1-driven pulmonary fibrosis. Methods Macrophage numbers in lungs of bleomycin-instilled mice treated or not with a specific PAR-1 antagonist (i.e. P1pal-12) were assessed by (immuno)histochemical analysis. Macrophage migration was assessed by trans-well migration assays while PAR-1 ligand production was addressed by qPCR. In vitro, fibroblasts were stimulated with macrophage-derived conditioned medium after which fibrotic marker expression and Smad-2 phosphorylation was analyzed by Western blot. Main results Macrophage numbers were significantly reduced in lungs of P1pal-12 treated animals after bleomycin instillation. In line with these data, PAR-1 stimulation increased monocyte/ macrophage recruitment in response to epithelium injury in in vitro trans-well assays. Moreover, macrophages induced fibroblasts migration, differentiation and the secretion of collage, which were inhibited in the presence of TGF-β receptor inhibitors. Interestingly, these profibrotic effects were partially inhibited by P1pal-12 treatment. Using shRNA mediated PAR-1 knock down in fibroblasts, we demonstrate that fibroblast PAR-1 contributes to TGF-β activation and production. Finally, we show that the macrophagedependent induction of PAR-1 driven TGF-β activation was mediated by FXa. Conclusions Our data identify novel mechanisms by which PAR-1 stimulation on different cell types can contribute to pulmonary fibrosis and pinpoint macrophages as key players in PAR-1 dependent development of pulmonary fibrosis. Lin Proefschrift 150626.indd 96 1-7-2015 8:35:45 97 PAR-1 regulates macrophage-mediated pulmonary fibrosis