Rationale: Recent findings suggesting transforming growth factor (TGF)-β₁ activation by mechanical stimuli in vitro raised the question of whether this phenomenon was relevant in vivo in the context of pulmonary fibrosis. Objectives: To explore the effect of mechanical stress on TGF-β₁ activation and its signaling pathway in rat and human fibrotic lung tissue using a novel ex vivo model. Methods: Rat lung fibrosis was induced using transient gene expression of active TGF-β₁. Lungs were harvested at Day 14 or 21 and submitted to various stimuli in a tissue bath equipped with a force transducer and servo-controlled arm. Measurements and Main Results: Fibrotic lung strips responded to tensile force by releasing active TGF-β₁ from latent stores with subsequent increase in tissue phospho-Smad2/3. In contrast, measurable active TGF-β₁ and phospho-Smad2/3 were not induced by mechanical stress in nonfibrotic lungs. Protease inhibition did not affect the release of active TGF-β₁. A TGF-β₁ receptor inhibitor, Rho-associated protein kinase inhibitor, and αv integrin inhibitor all attenuated mechanical stretch–induced phospho-Smad2/3 in fibrotic lung strips. Furthermore, the induction of phospho-Smad2/3 was enhanced in whole fibrotic rat lungs undergoing ventilation pressure challenge compared with control lungs. Last, tissue slices from human lung with usual interstitial pneumonia submitted to mechanical force showed an increase in TGF-β₁ activation and induction of phospho-Smad2/3 in contrast with human nonfibrotic lungs. Conclusions: Mechanical tissue stretch contributes to the development of pulmonary fibrosis via mechanotransduced activation of TGF-β₁ in rodent and human pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by myofibroblast proliferation. Transition of epithelial/mesothelial cells into myofibroblasts [epithelial‐to‐mesenchymal transition (EMT)] occurs under the influence of transforming growth factor (TGF)‐β1, with Snail being a major transcription factor. We study here the role of the heat‐shock protein HSP27 in fibrogenesis and EMT. In vitro , we have up‐ and down‐modulated HSP27 expression in mesothelial and epithelial cell lines and studied the expression of different EMT markers induced by TGF‐β1. In vivo , we inhibited HSP27 with the antisense oligonucleotide OGX‐427 (in phase II clinical trials as anticancer agent) in our rat subpleural/pulmonary fibrosis models. We demonstrate that HSP27 is strongly expressed during the fibrotic process in patients with IPF and in different in vivo models. We showed that HSP27 binds to and stabilizes Snail and consequently induces EMT. Conversely, HSP27 knockdown leads to Snail proteasomal degradation, thus inhibiting TGF‐β1‐induced EMT. Inhibition of HSP27 with OGX‐427 efficiently blocks EMT and fibrosis development. Controls in vivo were an empty adenovirus that did not induce fibrosis and a control antisense oligonucleotide. The present work opens the possibility of a new therapeutic use for HSP27 inhibitors against IPF, for which there is no conclusively effective treatment.—Wettstein, G., Bellaye, P.‐S., Kolb, M., Hammann, A., Crestani, B., Soler, P., Marchal‐Somme, J., Hazoume, A., Gauldie, J., Gunther, A., Micheau, O., Gleave, M., Camus, P., Garrido, C., Bonniaud, P. Inhibition of HSP27 blocks fibrosis development and EMT features by promoting Snail degradation. FASEB J. 27, 1549–1560 (2013). www.fasebj.org

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been associated with fibrotic lung disease, although exactly how they modulate this process remains unclear. Here we investigated the role of GRP78, the main UPR regulator, in an experimental model of lung injury and fibrosis. Grp78^+/−, Chop^−/− and wild type C57BL6/J mice were exposed to bleomycin by oropharyngeal intubation and lungs were examined at days 7 and 21. We demonstrate here that Grp78^+/− mice were strongly protected from bleomycin‐induced fibrosis, as shown by immunohistochemical analysis, collagen content and lung function measurements. In the inflammatory phase of this model, a reduced number of lung macrophages associated with an increased number of TUNEL‐positive cells were observed in Grp78^+/− mice. Dual immunohistochemical and in situ hybridization experiments showed that the macrophage population from the protected Grp78^+/− mice was also strongly positive for cleaved caspase‐3 and Chop mRNA, respectively. In contrast, the administration of bleomycin to Chop^−/− mice resulted in increased quasi‐static elastance and extracellular matrix deposition associated with an increased number of parenchymal arginase‐1‐positive macrophages that were negative for cleaved caspase‐3. The data presented indicate that the UPR is activated in fibrotic lung tissue and strongly localized to macrophages. GRP78‐ and CHOP‐mediated macrophage apoptosis was found to protect against bleomycin‐induced fibrosis. Overall, we demonstrate here that the fibrotic response to bleomycin is dependent on GRP78‐mediated events and provides evidence that macrophage polarization and apoptosis may play a role in this process. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Idiopathic pulmonary fibrosis (IPF) is characterized by progressive fibroblast and myofibroblast proliferation, and extensive deposition of extracellular matrix (ECM). Fibroblast growth factor‐1 (FGF‐1) belongs to the FGF family and has been shown to inhibit fibroblast collagen production and differentiation into myofibroblasts, and revert epithelial–mesenchymal transition by inhibiting TGF‐β1 signalling pathways. However, the precise role of FGF‐1 in pulmonary fibrosis has not yet been elucidated. In this study, we explore the mechanisms underlying the anti‐fibrogenic effect of FGF‐1 in pulmonary fibrosis in vitro and in vivo by prolonged transient overexpression of FGF‐1 (AdFGF‐1) and TGF‐β1 (AdTGF‐β1) using adenoviral vectors. In vivo, FGF‐1 overexpression markedly attenuated TGF‐β1‐induced pulmonary fibrosis in rat lungs when given both concomitantly, or delayed, by enhancing proliferation and hyperplasia of alveolar epithelial cells (AECs). AdFGF‐1 also attenuated the TGF‐β1 signalling pathway and induced FGFR1 expression in AECs. In vitro, AdFGF‐1 prevented the increase in α‐SMA and the decrease in E‐cadherin induced by AdTGF‐β1 in normal human lung fibroblasts, primary human pulmonary AECs, and A549 cells. Concomitantly, AdTGF‐β1‐induced Smad2 phosphorylation was significantly reduced by AdFGF‐1 in both cell types. AdFGF‐1 also attenuated the increase in TGFβR1 protein and mRNA levels in fibroblasts. In AECs, AdFGF‐1 decreased TGFβR1 protein by favouring TGFβR1 degradation through the caveolin‐1/proteasome pathway. Furthermore, FGFR1 expression was increased in AECs, whereas it was decreased in fibroblasts. In serum of IPF patients, FGF‐1 levels were increased compared to controls. Interestingly, FGF‐1 expression was restricted to areas of AEC hyperplasia, but not α‐SMA‐positive areas in IPF lung tissue. Our results demonstrate that FGF‐1 may have preventative and therapeutic effects on TGF‐β1‐driven pulmonary fibrosis via inhibiting myofibroblast differentiation, inducing AEC proliferation, regulating TGF‐β1 signalling by controlling TGFβR1 expression and degradation, and regulating FGFR1 expression. Thus, modulating FGF‐1 signalling represents a potential therapy for the treatment of pulmonary fibrosis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Experimental models are critical for the understanding of lung health and disease and are indispensable for drug development. However, the pathogenetic and clinical relevance of the models is often unclear. Further, the use of animals in biomedical research is controversial from an ethical perspective.The objective of this task force was to issue a statement with research recommendations about lung disease models by facilitating in-depth discussions between respiratory scientists, and to provide an overview of the literature on the available models. Focus was put on their specific benefits and limitations. This will result in more efficient use of resources and greater reduction in the numbers of animals employed, thereby enhancing the ethical standards and translational capacity of experimental research.The task force statement addresses general issues of experimental research (ethics, species, sex, age,ex vivoandin vitromodels, gene editing). The statement also includes research recommendations on modelling asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, lung infections, acute lung injury and pulmonary hypertension.The task force stressed the importance of using multiple models to strengthen validity of results, the need to increase the availability of human tissues and the importance of standard operating procedures and data quality.