NF-κB drives epithelial-mesenchymal mechanisms of lung fibrosis in a translational lung cell model.

In the progression phase of idiopathic pulmonary fibrosis (IPF), the normal alveolar structure of the lung is lost and replaced by remodeled fibrotic tissue and by bronchiolized cystic airspaces. Although these are characteristic features of IPF, knowledge of specific interactions between these pathological processes is limited. Here, the interaction of lung epithelial and lung mesenchymal cells was investigated in a coculture model of human primary airway epithelial cells (EC) and lung fibroblasts (FB). Single-cell RNA sequencing revealed that the starting EC population was heterogenous and enriched for cells with a basal cell signature. Furthermore, fractions of the initial EC and FB populations adopted distinct pro-fibrotic cell differentiation states upon cocultivation, resembling specific cell populations that were previously identified in lungs of patients with IPF. Transcriptomic analysis revealed active NF-κB signaling early in the cocultured EC and FB, and the identified NF-κB expression signatures were found in "HAS1 High FB" and "PLIN2+ FB" populations from IPF patient lungs. Pharmacological blockade of NF-κB signaling attenuated specific phenotypic changes of EC and prevented FB-mediated interleukin-6, interleukin-8, and CXC chemokine ligand 6 cytokine secretion, as well as collagen α-1(I) chain and α-smooth muscle actin accumulation. Thus, we identified NF-κB as a potential mediator, linking epithelial pathobiology with fibrogenesis.

The Antifibrotic Activity of Prostacyclin Receptor Agonism Is Mediated through Inhibition of YAP/TAZ.

Idiopathic pulmonary fibrosis is a life-threatening progressive disease characterized by loss of alveolar epithelial cells, inflammation, and aberrant fibroblast activation. The two currently approved therapies do not halt or reverse tissue remodeling, and therefore novel disease-modifying mechanisms are needed. Our results describe YAP/TAZ inhibition through prostacyclin (IP) receptor activation as a novel mechanism that suppresses profibrotic (myo)fibroblast activity. We investigated the antifibrotic properties of the selective IP receptor agonist ACT-333679 using primary human lung fibroblasts. ACT-333679 prevented transforming growth factor ß1-induced fibroblast-to-myofibroblast transition, proliferation, extracellular matrix synthesis, and IL-6 and PAI-1 secretion, and exerted relaxant effects in cell contraction assays. ACT-333679 treatment also reverted an established myofibroblast phenotype. Unbiased analysis of ACT-333679-induced whole-genome expression changes in transforming growth factor ß1-treated fibroblasts identified significant attenuation of genes regulated by YAP/TAZ, two transcriptional cofactors that are essential for fibrosis. We then demonstrated that ACT-333679, via elevation of cAMP, caused YAP/TAZ nuclear exclusion and subsequent suppression of YAP/TAZ-dependent profibrotic gene transcription. In summary, we offer a rationale for further exploring the potential of IP receptor agonists for the treatment of idiopathic pulmonary fibrosis.

Comparison of Macitentan and Bosentan on Right Ventricular Remodeling in a Rat Model of Non-vasoreactive Pulmonary Hypertension.

AIMS: We compared the efficacy of macitentan, a novel dual endothelin A/endothelin B receptor antagonist, with that of another dual endothelin receptor antagonist, bosentan, in a rat model of non-vasoreactive pulmonary hypertension (PH) with particular emphasis on right ventricular (RV) remodeling. METHODS AND RESULTS: Unlike monocrotaline or hypoxic/sugen rats, bleomycin-treated rats presented a non-vasoreactive PH characterized by the absence of pulmonary dilatation to adenosine. We therefore chose the bleomycin rat model to compare the effects of the maximally effective doses of macitentan and bosentan on pulmonary vascular and RV remodeling. Macitentan (100 mg·kg(-1)·d(-1)), but not bosentan (300 mg·kg(-1)·d(-1)), significantly prevented pulmonary vascular remodeling, RV hypertrophy, and cardiomyocyte diameter increase. Cardiac protection by macitentan was associated with a significant attenuation of genes related to cell hypertrophy and extracellular matrix remodeling. Microautoradiography and high performance liquid chromatography analysis showed greater distribution of macitentan than bosentan in the RV and pulmonary tissue. CONCLUSIONS: Macitentan was more efficacious than bosentan in preventing the development of pulmonary and RV hypertrophies in a model of non-vasoreactive PH. Greater ability to distribute into the tissue could contribute to the greater structural improvement by macitentan compared with bosentan.

A novel genomic signature with translational significance for human idiopathic pulmonary fibrosis.

The bleomycin-induced rodent lung fibrosis model is commonly used to study mechanisms of lung fibrosis and to test potential therapeutic interventions, despite the well recognized dissimilarities to human idiopathic pulmonary fibrosis (IPF). Therefore, in this study, we sought to identify genomic commonalities between the gene expression profiles from 100 IPF lungs and 108 control lungs that were obtained from the Lung Tissue Research Consortium, and rat lungs harvested at Days 3, 7, 14, 21, 28, 42, and 56 after bleomycin instillation. Surprisingly, the highest gene expression similarity between bleomycin-treated rat and IPF lungs was observed at Day 7. At this point of maximal rat-human commonality, we identified a novel set of 12 disease-relevant translational gene markers (C6, CTHRC1, CTSE, FHL2, GAL, GREM1, LCN2, MMP7, NELL1, PCSK1, PLA2G2A, and SLC2A5) that was able to separate almost all patients with IPF from control subjects in our cohort and in two additional IPF/control cohorts (GSE10667 and GSE24206). Furthermore, in combination with diffusing capacity of carbon monoxide measurements, four members of the translational gene marker set contributed to stratify patients with IPF according to disease severity. Significantly, pirfenidone attenuated the expression change of one (CTHRC1) translational gene marker in the bleomycin-induced lung fibrosis model, in transforming growth factor-ß1-treated primary human lung fibroblasts and transforming growth factor-ß1-treated human epithelial A549 cells. Our results suggest that a strategy focused on rodent model-human disease commonalities may identify genes that could be used to predict the pharmacological impact of therapeutic interventions, and thus facilitate the development of novel treatments for this devastating lung disease.

Sphingosine 1-phosphate (S1P) receptor agonists mediate pro-fibrotic responses in normal human lung fibroblasts via S1P2 and S1P3 receptors and Smad-independent signaling.

Synthetic sphingosine 1-phosphate receptor 1 modulators constitute a new class of drugs for the treatment of autoimmune diseases. Sphingosine 1-phosphate (S1P) signaling, however, is also involved in the development of fibrosis. Using normal human lung fibroblasts, we investigated the induction of fibrotic responses by the S1P receptor (S1PR) agonists S1P, FTY720-P, ponesimod, and SEW2871 and compared them with the responses induced by the known fibrotic mediator TGF-ß1. In contrast to TGF-ß1, S1PR agonists did not induce expression of the myofibroblast marker α-smooth muscle actin. However, TGF-ß1, S1P, and FTY720-P caused robust stimulation of extracellular matrix (ECM) synthesis and increased pro-fibrotic marker gene expression including connective tissue growth factor. Ponesimod showed limited and SEW2871 showed no pro-fibrotic potential in these readouts. Analysis of pro-fibrotic signaling pathways showed that in contrast to TGF-ß1, S1PR agonists did not activate Smad2/3 signaling but rather activated PI3K/Akt and ERK1/2 signaling to induce ECM synthesis. The strong induction of ECM synthesis by the nonselective agonists S1P and FTY720-P was due to the stimulation of S1P2 and S1P3 receptors, whereas the weaker induction of ECM synthesis at high concentrations of ponesimod was due to a low potency activation of S1P3 receptors. Finally, in normal human lung fibroblast-derived myofibroblasts that were generated by TGF-ß1 pretreatment, S1P and FTY720-P were effective stimulators of ECM synthesis, whereas ponesimod was inactive, because of the down-regulation of S1P3R expression in myofibroblasts. These data demonstrate that S1PR agonists are pro-fibrotic via S1P2R and S1P3R stimulation using Smad-independent pathways.