A drug screen with approved compounds identifies amlexanox as a novel Wnt/ß-catenin activator inducing lung epithelial organoid formation.

BACKGROUND AND PURPOSE: Emphysema is an incurable disease characterized by loss of lung tissue leading to impaired gas exchange. Wnt/ß-catenin signalling is reduced in emphysema, and exogenous activation of the pathway in experimental models in vivo and in human ex vivo lung tissue improves lung function and structure. We sought to identify a pharmaceutical able to activate Wnt/ß-catenin signalling and assess its potential to activate lung epithelial cells and repair. EXPERIMENTAL APPROACH: We screened 1216 human-approved compounds for Wnt/ß-catenin signalling activation using luciferase reporter cells and selected candidates based on their computationally predicted protein targets. We further performed confirmatory luciferase reporter and metabolic activity assays. Finally, we studied the regenerative potential in murine adult epithelial cell-derived lung organoids and in vivo using a murine elastase-induced emphysema model. KEY RESULTS: The primary screen identified 16 compounds that significantly induced Wnt/ß-catenin-dependent luciferase activity. Selected compounds activated Wnt/ß-catenin signalling without inducing cell toxicity or proliferation. Two compounds were able to promote organoid formation, which was reversed by pharmacological Wnt/ß-catenin inhibition, confirming the Wnt/ß-catenin-dependent mechanism of action. Amlexanox was used for in vivo evaluation, and preventive treatment resulted in improved lung function and structure in emphysematous mouse lungs. Moreover, gene expression of Hgf, an important alveolar repair marker, was increased, whereas disease marker Eln was decreased, indicating that amlexanox induces pro-regenerative signalling in emphysema. CONCLUSION AND IMPLICATIONS: Using a drug screen based on Wnt/ß-catenin activity, organoid assays and a murine emphysema model, amlexanox was identified as a novel potential therapeutic agent for emphysema.

Extracellular-Matrix-Reinforced Bioinks for 3D Bioprinting Human Tissue.

Recent advances in 3D bioprinting allow for generating intricate structures with dimensions relevant for human tissue, but suitable bioinks for producing translationally relevant tissue with complex geometries remain unidentified. Here, a tissue-specific hybrid bioink is described, composed of a natural polymer, alginate, reinforced with extracellular matrix derived from decellularized tissue (rECM). rECM has rheological and gelation properties beneficial for 3D bioprinting while retaining biologically inductive properties supporting tissue maturation ex vivo and in vivo. These bioinks are shear thinning, resist cell sedimentation, improve viability of multiple cell types, and enhance mechanical stability in hydrogels derived from them. 3D printed constructs generated from rECM bioinks suppress the foreign body response, are pro-angiogenic and support recipient-derived de novo blood vessel formation across the entire graft thickness in a murine model of transplant immunosuppression. Their proof-of-principle for generating human tissue is demonstrated by 3D bioprinting human airways composed of regionally specified primary human airway epithelial progenitor and smooth muscle cells. Airway lumens remained patent with viable cells for one month in vitro with evidence of differentiation into mature epithelial cell types found in native human airways. rECM bioinks are a promising new approach for generating functional human tissue using 3D bioprinting.

Highlights of the ERS Lung Science Conference and Sleep and Breathing Conference 2021 and the new ECMC members.

This article provides a brief description of some of the most remarkable sessions of the @EuroRespSoc Lung Science Conference and the Sleep and Breathing Conference 2021 and presents the new incoming members of the ECMC (@EarlyCareerERS) https://bit.ly/2RSDP40.

The Preparation of Decellularized Mouse Lung Matrix Scaffolds for Analysis of Lung Regenerative Cell Potential.

Lung transplantation is the only option for patients with end-stage lung disease, but there is a shortage of available lung donors. Furthermore, efficiency of lung transplantation has been limited due to primary graft dysfunction. Recent mouse models mimicking lung disease in humans have allowed for deepening our understanding of disease pathomechanisms. Moreover, new techniques such as decellularization and recellularization have opened up new possibilities to contribute to our understanding of the regenerative mechanisms involved in the lung. Stripping the lung of its native cells allows for unprecedented analyses of extracellular matrix and sets a physiologic platform to study the regenerative potential of seeded cells. A comprehensive understanding of the molecular pathways involved for lung development and regeneration in mouse models can be translated to regeneration strategies in higher organisms, including humans. Here we describe and discuss several techniques used for murine lung de- and recellularization, methods for evaluation of efficacy including histology, protein/RNA isolation at the whole lung, as well as lung slices level.

Increased Extracellular Vesicles Mediate WNT5A Signaling in Idiopathic Pulmonary Fibrosis.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo)fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored.Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF.Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies.Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-ß in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A.Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.

How to build a lung: latest advances and emerging themes in lung bioengineering.

Chronic respiratory diseases remain a major cause of morbidity and mortality worldwide. The only option at end-stage disease is lung transplantation, but there are not enough donor lungs to meet clinical demand. Alternative options to increase tissue availability for lung transplantation are urgently required to close the gap on this unmet clinical need. A growing number of tissue engineering approaches are exploring the potential to generate lung tissue ex vivo for transplantation. Both biologically derived and manufactured scaffolds seeded with cells and grown ex vivo have been explored in pre-clinical studies, with the eventual goal of generating functional pulmonary tissue for transplantation. Recently, there have been significant efforts to scale-up cell culture methods to generate adequate cell numbers for human-scale bioengineering approaches. Concomitantly, there have been exciting efforts in designing bioreactors that allow for appropriate cell seeding and development of functional lung tissue over time. This review aims to present the current state-of-the-art progress for each of these areas and to discuss promising new ideas within the field of lung bioengineering.

Two succeeding fibroblastic lineages drive dermal development and the transition from regeneration to scarring.

During fetal development, mammalian back-skin undergoes a natural transition in response to injury, from scarless regeneration to skin scarring. Here, we characterize dermal morphogenesis and follow two distinct embryonic fibroblast lineages, based on their history of expression of the engrailed 1 gene. We use single-cell fate-mapping, live three dimensional confocal imaging and in silico analysis coupled with immunolabelling to reveal unanticipated structural and regional complexity and dynamics within the dermis. We show that dermal development and regeneration are driven by engrailed 1-history-naive fibroblasts, whose numbers subsequently decline. Conversely, engrailed 1-history-positive fibroblasts possess scarring abilities at this early stage and their expansion later on drives scar emergence. The transition can be reversed, locally, by transplanting engrailed 1-naive cells. Thus, fibroblastic lineage replacement couples the decline of regeneration with the emergence of scarring and creates potential clinical avenues to reduce scarring.