Expression of Stress-Induced Genes in Bronchoalveolar Lavage Cells and Lung Fibroblasts from Healthy and COPD Subjects.

Chronic obstructive pulmonary disease (COPD) is commonly caused from smoking cigarettes that induce biological stress responses. Previously we found disorganized endoplasmic reticulum (ER) in fibroblasts from COPD with different responses to chemical stressors compared to healthy subjects. Here, we aimed to investigate differences in stress-related gene expressions within lung cells from COPD and healthy subjects. Bronchoalveolar lavage (BAL) cells were collected from seven COPD and 35 healthy subjects. Lung fibroblasts were derived from 19 COPD and 24 healthy subjects and exposed to cigarette smoke extract (CSE). Gene and protein expression and cell proliferation were investigated. Compared to healthy subjects, we found lower gene expression of CHOP in lung fibroblasts from COPD subjects. Exposure to CSE caused inhibition of lung fibroblast proliferation in both groups, though the changes in ER stress-related gene expressions (ATF6, IRE1, PERK, ATF4, CHOP, BCL2L1) and genes relating to proteasomal subunits mostly occurred in healthy lung fibroblasts. No differences were found in BAL cells. In this study, we have found that lung fibroblasts from COPD subjects have an atypical ER stress gene response to CSE, particularly in genes related to apoptosis. This difference in response to CSE may be a contributing factor to COPD progression.

Lung Fibroblasts from Chronic Obstructive Pulmonary Disease Subjects Have a Deficient Gene Expression Response to Cigarette Smoke Extract Compared to Healthy.

Background and aim: Cigarette smoking is the most common cause of chronic obstructive pulmonary disease (COPD) but more mechanistic studies are needed. Cigarette smoke extract (CSE) can elicit a strong response in many COPD-related cell types, but no studies have been performed in lung fibroblasts. Therefore, we aimed to investigate the effect of CSE on gene expression in lung fibroblasts from healthy and COPD subjects. Patients and methods: Primary lung fibroblasts, derived from six healthy and six COPD subjects (all current or ex-smokers), were either unstimulated (baseline) or stimulated with 30% CSE for 4 h prior to RNA isolation. The mRNA expression levels were measured using the NanoString nCounter Human Fibrosis V2 panel (760 genes). Pathway enrichment was assessed for unique gene ontology terms of healthy and COPD. Results: At baseline, a difference in the expression of 17 genes was found in healthy and COPD subjects. Differential expression of genes after CSE stimulation resulted in significantly less changes in COPD lung fibroblasts (70 genes) than in healthy (207 genes), with 51 genes changed in both. COPD maintained low NOTCH signaling throughout and upregulated JUN >80%, indicating an increase in apoptosis. Healthy downregulated the Mitogen-activated protein kinase (MAPK) signaling cascade, including a ≥50% reduction in FGF2, CRK, TGFBR1 and MEF2A. Healthy also downregulated KAT6A and genes related to cell proliferation, all together indicating possible cell senescence signaling. Conclusion: Overall, COPD lung fibroblasts responded to CSE stimulation with a very different and deficient expression profile compared to healthy. Highlighting that stimulated healthy cells are not an appropriate substitute for COPD cells which is important when investigating the mechanisms of COPD.

Innovative three-dimensional models for understanding mechanisms underlying lung diseases: powerful tools for translational research.

Chronic lung diseases result from alteration and/or destruction of lung tissue, inevitably causing decreased breathing capacity and quality of life for patients. While animal models have paved the way for our understanding of pathobiology and the development of therapeutic strategies for disease management, their translational capacity is limited. There is, therefore, a well-recognised need for innovative in vitro models to reflect chronic lung diseases, which will facilitate mechanism investigation and the advancement of new treatment strategies. In the last decades, lungs have been modelled in healthy and diseased conditions using precision-cut lung slices, organoids, extracellular matrix-derived hydrogels and lung-on-chip systems. These three-dimensional models together provide a wide spectrum of applicability and mimicry of the lung microenvironment. While each system has its own limitations, their advantages over traditional two-dimensional culture systems, or even over animal models, increases the value of in vitro models. Generating new and advanced models with increased translational capacity will not only benefit our understanding of the pathobiology of lung diseases but should also shorten the timelines required for discovery and generation of new therapeutics. This article summarises and provides an outline of the European Respiratory Society research seminar "Innovative 3D models for understanding mechanisms underlying lung diseases: powerful tools for translational research", held in Lisbon, Portugal, in April 2022. Current in vitro models developed for recapitulating healthy and diseased lungs are outlined and discussed with respect to the challenges associated with them, efforts to develop best practices for model generation, characterisation and utilisation of models and state-of-the-art translational potential.

Alveolar epithelial cells are competent producers of interstitial extracellular matrix with disease relevant plasticity in a human in vitro 3D model.

Alveolar epithelial cells (AEC) have been implicated in pathological remodelling. We examined the capacity of AEC to produce extracellular matrix (ECM) and thereby directly contribute towards remodelling in chronic lung diseases. Cryopreserved type 2 AEC (AEC2) from healthy lungs and chronic obstructive pulmonary disease (COPD) afflicted lungs were cultured in decellularized healthy human lung slices for 13 days. Healthy-derived AEC2 were treated with transforming growth factor ß1 (TGF-ß1) to evaluate the plasticity of their ECM production. Evaluation of phenotypic markers and expression of matrisome genes and proteins were evaluated by RNA-sequencing, mass spectrometry and immunohistochemistry. The AEC2 displayed an AEC marker profile similar to freshly isolated AEC2 throughout the 13-day culture period. COPD-derived AECs proliferated as healthy AECs with few differences in gene and protein expression while retaining increased expression of disease marker HLA-A. The AEC2 expressed basement membrane components and a complex set of interstitial ECM proteins. TGF-ß1 stimuli induced a significant change in interstitial ECM production from AEC2 without loss of specific AEC marker expression. This study reveals a previously unexplored potential of AEC to directly contribute to ECM turnover by producing interstitial ECM proteins, motivating a re-evaluation of the role of AEC2 in pathological lung remodelling.

Uncovering the cytotoxic effects of air pollution with multi-modal imaging of in vitro respiratory models.

Annually, an estimated seven million deaths are linked to exposure to airborne pollutants. Despite extensive epidemiological evidence supporting clear associations between poor air quality and a range of short- and long-term health effects, there are considerable gaps in our understanding of the specific mechanisms by which pollutant exposure induces adverse biological responses at the cellular and tissue levels. The development of more complex, predictive, in vitro respiratory models, including two- and three-dimensional cell cultures, spheroids, organoids and tissue cultures, along with more realistic aerosol exposure systems, offers new opportunities to investigate the cytotoxic effects of airborne particulates under controlled laboratory conditions. Parallel advances in high-resolution microscopy have resulted in a range of in vitro imaging tools capable of visualizing and analysing biological systems across unprecedented scales of length, time and complexity. This article considers state-of-the-art in vitro respiratory models and aerosol exposure systems and how they can be interrogated using high-resolution microscopy techniques to investigate cell-pollutant interactions, from the uptake and trafficking of particles to structural and functional modification of subcellular organelles and cells. These data can provide a mechanistic basis from which to advance our understanding of the health effects of airborne particulate pollution and develop improved mitigation measures.

Effects of hypoxia on bronchial and alveolar epithelial cells linked to pathogenesis in chronic lung disorders.

Introduction: Chronic lung disorders involve pathological alterations in the lung tissue with hypoxia as a consequence. Hypoxia may influence the release of inflammatory mediators and growth factors including vascular endothelial growth factor (VEGF) and prostaglandin (PG)E2. The aim of this work was to investigate how hypoxia affects human lung epithelial cells in combination with profibrotic stimuli and its correlation to pathogenesis. Methods: Human bronchial (BEAS-2B) and alveolar (hAELVi) epithelial cells were exposed to either hypoxia (1% O2) or normoxia (21% O2) during 24 h, with or without transforming growth factor (TGF)-ß1. mRNA expression of genes and proteins related to disease pathology were analysed with qPCR, ELISA or immunocytochemistry. Alterations in cell viability and metabolic activity were determined. Results: In BEAS-2B and hAELVi, hypoxia significantly dowregulated genes related to fibrosis, mitochondrial stress, oxidative stress, apoptosis and inflammation whereas VEGF receptor 2 increased. Hypoxia increased the expression of Tenascin-C, whereas both hypoxia and TGF-ß1 stimuli increased the release of VEGF, IL-6, IL-8 and MCP-1 in BEAS-2B. In hAELVi, hypoxia reduced the release of fibroblast growth factor, epidermal growth factor, PGE2, IL-6 and IL-8, whereas TGF-ß1 stimulus significantly increased the release of PGE2 and IL-6. TGF-ß1 stimulated BEAS-2B cells showed a decreased release of VEGF-A and IL-8, while TGF-ß1 stimulated hAELVi cells showed a decreased release of PGE2 and IL-8 during hypoxia compared to normoxia. Metabolic activity was significantly increased by hypoxia in both epithelial cell types. Discussion: In conclusion, our data indicate that bronchial and alveolar epithelial cells respond differently to hypoxia and profibrotic stimuli. The bronchial epithelium appears more responsive to changes in oxygen levels and remodelling processes compared to the alveoli, suggesting that hypoxia may be a driver of pathogenesis in chronic lung disorders.

A tunable physiomimetic stretch system evaluated with precision cut lung slices and recellularized human lung scaffolds.

Breathing exposes lung cells to continual mechanical stimuli, which is part of the microenvironmental signals directing cellular functions together with the extracellular matrix (ECM). Therefore, developing systems that incorporate both stimuli is urgent to fully understand cell behavior. This study aims to introduce a novel in vitro culture methodology combining a cyclic stretch that simulates in vivo breathing with 3D cell culture platforms in the form of decellularized lung slices (DLS) and precision cut lung slices (PCLS). To this end, we have constructed a device that mimics the amplitudes and frequencies of distensions seen in the breathing human lung. For its validation, we cultured H441 lung epithelial cells in human DLS exposed to 16 stretch cycles per minute with a 10% stretch amplitude. Cell viability (resazurin reduction), proliferation (Ki-67) and YAP1 activation were evaluated at 24 and 96 h by immunohistochemistry, while the expression of SFTPB, COL3A1, COL4A3 and LAMA5 was evaluated by qPCR. Cyclic stretch induced an increase in SFTPB expression after 24 h without a concomitant increase in the stretch responsive gene YAP1. Moreover, the ECM milieu lowered the expression of the basement membrane protein genes COL4A3 and LAMA5 compared to tissue culture plastic control cultures, but no effect was observed by the mechanical stimuli. The device also confirmed good compatibility with PCLS culture, showing preserved morphology and metabolism in rat PCLS after 72 h of mechanical stretch. Thus, we present a novel device and methodology for the easy assembling and study of lung tissue slice cultures subjected to physiomimetic mechanical stimuli, which shows promise for future studies of cell and tissue function in a lung ECM milieu with physiological or pathological mechanical stimuli.

Stretch increases alveolar type 1 cell number in fetal lungs through ROCK-Yap/Taz pathway.

Accurate fluid pressure in the fetal lung is critical for its development, especially at the beginning of the saccular stage when alveolar epithelial type 1 (AT1) and type 2 (AT2) cells differentiate from the epithelial progenitors. Despite our growing understanding of the role of physical forces in lung development, the molecular mechanisms that regulate the transduction of mechanical stretch to alveolar differentiation remain elusive. To simulate lung distension, we optimized both an ex vivo model with precision cut lung slices and an in vivo model of fetal tracheal occlusion. Increased mechanical tension showed to improve alveolar maturation and differentiation toward AT1. By manipulating ROCK pathway, we demonstrate that stretch-induced Yap/Taz activation promotes alveolar differentiation toward AT1 phenotype via ROCK activity. Our findings show that balanced ROCK-Yap/Taz signaling is essential to regulate AT1 differentiation in response to mechanical stretching of the fetal lung, which might be helpful in improving lung development and regeneration.

Crosstalk between Mast Cells and Lung Fibroblasts Is Modified by Alveolar Extracellular Matrix and Influences Epithelial Migration.

Mast cells play an important role in asthma, however, the interactions between mast cells, fibroblasts and epithelial cells in idiopathic pulmonary fibrosis (IPF) are less known. The objectives were to investigate the effect of mast cells on fibroblast activity and migration of epithelial cells. Lung fibroblasts from IPF patients and healthy individuals were co-cultured with LAD2 mast cells or stimulated with the proteases tryptase and chymase. Human lung fibroblasts and mast cells were cultured on cell culture plastic plates or decellularized human lung tissue (scaffolds) to create a more physiological milieu by providing an alveolar extracellular matrix. Released mediators were analyzed and evaluated for effects on epithelial cell migration. Tryptase increased vascular endothelial growth factor (VEGF) release from fibroblasts, whereas co-culture with mast cells increased IL-6 and hepatocyte growth factor (HGF). Culture in scaffolds increased the release of VEGF compared to culture on plastic. Migration of epithelial cells was reduced by IL-6, while HGF and conditioned media from scaffold cultures promoted migration. In conclusion, mast cells and tryptase increased fibroblast release of mediators that influenced epithelial migration. These data indicate a role of mast cells and tryptase in the interplay between fibroblasts, epithelial cells and the alveolar extracellular matrix in health and lung disease.

Pathological Insight into 5-HT2B Receptor Activation in Fibrosing Interstitial Lung Diseases.

Interstitial lung disease (ILD) encompasses a heterogeneous group of more than 200 conditions, of which primarily idiopathic pulmonary fibrosis (IPF), idiopathic nonspecific interstitial pneumonia, hypersensitivity pneumonitis, ILD associated with autoimmune diseases and sarcoidosis may present a progressive fibrosing (PF) phenotype. Despite different aetiology and histopathological patterns, the PF-ILDs have similarities regarding disease mechanisms with self-sustaining fibrosis, which suggests that the diseases may share common pathogenetic pathways. Previous studies show an enhanced activation of serotonergic signaling in pulmonary fibrosis, and the serotonin (5-HT)2 receptors have been implicated to have important roles in observed profibrotic actions. Our research findings in support by others, demonstrate antifibrotic effects with 5-HT2B receptor antagonists, alleviating several key events common for the fibrotic diseases such as myofibroblast differentiation and connective tissue deposition. In this review, we will address the potential role of 5-HT and in particular the 5-HT2B receptors in three PF-ILDs: ILD associated with systemic sclerosis (SSc-ILD), ILD associated with rheumatoid arthritis (RA-ILD) and IPF. Highlighting the converging pathways in these diseases discloses the 5-HT2B receptor as a potential disease target for PF-ILDs, which today have an urgent unmet need for therapeutic strategies.

Silver Nanoparticles Alter Cell Viability Ex Vivo and in Vitro and Induce Proinflammatory Effects in Human Lung Fibroblasts.

Silver nanoparticles (AgNPs) are commonly used in commercial and medical applications. However, AgNPs may induce toxicity, extracellular matrix (ECM) changes and inflammatory responses. Fibroblasts are key players in remodeling processes and major producers of the ECM. The aims of this study were to explore the effect of AgNPs on cell viability, both ex vivo in murine precision cut lung slices (PCLS) and in vitro in human lung fibroblasts (HFL-1), and immunomodulatory responses in fibroblasts. PCLS and HFL-1 were exposed to AgNPs with different sizes, 10 nm and 75 nm, at concentrations 2 µg/mL and 10 µg/mL. Changes in synthesis of ECM proteins, growth factors and cytokines were analyzed in HFL-1. Ag10 and Ag75 affected cell viability, with significantly reduced metabolic activities at 10 µg/mL in both PCLS and HFL-1 after 48 h. AgNPs significantly increased procollagen I synthesis and release of IL-8, prostaglandin E2, RANTES and eotaxin, whereas reduced IL-6 release was observed in HFL-1 after 72 h. Our data indicate toxic effects of AgNP exposure on cell viability ex vivo and in vitro with altered procollagen and proinflammatory cytokine secretion in fibroblasts over time. Hence, careful characterizations of AgNPs are of importance, and future studies should include timepoints beyond 24 h.

VEGF synthesis and VEGF receptor 2 expression in patients with bronchiolitis obliterans syndrome after lung transplantation.

OBJECTIVES: Chronic lung allograft dysfunction including Bronchiolitis obliterans syndrome (BOS) is common after lung transplantation. Histologically, BOS is recognized as fibrotic lesions with accumulated extracellular matrix (ECM) in small airways. Lung fibroblasts are major producers of ECM and vascular endothelial growth factor (VEGF). In this study we hypothesize that VEGF is involved in BOS development after lung transplantation. METHODS: We investigated the effect of profibrotic transforming growth factor (TGF-ß) on VEGF synthesis in lung fibroblasts isolated from distal lung tissue biopsies taken from patients at 3, 6 and 12 months after lung transplantation (n = 14). Co-expression of VEGF receptor (VEGFR) 2 and collagen marker prolyl4-hydroxylase (p4OH) were analyzed in lung tissue from patients with BOS (n = 11). RESULTS: VEGF synthesis from distal derived lung fibroblasts were significantly lower 3 months after lung transplantation (168.6 ± 133.7; n = 7) compared to non-transplanted subjects (451.8 ± 185.9; n = 9; p = 0.0033) and increased over time at 6 months (584.1 ± 264.9; n = 9; p = 0.0033) and 12 months (451.1 ± 207.5; n = 8; p = 0.0065) post transplantation. TGF-ß significantly induced VEGF synthesis at all time points. At 12 months post transplantation there was significantly less VEGF synthesis after TGF-ß stimulation in fibroblasts obtained from BOS patients (1170 ± 450.2; n = 4) compared to patients without any chronic rejection process (1980 ± 417.9; n = 4; p < 0.039). The numbers of cells expressing VEGFR2/p4OH were increased in patients with BOS (33.2 ± 10.9; n = 11) compared to control subjects (10.1 ± 9.9; n = 11; p < 0.001). CONCLUSIONS: Our results support that changes in VEGF/VEGFR2 axis could be involved in BOS development and marker of poor outcome.

Pressurized carbon dioxide as a potential tool for decellularization of pulmonary arteries for transplant purposes.

Vascular bio-scaffolds produced from decellularized tissue offer a promising material for treatment of several types of cardiovascular diseases. These materials have the potential to maintain the functional properties of the extracellular matrix (ECM), and allow for growth and remodeling in vivo. The most commonly used methods for decellularization are based on chemicals and enzymes combinations, which often damage the ECM and cause cytotoxic effects in vivo. Mild methods involving pressurized CO2-ethanol (EtOH)-based fluids, in a supercritical or near supercritical state, have been studied for decellularization of cardiovascular tissue, but results are controversial. Moreover, data are lacking on the amount and type of lipids remaining in the tissue. Here we show that pressurized CO2-EtOH-H2O fluids (average molar composition, ΧCO2 0.91) yielded close to complete removal of lipids from porcine pulmonary arteries, including a notably decrease of pro-inflammatory fatty acids. Pressurized CO2-limonene fluids (ΧCO2 0.88) and neat supercritical CO2 (scCO2) achieved the removal of 90% of triacylglycerides. Moreover, treatment of tissue with pressurized CO2-limonene followed by enzyme treatment, resulted in efficient DNA removal. The structure of elastic fibers was preserved after pressurized treatment, regardless solvent composition. In conclusion, pressurized CO2-ethanol fluids offer an efficient tool for delipidation in bio-scaffold production, while pressurized CO2-limonene fluids facilitate subsequent enzymatic removal of DNA.

Matrisome Properties of Scaffolds Direct Fibroblasts in Idiopathic Pulmonary Fibrosis.

In idiopathic pulmonary fibrosis (IPF) structural properties of the extracellular matrix (ECM) are altered and influence cellular responses through cell-matrix interactions. Scaffolds (decellularized tissue) derived from subpleural healthy and IPF lungs were examined regarding biomechanical properties and ECM composition of proteins (the matrisome). Scaffolds were repopulated with healthy fibroblasts cultured under static stretch with heavy isotope amino acids (SILAC), to examine newly synthesized proteins over time. IPF scaffolds were characterized by increased tissue density, stiffness, ultimate force, and differential expressions of matrisome proteins compared to healthy scaffolds. Collagens, proteoglycans, and ECM glycoproteins were increased in IPF scaffolds, however while specific basement membrane (BM) proteins such as laminins and collagen IV were decreased, nidogen-2 was also increased. Findings were confirmed with histology, clearly showing a disorganized BM. Fibroblasts produced scaffold-specific proteins mimicking preexisting scaffold composition, where 11 out of 20 BM proteins were differentially expressed, along with increased periostin and proteoglycans production. We demonstrate how matrisome changes affect fibroblast activity using novel approaches to study temporal differences, where IPF scaffolds support a disorganized BM and upregulation of disease-associated proteins. These matrix-directed cellular responses emphasize the IPF matrisome and specifically the BM components as important factors for disease progression.

Efficient methodology for the extraction and analysis of lipids from porcine pulmonary artery by supercritical fluid chromatography coupled to mass spectrometry.

Pulmonary artery grafts are needed as cardiovascular bioprosthetics. For successful tissue recellularization after transplantation, lipids have to be removed from the donor artery. Developing a selective process to remove lipids without damaging the extracellular matrix greatly depends on knowing the amount and type of lipid compounds in the specific tissue. Here we present an efficient methodology for the study of lipids present in porcine pulmonary arteries. The performance of six extraction methods to recover lipids from artery was evaluated. For this purpose, a supercritical fluid chromatography method coupled to quadrupole time-of-flight mass spectrometry detection (UHPSFC/QTOF-MS) was adapted. The method enabled separation of lipids of a wide range of polarity according to lipid class in less than 7 min. One dichloromethane-based extraction method was shown to be the most efficient one for the recovery of lipids from pulmonary artery. However, one MTBE-based extraction method was able to show the highest fatty acid extraction yields (to the expense of longer extraction times). Lipids were relative quantified according to class, and the major species within each class were identified. Triacylglycerols and glycerophospholipids were the most abundant classes, followed by sphingomyelins, monoacylglycerols and fatty acyls. The matrix effect exerted no interference on the analytical method, except for some few combinations of extraction method and lipid class. These results are of relevance for lipidomic studies from solid tissue, in particular for studies on pulmonary and cardiovascular diseases. Finally, our work sets the basis for the further development of a selective processes to remove lipids from pulmonary artery without damaging the tissue prior to transplantation.

Chronic Obstructive Pulmonary Disease and Lung Cancer: Underlying Pathophysiology and New Therapeutic Modalities.

Chronic obstructive pulmonary disease (COPD) and lung cancer are major lung diseases affecting millions worldwide. Both diseases have links to cigarette smoking and exert a considerable societal burden. People suffering from COPD are at higher risk of developing lung cancer than those without, and are more susceptible to poor outcomes after diagnosis and treatment. Lung cancer and COPD are closely associated, possibly sharing common traits such as an underlying genetic predisposition, epithelial and endothelial cell plasticity, dysfunctional inflammatory mechanisms including the deposition of excessive extracellular matrix, angiogenesis, susceptibility to DNA damage and cellular mutagenesis. In fact, COPD could be the driving factor for lung cancer, providing a conducive environment that propagates its evolution. In the early stages of smoking, body defences provide a combative immune/oxidative response and DNA repair mechanisms are likely to subdue these changes to a certain extent; however, in patients with COPD with lung cancer the consequences could be devastating, potentially contributing to slower postoperative recovery after lung resection and increased resistance to radiotherapy and chemotherapy. Vital to the development of new-targeted therapies is an in-depth understanding of various molecular mechanisms that are associated with both pathologies. In this comprehensive review, we provide a detailed overview of possible underlying factors that link COPD and lung cancer, and current therapeutic advances from both human and preclinical animal models that can effectively mitigate this unholy relationship.

Mast cells and mast cell tryptase enhance migration of human lung fibroblasts through protease-activated receptor 2.

BACKGROUND: Mast cells may activate fibroblasts and contribute to remodeling processes in the lung. However, the mechanism behind these actions needs to be further investigated. Fibroblasts are major regulators of on-going remodeling processes. Protease activated receptor 2 (PAR2) expressed by fibroblasts may be activated by serine proteases, such as the mast cell mediator tryptase. The objective in this study was to investigate the effects of mast cells and specifically mast cell tryptase on fibroblast migration and the role of PAR2 activation. METHODS: Human lung fibroblasts (HFL-1) were cultured together with human peripheral blood-derived mast cells or LAD2 mast cells and stimulated with either conditioned medium from LAD2 cells or tryptase. Analyses of immunological stimulation of mast cells by IgE/anti IgE in the co-culture system were also performed. The importance of PAR2 activation by mast cells and mast cell tryptase for the migratory effects of fibroblasts was investigated by pre-treatment with the PAR2 antagonist P2pal-18S. The expression of PAR2 was analyzed on fibroblasts and mast cells. RESULTS: The migratory capacity of HFL-1 cells was enhanced by blood-derived mast cells (p < 0.02), LAD2 cells (p < 0.001), conditioned medium (p < 0.05) and tryptase (p < 0.006). P2pal-18S decreased the induced migration caused by mast cells (p < 0.001) and tryptase (p < 0.001) and the expression of PAR2 was verified in HFL-1 cells. Mast cells immunologically stimulated with IgE/Anti IgE had no further effects on fibroblast migration. CONCLUSIONS: Mast cells and the mast cell mediator tryptase may have crucial roles in inducing lung fibroblast migration via PAR-2 activation, which may contribute to remodeling processes in chronic lung diseases.

Quantifying extracellular matrix turnover in human lung scaffold cultures.

Remodelling of the extracellular matrix is accomplished by altering the balance between matrix macromolecule production and degradation. However, it is not well understood how cells balance production of new matrix molecules and degradation of existing ones during tissue remodelling and regeneration. In this study, we used decellularized lung scaffolds repopulated with allogenic lung fibroblasts cultured with stable isotope labelled amino acids to quantify the balance between matrix production and degradation at a proteome-wide scale. Specific temporal dynamics of different matrisome proteins were found to correspond to the proliferative activity of the repopulating cells and the degree of extracellular deposition. The remodeling of the scaffold was characterized by an initial phase with cell proliferation and high production of cell adhesion proteins such as emilin-1 and fibronectin. Extended culture time resulted in increased levels of core matrisome proteins. In a comparison with monolayer cultures on plastic, culture in lung scaffolds lead to a pronounced accumulation of proteoglycans, such as versican and decorin, resulting in regeneration of an extracellular matrix with greater resemblance to native lung tissue compared to standard monolayer cultures. Collectively, the study presents a promising technique for increasing the understanding of cell- extracellular matrix interactions under healthy and diseased conditions.

Pulmonary fibrosis in vivo displays increased p21 expression reduced by 5-HT2B receptor antagonists in vitro - a potential pathway affecting proliferation.

Serotonin (5-hydroxytryptamine) has repeatedly been associated with the development of fibrotic disorders such as pulmonary fibrosis. By blocking the binding of 5-HT to 5-HT2B receptors with receptor antagonists, several pro-fibrotic mechanisms can be inhibited. Bleomycin-induced pulmonary fibrosis is a model used to evaluate pathological mechanisms and pharmacological interventions. Previously we have shown attenuated fibrosis in systemic bleomycin-treated mice following treatment with two 5-HT2B receptor antagonists (EXT5 and EXT9). Our aim is to further identify cellular effects and signaling pathways associated with the anti-fibrotic effects of EXT5/9. Gene expressions in lung tissues from systemic bleomycin-treated mice were examined, revealing significant increased expression of Cdkn1α (a gene coding for p21), particularly in distal regions of the lung. In vitro studies in human lung fibroblasts revealed increased levels of p21 (p = 0.0032) and pAkt (p = 0.12) following treatment with 5-HT (10 µM). The induction of p21 and pAkt appears to be regulated by 5-HT2B receptors, with diminished protein levels following EXT9-treatment (p21 p = 0.0024, pAkt p = 0.15). Additionally, 5-HT induced fibroblast proliferation, an event significantly reduced by EXT5 (10 µM) and EXT9 (10 µM). In conclusion, our results suggest that 5-HT2B receptor antagonism attenuates pulmonary fibrosis in part by anti-proliferative effects, associated with inhibited pAkt/p21 signaling pathway.

Effects of 5-Hydroxytryptamine Class 2 Receptor Antagonists on Bronchoconstriction and Pulmonary Remodeling Processes.

Serotonin [5-hydroxytryptamine (5-HT)] is associated with several chronic pulmonary diseases, recognizing 5-HT2 receptor antagonists as potential inhibitors of tissue remodeling. However, the effects of 5-HT2 receptors, especially 5-HT2B receptors on airway function and remodeling, are unclear. We investigated the role of 5-HT2B receptors on airway smooth muscle contractility and remodeling processes. Murine precision-cut lung slices were pretreated with 5-HT2B receptor antagonists (EXT5, EXT9, RS 127445, and PRX 08066), as well as ketanserin (5-HT2A/2C receptor antagonist) (1, 10 µmol/L), before addition of cumulative concentrations of 5-HT to induce bronchoconstriction. Remodeling effects after treatment with 10 µmol/L 5-HT and 5-HT2 receptor antagonists were further studied in distal lung tissue by examining release of profibrotic transforming growth factor (TGF)-ß1 and proliferation of human bronchial smooth muscle cells (HBSMCs). 5-HT-induced bronchoconstriction was significantly reduced by EXT5, EXT9, and ketanserin, but not by RS 127445 or PRX 08066. The 5-HT2B receptor antagonists significantly reduced TGF-ß1 release. 5-HT, in combination with TGF-ß1, increased proliferation of HBSMCs, a process reduced by EXT5 and EXT9. Our results indicate that EXT5 and EXT9 may relieve bronchoconstriction in murine airways and serve as an add-on effect in attenuating pulmonary remodeling by improving airway function. The antiproliferative effect on HBSMCs and the inhibition of TGF-ß1 release further support a role of 5-HT2B receptors in pathologic remodeling processes.