Chronic obstructive pulmonary disease (COPD) is a progressive condition characterized by airway remodeling, including emphysema and fibrosis. Proteoglycans and their glycosaminoglycan (GAG) chains are key components of the extracellular matrix and may be altered as the disease advances. This study analyzed lung tissue from COPD patients (GOLD stages II-III and IV), non-COPD smokers, and non-smokers to assess proteoglycan and GAG changes. While LC-MS revealed no alterations in chondroitin/dermatan sulfate (CS/DS) or heparan sulfate (HS) proteoglycan core proteins, the total GAG level increased in GOLD II-IV patients. HS displayed increased N- and 2-O-sulfation in GOLD IV, while CS/DS levels and 4-O-sulfation were enhanced across GOLD II-IV. These findings were supported by transcriptomic data indicating upregulation of CHST11, the main CS/DS 4-O-sulfotransferase. In line with previous findings, TGF-β signaling was shown to be enriched in COPD patients and to regulate the CHST11 expression. These results were confirmed by TGF-β stimulation of lung fibroblasts showing increased CS/DS levels, 4-O-sulfation, and CHST11 expression. In conclusion, COPD is associated with disease-stage-specific changes in GAG sulfation, particularly enhanced CS/DS 4-O-sulfation that is likely to be driven by TGF-β. These alterations may contribute to extracellular matrix remodeling and represent potential targets for therapeutic intervention to mitigate disease progression.

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-β1) 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-β1 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.

SARS-CoV-2 has been shown to predominantly infect the airways and the respiratory tract and too often have an unpredictable and different pathologic pattern compared to other respiratory diseases. Current clinical diagnostical tools in pulmonary medicine expose patients to harmful radiation, are too unspecific or even invasive. Proteomic analysis of exhaled breath particles (EBPs) in contrast, are non-invasive, sample directly from the pathological source and presents as a novel explorative and diagnostical tool. Patients with PCR-verified COVID-19 infection (COV-POS, n = 20), and patients with respiratory symptoms but with > 2 negative polymerase chain reaction (PCR) tests (COV-NEG, n = 16) and healthy controls (HCO, n = 12) were prospectively recruited. EBPs were collected using a “particles in exhaled air” (PExA 2.0) device. Particle per exhaled volume (PEV) and size distribution profiles were compared. Proteins were analyzed using liquid chromatography-mass spectrometry. A random forest machine learning classification model was then trained and validated on EBP data achieving an accuracy of 0.92. Significant increases in PEV and changes in size distribution profiles of EBPs was seen in COV-POS and COV-NEG compared to healthy controls. We achieved a deep proteome profiling of EBP across the three groups with proteins involved in immune activation, acute phase response, cell adhesion, blood coagulation, and known components of the respiratory tract lining fluid, among others. We demonstrated promising results for the use of an integrated EBP biomarker panel together with particle concentration for diagnosis of COVID-19 as well as a robust method for protein identification in EBPs. Our results demonstrate the promising potential for the use of EBP fingerprints in biomarker discovery and for diagnosing pulmonary diseases, rapidly and non-invasively with minimal patient discomfort.

Background: Alveolar type 2 epithelial cells (AEC2) secrete important surfactants and act as alveolar progenitor cells, but their involvement in lung extracellular matrix (ECM) production is not fully understood. The aim was to examine the capacity of AEC2 to produce ECM and their contribution to ECM remodeling by using a new culture model. Methods: The marker HT2-280 was used to isolate primary AEC2 from healthy donor lungs and from end-stage lungs from COPD patients. Cryopreserved AEC2 were then cultured in non-diseased human decellularized lung slices (DLS), without prior expansion on tissue culture plastic, for 13 days. AEC2-derived proteins were separated from proteins in the DLS using stable isotope labeling by amino acids in cell culture. Healthy AEC2 were treated with TGF-β1 to evaluate matrisome changes. Phenotypic markers, matrisome genes and proteins were evaluated by RNA-sequencing, mass spectrometry and immunohistochemistry. Results: AEC2 in the culture model showed similar marker profile as freshly isolated AEC2. The AEC2 matrisome profile expressed basement membrane components but also a complex set of interstitial ECM proteins. AEC2 from COPD patients resembled healthy AEC2 when cultured in healthy derived DLS, however, COPD-derived AEC2 retained expression of the disease marker HLA-A. TGF-β1 stimuli induced a pronounced increase in interstitial matrix production from healthy AEC2. Conclusions: This study reveal that AEC2 maintain their phenotype when cultured in human decellularized lung slices. The AEC directly contribute to ECM turnover and may thereby have a more active role in pathological lung remodelling.

Lung transplantion (LTx) recipients have low long-term survival and a high incidence of bronchiolitis obliterans syndrome (BOS), an inflammation of the small airways in chronic rejection of a lung allograft. There is great clinical need for a minimally invasive biomarker of BOS. Here, 644 different proteins were analyzed to detect biomarkers that distinguish BOS grade 0 from grades 1-3. The plasma of 46 double lung transplant patients was analyzed for proteins using a high-component, multiplex immunoassay that enables analysis of protein biomarkers. Proximity Extension Assay (PEA) consists of antibody probe pairs which bind to targets. The resulting polymerase chain reaction (PCR) reporter sequence can be quantified by real-time PCR. Samples were collected at baseline and 1-year post transplantation. Enzyme-linked immunosorbent assay (ELISA) was used to validate the findings of the PEA analysis across both time points and microarray datasets from other lung transplantation centers demonstrated the same findings. Significant decreases in the plasma protein levels of CRH, FERC2, IL-20RA, TNFB, and IGSF3 and an increase in MMP-9 and CTSL1 were seen in patients who developed BOS compared to those who did not. In this study, CRH is presented as a novel potential biomarker in the progression of disease because of its decreased levels in patients across all BOS grades. Additionally, biomarkers involving the remodeling of the extracellular matrix (ECM), such as MMP-9 and CTSL1, were increased in BOS patients.

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.

Despite improvements, lung transplantation remains hampered by both a scarcity of donor organs and by mortality following primary graft dysfunction (PGD). Since acute respiratory distress syndrome (ARDS) limits donor lungs utilization, we investigated cytokine adsorption as a means of treating ARDS donor lungs. We induced mild to moderate ARDS using lipopolysaccharide in 16 donor pigs. Lungs were then treated with or without cytokine adsorption during ex vivo lung perfusion (EVLP) and/or post-transplantation using extracorporeal hemoperfusion. The treatment significantly decreased cytokine levels during EVLP and decreased levels of immune cells post-transplantation. Histology demonstrated fewer signs of lung injury across both treatment periods and the incidence of PGD was significantly reduced among treated animals. Overall, cytokine adsorption was able to restore lung function and reduce PGD in lung transplantation. We suggest this treatment will increase the availability of donor lungs and increase the tolerability of donor lungs in the recipient.

Background: Screening decreases mortality among lung cancer patients but is not widely implemented, thus there is an unmet need for an easily accessible non-invasive method to enable early diagnosis. Particles in exhaled air offer a promising such diagnostic tool. We investigated the validity of a particles in exhaled air device (PExA) to measure the particle flow rate (PFR) and collect exhaled breath particles (EBP) to diagnose primary lung adenocarcinoma (LUAD).Methods: Seventeen patients listed for resection of LUAD stages IA-IIIA and 18 non-cancer surgical control patients were enrolled. EBP were collected before and after surgery for LUAD, and once for controls. Proteomic analysis was carried out using a proximity extension assay technology. Results were validated in both plasma from the same cohort and with microarray data from healthy lung tissue and LUAD tissue in the GSE10072 dataset.Results: Of the 92 proteins analyzed, levels of five proteins in EBP were significantly higher in the LUAD patients compared to controls. Levels of phospholipid transfer protein (PLTP) and hepatocyte growth factor receptor (MET) decreased in LUAD patients after surgery compared to control patients. PFR was significantly higher in the LUAD cohort at all timepoints compared to the control group. MET in plasma correlated significantly with MET in EBP.Conclusion: Collection of EBP and measuring of PFR has never been performed in patients with LUAD. In the present study PFR alone could distinguish between LUAD and patients without LUAD. PLTP and MET were identified as potential biomarkers to evaluate successful tumor excision.

Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation, small airway remodeling, and emphysema. Airway remodeling in patients with COPD involves both the airway epithelium and the subepithelial extracellular matrix (ECM). However, it is currently unknown how epithelial remodeling in COPD airways depends on the relative influence from inherent defects in the epithelial cells and alterations in the ECM. To address this, we analyzed global gene expression in COPD human bronchial epithelial cells (HBEC) and normal HBEC after repopulation on decellularized bronchial scaffolds derived from patients with COPD or donors without COPD. COPD HBEC grown on bronchial scaffolds showed an impaired ability to initiate ciliated-cell differentiation, which was evident on all scaffolds regardless of their origin. In addition, although normal HBEC were less affected by the disease state of the bronchial scaffolds, COPD HBEC showed a gene expression pattern indicating increased proliferation and a retained basal-cell phenotype when grown on COPD bronchial scaffolds compared with normal bronchial scaffolds. By using mass spectrometry, we identified 13 matrisome proteins as being differentially abundant between COPD bronchial scaffolds and normal bronchial scaffolds. These observations are consistent with COPD pathology and suggest that both epithelial cells and the ECM contribute to epithelial-cell remodeling in COPD airways.

In severe acute respiratory distress syndrome (ARDS), extracorporeal membrane oxygenation (ECMO) is a life-prolonging treatment, especially among COVID-19 patients. Evaluation of lung injury progression is challenging with current techniques. Diagnostic imaging or invasive diagnostics are risky given the difficulties of intra-hospital transportation, contraindication of biopsies, and the potential for the spread of infections, such as in COVID-19 patients. We have recently shown that particle flow rate (PFR) from exhaled breath could be a noninvasive, early detection method for ARDS during mechanical ventilation. We hypothesized that PFR could also measure the progress of lung injury during ECMO treatment. Lipopolysaccharide (LPS) was thus used to induce ARDS in pigs under mechanical ventilation. Eight were connected to ECMO, whereas seven animals were not. In addition, six animals received sham treatment with saline. Four human patients with ECMO and ARDS were also monitored. In the pigs, as lung injury ensued, the PFR dramatically increased and a particular spike followed the establishment of ECMO in the LPS-treated animals. PFR remained elevated in all animals with no signs of lung recovery. In the human patients, in the two that recovered, PFR decreased. In the two whose lung function deteriorated while on ECMO, there was increased PFR with no sign of recovery in lung function. The present results indicate that real-time monitoring of PFR may be a new, complementary approach in the clinic for measurement of the extent of lung injury and recovery over time in ECMO patients with ARDS.

Despite improvements, lung transplantation (LTx) remains hampered by both a scarcity of donor organs and by mortality following primary graft dysfunction (PGD). Since acute respiratory distress syndrome (ARDS) limits donor lungs utilization, we investigated cytokine filtration as a means of restoring ARDS donor lungs. We induced mild to moderate ARDS using lipopolysaccharide in 12 donor pigs. Lungs were then treated with or without cytokine filtration during ex vivo lung perfusion (EVLP) and post-transplantation using extracorporeal hemoperfusion. The treatment significantly decreased cytokine levels during EVLP and decreased levels of immune cells post-transplantation. Histology demonstrated fewer signs of lung injury across both treatment periods and the incidence of PGD was significantly reduced among treated animals. Overall, cytokine filtration was able to restore lung function and reduce PGD in lung transplantation. We suggest this treatment will increase the availability of donor lungs and increase the tolerability of donor lungs in the recipient.

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.

Chronic lung allograft dysfunction (CLAD) is a severely limiting factor to the long-term survival of lung transplant recipients (LTx). Detection of CLAD is hampered by a lack of clinically available markers. Exhaled breath particles (EBP) is proposed as a noninvasive means of identifying CLAD patients and observing their condition. This pilot study aims to capture the range of EBP expected in human samples and identify possible candidate markers for further study. EBP are collected using a PExA device. Samples were obtained from healthy subjects (a combined sample of 600 ng and a 200 ng from each) and from five patients (one grouped sample of 600 ng from three patients and two 200 ng samples from two unique patients). The samples were analyzed via tandem mass spectrometry (MS). Up to 650 proteins were identified in the EBP samples identified. The fifty proteins identified represented a breadth of biological function and displayed a variety of localizations. Ten could be traced back to immunological system processes and included a number of immunoglobulin constant regions. Sixteen of these proteins could be found in the extracellular region. EBP presents as a novel method for the non-invasive analysis of LTx with the potential to monitor for the development of CLAD. This pilot study characterizes the range of proteins that can be identified from samples across subjects and serves as the basis for the exploration of markers in LTx. The findings of this work serve as the establishing method for the ongoing effort to use EBP to identify and monitor CLAD in LTx recipients.

Acute respiratory distress syndrome (ARDS) is a common cause of death in the intensive care unit, with mortality rates of ~30–40%. To reduce invasive diagnostics such as bronchoalveolar lavage and time-consuming in-hospital transports for imaging diagnostics, we hypothesized that particle flow rate (PFR) pattern from the airways could be an early detection method and contribute to improving diagnostics and optimizing personalized therapies. Porcine models were ventilated mechanically. Lipopolysaccharide (LPS) was administered endotracheally and in the pulmonary artery to induce ARDS. PFR was measured using a customized particles in exhaled air (PExA 2.0) device. In contrast to control animals undergoing mechanical ventilation and receiving saline administration, animals who received LPS developed ARDS according to clinical guidelines and histologic assessment. Plasma levels of TNF-α and IL-6 increased significantly compared with baseline after 120 and 180 min, respectively. On the other hand, the PFR significantly increased and peaked 60 min after LPS administration, i.e., ~30 min before any ARDS stage was observed with other well-established outcome measurements such as hypoxemia, increased inspiratory pressure, and lower tidal volumes or plasma cytokine levels. The present results imply that PFR could be used to detect early biomarkers or as a clinical indicator for the onset of ARDS.

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-beta) 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-beta significantly induced VEGF synthesis at all time points. At 12 months post transplantation there was significantly less VEGF synthesis after TGF-beta 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.

Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. Using a commonly utilized detergent-based decellularization approach in human autopsy lungs resulted in disproportionate losses of GAGs with depletion of chondroitin sulfate/dermatan sulfate (CS/DS) > heparan sulfate (HS) > hyaluronic acid (HA). Specific changes in disaccharide composition of remaining GAGs were observed with disproportionate loss of NS and NS2S for HS groups and of 4S for CS/DS groups. No significant influence of smoking history, sex, time to autopsy, or age was observed in native vs. decellularized lungs. Notably, surface plasmon resonance demonstrated that GAGs remaining in decellularized lungs were unable to bind key matrix-associated growth factors FGF2, HGF, and TGF beta 1. Growth of lung epithelial, pulmonary vascular, and stromal cells cultured on the surface of or embedded within gels derived from decellularized human lungs was differentially and combinatorially enhanced by replenishing specific GAGs and FGF2. HGF, and TGF beta 1. In summary, lung decellularization results in loss and/or dysfunction of specific GAGs or side chains significantly affecting matrix-associated growth factor binding and lung cell metabolism. GAG and matrix-associated growth factor replenishment thus needs to be incorporated into schemes for investigations utilizing gels and other materials produced from decellularized human lungs. Statement of significance Despite progress in use of decellularized lung scaffolds in ex vivo lung bioengineering schemes, including use of gels and other materials derived from the scaffolds, the detailed composition and functional role of extracellular matrix (ECM) proteoglycans (PGs) and their glycosaminoglycan (GAG) chains remaining in decellularized lungs, is poorly understood. In the current studies, we demonstrate that glycosaminoglycans (GAGS) are significantly depleted during decellularization and those that remain are dysfunctional and unable to bind matrix-associated growth factors critical for cell growth and differentiation. Systematically repleting GAGs and matrix-associated growth factors to gels derived from decellularized human lung significantly and differentially affects cell growth. These studies highlight the importance of considering GAGs in decellularized lungs and their derivatives. (C) 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Background: Protease activated receptor 2 (PAR2) is highly expressed on various cells. Although the well documented effects of PAR2 in innate immune responses to inflammation, the knowledge of PAR2 in the pathogenesis of chronic lung diseases is limited. The objective was to investigate the role of PAR2 and its effect on fibroblasts regarding changes in phenotype profile and mediator release in fibroblast monoculture and in co-culture with mast cells. Methods: The experiments were performed in 2D in vitro cultures on cell culture plastic and in a 3D ex vivo model where the cells were seeded on decellularized human lung scaffolds in order to mimic a more physiological milieu for the cells to interact. Human lung fibroblasts (HFL-1) were co-cultured with LAD2 mast cells with and without the PAR2 antagonist, P2pal-18s. The fibroblast morphology, distribution of a-smooth muscle actin protein and mediator profile were analysed. Results: The PAR-2 antagonist induced an altered fibroblast phenotype to a more elongated shape and increased aSMA expression. The PAR2 antagonist significantly induced a different mediator profile, where Il-6 (p=0.003), VEGF (p=0.004) and HGF synthesis (p=0.001) increased in fibroblasts co-cultured with mast cells. The mediators were differently synthesised when the cells were co-cultured in human lung scaffolds compared to 2D culture on plastic. Conclusions: PAR2 antagonism promote important effects on human lung fibroblast function with alterations in mediator release and morphological changes. These data indicate an important role of PAR2 in acute as well as chronic inflammatory diseases in which fibroblasts and mast cells are involved.

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. (C) 2019 Elsevier B.V. All rights reserved.

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.