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Background: 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. Methods: Primary distally-derived lung fibroblasts obtained from IPF patients and healthy individuals were cultured with LAD2 mast cells or stimulated with the mast cell proteases tryptase and chymase for 72 hours. Human lung fibroblasts and mast cells were cultured on traditional cell culture plastics or decellularized human lung tissue slices (lung scaffolds) to create a more physiological milieu by providing an alveolar extracellular matrix. Released mediators were analyzed and further evaluated for effects on epithelial cell migration. Results: Tryptase increased IL-6, VEGF and HGF release whereas co-culture with mast cells increased IL-6 in healthy lung fibroblasts. VEGF-C release was significantly increased in IPF fibroblasts and further induced by mast cells compared to healthy fibroblasts. Tryptase induced fibroblast migration but mast cells did not promote a myofibroblast phenotype such as altered αSMA levels. Culture in lung scaffolds increased the release of HGF and VEGF compared to culture on plastic plates. Migration of A549 epithelial cells was significantly reduced by IL-6, while HGF and conditioned media from scaffold cultures with mast cells and fibroblasts promoted migration. Conclusions: Mast cells and tryptase increased fibroblast release of mediators that influenced epithelial migration. These data indicate a role of mast cells in the interplay between fibroblasts, epithelial cells and the alveolar extracellular matrix in the pathogenesis of lung diseases.

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.

Pulmonary vascular remodelling is a characteristic sign in different lung disorders. Vascular endothelial growth factor (VEGF) is a key mediator in vascular remodelling. The objective was to study the role of VEGF on fibroblast activity, such as extracellular matrix (ECM) synthesis, migration and proliferative capacity and to study if VEGF is affected in different lung disorders. Studies were performed in human lung fibroblasts (HFL-1) and primary distal fibroblasts cultures from control subjects, patients with COPD (GOLD IV) and patients with bronchiolitis obliterans syndrome (BOS). Lung fibroblasts were stimulated with TGF-b1. VEGF165 synthesis was measured in the cell culture medium. Changes in synthesis of the ECM proteins proteoglycans and procollagen I, proliferation rate and migration were analysed after stimulations with VEGF165 (1-10000 pg/ml). TGF-β1 significantly enhanced VEGF synthesis in all cell types. There were no differences in VEGF synthesis between fibroblasts from control subjects and COPD patients, whereas patients with BOS had a higher VEGF synthesis (p In conclusion, VEGF is promoting specific ECM synthesis and fibroblast activation. VEGF may have a crucial role in the interplay between fibroblasts and other cells in vascular remodelling processes in the distal lung.

Many lung-transplanted (LTx) patients develop obliterative bronchiolitis (OB) with deposition of extracellular matrix, accompanied by fibrocyte infiltration and increased lumen of vessels. In this study we hypothesize that fibrocytes, VEGF and prostacyclin (PGI2) are involved in remodelling processes after LTx. We investigated TGF-β activated peripheral fibroblast phenotypes regarding production of VEGF 165 /PGI2. Fibrocytes and fibroblast phenotypes were identified by prolyl4-hydroxylase (p4OH), CD45, VEGFR2 or PGIR. VEGF production from fibroblasts after LTx significantly decreased 3mo after LTx compared to healthy controls (p

Background: The mesenchymal stromal cells (MSC) are potent candidates for cell therapies, due to their immune-regulatory and regenerative properties. In most clinical trials bone marrow-derived MSC have been used, also for treatment of various pulmonary diseases. Bone marrow-derived MSC play an important role within the bone marrow, by providing and maintaining a functioning niche for the hematopoiesis. The functional role of lung-resident MSC, on the other hand, is still unknown. Exploring the proteins produced by lung-derived MSC might improve our understanding of the functional role of MSC within the lung. Therefore, we aimed to characterize the proteins produced by lung-derived MSC and to compare those findings to the proteins produced by MSC isolated from bone marrow. Methods: Proteins within the cell-layer and the conditioned medium of MSC isolated from lung biopsies and bone marrow aspirates were characterized using mass spectrometry-based quantitative proteomics. Results: Our results indicate that MSC isolated from lung biopsies and bone marrow aspirates are prominent producers of extracellular matrix proteins. Furthermore, these analyses revealed differences between lung- and bone marrow-derived MSC, both regarding proteins identified in the cell layer and within the conditioned medium. Conclusions: Lung-derived MSC are prominent extracellular matrix producers, and our results indicate that lung- and bone marrow-derived MSC produce different protein profiles.

Rationale: Idiopathic pulmonary fibrosis (IPF) is characterized by loss of lung function due to aberrant deposition of extracellular matrix (ECM) constituents. One such constituent is glycosaminoglycans (GAGs) consisting of unbranched polymers of repeating disaccharides. They are highly negatively charged which renders them the ability to interact with important mediators in the IPF pathogenesis. The abundance and localisation of GAGs in relation to tissue density is not known in IPF. The aim of this study was therefore to measure GAGs in lung samples from IPF patients and from control subjects. Methods: Lung tissue samples were collected from more or less dense areas in distal lungs from IPF patients (n=8) and from donor lungs (n=4). GAGs were isolated and quantified by HPLC after fluorochrome labelling. The total collagen content was quantified by the amount of hydroxyproline and fibrillar collagen was measured by picro-sirius red staining. Results: The total amount of heparan sulfate (HS), chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA) was significantly higher in denser areas of IPF lungs compared to less dense areas (p ≤ 0.01 for HS, p ≤ 0.05 for CS/DS and p ≤ 0.01 for HA) and also compared to control lungs. HA was significantly increased in less fibrotic areas compared to control lungs. Moreover, the amount of GAGs in IPF tissue correlated with the volume fraction of picro-sirius red stain but not with the hydroxyproline content. Conclusions: Our results show that GAGs are quantatively elevated in fibrotic lesions characterized by high tissue density and increased levels of fibrillar collagen in IPF. This may create an altered ECM niche that promotes an active remodelling process.