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. 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. 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. 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.

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.

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.

Background and objective: Involvement of pulmonary vascular remodelling is a characteristic sign in COPD. Vascular mediators such as vascular endothelial growth factor (VEGF) and prostacyclin may regulate fibroblast activity. The objective was to study the synthesis of VEGF and interactions with prostacyclin and transforming growth factor (TGF)‐β₁ in lung fibroblasts from patients with COPD and healthy control subjects. To further explore the autocrine role of synthesized VEGF on fibroblast activity, studies were performed in human lung fibroblasts (HFL‐1).Methods: Primary distal lung fibroblast cultures were established from healthy individuals and from COPD patients (GOLD stage IV). Lung fibroblasts were stimulated with the prostacyclin analogue iloprost and the profibrotic stimuli TGF‐β₁. VEGF synthesis was measured in the cell culture medium. Changes in proliferation rate, migration and synthesis of the extracellular matrix (ECM) proteins proteoglycans were analysed after stimulations with VEGF‐A isoform 165 (VEGF₁₆₅; 1–10 000 pg/mL) in HFL‐1.Results: Iloprost and TGF‐β₁ significantly increased VEGF synthesis in both fibroblasts from COPD patients and control subjects. TGF‐β₁‐induced VEGF synthesis was significantly reduced by the cyclooxygenase inhibitor indomethacin in fibroblasts from COPD patients. VEGF significantly increased proliferation rate and migration capacity in HFL‐1. VEGF also significantly increased synthesis of the ECM proteins biglycan and perlecan. The VEGF receptors (VEGFR), VEGFR1, VEGFR2 and VEGFR3, were all expressed in primary lung fibroblasts and HFL‐1.Conclusion: VEGF is synthesized in high amounts by distal lung fibroblasts and may have a crucial role in ongoing vascular remodelling processes in the distal lung compartments.

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, Χ_CO2 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 (Χ_CO2 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.