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.

BackgroundMast 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.MethodsHuman 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.ResultsThe 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.ConclusionsMast 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.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0269-3) contains supplementary material, which is available to authorized users.

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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.

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 co 2 -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 co 2 -etoH-H 2 O 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 co 2 -limonene fluids (Χ CO2 0.88) and neat supercritical CO 2 (scco 2 ) achieved the removal of 90% of triacylglycerides. Moreover, treatment of tissue with pressurized co 2 -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 co 2 -ethanol fluids offer an efficient tool for delipidation in bio-scaffold production, while pressurized CO 2 -limonene fluids facilitate subsequent enzymatic removal of DNA.Cardiovascular diseases (CVDs) are responsible for 17.9 million deaths per year in the world (31% of total deaths) 1 . In 2015, the global prevalence of arterial hypertension (AHT), the most prevalent risk factor for CVD development, was estimated to be around 30-45% of the adult population, increasing up to 60% in people above 60 years of age 2 . Moreover, the prevalence of AHT is estimated to increase by 15-20% in 2025 2 . Pulmonary arterial hypertension (PAH), a sub-form of AHT, is characterized by breakdown of elastic fibers and alterations in the cross-linking of collagen, resulting in remodeling the extracellular matrix (ECM) in pulmonary arteries 3,4 . Hypertrophic remodeling of the media and endothelial cell dysfunction result in a high vascular resistance and thrombosis 4,5 , potentially leading to right ventricular failure and death in severely affected patients.Organ or tissue transplantation is the last option proposed for such CVDs-affected patients with a poor prognosis. However, the lack of compatible organs and tissues constitutes a major limitation. Even though the global rate of transplantation increased by 7.25% between 2015 and 2016, reaching a rate of 15.5 organs transplanted per hour 6 , less than 10% of the transplant needs are covered. Consequently, patients often have to wait long time for transplantation, resulting in worsening of their medical condition. Furthermore, those that are offered a transplantation require life-long immune therapy to redu...

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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 minutes. 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.