Publications by authors named "Gunilla Westergren Thorsson"

103 Publications

Impaired Differentiation of COPD Bronchial Epithelial Cells Grown on Bronchial Scaffolds.

Am J Respir Cell Mol Biol 2021 Apr 21. Epub 2021 Apr 21.

AstraZeneca, Gothenburg, Sweden;

Chronic obstructive pulmonary disease (COPD) is characterized by airway inflammation, small airway remodeling and emphysema. Airway remodeling in COPD patients 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 and normal human bronchial epithelial cells (HBEC) after repopulation on decellularized bronchial scaffolds derived from COPD patients or non-COPD donors. 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, while 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 compared to normal bronchial scaffolds. Mass spectrometry identified thirteen matrisome proteins as differentially abundant between COPD 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. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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http://dx.doi.org/10.1165/rcmb.2019-0395OCDOI Listing
April 2021

The structure of human dermatan sulfate epimerase 1 emphasizes the importance of C5-epimerization of glucuronic acid in higher organisms.

Chem Sci 2021 Feb 8;12(5):1869-1885. Epub 2020 Dec 8.

Department of Experimental Medical Science , Lund University , Lund , Sweden . Email:

Dermatan sulfate epimerase 1 (DS-epi1, EC 5.1.3.19) catalyzes the conversion of d-glucuronic acid to l-iduronic acid on the polymer level, a key step in the biosynthesis of the glycosaminoglycan dermatan sulfate. Here, we present the first crystal structure of the catalytic domains of DS-epi1, solved at 2.4 Å resolution, as well as a model of the full-length luminal protein obtained by a combination of macromolecular crystallography and targeted cross-linking mass spectrometry. Based on docking studies and molecular dynamics simulations of the protein structure and a chondroitin substrate, we suggest a novel mechanism of DS-epi1, involving a His/double-Tyr motif. Our work uncovers detailed information about the domain architecture, active site, metal-coordinating center and pattern of -glycosylation of the protein. Additionally, the structure of DS-epi1 reveals a high structural similarity to proteins from several families of bacterial polysaccharide lyases. DS-epi1 is of great importance in a range of diseases, and the structure provides a necessary starting point for design of active site inhibitors.
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http://dx.doi.org/10.1039/d0sc05971dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8006597PMC
February 2021

Human Primary Airway Basal Cells Display a Continuum of Molecular Phases from Health to Disease in COPD.

Am J Respir Cell Mol Biol 2021 Mar 31. Epub 2021 Mar 31.

Lund University, 5193, Molecular Medicine and Gene Therapy, Lund, Sweden;

Airway basal cells are crucial for regeneration of the human lung airway epithelium, and are thought to be important contributors to Chronic Obstructive Pulmonary Disease (COPD) and other lung disorders. In order to reveal how basal cells contribute to disease, and to discover novel therapeutic targets, these basal cells need to be further characterized. In this study, we optimized a flow cytometry-based cell sorting protocol for primary human airway basal cells dependent on cell size and Nerve-Growth Factor Receptor (NGFR) expression. The basal cell population was found to be molecularly and functionally heterogeneous in contrast to cultured basal cells. In addition, significant differences were found such as KRT14 expression exclusively existing in cultured cells. Also, colony-forming capacity was significantly increased in cultured cells showing a clonal enrichment in vitro. Next, by single cell RNA sequencing on primary basal cells from healthy donors and GOLD stage IV COPD patients, the gene expression revealed a continuum ranging from healthy basal cell signatures to diseased basal cell phenotypes. We identified several upregulated genes that may indicate COPD, such as stress response related genes GADD45B and AHSA1, along with genes involved in the response to hypoxia such as CITED2 and SOD1. Taken together, the presence of healthy basal cells in stage IV COPD demonstrates the potential for regeneration through the discovery of novel therapeutic targets. In addition, we show the importance of studying primary basal cells when investigating disease mechanisms as well as for developing future cell-based therapies in the human lung.
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http://dx.doi.org/10.1165/rcmb.2020-0464OCDOI Listing
March 2021

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

Int J Mol Sci 2021 Jan 6;22(2). Epub 2021 Jan 6.

Unit of Lung Biology, Department of Experimental Medical Sciences, Lund University, 221 84 Lund, Sweden.

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.
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http://dx.doi.org/10.3390/ijms22020506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7825515PMC
January 2021

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

Int J Mol Sci 2020 Dec 28;22(1). Epub 2020 Dec 28.

Lung Biology, Department of Experimental Medical Science, Lund University, BMC C12, 22184 Lund, Sweden.

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) receptors have been implicated to have important roles in observed profibrotic actions. Our research findings in support by others, demonstrate antifibrotic effects with 5-HT 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-HT 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-HT receptor as a potential disease target for PF-ILDs, which today have an urgent unmet need for therapeutic strategies.
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http://dx.doi.org/10.3390/ijms22010225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7796180PMC
December 2020

Stem Cells, Cell Therapies, and Bioengineering in Lung Biology and Disease 2019.

ERJ Open Res 2020 Oct 19;6(4). Epub 2020 Oct 19.

Hastings Center for Pulmonary Research, Dept of Medicine, University of Southern California, Los Angeles, CA, USA.

A workshop entitled "Stem Cells, Cell Therapies and Bioengineering in Lung Biology and Diseases" was hosted by the University of Vermont Larner College of Medicine in collaboration with the National Heart, Lung and Blood Institute, the Alpha-1 Foundation, the Cystic Fibrosis Foundation, the International Society for Cell and Gene Therapy and the Pulmonary Fibrosis Foundation. The event was held from July 15 to 18, 2019 at the University of Vermont, Burlington, Vermont. The objectives of the conference were to review and discuss the current status of the following active areas of research: 1) technological advancements in the analysis and visualisation of lung stem and progenitor cells; 2) evaluation of lung stem and progenitor cells in the context of their interactions with the niche; 3) progress toward the application and delivery of stem and progenitor cells for the treatment of lung diseases such as cystic fibrosis; 4) progress in induced pluripotent stem cell models and application for disease modelling; and 5) the emerging roles of cell therapy and extracellular vesicles in immunomodulation of the lung. This selection of topics represents some of the most dynamic research areas in which incredible progress continues to be made. The workshop also included active discussion on the regulation and commercialisation of regenerative medicine products and concluded with an open discussion to set priorities and recommendations for future research directions in basic and translation lung biology.
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http://dx.doi.org/10.1183/23120541.00123-2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569162PMC
October 2020

Converging pathways in pulmonary fibrosis and Covid-19 - The fibrotic link to disease severity.

Respir Med X 2020 Nov 9;2:100023. Epub 2020 Oct 9.

Lung Biology Unit, Department of Experimental Medical Science, Lund University, Lund, Sweden.

As Covid-19 affects millions of people worldwide, the global health care will encounter an increasing burden of the aftermaths of the disease. Evidence shows that up to a fifth of the patients develop fibrotic tissue in the lung. The SARS outbreak in the early 2000 resulted in chronic pulmonary fibrosis in a subset (around 4%) of the patients, and correlated to reduced lung function and forced expiratory volume (FEV). The similarities between corona virus infections causing SARS and Covid-19 are striking, except that the novel coronavirus, SARS-CoV-2, has proven to have an even higher communicability. This would translate into a large number of patients seeking care for clinical signs of pulmonary fibrosis, given that the Covid-19 pandemic has up till now (Sept 2020) affected around 30 million people. The SARS-CoV-2 is dependent on binding to the angiotensin converting enzyme 2 (ACE2), which is part of the renin-angiotensin system (RAS). Downregulation of ACE2 upon virus binding disturbs downstream activities of RAS resulting in increased inflammation and development of fibrosis. The poor prognosis and risk of developing pulmonary fibrosis are therefore associated with the increased expression of ACE2 in risk groups, such as obesity, heart disorders and aging, conferring plenty of binding opportunity for the virus and subsequently the internalization of ACE2, thus devoiding the enzyme from acting counter-inflammatory and antifibrotic. Identifying pathways that are associated with Covid-19 severity that result in pulmonary fibrosis may enable early diagnosis and individualized treatment for these patients to prevent or reduce irreversible fibrotic damage to the lung.
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http://dx.doi.org/10.1016/j.yrmex.2020.100023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546672PMC
November 2020

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

Respir Med 2020 05 21;166:105944. Epub 2020 Mar 21.

Unit of Lung Biology, Dept. of Experimental Medical Science, Lund University, Sweden.

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-β) 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-β significantly induced VEGF synthesis at all time points. At 12 months post transplantation there was significantly less VEGF synthesis after TGF-β 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.
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http://dx.doi.org/10.1016/j.rmed.2020.105944DOI Listing
May 2020

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

Sci Rep 2020 03 4;10(1):4031. Epub 2020 Mar 4.

Centre for Analysis and Synthesis, Department of Chemistry, Lund University, SE-22100, Lund, Sweden.

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-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-EtOH-HO fluids (average molar composition, Χ 0.91) yielded close to complete removal of lipids from porcine pulmonary arteries, including a notably decrease of pro-inflammatory fatty acids. Pressurized CO-limonene fluids (Χ 0.88) and neat supercritical CO (scCO) achieved the removal of 90% of triacylglycerides. Moreover, treatment of tissue with pressurized CO-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-ethanol fluids offer an efficient tool for delipidation in bio-scaffold production, while pressurized CO-limonene fluids facilitate subsequent enzymatic removal of DNA.
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http://dx.doi.org/10.1038/s41598-020-60827-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7055267PMC
March 2020

Comparison of Normal and Metaplastic Epithelium in Patients with Stable versus Persistently Symptomatic Severe Asthma Using Laser-Capture Microdissection and Data-Independent Acquisition-Mass Spectrometry.

Am J Pathol 2019 12;189(12):2358-2365

Division of Lung Biology, Department of Experimental Medical Science, Lund University, Lund, Sweden.

A proportion of patients with severe asthma (SA) show poor responses to traditional asthma medications; however, it remains unknown why some patients remain persistently symptomatic. Our objective was to explore the use of laser-capture microdissection of specific epithelial structures combined with quantitative data-independent acquisition mass spectrometry to elucidate differences in protein composition in patients with SA with varying symptom control. Unbiased label-free quantitative proteome analyses were performed on laser-capture-microdissected areas of specific epithelial structures from patients with SA with varying degrees of symptom control. A total of 1993 stable SA and 1652 symptomatic SA proteins in normal epithelium and 1458 stable SA and 1647 symptomatic SA proteins in metaplastic epithelium were quantified. When comparing proteome profiles based on symptom control, 33 proteins in patients with stable SA (≥twofold change; P ≤ 0.05) and 13 proteins in patients with persistently symptomatic SA (≥twofold change; P ≤ 0.05) were enriched significantly. When comparing proteome profiles based on epithelial status, 21 proteins in normal epithelium (≥twofold change; P ≤ 0.05) and 6 proteins in metaplastic epithelium (≥twofold change; P ≤ 0.05) were enriched significantly. New treatment strategies are needed for patients with severe asthma and exploratory studies of unbiased nature such as this may help when searching for new mechanisms and potential targets involved in the disease pathology.
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http://dx.doi.org/10.1016/j.ajpath.2019.08.003DOI Listing
December 2019

Functional role of glycosaminoglycans in decellularized lung extracellular matrix.

Acta Biomater 2020 01 18;102:231-246. Epub 2019 Nov 18.

University of Vermont, Larner College of Medicine, Burlington, VT, United States. Electronic address:

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 TGFβ1. 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 TGFβ1. 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.
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http://dx.doi.org/10.1016/j.actbio.2019.11.029DOI Listing
January 2020

Matrisome Properties of Scaffolds Direct Fibroblasts in Idiopathic Pulmonary Fibrosis.

Int J Mol Sci 2019 Aug 17;20(16). Epub 2019 Aug 17.

Lung Biology, Department of Experimental Medical Sciences, Lund University, BMC C12, Lund 221 84, Sweden.

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.
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http://dx.doi.org/10.3390/ijms20164013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719040PMC
August 2019

Glycosaminoglycans: A Link Between Development and Regeneration in the Lung.

Stem Cells Dev 2019 07 11;28(13):823-832. Epub 2019 Jun 11.

Experimental Medical Sciences, Lung Biology, Lund, Sweden.

What can we learn from embryogenesis to increase our understanding of how regeneration of damaged adult lung tissue could be induced in serious lung diseases such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), and asthma? The local tissue niche determines events in both embryogenesis and repair of the adult lung. Important constituents of the niche are extracellular matrix (ECM) molecules, including proteoglycans and glycosaminoglycans (GAGs). GAGs, strategically located in the pericellular and extracellular space, bind developmentally active growth factors (GFs) and morphogens such as fibroblast growth factors (FGFs), transforming growth factor-β (TGF-β), and bone morphogenetic proteins (BMPs) aside from cytokines. These interactions affect activities in many cells, including stem cells, important in development and tissue regeneration. Moreover, it is becoming clear that the "inherent code," such as sulfation of disaccharides of GAGs, is a strong determinant of cellular outcome. Sulfation patterns, deacetylations, and epimerizations of GAG chains function as tuning forks in gradient formation of morphogens, growth factors, and cytokines. Learning to tune these fine instruments, that is, interactions between GFs, chemokines, and cytokines with the specific disaccharide code of GAGs in the adult lung, could become the key to unlock inherent regenerative forces to override pathological remodeling. This review aims to provide an overview of the role GAGs play during development and similar events in regenerative efforts in the adult lung.
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http://dx.doi.org/10.1089/scd.2019.0009DOI Listing
July 2019

The proportion of alveolar type 1 cells decreases in murine hypoplastic congenital diaphragmatic hernia lungs.

PLoS One 2019 17;14(4):e0214793. Epub 2019 Apr 17.

Department of Development and Regeneration, Division Organ Systems, KU Leuven, Leuven, Belgium.

Background: Pulmonary hypoplasia, characterized by incomplete alveolar development, remains a major cause of mortality and morbidity in congenital diaphragmatic hernia. Recently demonstrated to differentiate from a common bipotent progenitor during development, the two cell types that line the alveoli type 1 and type 2 alveolar cells have shown to alter their relative ratio in congenital diaphragmatic hernia lungs.

Objective: We used the nitrofen/bisdiamine mouse model to induce congenital diaphragmatic hernia and accurately assess the status of alveolar epithelial cell differentiation in relation to the common bipotent progenitors.

Study Design: Pregnant Swiss mice were gavage-fed with nitrofen/bisdiamine or vehicle at embryonic day 8.5. The administered dose was optimized by assessing the survival, congenital diaphragmatic hernia and facial abnormality rates of the exposed mouse pups. NanoCT was performed on embryonic day 11.5 and 16.5 to assess the embryonic and early canalicular stages of lung development. At embryonic day 17.5 corresponding to late canalicular stage, congenital diaphragmatic hernia lungs were characterized by measuring the lung weight/body weight ratio, morphometry, epithelial cell marker gene expression levels and alveolar cell type quantification.

Results: Nitrofen/bisdiamine associated congenital diaphragmatic hernia lungs showed delayed development, hypoplasia with morphologic immaturity and thickened alveolar walls. Expression levels of distal epithelial progenitor marker Id2 increased, alveolar type 1 cell markers Pdpn and Hopx decreased, while type 2 cell markers pro-SPC and Muc1 remained constant during the canalicular stage. The number of Pdpn+ type 1 alveolar cells also decreased in congenital diaphragmatic hernia lungs.

Conclusion: The mouse nitrofen/bisdiamine model is a potential model allowing the study of congenital diaphragmatic hernia lung development from early stages using a wide array of methods. Based on this model, the alveolar epithelium showed a decrease in the number of alveolar type 1 cell in congenital diaphragmatic hernia lungs while type 2 cell population remains unchanged.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214793PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6469843PMC
January 2020

Recombinant dermatan sulfate is a potent activator of heparin cofactor II-dependent inhibition of thrombin.

Glycobiology 2019 06;29(6):446-451

Department of Experimental Medical Science, BMC C12, Lund University, Lund, Sweden.

The glycosaminoglycan dermatan sulfate (DS) is a well-known activator of heparin cofactor II-dependent inactivation of thrombin. In contrast to heparin, dermatan sulfate has never been prepared recombinantly from material of non-animal origin. Here we report on the enzymatic synthesis of structurally well-defined DS with high anticoagulant activity. Using a microbial K4 polysaccharide and the recombinant enzymes DS-epimerase 1, dermatan 4-O-sulfotransferase 1, uronyl 2-O-sulfotransferase and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, several new glycostructures have been prepared, such as a homogenously sulfated IdoA-GalNAc-4S polymer and its 2-O-, 6-O- and 2,6-O-sulfated derivatives. Importantly, the recombinant highly 2,4-O-sulfated DS inhibits thrombin via heparin cofactor II, approximately 20 times better than heparin, enabling manipulation of vascular and extravascular coagulation. The potential of this method can be extended to preparation of specific structures that are of importance for binding and activation of cytokines, and control of inflammation and metastasis, involving extravasation and migration.
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http://dx.doi.org/10.1093/glycob/cwz019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7008404PMC
June 2019

Expression, activity and localization of lysosomal sulfatases in Chronic Obstructive Pulmonary Disease.

Sci Rep 2019 02 13;9(1):1991. Epub 2019 Feb 13.

Respiratory Medicine and Allergology, Department of Clinical Sciences Lund, Lund University, Lund, Sweden.

Chronic obstructive pulmonary disease (COPD) is a leading cause of death world-wide. Recently, we showed that COPD is associated with gene polymorphisms in SUMF1, a master regulator of sulfatases. Sulfatases are involved in extracellular matrix remodeling and activated by SUMF1, but their role in the lung is poorly described. We aimed to examine how sulfatases are affected in the airways of patients with COPD compared to ever smokers and never smokers. We observed that mRNA expression of the sulfatases GALNS, GNS and IDS was increased, while protein expression of many sulfatases was decreased in COPD fibroblasts. Several sulfatases, including GALNS, IDS, and SGSH, showed increased activity in COPD fibroblasts. Examination of different sulfatases by immunofluorescence showed that IDS, ARSB, GNS and SGSH in fibroblasts were localized to sites other than their reported destination. Using a master panel from different organs, RNA expression of all sulfatases could be observed in lung tissue. Additionally, immunohistochemistry on lung biopsies indicated differing expression of sulfatases in COPD patients. In conclusion, mRNA, protein expression, sulfatase activity levels, and localization of sulfatases are altered in lung fibroblasts and lung tissue from COPD patients and may be mechanistically important in COPD pathogenesis. This could contribute to the understanding of the disease mechanism in COPD and in the long run, to lead to more individualized therapies.
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http://dx.doi.org/10.1038/s41598-018-37958-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6374378PMC
February 2019

The Pulmonary Extracellular Matrix Is a Bactericidal Barrier Against in Chronic Obstructive Pulmonary Disease (COPD): Implications for an Innate Host Defense Function of Collagen VI.

Front Immunol 2018 31;9:1988. Epub 2018 Aug 31.

Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden.

Non-typeable (NTHi) is a Gram-negative human commensal commonly residing in the nasopharynx of preschool children. It occasionally causes upper respiratory tract infection such as acute otitis media, but can also spread to the lower respiratory tract causing bronchitis and pneumonia. There is increasing recognition that NTHi has an important role in chronic lower respiratory tract inflammation, particularly in persistent infection in patients suffering from chronic obstructive pulmonary disease (COPD). Here, we set out to assess the innate protective effects of collagen VI, a ubiquitous extracellular matrix component, against NTHi infection , collagen VI rapidly kills bacteria through pore formation and membrane rupture, followed by exudation of intracellular content. This effect is mediated by specific binding of the von Willebrand A (VWA) domains of collagen VI to the NTHi surface adhesins protein E (PE) and autotransporter protein (Hap). Similar observations were made specimens from murine airways and COPD patient biopsies. NTHi bacteria adhered to collagen fibrils in the airway mucosa and were rapidly killed by membrane destabilization. The significance in host-pathogen interplay of one of these molecules, PE, was highlighted by the observation that it confers partial protection from bacterial killing. Bacteria lacking PE were more prone to antimicrobial activity than NTHi expressing PE. Altogether the data shed new light on the carefully orchestrated molecular events of the host-pathogen interplay in COPD and emphasize the importance of the extracellular matrix as a novel branch of innate host defense.
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http://dx.doi.org/10.3389/fimmu.2018.01988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6127292PMC
September 2019

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

Cell Commun Signal 2018 09 15;16(1):59. Epub 2018 Sep 15.

Unit of Lung Biology, Department of Experimental Medical Sciences, Lund University, BMC C12, 221 84, Lund, Sweden.

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.
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http://dx.doi.org/10.1186/s12964-018-0269-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139170PMC
September 2018

Dermatan sulfate epimerase 1 and dermatan 4--sulfotransferase 1 form complexes that generate long epimerized 4--sulfated blocks.

J Biol Chem 2018 08 5;293(35):13725-13735. Epub 2018 Jul 5.

From the Department of Experimental Medical Science, Lund University, SE-221 00, Lund, Sweden,

During the biosynthesis of chondroitin/dermatan sulfate (CS/DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. , concomitant with epimerization, dermatan 4--sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid-containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid-forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.
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http://dx.doi.org/10.1074/jbc.RA118.003875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6120188PMC
August 2018

Quantifying extracellular matrix turnover in human lung scaffold cultures.

Sci Rep 2018 04 3;8(1):5409. Epub 2018 Apr 3.

Division of Infection Medicine, Department Clinical Sciences, Lund University, Lund, Sweden.

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.
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http://dx.doi.org/10.1038/s41598-018-23702-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5882971PMC
April 2018

Quantitative proteomic characterization of the lung extracellular matrix in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

J Proteomics 2018 10 1;189:23-33. Epub 2018 Mar 1.

Department of Clinical Sciences, Division of Infection Medicine Proteomics, Lund University, Lund, Sweden.

Remodeling of the extracellular matrix (ECM) is a common feature in lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Here, we applied a sequential tissue extraction strategy to describe disease-specific remodeling of human lung tissue in disease, using end-stages of COPD and IPF. Our strategy was based on quantitative comparison of the disease proteomes, with specific focus on the matrisome, using data-independent acquisition and targeted data analysis (SWATH-MS). Our work provides an in-depth proteomic characterization of human lung tissue during impaired tissue remodeling. In addition, we show important quantitative and qualitative effects of the solubility of matrisome proteins. COPD was characterized by a disease-specific increase in ECM regulators, metalloproteinase inhibitor 3 (TIMP3) and matrix metalloproteinase 28 (MMP-28), whereas for IPF, impairment in cell adhesion proteins, such as collagen VI and laminins, was most prominent. For both diseases, we identified increased levels of proteins involved in the regulation of endopeptidase activity, with several proteins belonging to the serpin family. The established human lung quantitative proteome inventory and the construction of a tissue-specific protein assay library provides a resource for future quantitative proteomic analyses of human lung tissues. SIGNIFICANCE: We present a sequential tissue extraction strategy to determine changes in extractability of matrisome proteins in end-stage COPD and IPF compared to healthy control tissue. Extensive quantitative analysis of the proteome changes of the disease states revealed altered solubility of matrisome proteins involved in ECM regulators and cell-ECM communication. The results highlight disease-specific remodeling mechanisms associated with COPD and IPF.
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http://dx.doi.org/10.1016/j.jprot.2018.02.027DOI Listing
October 2018

Endoplasmic reticulum, Golgi, and lysosomes are disorganized in lung fibroblasts from chronic obstructive pulmonary disease patients.

Physiol Rep 2018 03;6(5)

Department of Clinical Sciences Lund, Respiratory Medicine and Allergology, Lund University, Lund, Sweden.

Chronic Obstructive Pulmonary Disease (COPD) is often caused by smoking and other stressors. This causes oxidative stress, which induces numerous changes on both the transcriptome and proteome of the cell. We aimed to examine if the endomembrane pathway, including the endoplasmic reticulum (ER), Golgi, and lysosomes, was disrupted in fibroblasts from COPD patients as opposed to healthy ever-smokers or never-smokers, and if the response to stress differed. Different cellular compartments involved in the endomembrane pathway, as well as mRNA expression and apoptosis, were examined before and after the addition of stress in lung fibroblasts from never-smokers, ever-smokers, and patients with COPD. We found that the ER, Golgi, and lysosomes were disorganized in fibroblasts from COPD patients under baseline conditions. After a time course with ER stress inducing chemicals, changes to the phenotypes of cellular compartments in COPD patient fibroblasts were observed, and the expression of the ER stress-induced gene ERP72 was upregulated more in the COPD patient's cells compared to ever-smokers or never-smokers. Lastly, a tendency of increased active Caspase-3 was observed in COPD fibroblasts. Our results show that COPD patients have phenotypic changes in the lung fibroblasts endomembrane pathway, and respond differently to stress. Furthermore, these fibroblasts were cultured for several weeks outside the body, but they were not able to regain proper ER structure, indicating that the internal changes to the endomembrane system are permanent in smokers. This vulnerability to cellular stress might be a cause as to why some smokers develop COPD.
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http://dx.doi.org/10.14814/phy2.13584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827558PMC
March 2018

Bronchial extracellular matrix from COPD patients induces altered gene expression in repopulated primary human bronchial epithelial cells.

Sci Rep 2018 02 22;8(1):3502. Epub 2018 Feb 22.

Bioscience Regeneration Department, Respiratory, Inflammation and Autoimmunity, IMED Biotech Unit, AstraZeneca, Gothenburg, Sweden.

Chronic obstructive pulmonary disease (COPD) is a serious global health problem characterized by chronic airway inflammation, progressive airflow limitation and destruction of lung parenchyma. Remodeling of the bronchial airways in COPD includes changes in both the bronchial epithelium and the subepithelial extracellular matrix (ECM). To explore the impact of an aberrant ECM on epithelial cell phenotype in COPD we developed a new ex vivo model, in which normal human bronchial epithelial (NHBE) cells repopulate and differentiate on decellularized human bronchial scaffolds derived from COPD patients and healthy individuals. By using transcriptomics, we show that bronchial ECM from COPD patients induces differential gene expression in primary NHBE cells when compared to normal bronchial ECM. The gene expression profile indicated altered activity of upstream mediators associated with COPD pathophysiology, including hepatocyte growth factor, transforming growth factor beta 1 and platelet-derived growth factor B, which suggests that COPD-related changes in the bronchial ECM contribute to the defective regenerative ability in the airways of COPD patients.
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http://dx.doi.org/10.1038/s41598-018-21727-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823945PMC
February 2018

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

Am J Pathol 2018 05 16;188(5):1113-1119. Epub 2018 Feb 16.

Lung Biology Group, Department of Experimental Medical Science, Lund University, Lund, Sweden.

Serotonin [5-hydroxytryptamine (5-HT)] is associated with several chronic pulmonary diseases, recognizing 5-HT receptor antagonists as potential inhibitors of tissue remodeling. However, the effects of 5-HT receptors, especially 5-HT receptors on airway function and remodeling, are unclear. We investigated the role of 5-HT receptors on airway smooth muscle contractility and remodeling processes. Murine precision-cut lung slices were pretreated with 5-HT receptor antagonists (EXT5, EXT9, RS 127445, and PRX 08066), as well as ketanserin (5-HT 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-HT receptor antagonists were further studied in distal lung tissue by examining release of profibrotic transforming growth factor (TGF)-β1 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-HT receptor antagonists significantly reduced TGF-β1 release. 5-HT, in combination with TGF-β1, 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-β1 release further support a role of 5-HT receptors in pathologic remodeling processes.
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http://dx.doi.org/10.1016/j.ajpath.2018.01.006DOI Listing
May 2018

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

Sci Rep 2018 01 31;8(1):1927. Epub 2018 Jan 31.

Lung Biology, Department of Experimental Medical Science, Lund University, Lund, Sweden.

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-HT 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-HT 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-HT 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-HT receptor antagonism attenuates pulmonary fibrosis in part by anti-proliferative effects, associated with inhibited pAkt/p21 signaling pathway.
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http://dx.doi.org/10.1038/s41598-018-20430-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792547PMC
January 2018

Temporal trend of autonomic nerve function and HSP27, MIF and PAI-1 in type 1 diabetes.

J Clin Transl Endocrinol 2017 Jun 20;8:15-21. Epub 2017 Mar 20.

Department of Translational Medicine - Hand Surgery, Lund University, Skåne University Hospital, Jan Waldenströms gata 5, 205 02 Malmö, Sweden.

Aim: Diabetes mellitus type 1 (T1D) has numerous complications including autonomic neuropathy, dysfunction of the autonomous nervous system. This study focuses on Heat Shock Protein 27 (HSP27), Macrophage Migration Inhibitory Factor (MIF), Plasminogen Activator Inhibitor-1 (PAI-1) and HbA1c and their possible roles in effects of diabetes on the autonomic nervous system.

Methods: Patients with T1D (n = 32, 41% women) were recruited in 1985 and followed up on four occasions (1989, 1993, 1998, and 2005). Autonomic function was tested using expiration/inspiration (E/I-ratio). Blood samples, . HSP27 (last three occasions), MIF, PAI-1 (last two occasions) and HbA1c (five occasions), were analyzed.

Results: Autonomic nerve function deteriorated over time during the 20-year-period, but levels of HSP27, MIF, and PAI-1 were not associated with cardiovascular autonomic neuropathy. MIF and PAI-1 were lower in T1D than in healthy controls in 2005. Increased HbA1c correlated with a decrease in E/I-ratio.

Conclusions: Neither the neuroprotective substance HSP27 nor the inflammatory substances, MIF and PAI-1 were associated with measures of cardiovascular autonomic nerve function, but a deterioration of such function was observed in relation to increasing HbA1c in T1D during a 20-year follow-up period. Improved glucose control might be associated with protection against autonomic neuropathy in T1D.
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http://dx.doi.org/10.1016/j.jcte.2017.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651332PMC
June 2017

Quantitative proteomic characterization of lung-MSC and bone marrow-MSC using DIA-mass spectrometry.

Sci Rep 2017 08 24;7(1):9316. Epub 2017 Aug 24.

Department of Experimental Medical Science, Lung Biology Unit, Lund University, 22184, Lund, Sweden.

Mesenchymal stromal cells (MSC) are ideal candidates for cell therapies, due to their immune-regulatory and regenerative properties. We have previously reported that lung-derived MSC are tissue-resident cells with lung-specific properties compared to bone marrow-derived MSC. Assessing relevant molecular differences between lung-MSC and bone marrow-MSC is important, given that such differences may impact their behavior and potential therapeutic use. Here, we present an in-depth mass spectrometry (MS) based strategy to investigate the proteomes of lung-MSC and bone marrow-MSC. The MS-strategy relies on label free quantitative data-independent acquisition (DIA) analysis and targeted data analysis using a MSC specific spectral library. We identified several significantly differentially expressed proteins between lung-MSC and bone marrow-MSC within the cell layer (352 proteins) and in the conditioned medium (49 proteins). Bioinformatics analysis revealed differences in regulation of cell proliferation, which was functionally confirmed by decreasing proliferation rate through Cytochrome P450 stimulation. Our study reveals important differences within proteome and matrisome profiles between lung- and bone marrow-derived MSC that may influence their behavior and affect the clinical outcome when used for cell-therapy.
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http://dx.doi.org/10.1038/s41598-017-09127-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570998PMC
August 2017

VEGF synthesis is induced by prostacyclin and TGF-β in distal lung fibroblasts from COPD patients and control subjects: Implications for pulmonary vascular remodelling.

Respirology 2018 01 22;23(1):68-75. Epub 2017 Aug 22.

Unit of Lung Biology, Department of Experimental Medical Sciences, Lund University, Lund, Sweden.

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)-β 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-β . 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 (VEGF ; 1-10 000 pg/mL) in HFL-1.

Results: Iloprost and TGF-β significantly increased VEGF synthesis in both fibroblasts from COPD patients and control subjects. TGF-β -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.
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http://dx.doi.org/10.1111/resp.13142DOI Listing
January 2018

REST suppression mediates neural conversion of adult human fibroblasts via microRNA-dependent and -independent pathways.

EMBO Mol Med 2017 08;9(8):1117-1131

Division of Neurobiology and Lund Stem Cell Center, Department of Experimental Medical Science, Wallenberg Neuroscience Center, Lund University, Lund, Sweden

Direct conversion of human fibroblasts into mature and functional neurons, termed induced neurons (iNs), was achieved for the first time 6 years ago. This technology offers a promising shortcut for obtaining patient- and disease-specific neurons for disease modeling, drug screening, and other biomedical applications. However, fibroblasts from adult donors do not reprogram as easily as fetal donors, and no current reprogramming approach is sufficiently efficient to allow the use of this technology using patient-derived material for large-scale applications. Here, we investigate the difference in reprogramming requirements between fetal and adult human fibroblasts and identify REST as a major reprogramming barrier in adult fibroblasts. Via functional experiments where we overexpress and knockdown the REST-controlled neuron-specific microRNAs miR-9 and miR-124, we show that the effect of REST inhibition is only partially mediated via microRNA up-regulation. Transcriptional analysis confirmed that REST knockdown activates an overlapping subset of neuronal genes as microRNA overexpression and also a distinct set of neuronal genes that are not activated via microRNA overexpression. Based on this, we developed an optimized one-step method to efficiently reprogram dermal fibroblasts from elderly individuals using a single-vector system and demonstrate that it is possible to obtain iNs of high yield and purity from aged individuals with a range of familial and sporadic neurodegenerative disorders including Parkinson's, Huntington's, as well as Alzheimer's disease.
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http://dx.doi.org/10.15252/emmm.201607471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5538296PMC
August 2017