Publications by authors named "Grazyna Kwapiszewska"

94 Publications

Dysbalance of ACE2 levels - a possible cause for severe COVID-19 outcome in COPD.

J Pathol Clin Res 2021 May 12. Epub 2021 May 12.

Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to healthcare systems worldwide. Binding of the virus to angiotensin-converting enzyme 2 (ACE2) is an important step in the infection mechanism. However, it is unknown if ACE2 expression in patients with chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary arterial hypertension (IPAH), or pulmonary fibrosis (PF), is changed as compared to controls. We used lung samples from patients with COPD (n = 28), IPAH (n = 10), and PF (n = 10) as well as healthy control donor (n = 10) tissue samples to investigate the expression of ACE2 and related cofactors that might influence the course of SARS-CoV-2 infection. Expression levels of the ACE2 receptor, the putative receptor CD147/BSG, and the viral entry cofactors TMPRSS2 (transmembrane serine protease 2), EZR, and FURIN were determined by quantitative PCR and in open-access RNA sequencing datasets. Immunohistochemical and single-cell RNA sequencing (scRNAseq) analyses were used for localization and coexpression, respectively. Soluble ACE2 (sACE2) plasma levels were analyzed by enzyme-linked immunosorbent assay. In COPD as compared to donor, IPAH, and PF lung tissue, gene expression of ACE2, TMPRSS2, and EZR was significantly elevated, but circulating sACE2 levels were significantly reduced in COPD and PF plasma compared to healthy control and IPAH plasma samples. Lung tissue expressions of FURIN and CD147/BSG were downregulated in COPD. None of these changes were associated with changes in pulmonary hemodynamics. Histological analysis revealed coexpression of ACE2, TMPRSS2, and Ezrin in bronchial regions and epithelial cells. This was confirmed by scRNAseq analysis. There were no significant expression changes of the analyzed molecules in the lung tissue of IPAH and idiopathic PF as compared to control. In conclusion, we reveal increased ACE2 and TMPRSS2 expression in lung tissue with a concomitant decrease of protective sACE2 in COPD patients. These changes represent the possible risk factors for an increased susceptibility of COPD patients to SARS-CoV-2 infection.
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http://dx.doi.org/10.1002/cjp2.224DOI Listing
May 2021

Pulmonary fibrosis in Fra-2 transgenic mice is associated with decreased numbers of alveolar macrophages and increased susceptibility to pneumococcal pneumonia.

Am J Physiol Lung Cell Mol Physiol 2021 May 3;320(5):L916-L925. Epub 2021 Mar 3.

Division of Pulmonary Inflammation, Charité - Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.

Idiopathic pulmonary fibrosis (IPF) is a deadly condition characterized by progressive respiratory dysfunction. Exacerbations due to airway infections are believed to promote disease progression, and presence of in the lung microbiome has been associated with the progression of IPF and mortality. The aim of this study was to analyze the effect of lung fibrosis on susceptibility to pneumococcal pneumonia and bacteremia. The effects of subclinical (low dose) infection with were studied in a well characterized fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of spontaneous, progressive pulmonary fibrosis. Forty-eight hours after transnasal infection with , bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood, and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Lung compliance and arterial blood gases were assessed. In contrast to wildtype mice, low dose lung infection with in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load, and bacteremia. BAL alveolar macrophages were reduced in Fra-2 TG mice compared to the corresponding WT mice. Proinflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, and CXCL1) were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Lung compliance was decreased in Fra-2 TG mice following low dose infection with . Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.
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http://dx.doi.org/10.1152/ajplung.00505.2020DOI Listing
May 2021

Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy.

Histochem Cell Biol 2021 May 6;155(5):593-603. Epub 2021 Jan 6.

Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Division of Cell Biology, Histology and Embryology, Medical University of Graz, Neue Stiftingtalstraße 6/II, 8010, Graz, Austria.

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.
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http://dx.doi.org/10.1007/s00418-020-01952-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8134286PMC
May 2021

Identification of a Repair-Supportive Mesenchymal Cell Population during Airway Epithelial Regeneration.

Cell Rep 2020 12;33(12):108549

Key Laboratory of Interventional Pulmonology of Zhejiang Province, Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325015, China; Department of Internal Medicine, Universities of Giessen and Marburg Lung Center (UGMLC), Cardio-Pulmonary Institute (CPI), Member of the German Center for Lung Research (DZL), Justus-Liebig University Giessen, 35392 Giessen, Germany; Institute for Lung Health (ILH), 35392 Giessen, Germany. Electronic address:

Tissue regeneration requires coordinated and dynamic remodeling of stem and progenitor cells and the surrounding niche. Although the plasticity of epithelial cells has been well explored in many tissues, the dynamic changes occurring in niche cells remain elusive. Here, we show that, during lung repair after naphthalene injury, a population of PDGFRα cells emerges in the non-cartilaginous conducting airway niche, which is normally populated by airway smooth muscle cells (ASMCs). This cell population, which we term "repair-supportive mesenchymal cells" (RSMCs), is distinct from conventional ASMCs, which have previously been shown to contribute to epithelial repair. Gene expression analysis on sorted lineage-labeled cells shows that RSMCs express low levels of ASMC markers, but high levels of the pro-regenerative marker Fgf10. Organoid co-cultures demonstrate an enhanced ability for RSMCs in supporting club-cell growth. Our study highlights the dynamics of mesenchymal cells in the airway niche and has implications for chronic airway-injury-associated diseases.
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http://dx.doi.org/10.1016/j.celrep.2020.108549DOI Listing
December 2020

Machine Learning Analysis of the Bleomycin Mouse Model Reveals the Compartmental and Temporal Inflammatory Pulmonary Fingerprint.

iScience 2020 Dec 18;23(12):101819. Epub 2020 Nov 18.

Ludwig Boltzmann Institute for Lung Vascular Research, Graz 8010, Austria.

The bleomycin mouse model is the extensively used model to study pulmonary fibrosis; however, the inflammatory cell kinetics and their compartmentalization is still incompletely understood. Here we assembled historical flow cytometry data, totaling 303 samples and 16 inflammatory-cell populations, and applied advanced data modeling and machine learning methods to conclusively detail these kinetics. Three days post-bleomycin, the inflammatory profile was typified by acute innate inflammation, pronounced neutrophilia, especially of SiglecF neutrophils, and alveolar macrophage loss. Between 14 and 21 days, rapid responders were increasingly replaced by T and B cells and monocyte-derived alveolar macrophages. Multicolour imaging revealed the spatial-temporal cell distribution and the close association of T cells with deposited collagen. Unbiased immunophenotyping and data modeling exposed the dynamic shifts in immune-cell composition over the course of bleomycin-triggered lung injury. These results and workflow provide a reference point for future investigations and can easily be applied in the analysis of other datasets.
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http://dx.doi.org/10.1016/j.isci.2020.101819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725744PMC
December 2020

Editorial: Multitasking Biomolecules in Human Pathologies: Known Players on Their Unexpected Journeys.

Front Med (Lausanne) 2020 21;7:478. Epub 2020 Aug 21.

Department of Biochemistry, Universities of Giessen and Marburg Lung Center, Giessen, Germany.

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http://dx.doi.org/10.3389/fmed.2020.00478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7471247PMC
August 2020

Stiffness of the Extracellular Matrix: A Regulator of Prostaglandins in Pulmonary Fibrosis?

Am J Respir Cell Mol Biol 2020 12;63(6):721-722

Ludwig Boltzmann Institute for Lung Vascular Research Graz, Austria.

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http://dx.doi.org/10.1165/rcmb.2020-0398EDDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7790137PMC
December 2020

Endothelial Basement Membrane Components and Their Products, Matrikines: Active Drivers of Pulmonary Hypertension?

Cells 2020 09 3;9(9). Epub 2020 Sep 3.

Otto Loewi Research Center, Division of Physiology, Medical University of Graz, 8010 Graz, Austria.

Pulmonary arterial hypertension (PAH) is a vascular disease that is characterized by elevated pulmonary arterial pressure (PAP) due to progressive vascular remodeling. Extracellular matrix (ECM) deposition in pulmonary arteries (PA) is one of the key features of vascular remodeling. Emerging evidence indicates that the basement membrane (BM), a specialized cluster of ECM proteins underlying the endothelium, may be actively involved in the progression of vascular remodeling. The BM and its steady turnover are pivotal for maintaining appropriate vascular functions. However, the pathologically elevated turnover of BM components leads to an increased release of biologically active short fragments, which are called matrikines. Both BM components and their matrikines can interfere with pivotal biological processes, such as survival, proliferation, adhesion, and migration and thus may actively contribute to endothelial dysfunction. Therefore, in this review, we summarize the emerging role of the BM and its matrikines on the vascular endothelium and further discuss its implications on lung vascular remodeling in pulmonary hypertension.
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http://dx.doi.org/10.3390/cells9092029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563239PMC
September 2020

Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries.

Cells 2020 08 28;9(9). Epub 2020 Aug 28.

Ludwig Boltzmann Institute for Lung Vascular Research, Neue Stiftingtalstraße 6, 8010 Graz, Austria.

Endothelial dysfunction is one of the hallmarks of different vascular diseases, including pulmonary arterial hypertension (PAH). Ion channelome changes have long been connected to vascular remodeling in PAH, yet only recently has the focus shifted towards Ca-activated Cl channels (CaCC). The most prominent member of the CaCC TMEM16A has been shown to contribute to the pathogenesis of idiopathic PAH (IPAH) in pulmonary arterial smooth muscle cells, however its role in the homeostasis of healthy human pulmonary arterial endothelial cells (PAECs) and in the development of endothelial dysfunction remains underrepresented. Here we report enhanced TMEM16A activity in IPAH PAECs by whole-cell patch-clamp recordings. Using adenoviral-mediated TMEM16A increase in healthy primary human PAECs in vitro and in human pulmonary arteries ex vivo, we demonstrate the functional consequences of the augmented TMEM16A activity: alterations of Ca dynamics and eNOS activity as well as decreased NO production, PAECs proliferation, wound healing, tube formation and acetylcholine-mediated relaxation of human pulmonary arteries. We propose that the ERK1/2 pathway is specifically affected by elevated TMEM16A activity, leading to these pathological changes. With this work we introduce increased TMEM16A activity in the cell membrane of human PAECs for the development of endothelial dysfunction in PAH.
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http://dx.doi.org/10.3390/cells9091984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7563136PMC
August 2020

TWIST1 Drives Smooth Muscle Cell Proliferation in Pulmonary Hypertension via Loss of GATA-6 and BMPR2.

Am J Respir Crit Care Med 2020 11;202(9):1283-1296

Institute of Physiology, Charité-Universitätsmedizin Berlin, Berlin, Germany; and.

The bHLH (basic helix-loop-helix) transcription factor TWIST1 (Twist-related protein 1) controls cell proliferation and differentiation in tissue development and disease processes. Recently, endothelial TWIST1 has been linked to pulmonary hypertension (PH) and endothelial-to-mesenchymal transition, yet the role of TWIST1 in smooth muscle cells (SMCs) remains so far unclear. To define the role of TWIST1 in SMCs in the pathogenesis of PH. SMC-specific TWIST1-deficient mice, SMC-specific TWIST1 silencing in rats, mass spectrometry, immunoprecipitation, and chromatin immunoprecipitation were used to delineate the role of SMC TWIST1 in PH. In pulmonary vessels from patients with PH and rodent PH models, TWIST1 expression was markedly increased and predominantly localized to SMCs. SMC-specific TWIST1 deficiency or silencing attenuated the development of PH and distal vessel muscularization in chronically hypoxic mice and in monocrotaline-treated rats. , TWIST1 inhibition or silencing prevented pulmonary artery SMC proliferation and migration. Mechanistically, the observed effects were mediated, at least in part, by TWIST1-dependent degradation of GATA-6 (GATA-binding protein 6). BMPR2 (bone morphogenetic protein receptor-2) was identified as a novel downstream target of GATA-6, which directly binds to its promoter. Inhibition of TWIST1 promoted the recruitment of GATA-6 to the promoter and restored BMPR2 functional expression. Our findings identify a key role for SMC TWIST1 in the pathogenesis of lung vascular remodeling and in PH that is partially mediated via reduced GATA-6-dependent expression. Inhibition of SMC TWIST1 may constitute a new therapeutic strategy for the treatment of PH.
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http://dx.doi.org/10.1164/rccm.201909-1884OCDOI Listing
November 2020

CDK4/6 inhibition enhances pulmonary inflammatory infiltration in bleomycin-induced lung fibrosis.

Respir Res 2020 Jul 2;21(1):167. Epub 2020 Jul 2.

Ludwig Boltzmann Institute for Lung Vascular Research, Neue Stiftingtalstrasse 6/VI, 8010, Graz, Austria.

Inhibitors of cyclin-dependent kinases 4/6 (CDK4/6) block cell cycle progression and are commonly used for treatment of several forms of cancer. Due to their anti-proliferative mode of action, we hypothesized that palbociclib could attenuate the development of bleomycin-induced lung fibrosis. In a preclinical setting, mice were treated with bleomycin and then co-treated with or without palbociclib. Lung function, collagen deposition and pulmonary inflammation were analysed after 14 days.Bleomycin treatment led to an increase of pulmonary fibrosis and inflammation, and concomitant decline of lung function. Palbociclib treatment significantly decreased collagen deposition in the lung after bleomycin treatment, but did not ameliorate lung function. Importantly, palbociclib augmented inflammatory cell recruitment (including macrophages and T cells) in the bronchoalveolar lavage fluid.This study supports the recent alert from the Food and Drug Administration (FDA) that use of CDK4/6 inhibitors, such as palbociclib, may have severe pulmonary adverse effects. Our study showing heightened pulmonary inflammation following palbociclib treatment highlights the risk of severe inflammatory adverse effects in the lung. This is of special interest in patients with known pulmonary risk factors and emphasizes the need of careful monitoring all patients treated with CDK4/6 inhibitors for signs of lung inflammation.
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http://dx.doi.org/10.1186/s12931-020-01433-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7331186PMC
July 2020

Basement Membrane Remodeling Controls Endothelial Function in Idiopathic Pulmonary Arterial Hypertension.

Am J Respir Cell Mol Biol 2020 07;63(1):104-117

Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria.

The extracellular matrix (ECM) increasingly emerges as an active driver in several diseases, including idiopathic pulmonary arterial hypertension (IPAH). The basement membrane (BM) is a specialized class of ECM proteins. In pulmonary arteries, the BM is in close contact and direct proximity to vascular cells, including endothelial cells. So far, the role of the BM has remained underinvestigated in IPAH. Here, we aimed to shed light on the involvement of the BM in IPAH, by addressing its structure, composition, and function. On an ultrastructural level, we observed a marked increase in BM thickness in IPAH pulmonary vessels. BM composition was distinct in small and large vessels and altered in IPAH. Proteoglycans were mostly responsible for distinction between smaller and larger vessels, whereas BM collagens and laminins were more abundantly expressed in IPAH. Type IV collagen and laminin both strengthened endothelial barrier integrity. However, only type IV collagen concentration dependently increased cell adhesion of both donor and IPAH-derived pulmonary arterial endothelial cells (PAECs) and induced nuclear translocation of mechanosensitive transcriptional coactivator of the hippo pathway YAP (Yes-activated protein). On the other hand, laminin caused cytoplasmic retention of YAP in IPAH PAECs. Accordingly, silencing of COL4A5 and LAMC1, respectively, differentially affected tight junction formation and barrier integrity in both donor and IPAH PAECs. Collectively, our results highlight the importance of a well-maintained BM homeostasis. By linking changes in BM structure and composition to altered endothelial cell function, we here suggest an active involvement of the BM in IPAH pathogenesis.
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http://dx.doi.org/10.1165/rcmb.2019-0303OCDOI Listing
July 2020

PDGFRα and αSMA mark two distinct mesenchymal cell populations involved in parenchymal and vascular remodeling in pulmonary fibrosis.

Am J Physiol Lung Cell Mol Physiol 2020 04 5;318(4):L684-L697. Epub 2020 Feb 5.

Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria.

Pulmonary fibrosis is characterized by pronounced collagen deposition and myofibroblast expansion, whose origin and plasticity remain elusive. We utilized a fate-mapping approach to investigate α-smooth muscle actin (αSMA)+ and platelet-derived growth factor receptor α (PDGFRα)+ cells in two lung fibrosis models, complemented by cell type-specific next-generation sequencing and investigations on human lungs. Our data revealed that αSMA+ and PDGFRα+ cells mark two distinct mesenchymal lineages with minimal transdifferentiation potential during lung fibrotic remodeling. Parenchymal and perivascular fibrotic regions were populated predominantly with PDGFRα+ cells expressing collagen, while αSMA+ cells in the parenchyma and vessel wall showed variable expression of collagen and the contractile protein desmin. The distinct gene expression profile found in normal conditions was retained during pathologic remodeling. Cumulatively, our findings identify αSMA+ and PDGFRα+ cells as two separate lineages with distinct gene expression profiles in adult lungs. This cellular heterogeneity suggests that anti-fibrotic therapy should target diverse cell populations.
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http://dx.doi.org/10.1152/ajplung.00128.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189793PMC
April 2020

FHL-1 is not involved in pressure overload-induced maladaptive right ventricular remodeling and dysfunction.

Basic Res Cardiol 2020 01 24;115(2):17. Epub 2020 Jan 24.

Member of the German Center for Lung Research (DZL), Excellence Cluster Cardio-Pulmonary System (ECCPS), Universities of Giessen and Marburg Lung Center (UGMLC), Justus-Liebig-University Giessen, Aulweg 130, 35392, Giessen, Germany.

Aims: The cytoskeletal signaling protein four and-a-half LIM domains 1 (FHL-1) has recently been identified as a novel key player in pulmonary hypertension as well as in left heart diseases. In this regard, FHL-1 has been implicated in dysregulated hypertrophic signaling in pulmonary arterial smooth muscle cells leading to pulmonary hypertension. In mice, FHL-1-deficiency (FHL-1) led to an attenuated hypertrophic signaling associated with a blunted hypertrophic response of the pressure-overloaded left ventricle (LV). However, the role of FHL-1 in right heart hypertrophy has not yet been addressed.

Methods And Results: We investigated FHL-1 expression in C57Bl/6 mice subjected to chronic biomechanical stress and found it to be enhanced in the right ventricle (RV). Next, we subjected FHL-1 and corresponding wild-type mice to pressure overload of the RV by pulmonary arterial banding for various time points. However, in contrast to the previously published study in LV-pressure overload, which was confirmed here, RV hypertrophy and hypertrophic signaling was not diminished in FHL-1 mice. In detail, right ventricular pressure overload led to hypertrophy, dilatation and fibrosis of the RV from both FHL-1 and wild-type mice. RV remodeling was associated with impaired RV function as evidenced by reduced tricuspid annular plane systolic excursion. Additionally, PAB induced upregulation of natriuretic peptides and slight downregulation of phospholamban and ryanodine receptor 2 in the RV. However, there was no difference between genotypes in the degree of expression change.

Conclusion: FHL-1 pathway is not involved in the control of adverse remodeling in the pressure overloaded RV.
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http://dx.doi.org/10.1007/s00395-019-0767-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981327PMC
January 2020

Loss of LRP1 promotes acquisition of contractile-myofibroblast phenotype and release of active TGF-β1 from ECM stores.

Matrix Biol 2020 06 11;88:69-88. Epub 2019 Dec 11.

Departments of Biochemistry and Internal Medicine, Universities of Giessen and Marburg Lung Center, Giessen, Germany. Electronic address:

In healing tissue, fibroblasts differentiate to α-smooth muscle actin (SMA)-expressing contractile-myofibroblasts, which pull the wound edges together ensuring proper tissue repair. Uncontrolled expansion of the myofibroblast population may, however, lead to excessive tissue scarring and finally to organ dysfunction. Here, we demonstrate that the loss of low-density lipoprotein receptor-related protein (LRP) 1 overactivates the JNK1/2-c-Jun-Fra-2 signaling pathway leading to the induction of α-SMA and periostin expression in human lung fibroblasts (hLF). These changes are accompanied by increased contractility of the cells and the integrin- and protease-dependent release of active transforming growth factor (TGF)-β1 from the extracellular matrix (ECM) stores. Liberation of active TGF-β1 from the ECM further enhances α-SMA and periostin expression thus accelerating the phenotypic switch of hLF. Global gene expression profiling of LRP1-depleted hLF revealed that the loss of LRP1 affects cytoskeleton reorganization, cell-ECM contacts, and ECM production. In line with these findings, fibrotic changes in the skin and lung of Fra-2 transgenic mice were associated with LRP1 depletion and c-Jun overexpression. Altogether, our results suggest that dysregulation of LRP1 expression in fibroblasts in healing tissue may lead to the unrestrained expansion of contractile myofibroblasts and thereby to fibrosis development. Further studies identifying molecules, which regulate LRP1 expression, may provide new therapeutic options for largely untreatable human fibrotic diseases.
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http://dx.doi.org/10.1016/j.matbio.2019.12.001DOI Listing
June 2020

Inhibiting eicosanoid degradation exerts antifibrotic effects in a pulmonary fibrosis mouse model and human tissue.

J Allergy Clin Immunol 2020 03 5;145(3):818-833.e11. Epub 2019 Dec 5.

Division of Pharmacology, Otto Loewi Research Center, Medical University of Graz, Graz, Austria. Electronic address:

Background: Idiopathic pulmonary fibrosis (IPF) is a disease with high 5-year mortality and few therapeutic options. Prostaglandin (PG) E exhibits antifibrotic properties and is reduced in bronchoalveolar lavage from patients with IPF. 15-Prostaglandin dehydrogenase (15-PGDH) is the key enzyme in PGE metabolism under the control of TGF-β and microRNA 218.

Objective: We sought to investigate the expression of 15-PGDH in IPF and the therapeutic potential of a specific inhibitor of this enzyme in a mouse model and human tissue.

Methods: In vitro studies, including fibrocyte differentiation, regulation of 15-PGDH, RT-PCR, and Western blot, were performed using peripheral blood from healthy donors and patients with IPF and A549 cells. Immunohistochemistry, immunofluorescence, 15-PGDH activity assays, and in situ hybridization as well as ex vivo IPF tissue culture experiments were done using healthy donor and IPF lungs. Therapeutic effects of 15-PGDH inhibition were studied in the bleomycin mouse model of pulmonary fibrosis.

Results: We demonstrate that 15-PGDH shows areas of increased expression in patients with IPF. Inhibition of this enzyme increases PGE levels and reduces collagen production in IPF precision cut lung slices and in the bleomycin model. Inhibitor-treated mice show amelioration of lung function, decreased alveolar epithelial cell apoptosis, and fibroblast proliferation. Pulmonary fibrocyte accumulation is also decreased by inhibitor treatment in mice, similar to PGE that inhibits fibrocyte differentiation from blood of healthy donors and patients with IPF. Finally, microRNA 218-5p, which is downregulated in patients with IPF, suppressed 15-PGDH expression in vivo and in vitro.

Conclusions: These findings highlight the role of 15-PGDH in IPF and suggest 15-PGDH inhibition as a promising therapeutic approach.
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http://dx.doi.org/10.1016/j.jaci.2019.11.032DOI Listing
March 2020

Hot topics in the mechanisms of pulmonary arterial hypertension disease: cancer-like pathobiology, the role of the adventitia, systemic involvement, and right ventricular failure.

Pulm Circ 2019 Oct-Dec;9(4):2045894019889775. Epub 2019 Nov 20.

Institute of Physiology, Charité - Universitaetsmedizin Berlin, Berlin, Germany.

In order to intervene appropriately and develop disease-modifying therapeutics for pulmonary arterial hypertension, it is crucial to understand the mechanisms of disease pathogenesis and progression. We herein discuss four topics of disease mechanisms that are currently highly debated, yet still unsolved, in the field of pulmonary arterial hypertension. Is pulmonary arterial hypertension a cancer-like disease? Does the adventitia play an important role in the initiation of pulmonary vascular remodeling? Is pulmonary arterial hypertension a systemic disease? Does capillary loss drive right ventricular failure? While pulmonary arterial hypertension does not replicate all features of cancer, anti-proliferative cancer therapeutics might still be beneficial in pulmonary arterial hypertension if monitored for safety and tolerability. It was recognized that the adventitia as a cell-rich compartment is important in the disease pathogenesis of pulmonary arterial hypertension and should be a therapeutic target, albeit the data are inconclusive as to whether the adventitia is involved in the initiation of neointima formation. There was agreement that systemic diseases can lead to pulmonary arterial hypertension and that pulmonary arterial hypertension can have systemic effects related to the advanced lung pathology, yet there was less agreement on whether idiopathic pulmonary arterial hypertension is a systemic disease per se. Despite acknowledging the limitations of exactly assessing vascular density in the right ventricle, it was recognized that the failing right ventricle may show inadequate vascular adaptation resulting in inadequate delivery of oxygen and other metabolites. Although the debate was not meant to result in a definite resolution of the specific arguments, it sparked ideas about how we might resolve the discrepancies by improving our disease modeling (rodent models, large-animal studies, studies of human cells, tissues, and organs) as well as standardization of the models. Novel experimental approaches, such as lineage tracing and better three-dimensional imaging of experimental as well as human lung and heart tissues, might unravel how different cells contribute to the disease pathology.
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http://dx.doi.org/10.1177/2045894019889775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868582PMC
November 2019

Kinases as potential targets for treatment of pulmonary hypertension and right ventricular dysfunction.

Br J Pharmacol 2021 01 4;178(1):31-53. Epub 2020 Feb 4.

Department of Internal Medicine, Justus-Liebig University Giessen, Giessen, Germany.

Pulmonary hypertension (PH) is a progressive pulmonary vasculopathy that causes chronic right ventricular pressure overload and often leads to right ventricular failure. Various kinase inhibitors have been studied in the setting of PH and either improved or worsened the disease, highlighting the importance of understanding the specific role of the respective kinases in a spatiotemporal cellular context. In this review, we will summarize the knowledge on the role of kinases in PH and focus on druggable targets for which certain criteria are met: (a) deregulation of the kinase in PH; (b) small-molecule inhibitors are available (e.g. from the oncology field); (c) preclinical studies have shown their efficacy in PH models; and (d) when available, therapeutic exploitation in human PH has been initiated. Along this line, clinical considerations such as personalized medicine approaches to predict therapy response and adverse side events such as cardiotoxicity together with their clinical management are discussed. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.
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http://dx.doi.org/10.1111/bph.14919DOI Listing
January 2021

IL-1 receptor blockade skews inflammation towards Th2 in a mouse model of systemic sclerosis.

Eur Respir J 2019 09 29;54(3). Epub 2019 Sep 29.

Otto Loewi Research Center, Medical University of Graz, Graz, Austria

The interleukin (IL)-1 family of cytokines is strongly associated with systemic sclerosis (SSc) and pulmonary involvement, but the molecular mechanisms are poorly understood. The aim of this study was to assess the role of IL-1α and IL-1β in pulmonary vascular and interstitial remodelling in a mouse model of SSc.IL-1α and IL-1β were localised in lungs of SSc patients and in the fos-related antigen-2 (Fra-2) transgenic (TG) mouse model of SSc. Lung function, haemodynamic parameters and pulmonary inflammation were measured in Fra-2 TG mice with or without 8 weeks of treatment with the IL-1 receptor antagonist anakinra (25 mg·kg·day). Direct effects of IL-1 on pulmonary arterial smooth muscle cells (PASMCs) and parenchymal fibroblasts were investigated Fra-2 TG mice exhibited increased collagen deposition in the lung, restrictive lung function and enhanced muscularisation of the vasculature with concomitant pulmonary hypertension reminiscent of the changes in SSc patients. Immunoreactivity of IL-1α and IL-1β was increased in Fra-2 TG mice and in patients with SSc. IL-1 stimulation reduced collagen expression in PASMCs and parenchymal fibroblasts distinct signalling pathways. Blocking IL-1 signalling in Fra-2 TG worsened pulmonary fibrosis and restriction, enhanced T-helper cell type 2 (Th2) inflammation, and increased the number of pro-fibrotic, alternatively activated macrophages.Our data suggest that blocking IL-1 signalling as currently investigated in several clinical studies might aggravate pulmonary fibrosis in specific patient subsets due to Th2 skewing of immune responses and formation of alternatively activated pro-fibrogenic macrophages.
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http://dx.doi.org/10.1183/13993003.00154-2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6860995PMC
September 2019

Metformin induces lipogenic differentiation in myofibroblasts to reverse lung fibrosis.

Nat Commun 2019 07 5;10(1):2987. Epub 2019 Jul 5.

Key Laboratory of Interventional Pulmonology of Zhejiang Province, Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, 325035, Wenzhou, China.

Idiopathic pulmonary fibrosis (IPF) is a fatal disease in which the intricate alveolar network of the lung is progressively replaced by fibrotic scars. Myofibroblasts are the effector cells that excessively deposit extracellular matrix proteins thus compromising lung structure and function. Emerging literature suggests a correlation between fibrosis and metabolic alterations in IPF. In this study, we show that the first-line antidiabetic drug metformin exerts potent antifibrotic effects in the lung by modulating metabolic pathways, inhibiting TGFβ1 action, suppressing collagen formation, activating PPARγ signaling and inducing lipogenic differentiation in lung fibroblasts derived from IPF patients. Using genetic lineage tracing in a murine model of lung fibrosis, we show that metformin alters the fate of myofibroblasts and accelerates fibrosis resolution by inducing myofibroblast-to-lipofibroblast transdifferentiation. Detailed pathway analysis revealed a two-arm mechanism by which metformin accelerates fibrosis resolution. Our data report an antifibrotic role for metformin in the lung, thus warranting further therapeutic evaluation.
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http://dx.doi.org/10.1038/s41467-019-10839-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611870PMC
July 2019

Role of the Aryl Hydrocarbon Receptor/ARNT/Cytochrome P450 System in Pulmonary Vascular Diseases.

Circ Res 2019 07 26;125(3):356-366. Epub 2019 Jun 26.

Department of Pulmonary Medicine, Amsterdam University Medical Centers, the Netherlands (N.F.V.).

Rationale: CYPs (cytochrome p450) are critically involved in the metabolism of xenobiotics and toxins. Given that pulmonary hypertension is strongly associated with environmental exposure, we hypothesize that CYPs play a role in the development and maintenance of pathological vascular remodeling.

Objective: We sought to identify key CYPs that could link drug or hormone metabolism to the development of pulmonary hypertension.

Methods And Results: We searched in Medline (PubMed) database, as well as http://www.clinicaltrials.gov, for CYPs associated with many key aspects of pulmonary arterial hypertension including, but not limited to, severe pulmonary hypertension, estrogen metabolism, inflammation mechanisms, quasi-malignant cell growth, drug susceptibility, and metabolism of the pulmonary arterial hypertension-specific drugs.

Conclusions: We postulate a hypothesis where the AhR (aryl hydrocarbon receptor) mediates aberrant cell growth via expression of different CYPs associated with estrogen metabolism and inflammation.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315054DOI Listing
July 2019

Echocardiographic Measurement of Right Ventricular Diastolic Parameters in Mouse.

J Vis Exp 2019 04 27(146). Epub 2019 Apr 27.

Ludwig Boltzmann Institute for Lung Vascular Research.

Diastolic dysfunction is a prominent feature of right ventricular (RV) remodeling associated with conditions of pressure overload. However, the RV diastolic function is rarely quantified in experimental studies. This might be due to technical difficulties in the visualization of the RV in the apical four-chamber view in rodents. Here we describe two positions facilitating the visualization of the apical four-chamber view in mice to assess the RV diastolic function. The apical four-chamber view is enabled by tilting the mouse fixation platform to the left and caudally (LeCa) or to the right and cranially (RiCr). Both positions provide images of comparable quality. The results of the RV diastolic function obtained from two positions are not significantly different. Both positions are comparably easy to perform. This protocol can be incorporated into published protocols and enables detailed investigations of the RV function.
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http://dx.doi.org/10.3791/58021DOI Listing
April 2019

Targeting TMEM16A to reverse vasoconstriction and remodelling in idiopathic pulmonary arterial hypertension.

Eur Respir J 2019 06 5;53(6). Epub 2019 Jun 5.

Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria

Our systematic analysis of anion channels and transporters in idiopathic pulmonary arterial hypertension (IPAH) showed marked upregulation of the Cl channel TMEM16A gene. We hypothesised that TMEM16A overexpression might represent a novel vicious circle in the molecular pathways causing pulmonary arterial hypertension (PAH).We investigated healthy donor lungs (n=40) and recipient lungs with IPAH (n=38) for the expression of anion channel and transporter genes in small pulmonary arteries and pulmonary artery smooth muscle cells (PASMCs).In IPAH, TMEM16A was strongly upregulated and patch-clamp recordings confirmed an increased Cl current in PASMCs (n=9-10). These cells were depolarised and could be repolarised by TMEM16A inhibitors or knock-down experiments (n=6-10). Inhibition/knock-down of TMEM16A reduced the proliferation of IPAH-PASMCs (n=6). Conversely, overexpression of TMEM16A in healthy donor PASMCs produced an IPAH-like phenotype. Chronic application of benzbromarone in two independent animal models significantly decreased right ventricular pressure and reversed remodelling of established pulmonary hypertension.Our findings suggest that increased TMEM16A expression and activity comprise an important pathologic mechanism underlying the vasoconstriction and remodelling of pulmonary arteries in PAH. Inhibition of TMEM16A represents a novel therapeutic approach to reverse remodelling in PAH.
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http://dx.doi.org/10.1183/13993003.00965-2018DOI Listing
June 2019

LRP1 promotes synthetic phenotype of pulmonary artery smooth muscle cells in pulmonary hypertension.

Biochim Biophys Acta Mol Basis Dis 2019 06 22;1865(6):1604-1616. Epub 2019 Mar 22.

Department of Biochemistry, Universities of Giessen and Marburg Lung Center, Giessen, Germany. Electronic address:

Pulmonary hypertension (PH) is characterized by a thickening of the distal pulmonary arteries caused by medial hypertrophy, intimal proliferation and vascular fibrosis. Low density lipoprotein receptor-related protein 1 (LRP1) maintains vascular homeostasis by mediating endocytosis of numerous ligands and by initiating and regulating signaling pathways. Here, we demonstrate the increased levels of LRP1 protein in the lungs of idiopathic pulmonary arterial hypertension (IPAH) patients, hypoxia-exposed mice, and monocrotaline-treated rats. Platelet-derived growth factor (PDGF)-BB upregulated LRP1 expression in pulmonary artery smooth muscle cells (PASMC). This effect was reversed by the PDGF-BB neutralizing antibody or the PDGF receptor antagonist. Depletion of LRP1 decreased proliferation of donor and IPAH PASMC in a β1-integrin-dependent manner. Furthermore, LRP1 silencing attenuated the expression of fibronectin and collagen I and increased the levels of α-smooth muscle actin and myocardin in donor, but not in IPAH, PASMC. In addition, smooth muscle cell (SMC)-specific LRP1 knockout augmented α-SMA expression in pulmonary vessels and reduced SMC proliferation in 3D ex vivo murine lung tissue cultures. In conclusion, our results indicate that LRP1 promotes the dedifferentiation of PASMC from a contractile to a synthetic phenotype thus suggesting its contribution to vascular remodeling in PH.
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http://dx.doi.org/10.1016/j.bbadis.2019.03.012DOI Listing
June 2019

Editorial: Molecular Mechanisms in Pulmonary Hypertension and Right Ventricle Dysfunction.

Front Physiol 2018 10;9:1777. Epub 2018 Dec 10.

Department of Biomedical and Molecular Sciences, Queen's University, Kingston, ON, Canada.

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http://dx.doi.org/10.3389/fphys.2018.01777DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298242PMC
December 2018

Disconnect between Fibrotic Response and Right Ventricular Dysfunction.

Am J Respir Crit Care Med 2019 06;199(12):1550-1560

1 Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria.

Remodeling and fibrosis of the right ventricle (RV) may cause RV dysfunction and poor survival in patients with pulmonary hypertension. To investigate the consequences of RV fibrosis modulation and the accompanying cellular changes on RV function. Expression of fibrotic markers was assessed in the RV of patients with pulmonary hypertension, the murine pulmonary artery banding, and rat monocrotaline and Sugen5416/hypoxia models. Invasive hemodynamic and echocardiographic assessment was performed on galectin-3 knockout or inhibitor-treated mice. Established fibrosis was characterized by marked expression of galectin-3 and an enhanced number of proliferating RV fibroblasts. Galectin-3 genetic and pharmacologic inhibition or antifibrotic treatment with pirfenidone significantly diminished RV fibrosis progression in the pulmonary artery banding model, without improving RV functional parameters. RV fibrotic regions were populated with mesenchymal cells coexpressing vimentin and PDGFRα (platelet-derived growth factor receptor-α), but generally lacked αSMA (α-smooth muscle actin) positivity. Serum levels of galectin-3 were increased in patients with idiopathic pulmonary arterial hypertension but did not correlate with cardiac function. No changes of galectin-3 expression were observed in the lungs. We identified extrapulmonary galectin-3 as an important mediator that drives RV fibrosis in pulmonary hypertension through the expansion of PDGFRα/vimentin-expressing cardiac fibroblasts. However, interventions effectively targeting fibrosis lack significant beneficial effects on RV function.
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http://dx.doi.org/10.1164/rccm.201809-1737OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580669PMC
June 2019

Long non-coding RNAs influence the transcriptome in pulmonary arterial hypertension: the role of PAXIP1-AS1.

J Pathol 2019 03 16;247(3):357-370. Epub 2019 Jan 16.

Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria.

In idiopathic pulmonary arterial hypertension (IPAH), global transcriptional changes induce a smooth muscle cell phenotype characterised by excessive proliferation, migration, and apoptosis resistance. Long non-coding RNAs (lncRNAs) are key regulators of cellular function. Using a compartment-specific transcriptional profiling approach, we sought to investigate the link between transcriptional reprogramming by lncRNAs and the maladaptive smooth muscle cell phenotype in IPAH. Transcriptional profiling of small remodelled arteries from 18 IPAH patients and 17 controls revealed global perturbations in metabolic, neuronal, proliferative, and immunological processes. We demonstrated an IPAH-specific lncRNA expression profile and identified the lncRNA PAXIP1-AS1 as highly abundant. Comparative transcriptomic analysis and functional assays revealed an intrinsic role for PAXIP1-AS1 in orchestrating the hyperproliferative and migratory actions of IPAH smooth muscle cells. Further, we showed that PAXIP1-AS1 mechanistically interferes with the focal adhesion axis via regulation of expression and phosphorylation of its downstream target paxillin. Overall, we show that changes in the lncRNA transcriptome contribute to the disease-specific transcriptional landscape in IPAH. Our results suggest that lncRNAs, such as PAXIP1-AS1, can modulate smooth muscle cell function by affecting multiple IPAH-specific transcriptional programmes. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6900182PMC
March 2019