Publications by authors named "Przemyslaw Blyszczuk"

56 Publications

Dickkopf-1 is downregulated in blood platelets of axial spondyloarthritis patients.

Arthritis Rheumatol 2021 Mar 28. Epub 2021 Mar 28.

Department of Clinical Immunology, Institute of Pediatrics, Jagiellonian University Medical College, Wielicka Str. 265, 30-663, Krakow, Poland.

Objective: Axial spondyloarthritis (axSpA) is a chronic autoinflammatory disease with new bone formation, which is controlled by the Wnt/β-catenin signaling. Dickkopf-1 (Dkk-1) is an inhibitor of the Wnt pathway and platelets represent a major source of Dkk-1 in humans. In a current study, we investigated whether the serum level of Dkk-1 and platelets expression of DKK1 mRNA and Dkk-1 protein are affected in axSpA patients when compared with healthy controls.

Methods: We enrolled 41 patients with axSpA and 35 healthy controls. Total serum Dkk-1 level was measured by quantitative ELISA in all patients and controls. Platelets DKK1 mRNA was analyzed by qRT-PCR in 20 axSpA patients and 20 controls, and Dkk-1 protein level by immunoblot in 20 axSpA patients and 18 controls.

Results: We found a lower concentration of Dkk-1 in serum of axSpA patients in comparison to controls. Furthermore, the expression of Dkk-1 was significantly reduced in axSpA platelets both at the transcriptional and protein level.

Conclusion: Our preliminary observation suggests that dysfunction of the megakaryocyte-blood platelet axis might be responsible for reduced serum Dkk-1 in axSpA patients. Dkk-1 is downregulated in platelets of axSpA patients, which might translate into new bone formation.
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http://dx.doi.org/10.1002/art.41739DOI Listing
March 2021

CD52 regulates monocyte adhesion and interferon type I signalling in systemic sclerosis patients.

Arthritis Rheumatol 2021 Mar 24. Epub 2021 Mar 24.

Department of Rheumatology, Center of Experimental Rheumatology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.

Objectives: Systemic sclerosis (SSc) is characterised by dysregulation of type I interferon (IFN-I) signalling. CD52 is known for its immunosuppressive functions in T-cells. We aimed to investigate the role of CD52 in monocyte adhesion and IFN-I signalling in SSc.

Methods: Transcriptome profiles of circulating CD14 monocytes from lcSSc, dcSS and healthy controls were analysed by RNA sequencing. Levels of CD52, CD11b/integrin α and CD18/integrin β in whole blood was assessed by flow cytometry. CD52 expression was analysed in relation to disease phenotype (early, lcSSc, dcSS) and autoantibody profiles. The impact of overexpression, knockdown, and antibody blocking of CD52 were analysed by gene and protein expressions and functional assays.

Results: Pathway enrichment analysis indicated an increase in adhesion- and IFN-I-related genes in SSc monocytes. These cells displayed up-regulated CD11b/CD18, reduced CD52 expression and enhanced adhesion to ICAM1 and endothelial cells. CD52 expression was consistent with SSc subtypes, immunosuppressive treatment and autoantibody profiles of SSc patients and monocyte adhesion properties. Overexpression of CD52 decreased CD18 levels and monocyte adhesion, while knockdown of CD52 increased monocyte adhesion. Treatment with the anti-CD52 antibody Alemtuzumab increased monocyte adhesion, CD11b/CD18 expression, and enhanced IFN-I responses. Monocytic CD52 expression was up-regulated by IL-4/IL-13 via STAT6 pathway and down-regulated by LPS, IFN-α, IFN-β or IFN-γ in JAK1 and histone deacetylates (HDAC) IIa-dependent manner.

Conclusions: Down-regulation of anti-adhesive CD52 antigen in CD14 monocytes represents a novel mechanism in the pathogenesis of SSc. Targeting of the IFN-HDAC-CD52 axis in monocytes might represent a new therapeutic option for early SSc patients.
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http://dx.doi.org/10.1002/art.41737DOI Listing
March 2021

The AP-1 Transcription Factor Fosl-2 Regulates Autophagy in Cardiac Fibroblasts during Myocardial Fibrogenesis.

Int J Mol Sci 2021 Feb 13;22(4). Epub 2021 Feb 13.

Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, University of Zurich, 8952 Schlieren, Switzerland.

Background: Pathological activation of cardiac fibroblasts is a key step in development and progression of cardiac fibrosis and heart failure. This process has been associated with enhanced autophagocytosis, but molecular mechanisms remain largely unknown.

Methods And Results: Immunohistochemical analysis of endomyocardial biopsies showed increased activation of autophagy in fibrotic hearts of patients with inflammatory cardiomyopathy. In vitro experiments using mouse and human cardiac fibroblasts confirmed that blockade of autophagy with Bafilomycin A1 inhibited fibroblast-to-myofibroblast transition induced by transforming growth factor (TGF)-β. Next, we observed that cardiac fibroblasts obtained from mice overexpressing transcription factor Fos-related antigen 2 (Fosl-2tg) expressed elevated protein levels of autophagy markers: the lipid modified form of microtubule-associated protein 1A/1B-light chain 3B (LC3BII), Beclin-1 and autophagy related 5 (Atg5). In complementary experiments, silencing of Fosl-2 with antisense GapmeR oligonucleotides suppressed production of type I collagen, myofibroblast marker alpha smooth muscle actin and autophagy marker Beclin-1 in cardiac fibroblasts. On the other hand, silencing of either LC3B or Beclin-1 reduced Fosl-2 levels in TGF-β-activated, but not in unstimulated cells. Using a cardiac hypertrophy model induced by continuous infusion of angiotensin II with osmotic minipumps, we confirmed that mice lacking either Fosl-2 (Ccl19CreFosl2flox/flox) or Atg5 (Ccl19CreAtg5flox/flox) in stromal cells were protected from cardiac fibrosis.

Conclusion: Our findings demonstrate that Fosl-2 regulates autophagocytosis and the TGF-β-Fosl-2-autophagy axis controls differentiation of cardiac fibroblasts. These data provide a new insight for the development of pharmaceutical targets in cardiac fibrosis.
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http://dx.doi.org/10.3390/ijms22041861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917643PMC
February 2021

Angiotensin II receptor 1 controls profibrotic Wnt/β-catenin signalling in experimentalautoimmune myocarditis.

Cardiovasc Res 2021 Feb 12. Epub 2021 Feb 12.

Department of Clinical Immunology, Jagiellonian University Medical College, Cracow, Poland.

Aims: Angiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases, however the underlaying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis.

Methods And Results: EAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund's adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21, but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimera experiments proved that ATR1 signalling in the bone-marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-β) mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-β-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-β induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-β failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)β and nuclear β-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, β-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts.

Conclusions: Ang II-ATR1 signalling is critical for development of postinflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpins the importance of Ang II-ATR1 in effective TGF-β downstream signalling response including activation of profibrotic Wnt/β-catenin pathway.

Translational Perspective: Myocardial fibrosis causes impaired cardiac function in inflammatory heart diseases. It has been believed that targeting angiotensin II receptor 1 (ATR1) could not only lower blood pressure, but also effectively block both, immune and fibrotic processes in the heart. By using an experimental autoimmune myocarditis model and ATR1-deficient mice, we show that the specific ATR-dependent mechanism is rather limited to control profibrotic, but not inflammatory response. On the molecular level, ATR1 plays a central role in activation of the canonical Wnt pathway in profibrotic response. Thus, these data help to understand how the actual angiotensin II-ATR1 signalling contributes to immunofibrotic heart diseases.
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http://dx.doi.org/10.1093/cvr/cvab039DOI Listing
February 2021

Editorial: Cutting Edge Methodologies in Experimental Cardiovascular Research.

Front Cardiovasc Med 2020 14;7:621900. Epub 2020 Dec 14.

Department of Rheumatology, Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland.

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http://dx.doi.org/10.3389/fcvm.2020.621900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767825PMC
December 2020

Complexity of TNF-α Signaling in Heart Disease.

J Clin Med 2020 Oct 12;9(10). Epub 2020 Oct 12.

Department of Clinical Immunology, Jagiellonian University Medical College, 30-663 Cracow, Poland.

Heart disease is a leading cause of death with unmet clinical needs for targeted treatment options. Tumor necrosis factor alpha (TNF-α) represents a master pro-inflammatory cytokine that plays an important role in many immunopathogenic processes. Anti-TNF-α therapy is widely used in treating autoimmune inflammatory disorders, but in case of patients with heart disease, this treatment was unsuccessful or even harmful. The underlying reasons remain elusive until today. This review summarizes the effects of anti-TNF-α treatment in patients with and without heart disease and describes the involvement of TNF-α signaling in a number of animal models of cardiovascular diseases. We specifically focused on the role of TNF-α in specific cardiovascular conditions and in defined cardiac cell types. Although some mechanisms, mainly in disease development, are quite well known, a comprehensive understanding of TNF-α signaling in the failing heart is still incomplete. Published data identify pathogenic and cardioprotective mechanisms of TNF-α in the affected heart and highlight the differential role of two TNF-α receptors pointing to the complexity of the TNF-α signaling. In the light of these findings, it seems that targeting the TNF-α pathway in heart disease may show therapeutic benefits, but this approach must be more specific and selectively block pathogenic mechanisms. To this aim, more research is needed to better understand the molecular mechanisms of TNF-α signaling in the failing heart.
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http://dx.doi.org/10.3390/jcm9103267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7601316PMC
October 2020

The AP1 Transcription Factor Fosl2 Promotes Systemic Autoimmunity and Inflammation by Repressing Treg Development.

Cell Rep 2020 06;31(13):107826

Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland; Faculty of Medicine, University of Zurich, Zurich, Switzerland. Electronic address:

Regulatory T cells (Tregs) represent a major population in the control of immune homeostasis and autoimmunity. Here we show that Fos-like 2 (Fosl2), a TCR-induced AP1 transcription factor, represses Treg development and controls autoimmunity. Mice overexpressing Fosl2 (Fosl2) indeed show a systemic inflammatory phenotype, with immune infiltrates in multiple organs. This phenotype is absent in Fosl2 × Rag2 mice lacking T and B cells, and Fosl2 induces T cell-intrinsic reduction of Treg development that is responsible for the inflammatory phenotype. Fosl2 T cells can transfer inflammation, which is suppressed by the co-delivery of Tregs, while Fosl2 deficiency in T cells reduces the severity of autoimmunity in the EAE model. We find that Fosl2 could affect expression of FoxP3 and other Treg development genes. Our data highlight the importance of AP1 transcription factors, in particular Fosl2, during T cell development to determine Treg differentiation and control autoimmunity.
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http://dx.doi.org/10.1016/j.celrep.2020.107826DOI Listing
June 2020

Long noncoding RNA H19X is a key mediator of TGF-β-driven fibrosis.

J Clin Invest 2020 09;130(9):4888-4905

Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland.

TGF-β is a master regulator of fibrosis, driving the differentiation of fibroblasts into apoptosis-resistant myofibroblasts and sustaining the production of extracellular matrix (ECM) components. Here, we identified the nuclear long noncoding RNA (lncRNA) H19X as a master regulator of TGF-β-driven tissue fibrosis. H19X was consistently upregulated in a wide variety of human fibrotic tissues and diseases and was strongly induced by TGF-β, particularly in fibroblasts and fibroblast-related cells. Functional experiments following H19X silencing revealed that H19X was an obligatory factor for TGF-β-induced ECM synthesis as well as differentiation and survival of ECM-producing myofibroblasts. We showed that H19X regulates DDIT4L gene expression, specifically interacting with a region upstream of the DDIT4L gene and changing the chromatin accessibility of a DDIT4L enhancer. These events resulted in transcriptional repression of DDIT4L and, in turn, in increased collagen expression and fibrosis. Our results shed light on key effectors of TGF-β-induced ECM remodeling and fibrosis.
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http://dx.doi.org/10.1172/JCI135439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456219PMC
September 2020

Activated Cardiac Fibroblasts Control Contraction of Human Fibrotic Cardiac Microtissues by a β-Adrenoreceptor-Dependent Mechanism.

Cells 2020 05 20;9(5). Epub 2020 May 20.

Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Wagistr. 14, 8952 Schlieren, Switzerland.

Cardiac fibrosis represents a serious clinical problem. Development of novel treatment strategies is currently restricted by the lack of the relevant experimental models in a human genetic context. In this study, we fabricated self-aggregating, scaffold-free, 3D cardiac microtissues using human inducible pluripotent stem cell (iPSC)-derived cardiomyocytes and human cardiac fibroblasts. Fibrotic condition was obtained by treatment of cardiac microtissues with profibrotic cytokine transforming growth factor β1 (TGF-β1), preactivation of foetal cardiac fibroblasts with TGF-β1, or by the use of cardiac fibroblasts obtained from heart failure patients. In our model, TGF-β1 effectively induced profibrotic changes in cardiac fibroblasts and in cardiac microtissues. Fibrotic phenotype of cardiac microtissues was inhibited by treatment with TGF-β-receptor type 1 inhibitor SD208 in a dose-dependent manner. We observed that fibrotic cardiac microtissues substantially increased the spontaneous beating rate by shortening the relaxation phase and showed a lower contraction amplitude. Instead, no changes in action potential profile were detected. Furthermore, we demonstrated that contraction of human cardiac microtissues could be modulated by direct electrical stimulation or treatment with the β-adrenergic receptor agonist isoproterenol. However, in the absence of exogenous agonists, the β-adrenoreceptor blocker nadolol decreased beating rate of fibrotic cardiac microtissues by prolonging relaxation time. Thus, our data suggest that in fibrosis, activated cardiac fibroblasts could promote cardiac contraction rate by a direct stimulation of β-adrenoreceptor signalling. In conclusion, a model of fibrotic cardiac microtissues can be used as a high-throughput model for drug testing and to study cellular and molecular mechanisms of cardiac fibrosis.
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http://dx.doi.org/10.3390/cells9051270DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290967PMC
May 2020

Haploinsufficient and Mice Are Not Protected from Cardiac Inflammation and Postinflammatory Fibrosis in Experimental Autoimmune Myocarditis.

Cells 2020 03 12;9(3). Epub 2020 Mar 12.

Department of Clinical Immunology, Jagiellonian University Medical College, 30-663 Cracow, Poland.

Progressive cardiac fibrosis is a common cause of heart failure. Rho-associated, coiled-coil-containing protein kinases (ROCKs) have been shown to enhance fibrotic processes in the heart and in other organs. In this study, using wild-type, and haploinsufficient mice and mouse model of experimental autoimmune myocarditis (EAM) we addressed the role of ROCK1 and ROCK2 in development of myocarditis and postinflammatory fibrosis. We found that myocarditis severity was comparable in wild-type, and mice at day 21 of EAM. During the acute stage of the disease, hearts of mice showed unaffected numbers of CD11bCD36 macrophages, CD11bCD36Ly6GLy6c neutrophils, CD11bCD36Ly6GLy6c inflammatory monocytes, CD11bCD36Ly6GLy6c monocytes, CD11bSiglecF eosinophils, CD11bCD11c inflammatory dendritic cells and type I collagen-producing fibroblasts. Isolated cardiac fibroblasts treated with transforming growth factor-beta (TGF-β) showed attenuated Smad2 and extracellular signal-regulated kinase (Erk) phosphorylations that were associated with impaired upregulation of smooth muscle actin alpha (αSMA) protein. In contrast to cardiac fibroblasts, expanded heart inflammatory myeloid cells showed unaffected Smad2 activation but enhanced Erk phosphorylation following TGF-β treatment. inflammatory cells responded to TGF-β by a reduced transcriptional profibrotic response and failed to upregulate αSMA and fibronectin at the protein levels. Unexpectedly, in the EAM model wild-type, and mice developed a similar extent of cardiac fibrosis at day 40. In addition, hearts of the wild-type and mice showed comparable levels of cardiac vimentin, periostin and αSMA. In conclusion, despite the fact that ROCK1 regulates TGF-β-dependent profibrotic response, neither ROCK1 nor ROCK2 is critically involved in the development of postinflammatory fibrosis in the EAM model.
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http://dx.doi.org/10.3390/cells9030700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140701PMC
March 2020

Sirt6 deletion in bone marrow-derived cells increases atherosclerosis - Central role of macrophage scavenger receptor 1.

J Mol Cell Cardiol 2020 02 21;139:24-32. Epub 2020 Jan 21.

Center for Molecular Cardiology, University of Zurich, Zurich, Switzerland; Department of Cardiology, University Heart Center, Zurich University Hospital, Zurich, Switzerland. Electronic address:

Aims: Sirtuin 6 (Sirt6) is a NAD-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrow-restricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model.

Methods And Results: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe mice fed a high-cholesterol diet for 12 weeks (n = 12-14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels.

Conclusions: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.
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http://dx.doi.org/10.1016/j.yjmcc.2020.01.002DOI Listing
February 2020

Pathogenesis of ischaemic and non-ischaemic heart diseases in rheumatoid arthritis.

RMD Open 2020 01;6(1)

Department of Rheumatology, University of Debrecen, Faculty of Medicine, Debrecen, Hungary.

Rheumatoid arthritis (RA) is characterised by a chronic inflammatory condition of the joints, but the comorbidities of RA predominantly contribute to the reduced lifespan associated with this disease. Clinical data indicate that cardiovascular disease is the major comorbidity associated with mortality in RA. In this review, we aimed to describe the pathogenesis of heart failure in RA. First, we emphasised the fundamental differences between ischaemic and non-ischaemic heart diseases and referred to their relevance in excessive cardiovascular-dependent mortality in RA. Second, we highlighted aspects of asymptomatic changes in cardiac tissue and in coronary blood vessels that are commonly found in patients with diagnosed RA. Third, we focused on high-grade systemic inflammation as a key trigger of ischaemic and non-ischaemic heart diseases in RA, and described the implication of conventional and biologic antirheumatic medications on the development and progression of heart disease. In particular, we discussed the roles of tumour necrosis factor-alpha (TNF-α) and anti-TNF-α therapies on the development and progression of ischaemic and non-ischaemic heart diseases in RA.
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http://dx.doi.org/10.1136/rmdopen-2019-001032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7046979PMC
January 2020

Heart non-specific effector CD4 T cells protect from postinflammatory fibrosis and cardiac dysfunction in experimental autoimmune myocarditis.

Basic Res Cardiol 2019 12 20;115(1). Epub 2019 Dec 20.

Department of Rheumatology, Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland.

Heart-specific CD4 T cells have been implicated in development and progression of myocarditis in mice and in humans. Here, using mouse models of experimental autoimmune myocarditis (EAM) we investigated the role of heart non-specific CD4 T cells in the progression of the disease. Heart non-specific CD4 T cells were obtained from DO11.10 mice expressing transgenic T cell receptor recognizing chicken ovalbumin. We found that heart infiltrating CD4 T cells expressed exclusively effector (T) phenotype in the EAM model and in hearts of patients with lymphocytic myocarditis. Adoptive transfer experiments showed that while heart-specific T infiltrated the heart shortly after injection, heart non-specific T effectively accumulated during myocarditis and became the major heart-infiltrating CD4 T cell subset at later stage. Restimulation of co-cultured heart-specific and heart non-specific CD4 T cells with alpha-myosin heavy chain antigen showed mainly Th1/Th17 response for heart-specific T and up-regulation of a distinct set of extracellular signalling molecules in heart non-specific T. Adoptive transfer of heart non-specific T in mice with myocarditis did not affect inflammation severity at the peak of disease, but protected the heart from adverse post-inflammatory fibrotic remodelling and cardiac dysfunction at later stages of disease. Furthermore, mouse and human T stimulated in vitro with common gamma cytokines suppressed expression of profibrotic genes, reduced amount of α-smooth muscle actin filaments and decreased contraction of cardiac fibroblasts. In this study, we provided a proof-of-concept that heart non-specific T cells could effectively contribute to myocarditis and protect the heart from the dilated cardiomyopathy outcome.
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http://dx.doi.org/10.1007/s00395-019-0766-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925074PMC
December 2019

Macrophage NCOR1 protects from atherosclerosis by repressing a pro-atherogenic PPARγ signature.

Eur Heart J 2020 03;41(9):995-1005

Center for Molecular Cardiology, University of Zurich, Wagistrasse 12, 8952 Schlieren, Switzerland.

Aims: Nuclear receptors and their cofactors regulate key pathophysiological processes in atherosclerosis development. The transcriptional activity of these nuclear receptors is controlled by the nuclear receptor corepressors (NCOR), scaffolding proteins that form the basis of large corepressor complexes. Studies with primary macrophages demonstrated that the deletion of Ncor1 increases the expression of atherosclerotic molecules. However, the role of nuclear receptor corepressors in atherogenesis is unknown.

Methods And Results: We generated myeloid cell-specific Ncor1 knockout mice and crossbred them with low-density lipoprotein receptor (Ldlr) knockouts to study the role of macrophage NCOR1 in atherosclerosis. We demonstrate that myeloid cell-specific deletion of nuclear receptor corepressor 1 (NCOR1) aggravates atherosclerosis development in mice. Macrophage Ncor1-deficiency leads to increased foam cell formation, enhanced expression of pro-inflammatory cytokines, and atherosclerotic lesions characterized by larger necrotic cores and thinner fibrous caps. The immunometabolic effects of NCOR1 are mediated via suppression of peroxisome proliferator-activated receptor gamma (PPARγ) target genes in mouse and human macrophages, which lead to an enhanced expression of the CD36 scavenger receptor and subsequent increase in oxidized low-density lipoprotein uptake in the absence of NCOR1. Interestingly, in human atherosclerotic plaques, the expression of NCOR1 is reduced whereas the PPARγ signature is increased, and this signature is more pronounced in ruptured compared with non-ruptured carotid plaques.

Conclusions: Our findings show that macrophage NCOR1 blocks the pro-atherogenic functions of PPARγ in atherosclerosis and suggest that stabilizing the NCOR1-PPARγ binding could be a promising strategy to block the pro-atherogenic functions of plaque macrophages and lesion progression in atherosclerotic patients.
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http://dx.doi.org/10.1093/eurheartj/ehz667DOI Listing
March 2020

Experimental Mouse Model of Bleomycin-Induced Skin Fibrosis.

Curr Protoc Immunol 2019 09;126(1):e88

Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland.

Systemic sclerosis (SSc) refers to an autoimmune disease, which is manifested by inflammation, vasculopathy, and fibrosis of the skin and internal organs. There are a number of different animal models recapitulating specific aspects of SSc. The experimental mouse model of bleomycin-induced skin fibrosis is commonly used to study the pathogenesis observed in SSc. In this model, repetitive intradermal injections of the cytotoxic agent bleomycin trigger progressive skin thickening, associated with excessive accumulation of collagen, infiltration of immune cells, and formation of α-smooth muscle actin (α-SMA)-positive myofibroblasts. In this article, we provide a detailed protocol for the induction of skin fibrosis in experimental mice by bleomycin. Moreover, we describe procedures for processing and analyzing affected skin tissue, provide troubleshooting, highlight advantages and limitations of the presented model, and critically discuss representative results. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Intradermal bleomycin injections to induce skin fibrosis in mice Support Protocol: Mouse tissue collection for fibrosis evaluation and for other molecular assays Basic Protocol 2: Evaluation of mouse skin thickness using Masson's trichrome staining Basic Protocol 3: Measurement of hydroxyproline content in skin tissue using a colorimetric assay Basic Protocol 4: Evaluation of myofibroblasts in mouse skin by immunohistochemistry.
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http://dx.doi.org/10.1002/cpim.88DOI Listing
September 2019

Identification and Isolation of Cardiac Fibroblasts From the Adult Mouse Heart Using Two-Color Flow Cytometry.

Front Cardiovasc Med 2019 1;6:105. Epub 2019 Aug 1.

Department of Rheumatology, Center of Experimental Rheumatology, University Hospital Zurich, Zurich, Switzerland.

Cardiac fibroblasts represent a main stromal cell type in the healthy myocardium. Activation of cardiac fibroblasts has been implicated in the pathogenesis of many heart diseases. Profibrotic stimuli activate fibroblasts, which proliferate and differentiate into pathogenic myofibroblasts causing a fibrotic phenotype in the heart. Cardiac fibroblasts are characterized by production of type I collagen, but non-transgenic methods allowing their identification and isolation require further improvements. Herein, we present a new and simple flow cytometry-based method to identify and isolate cardiac fibroblasts from the murine heart. Wild-type and reporter mice expressing enhanced green fluorescent protein (EGFP) under the murine alpha1(I) collagen promoter (Col1a1-EGFP) were used in this study. Hearts were harvested and dissociated into single cell suspensions using enzymatic digestion. Cardiac cells were stained with the erythrocyte marker Ter119, the pan-leukocyte marker CD45, the endothelial cell marker CD31 and gp38 (known also as podoplanin). Fibroblasts were defined in a two-color flow cytometry analysis as a lineage-negative (Lin: Ter119CD45CD31) and gp38-positive (gp38) population. Analysis of hearts isolated from Col1a1-EGFP reporter mice showed that cardiac Lingp38 cells corresponded to type I collagen-producing cells. Lingp38 cells were partially positive for the mesenchymal markers CD44, CD140a, Sca-1 and CD90.2. Sorted Lingp38 cells were successfully expanded for up to four passages. Lingp38 cells activated by Transforming Growth Factor Beta 1 (TGF-β1) upregulated myofibroblast-specific genes and proteins, developed stress fibers positive for alpha smooth muscle actin (αSMA) and showed increased contractility in the collagen gel contraction assay. Two-color flow cytometry analysis using the selected cell surface antigens allows for the identification of collagen-producing fibroblasts in unaffected mouse hearts without using specific reporter constructs. This strategy opens new perspectives to study the physiology and pathophysiology of cardiac fibroblasts in mouse models.
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http://dx.doi.org/10.3389/fcvm.2019.00105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6686717PMC
August 2019

Myocarditis in Humans and in Experimental Animal Models.

Front Cardiovasc Med 2019 16;6:64. Epub 2019 May 16.

Department of Clinical Immunology, Jagiellonian University Medical College, Cracow, Poland.

Myocarditis is defined as an inflammation of the cardiac muscle. In humans, various infectious and non-infectious triggers induce myocarditis with a broad spectrum of histological presentations and clinical symptoms of the disease. Myocarditis often resolves spontaneously, but some patients develop heart failure and require organ transplantation. The need to understand cellular and molecular mechanisms of inflammatory heart diseases led to the development of mouse models for experimental myocarditis. It has been shown that pathogenic agents inducing myocarditis in humans can often trigger the disease in mice. Due to multiple etiologies of inflammatory heart diseases in humans, a number of different experimental approaches have been developed to induce myocarditis in mice. Accordingly, experimental myocarditis in mice can be induced by infection with cardiotropic agents, such as coxsackievirus B3 and protozoan parasite or by activating autoimmune responses against heart-specific antigens. In certain models, myocarditis is followed by the phenotype of dilated cardiomyopathy and the end stage of heart failure. This review describes the most commonly used mouse models of experimental myocarditis with a focus on the role of the innate and adaptive immune systems in induction and progression of the disease. The review discusses also advantages and limitations of individual mouse models in the context of the clinical manifestation and the course of the disease in humans. Finally, animal-free alternatives in myocarditis research are outlined.
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http://dx.doi.org/10.3389/fcvm.2019.00064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6532015PMC
May 2019

WNT3a and WNT5a Transported by Exosomes Activate WNT Signaling Pathways in Human Cardiac Fibroblasts.

Int J Mol Sci 2019 Mar 21;20(6). Epub 2019 Mar 21.

Department of Clinical Immunology, Jagiellonian University Medical College, 30-663 Cracow, Poland.

WNT signaling plays an important role in fibrotic processes in the heart. Recently, exosomes have been proposed as novel extracellular transporters for WNT proteins. In this study, we analyzed whether WNT3a and WNT5a carried by exosomes could activate downstream molecular pathways in human cardiac fibroblasts. Exosomes were isolated from conditioned medium of control, WNT3a- and WNT5a-producing L cells by differential ultracentrifugations. Obtained exosomes showed size ranging between 20⁻150 nm and expressed exosomal markers ALG-2-interacting protein X (ALIX) and CD63. Treatment with WNT3a-rich exosomes inhibited activity of glycogen synthase kinase 3β (GSK3β), induced nuclear translocation of β-catenin, and activated T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors as well as expression of WNT/β-catenin responsive genes in cardiac fibroblasts, but did not coactivate extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1) signaling pathways. In contrast, exosomes produced by WNT5a-producing L cells failed to activate β-catenin-dependent response, but successfully triggered phosphorylation of ERK1/2 and JNK and stimulated IL-6 production. In conclusion, exosomes containing WNT proteins can functionally contribute to cardiac fibrosis by activating profibrotic WNT pathways on cardiac fibroblasts and may represent a novel mechanism of spreading profibrotic signals in the heart.
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http://dx.doi.org/10.3390/ijms20061436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6472055PMC
March 2019

Sera of patients with axial spondyloarthritis (axSpA) enhance osteoclastogenic potential of monocytes isolated from healthy individuals.

BMC Musculoskelet Disord 2018 Dec 6;19(1):434. Epub 2018 Dec 6.

Department of Clinical Immunology, Institute of Paediatrics, Jagiellonian University Medical College, 265 Wielicka Str., 30-663, Krakow, Poland.

Background: Axial spondyloarthritis (axSpA) is characterized by significant bone loss caused by dysregulation of physiological bone turnover, possibly resulting from intensified differentiation of osteoclasts. The aim of this study was to reevaluate the levels of osteoclastogenesis-mediating factors: soluble RANKL, M-CSF, OPG and other cytokines in sera of untreated, with sDMARDs and/or bDMARDs, axSpA patients and to test whether these sera influence differentiation of healthy monocytes towards osteoclast lineage.

Methods: Bone remodeling molecules (RANKL, M-CSF, OPG, IL-6, OSM, IL-17A, TGFβ, and TNFα) were evaluated in 27 patients with axSpA and 23 age and sex-matched controls. Disease activity (BASDAI, ASDAS) and inflammatory markers (ESR, CRP) were assessed. Monocytes obtained from healthy individuals were cultured in vitro in presence of sera from 11 randomly chosen axSpA patients and 10 controls, with addition of exogenous M-CSF and/or RANKL or without. Osteoclastic differentiation was assessed analyzing osteoclast markers (cathepsin K and RANK at mRNA level) and with osteoclast-specific staining.

Results: axSpA patients' sera levels of soluble RANKL were significantly lower and M-CSF, IL-6, OSM, IL-17A and TNFα significantly higher in comparison to controls, whereas of OPG and TGFβ were comparable in both groups. Numbers of generated in vitro osteoclasts and cathepsin K mRNA levels did not differ between cultures supplemented with sera of healthy and axSpA patients, both in the absence and presence of M-CSF. Instead, addition of exogenous RANKL boosted osteoclastogenesis, which was significantly higher in cultures with axSpA sera. Furthermore, sera from axSpA patients induced substantially higher levels of RANK mRNA, independently of M-CSF and RANKL stimulation.

Conclusion: We show that, paradoxically, serum levels of soluble RANKL observed in axSpA are in fact significantly lower in comparison to healthy blood donors. Our results indicate that sera of axSpA patients - in contrary to healthy subjects - contain circulating, soluble factors (presumably IL-6, OSM, IL-17A, TNFα and others) able to stimulate healthy monocytes responsiveness to even relative low RANKL serum levels, by inducing high RANK mRNA expression and - as a net effect - boosting their osteoclastogenic potential. We suggest also that locally produced RANKL in axSpA may induce overactive osteoclasts from their precursors.
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http://dx.doi.org/10.1186/s12891-018-2356-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284314PMC
December 2018

Crosstalk between the TGF-β and WNT signalling pathways during cardiac fibrogenesis.

Acta Biochim Pol 2018 24;65(3):341-349. Epub 2018 Jul 24.

Department of Clinical Immunology, Jagiellonian University Medical College, Kraków, Poland.

Cardiac fibrosis is referred to as an excessive accumulation of stromal cells and extracellular matrix proteins in the myocardium. Progressive fibrosis causes stiffening of the cardiac tissue and affects conduction of electrical impulses, leading to heart failures in a broad range of cardiac conditions. At the cellular level, activation of the cardiac stromal cells and myofibroblast formation are considered as hallmarks of fibrogenesis. At the molecular level, transforming growth factor β (TGF-β) is traditionally considered as a master regulator of the profibrotic processes. More recently, the WNT signalling pathway has also been found to be implicated in the development of myocardial fibrosis. In this review, we summarize current knowledge on the involvement of TGF-β and WNT downstream molecular pathways to cardiac fibrogenesis and describe a crosstalk between these two profibrotic pathways. TGF-β and WNT ligands bind to different receptors and trigger various outputs. However, a growing body of evidence points to cross-regulation between these two pathways. It has been recognized that in cardiac pathologies TGF-β activates WNT/β-catenin signalling, which in turn stabilizes the TGF-β/Smad response. Furthermore both, the non-canonical TGF-β and non-canonical WNT signalling pathways, activate the same mitogen-activated protein kinases (MAPKs): the extracellular signal-regulated kinase (Erk), the c-Jun N-terminal kinases (JNKs) and p38. The crosstalk between TGF-β and WNT pathways seems to play an essential role in switching on the genetic machinery initiating profibrotic changes in the heart. Better understanding of these mechanisms will open new opportunities for development of targeted therapeutic approaches against cardiac fibrosis in the future.
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http://dx.doi.org/10.18388/abp.2018_2635DOI Listing
December 2018

Toll-like receptor 2: On the crossroad between immune response, cardiac fibrosis and liquorice candy.

Int J Cardiol 2018 09 22;267:163-164. Epub 2018 May 22.

Center of Experimental Rheumatology, Department of Rheumatology, University Hospital Zurich, Switzerland; Department of Clinical Immunology, Jagiellonian University Medical College, Cracow, Poland. Electronic address:

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http://dx.doi.org/10.1016/j.ijcard.2018.05.071DOI Listing
September 2018

Connexin43 Controls the Myofibroblastic Differentiation of Bronchial Fibroblasts from Patients with Asthma.

Am J Respir Cell Mol Biol 2017 07;57(1):100-110

1 Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Kraków, Poland.

Pathologic accumulation of myofibroblasts in asthmatic bronchi is regulated by extrinsic stimuli and by the intrinsic susceptibility of bronchial fibroblasts to transforming growth factor-β (TGF-β). The specific function of gap junctions and connexins in this process has remained unknown. Here, we investigated the role of connexin43 (Cx43) in TGF-β-induced myofibroblastic differentiation of fibroblasts derived from bronchoscopic biopsy specimens of patients with asthma and donors without asthma. Asthmatic fibroblasts expressed considerably higher levels of Cx43 and were more susceptible to TGF-β-induced myofibroblastic differentiation than were their nonasthmatic counterparts. TGF-β efficiently up-regulated Cx43 levels and activated the canonical Smad pathway in asthmatic cells. Ectopic Cx43 expression in nonasthmatic (Cx43) fibroblasts increased their predilection to TGF-β-induced Smad2 activation and fibroblast-myofibroblast transition. Transient Cx43 silencing in asthmatic (Cx43) fibroblasts by Cx43 small interfering RNA attenuated the TGF-β-triggered Smad2 activation and myofibroblast formation. Direct interactions of Smad2 and Cx43 with β-tubulin were demonstrated by co-immunoprecipitation assay, whereas the sensitivity of these interactions to TGF-β signaling was confirmed by Förster Resonance Energy Transfer analyses. Furthermore, inhibition of the TGF-β/Smad pathway attenuated TGF-β-triggered Cx43 up-regulation and myofibroblast differentiation of asthmatic fibroblasts. Chemical inhibition of gap junctional intercellular communication with 18 α-glycyrrhetinic acid did not affect the initiation of fibroblast-myofibroblast transition in asthmatic fibroblasts but interfered with the maintenance of their myofibroblastic phenotype. Collectively, our data identified Cx43 as a new player in the feedback mechanism regulating TGF-β/Smad-dependent differentiation of bronchial fibroblasts. Thus, our observations point to Cx43 as a novel profibrotic factor in asthma progression.
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http://dx.doi.org/10.1165/rcmb.2015-0255OCDOI Listing
July 2017

Lidocaine Enhances Contractile Function of Ischemic Myocardial Regions in Mouse Model of Sustained Myocardial Ischemia.

PLoS One 2016 3;11(5):e0154699. Epub 2016 May 3.

Cardioimmunology, Center of Molecular Cardiology, University of Zurich, Wagistr. 12, CH-8952, Schlieren, Switzerland.

Rationale: Perioperative myocardial ischemia is common in high-risk patients. The use of interventional revascularisation or even thrombolysis is limited in this patient subset due to exceedingly high bleeding risks. Blockade of voltage-gated sodium channels (VGSC) with lidocaine had been suggested to reduce infarct size and cardiomyocyte cell death in ischemia/reperfusion models. However, the impact of lidocaine on cardiac function during sustained ischemia still remains unclear.

Methods: Sustained myocardial ischemia was induced by ligation of the left anterior descending artery in 12-16 weeks old male BALB/c mice. Subcutaneous lidocaine (30 mg/kg) was used to block VGSC. Cardiac function was quantified at baseline and at 72h by conventional and speckle-tracking based echocardiography to allow high-sensitivity in vivo phenotyping. Infarct size and cardiomyocyte cell death were assessed post mortem histologically and indirectly using troponin measurements.

Results: Ischemia strongly impaired both, global systolic and diastolic function, which were partially rescued in lidocaine treated in mice. No differences regarding infarct size and cardiomyocyte cell death were observed. Mechanistically, and as shown with speckle-tracking analysis, lidocaine specifically improves residual contractility in the ischemic but not in the remote, non-ischemic myocardium.

Conclusion: VGSC blockade with lidocaine rescues function of ischemic myocardium as a potential bridging to revascularisation in the setting of perioperative myocardial ischemia.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154699PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854463PMC
July 2017

Transforming growth factor-β-dependent Wnt secretion controls myofibroblast formation and myocardial fibrosis progression in experimental autoimmune myocarditis.

Eur Heart J 2017 May;38(18):1413-1425

Research of Systemic Autoimmune Diseases, Division of Rheumatology, University Hospital Zurich, Wagistr. 14, CH-8952 Schlieren, Switzerland.

Aims: Myocardial fibrosis critically contributes to cardiac dysfunction in inflammatory dilated cardiomyopathy (iDCM). Activation of transforming growth factor-β (TGF-β) signalling is a key-step in promoting tissue remodelling and fibrosis in iDCM. Downstream mechanisms controlling these processes, remain elusive.

Methods And Results: Experimental autoimmune myocarditis (EAM) was induced in BALB/c mice with heart-specific antigen and adjuvant. Using heart-inflammatory precursors, as well as mouse and human cardiac fibroblasts, we demonstrated rapid secretion of Wnt proteins and activation of Wnt/β-catenin pathway in response to TGF-β signalling. Inactivation of extracellular Wnt with secreted Frizzled-related protein 2 (sFRP2) or inhibition of Wnt secretion with Wnt-C59 prevented TGF-β-mediated transformation of inflammatory precursors and cardiac fibroblasts into pathogenic myofibroblasts. Inhibition of T-cell factor (TCF)/β-catenin-mediated transcription with ICG-001 or genetic loss of β-catenin also prevented TGF-β-induced myofibroblasts formation. Furthermore, blocking of Smad-independent TGF-β-activated kinase 1 (TAK1) pathway completely abrogated TGF-β-induced Wnt secretion. Activation of Wnt pathway in the absence of TGF-β, however, failed to transform precursors into myofibroblasts. The critical role of Wnt axis for cardiac fibrosis in iDCM is also supported by elevated Wnt-1/Wnt-5a levels in human samples from hearts with myocarditis. Accordingly, and as an in vivo proof of principle, inhibition of Wnt secretion or TCF/β-catenin-mediated transcription abrogated the development of post-inflammatory fibrosis in EAM.

Conclusion: We identified TAK1-mediated rapid Wnt protein secretion as a novel downstream key mechanism of TGF-β-mediated myofibroblast differentiation and myocardial fibrosis progression in human and mouse myocarditis. Thus, pharmacological targeting of Wnts might represent a promising therapeutic approach against iDCM in the future.
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http://dx.doi.org/10.1093/eurheartj/ehw116DOI Listing
May 2017

Absence of nonhematopoietic MHC class II expression protects mice from experimental autoimmune myocarditis.

Eur J Immunol 2016 Mar 22;46(3):656-64. Epub 2015 Dec 22.

Department of Biochemistry, CIIL, University of Lausanne, Epalinges, Switzerland.

Experimental autoimmune myocarditis (EAM) is a CD4(+) T-cell-mediated model of human inflammatory dilated cardiomyopathies. Heart-specific CD4(+) T-cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation-induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV-/- K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α-myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV-/- K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4(+) T cells and in expression of IFN-γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN-γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN-γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis.
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http://dx.doi.org/10.1002/eji.201545945DOI Listing
March 2016

Deletion of Rictor in brain and fat alters peripheral clock gene expression and increases blood pressure.

Hypertension 2015 Aug 22;66(2):332-9. Epub 2015 Jun 22.

From the Research Unit, Department of Internal Medicine (K.D., I.B., S.G., R.H, E.B., E.H.) and Division of Surgical Research (M.A.), University Hospital Zurich, Zurich, Switzerland; Center of Competence Multimorbidity and University Research Priority Program "Dynamics of Healthy Aging" (K.D., I.B., S.G., R.H, E.B., E.H.), Laboratory for Animal Model Pathology, Institute of Veterinary Pathology, Vetsuisse faculty (G.P.), Zurich Integrative Rodent Physiology (P.S.), Institute of Pharmacology and Toxicology (A.A., S.A.B.), Horten Center for Patient-Oriented Research and Knowledge Transfer (U.H.), Cardioimmunology, Center of Molecular Cardiology (P.B.), and Zurich Center for Integrative Human Physiology (E.B.), University of Zurich, Zurich, Switzerland; and Biozentrum, University of Basel, Basel, Switzerland (M.N.H.).

The mammalian target of rapamycin complex 2 (mTORC2) contains the essential protein RICTOR and is activated by growth factors. mTORC2 in adipose tissue contributes to the regulation of glucose and lipid metabolism. In the perivascular adipose tissue, mTORC2 ensures normal vascular reactivity by controlling expression of inflammatory molecules. To assess whether RICTOR/mTORC2 contributes to blood pressure regulation, we applied a radiotelemetry approach in control and Rictor knockout (Rictor(aP2KO)) mice generated using adipocyte protein-2 gene promoter-driven CRE recombinase expression to delete Rictor. The 24-hour mean arterial pressure was increased in Rictor(aP2KO) mice, and the physiological decline in mean arterial pressure during the dark period was impaired. In parallel, heart rate and locomotor activity were elevated during the dark period with a pattern similar to blood pressure changes. This phenotype was associated with mild cardiomyocyte hypertrophy, decreased cardiac natriuretic peptides, and their receptor expression in adipocytes. Moreover, clock gene expression was reduced or phase-shifted in perivascular adipose tissue. No differences in clock gene expression were observed in the master clock suprachiasmatic nucleus, although Rictor gene expression was also lower in brain of Rictor(aP2KO) mice. Thus, this study highlights the importance of RICTOR/mTORC2 for interactions between vasculature, adipocytes, and brain to tune physiological outcomes, such as blood pressure and locomotor activity.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.115.05398DOI Listing
August 2015

Novel therapeutic options in inflammatory cardiomyopathy.

Swiss Med Wkly 2013 27;143:w13841. Epub 2013 Aug 27.

Cardioimmunology, Cardiovascular Research, Institute of Physiology , Zürich, SWITZERLAND;

Inflammatory dilated cardiomyopathy (iDCM) denotes cardiac dysfunction due to myocardial inflammation (myocarditis). Progressively impaired cardiac contractility, fibrosis and dilation of heart chambers characterise iDCM phenotypically, and are associated with poor clinical prognosis. Cardiotropic infections followed by autoimmune responses against heart tissue are the most common cause of iDCM. The pathophysiology of iDCM is still poorly understood. Nevertheless, our understanding of the molecular mechanisms of inflammatory heart failure advanced during recent years. In fact, recent mechanistic insights might open the view for novel diagnostic and therapeutic approaches for iDCM patients in the future. In this review we update our knowledge on disease mechanisms, summarise current clinical approaches for iDCM patients, and discuss future therapeutic options.
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http://dx.doi.org/10.4414/smw.2013.13841DOI Listing
June 2014

Innate signaling promotes formation of regulatory nitric oxide-producing dendritic cells limiting T-cell expansion in experimental autoimmune myocarditis.

Circulation 2013 Jun 13;127(23):2285-94. Epub 2013 May 13.

Cardioimmunology, Cardiovascular Research, Institute of Physiology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

Background: Activation of innate pattern-recognition receptors promotes CD4+ T-cell-mediated autoimmune myocarditis and subsequent inflammatory cardiomyopathy. Mechanisms that counterregulate exaggerated heart-specific autoimmunity are poorly understood.

Methods And Results: Experimental autoimmune myocarditis was induced in BALB/c mice by immunization with α-myosin heavy chain peptide and complete Freund's adjuvant. Together with interferon-γ, heat-killed Mycobacterium tuberculosis, an essential component of complete Freund's adjuvant, converted CD11b(hi)CD11c(-) monocytes into tumor necrosis factor-α- and nitric oxide synthase 2-producing dendritic cells (TipDCs). Heat-killed M. tuberculosis stimulated production of nitric oxide synthase 2 via Toll-like receptor 2-mediated nuclear factor-κB activation. TipDCs limited antigen-specific T-cell expansion through nitric oxide synthase 2-dependent nitric oxide production. Moreover, they promoted nitric oxide synthase 2 production in hematopoietic and stromal cells in a paracrine manner. Consequently, nitric oxide synthase 2 production by both radiosensitive hematopoietic and radioresistant stromal cells prevented exacerbation of autoimmune myocarditis in vivo.

Conclusions: Innate Toll-like receptor 2 stimulation promotes formation of regulatory TipDCs, which confine autoreactive T-cell responses in experimental autoimmune myocarditis via nitric oxide. Therefore, activation of innate pattern-recognition receptors is critical not only for disease induction but also for counterregulatory mechanisms, protecting the heart from exaggerated autoimmunity.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.112.000434DOI Listing
June 2013

Abnormal high-density lipoprotein induces endothelial dysfunction via activation of Toll-like receptor-2.

Immunity 2013 Apr 7;38(4):754-68. Epub 2013 Mar 7.

Cardiovascular Center, Cardiology, University Hospital Zurich and Cardiovascular Research, Institute of Physiology, University of Zurich, Zurich, Switzerland.

Endothelial injury and dysfunction (ED) represent a link between cardiovascular risk factors promoting hypertension and atherosclerosis, the leading cause of death in Western populations. High-density lipoprotein (HDL) is considered antiatherogenic and known to prevent ED. Using HDL from children and adults with chronic kidney dysfunction (HDL(CKD)), a population with high cardiovascular risk, we have demonstrated that HDL(CKD) in contrast to HDL(Healthy) promoted endothelial superoxide production, substantially reduced nitric oxide (NO) bioavailability, and subsequently increased arterial blood pressure (ABP). We have identified symmetric dimethylarginine (SDMA) in HDL(CKD) that causes transformation from physiological HDL into an abnormal lipoprotein inducing ED. Furthermore, we report that HDL(CKD) reduced endothelial NO availability via toll-like receptor-2 (TLR-2), leading to impaired endothelial repair, increased proinflammatory activation, and ABP. These data demonstrate how SDMA can modify the HDL particle to mimic a damage-associated molecular pattern that activates TLR-2 via a TLR-1- or TLR-6-coreceptor-independent pathway, linking abnormal HDL to innate immunity, ED, and hypertension.
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http://dx.doi.org/10.1016/j.immuni.2013.02.009DOI Listing
April 2013

Selective in vivo visualization of immune-cell infiltration in a mouse model of autoimmune myocarditis by fluorine-19 cardiac magnetic resonance.

Circ Cardiovasc Imaging 2013 Mar 23;6(2):277-84. Epub 2013 Jan 23.

Department of Radiology, University Hospital (CHUV), Lausanne, Switzerland.

Background: The goal of this study was to characterize the performance of fluorine-19 ((19)F) cardiac magnetic resonance (CMR) for the specific detection of inflammatory cells in a mouse model of myocarditis. Intravenously administered perfluorocarbons are taken up by infiltrating inflammatory cells and can be detected by (19)F-CMR. (19)F-labeled cells should, therefore, generate an exclusive signal at the inflamed regions within the myocardium.

Methods And Results: Experimental autoimmune myocarditis was induced in BALB/c mice. After intravenous injection of 2×200 µL of a perfluorocarbon on day 19 and 20 (n=9) after immunization, in vivo (19)F-CMR was performed at the peak of myocardial inflammation (day 21). In 5 additional animals, perfluorocarbon combined with FITC (fluorescein isothiocyanate) was administered for postmortem immunofluorescence and flow-cytometry analyses. Control experiments were performed in 9 animals. In vivo (19)F-CMR detected myocardial inflammation in all experimental autoimmune myocarditis-positive animals. Its resolution was sufficient to identify even small inflammatory foci, that is, at the surface of the right ventricle. Postmortem immunohistochemistry and flow cytometry confirmed the presence of perfluorocarbon in macrophages, dendritic cells, and granulocytes, but not in lymphocytes. The myocardial volume of elevated (19)F signal (rs=0.96; P<0.001), the (19)F signal-to-noise ratio (rs=0.92; P<0.001), and the (19)F signal integral (rs=0.96; P<0.001) at day 21 correlated with the histological myocarditis severity score.

Conclusions: In vivo (19)F-CMR was successfully used to visualize the inflammation specifically and robustly in experimental autoimmune myocarditis, and thus allowed for an unprecedented insight into the involvement of inflammatory cells in the disease process.
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http://dx.doi.org/10.1161/CIRCIMAGING.112.000125DOI Listing
March 2013