Publications by authors named "Dennis Wolf"

70 Publications

A DARPin targeting activated Mac-1 is a novel diagnostic tool and potential anti-inflammatory agent in myocarditis, sepsis and myocardial infarction.

Basic Res Cardiol 2021 Mar 15;116(1):17. Epub 2021 Mar 15.

Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

The monocyte β-integrin Mac-1 is crucial for leukocyte-endothelium interaction, rendering it an attractive therapeutic target for acute and chronic inflammation. Using phage display, a Designed-Ankyrin-Repeat-Protein (DARPin) was selected as a novel binding protein targeting and blocking the α I-domain, an activation-specific epitope of Mac-1. This DARPin, named F7, specifically binds to activated Mac-1 on mouse and human monocytes as determined by flow cytometry. Homology modelling and docking studies defined distinct interaction sites which were verified by mutagenesis. Intravital microscopy showed reduced leukocyte-endothelium adhesion in mice treated with this DARPin. Using mouse models of sepsis, myocarditis and ischaemia/reperfusion injury, we demonstrate therapeutic anti-inflammatory effects. Finally, the activated Mac-1-specific DARPin is established as a tool to detect monocyte activation in patients receiving extra-corporeal membrane oxygenation, as well as suffering from sepsis and ST-elevation myocardial infarction. The activated Mac-1-specific DARPin F7 binds preferentially to activated monocytes, detects inflammation in critically ill patients, and inhibits monocyte and neutrophil function as an efficient new anti-inflammatory agent.
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http://dx.doi.org/10.1007/s00395-021-00849-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960600PMC
March 2021

Inflammatory Cell Recruitment in Cardiovascular Disease.

Front Cell Dev Biol 2021 18;9:635527. Epub 2021 Feb 18.

Department of Cardiology and Angiology I, University Heart Center Freiburg, Freiburg, Germany.

Atherosclerosis, the main underlying pathology for myocardial infarction and stroke, is a chronic inflammatory disease of middle-sized to large arteries that is initiated and maintained by leukocytes infiltrating into the subendothelial space. It is now clear that the accumulation of pro-inflammatory leukocytes drives progression of atherosclerosis, its clinical complications, and directly modulates tissue-healing in the infarcted heart after myocardial infarction. This inflammatory response is orchestrated by multiple soluble mediators that enhance inflammation systemically and locally, as well as by a multitude of partially tissue-specific molecules that regulate homing, adhesion, and transmigration of leukocytes. While numerous experimental studies in the mouse have refined our understanding of leukocyte accumulation from a conceptual perspective, only a few anti-leukocyte therapies have been directly validated in humans. Lack of tissue-tropism of targeted factors required for leukocyte accumulation and unspecific inhibition strategies remain the major challenges to ultimately translate therapies that modulate leukocytes accumulation into clinical practice. Here, we carefully describe receptor and ligand pairs that guide leukocyte accumulation into the atherosclerotic plaque and the infarcted myocardium, and comment on potential future medical therapies.
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http://dx.doi.org/10.3389/fcell.2021.635527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7930487PMC
February 2021

ApoB-Specific CD4 T Cells in Mouse and Human Atherosclerosis.

Cells 2021 Feb 19;10(2). Epub 2021 Feb 19.

Department of Cardiology and Angiology I, University Heart Center Freiburg, Hugstetterstraße 55, 79106 Freiburg, Germany.

Atherosclerosis is a chronic inflammatory condition of the arterial wall that leads to the formation of vessel-occluding plaques within the subintimal space of middle-sized and larger arteries. While traditionally understood as a myeloid-driven lipid-storage disease, growing evidence suggests that the accumulation of low-density lipoprotein cholesterol (LDL-C) ignites an autoimmune response with CD4 T-helper (T) cells that recognize self-peptides from Apolipoprotein B (ApoB), the core protein of LDL-C. These autoreactive CD4 T cells home to the atherosclerotic plaque, clonally expand, instruct other cells in the plaque, and induce clinical plaque instability. Recent developments in detecting antigen-specific cells at the single cell level have demonstrated that ApoB-reactive CD4 T cells exist in humans and mice. Their phenotypes and functions deviate from classical immunological concepts of distinct and terminally differentiated T immunity. Instead, ApoB-specific CD4 T cells have a highly plastic phenotype, can acquire several, partially opposing and mixed transcriptional programs simultaneously, and transit from one T subset into another over time. In this review, we highlight adaptive immune mechanisms in atherosclerosis with a focus on CD4 T cells, introduce novel technologies to detect ApoB-specific CD4 T cells at the single cell level, and discuss the potential impact of ApoB-driven autoimmunity in atherosclerosis.
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http://dx.doi.org/10.3390/cells10020446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7922692PMC
February 2021

Deficiency of Endothelial CD40 Induces a Stable Plaque Phenotype and Limits Inflammatory Cell Recruitment to Atherosclerotic Lesions in Mice.

Thromb Haemost 2021 Feb 22. Epub 2021 Feb 22.

Department of Cardiology and Angiology I, University Heart Center Freiburg-Bad Krozingen, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Objectives:  The co-stimulatory CD40L-CD40 dyad exerts a critical role in atherosclerosis by modulating leukocyte accumulation into developing atherosclerotic plaques. The requirement for cell-type specific expression of both molecules, however, remains elusive. Here, we evaluate the contribution of CD40 expressed on endothelial cells (ECs) in a mouse model of atherosclerosis.

Methods And Results:  Atherosclerotic plaques of apolipoprotein E-deficient ( ) mice and humans displayed increased expression of CD40 on ECs compared with controls. To interrogate the role of CD40 on ECs in atherosclerosis, we induced EC-specific (BmxCre-driven) deficiency of CD40 in mice. After feeding a chow diet for 25 weeks, EC-specific deletion of CD40 (iEC-CD40) ameliorated plaque lipid deposition and lesional macrophage accumulation but increased intimal smooth muscle cell and collagen content, while atherosclerotic lesion size did not change. Leukocyte adhesion to the vessel wall was impaired in iEC-CD40-deficient mice as demonstrated by intravital microscopy. In accord, expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) in the vascular endothelium declined after deletion of CD40. In vitro, antibody-mediated inhibition of human endothelial CD40 significantly abated monocyte adhesion on ECs.

Conclusion:  Endothelial deficiency of CD40 in mice promotes structural features associated with a stable plaque phenotype in humans and decreases leukocyte adhesion. These results suggest that endothelial-expressed CD40 contributes to inflammatory cell migration and consecutive plaque formation in atherogenesis.
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http://dx.doi.org/10.1055/a-1397-1858DOI Listing
February 2021

The Use and Outcomes of Cerebral Protection Devices for Patients Undergoing Transfemoral Transcatheter Aortic Valve Replacement in Clinical Practice.

JACC Cardiovasc Interv 2021 01;14(2):161-168

University Heart Center Freiburg, Department of Cardiology and Angiology I, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Objectives: This study hypothesized that cerebral protection prevents strokes in patients undergoing transfemoral transcatheter aortic valve replacement (TAVR) in clinical practice.

Background: Preventing strokes is an important aim in TAVR procedures. Embolic protection devices may protect against cardiac embolism during TAVR, but their use and outcomes in clinical practice remain controversial.

Methods: Isolated transfemoral TAVR procedures performed in Germany with or without cerebral protection devices were extracted from a comprehensive nationwide billing dataset.

Results: A total of 41,654 TAVR procedures performed between 2015 and 2017 were analyzed. The overall share of procedures incorporating cerebral protection devices was 3.8%. Patients receiving cerebral protection devices were at increased operative risk (European System for Cardiac Operative Risk Evaluation score 13.8 vs. 14.7; p < 0.001) but of lower age (81.1 vs. 80.6 years; p = 0.001). To compare outcomes that may be related to the use of cerebral protection devices, a propensity score comparison was performed. The use of a cerebral protection device did not reduce the risk for stroke (adjusted risk difference [aRD]: +0.88%; 95% confidence interval [CI]: -0.07% to 1.83%; p = 0.069) or the risk for developing delirium (aRD: +1.31%; 95% CI: -0.28% to 2.89%; p = 0.106) as a sign of acute brain failure. Although brain damage could not be prevented, in-hospital mortality was lower in the group receiving a cerebral protection device (aRD: -0.76%; 95% CI: -1.46% to -0.06%; p = 0.034).

Conclusions: In this large national database, cerebral embolic protection devices were infrequently used during TAVR procedures. Device use was associated with lower mortality but not a reduction in stroke or delirium. Future studies are needed to confirm these findings.
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http://dx.doi.org/10.1016/j.jcin.2020.09.047DOI Listing
January 2021

Inhibition of macrophage proliferation dominates plaque regression in response to cholesterol lowering.

Basic Res Cardiol 2020 12 9;115(6):78. Epub 2020 Dec 9.

Department of Cardiology and Angiology I, University Heart Center Freiburg-Bad Krozingen and Faculty of Medicine, University of Freiburg, 55 Hugstetter St, 79106, Freiburg, Germany.

Statins induce plaque regression characterized by reduced macrophage content in humans, but the underlying mechanisms remain speculative. Studying the translational APOE*3-Leiden.CETP mouse model with a humanized lipoprotein metabolism, we find that systemic cholesterol lowering by oral atorvastatin or dietary restriction inhibits monocyte infiltration, and reverses macrophage accumulation in atherosclerotic plaques. Contrary to current believes, none of (1) reduced monocyte influx (studied by cell fate mapping in thorax-shielded irradiation bone marrow chimeras), (2) enhanced macrophage egress (studied by fluorescent bead labeling and transfer), or (3) atorvastatin accumulation in murine or human plaque (assessed by mass spectrometry) could adequately account for the observed loss in macrophage content in plaques that undergo phenotypic regression. Instead, suppression of local proliferation of macrophages dominates phenotypic plaque regression in response to cholesterol lowering: the lower the levels of serum LDL-cholesterol and lipid contents in murine aortic and human carotid artery plaques, the lower the rates of in situ macrophage proliferation. Our study identifies macrophage proliferation as the predominant turnover determinant and an attractive target for inducing plaque regression.
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http://dx.doi.org/10.1007/s00395-020-00838-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7725697PMC
December 2020

Heterogeneity of T Cells in Atherosclerosis Defined by Single-Cell RNA-Sequencing and Cytometry by Time of Flight.

Arterioscler Thromb Vasc Biol 2021 Feb 3;41(2):549-563. Epub 2020 Dec 3.

Department of Cardiology, Clinic III for Internal Medicine, University of Cologne, Germany. Department of Cardiology and Angiology I, University Heart Center Freiburg, Faculty of Medicine, University of Freiburg, Germany.

The infiltration and accumulation of pro- and anti-inflammatory leukocytes within the intimal layer of the arterial wall is a hallmark of developing and progressing atherosclerosis. While traditionally perceived as macrophage- and foam cell-dominated disease, it is now established that atherosclerosis is a partial autoimmune disease that involves the recognition of peptides from ApoB (apolipoprotein B), the core protein of LDL (low-density lipoprotein) cholesterol particles, by CD4 T-helper cells and autoantibodies against LDL and ApoB. Autoimmunity in the atherosclerotic plaque has long been understood as a pathogenic T-helper type-1 driven response with proinflammatory cytokine secretion. Recent developments in high-parametric cell immunophenotyping by mass cytometry, single-cell RNA-sequencing, and in tools exploring antigen-specificity have established the existence of several unforeseen layers of T-cell diversity with mixed T1 and T regulatory cells transcriptional programs and unpredicted fates. These findings suggest that pathogenic ApoB-reactive T cells evolve from atheroprotective and immunosuppressive CD4 T regulatory cells that lose their protective properties over time. Here, we discuss T-cell heterogeneity in atherosclerosis with a focus on plasticity, antigen-specificity, exhaustion, maturation, tissue residency, and its potential use in clinical prediction.
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http://dx.doi.org/10.1161/ATVBAHA.120.312137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837690PMC
February 2021

Impact of Preprocedural Aortic Valve Calcification on Conduction Disturbances after Transfemoral Aortic Valve Replacement.

Cardiology 2021 23;146(2):228-237. Epub 2020 Sep 23.

University Heart Center Freiburg, Department of Cardiology and Angiology I, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Aim: The present study analyzes in depth the impact of different calcification patterns on disturbances of the conduction system in transcatheter aortic valve replacement (TAVR) patients.

Methods And Results: A total of 169 preprocedural TAVR multislice computed tomography scans from consecutive transfemoral (TF) TAVRs performed between 2014 and 2017 using either Edwards SAPIEN or Medtronic Evolut R valves were retrospectively evaluated. The volume, distribution, and orientation of annular and valvular aortic valve calcification were measured and their impact on postoperative conduction disturbances was determined using linear and logistic regression analyses. The total volume of calcification and distribution at the aortic annulus or valve did not influence the conduction system. Oval calcification of the left aortic cusp was independently associated with an elevated risk for an increase in atrioventricular block degree (+0.6, p = 0.03). Moreover, orthogonal calcifications at the level of the aortic annulus were associated with an increased risk for QRS prolongation (+26 ms, p = 0.004) and an increased risk for permanent pacemaker implantation (OR 4.3, p = 0.03) after TF TAVR. This was more pronounced in patients undergoing TF TAVR using a balloon-expandable Edwards SAPIEN 3 valve (QRS +38.195 ms, p < 0.001; OR permanent pacemaker 15.48, p = 0.013).

Conclusion: Orthogonal annular calcification confers an increased risk for conduction disturbances after TAVR. This is even more pronounced after implantation of balloon-expandable valves.
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http://dx.doi.org/10.1159/000509389DOI Listing
September 2020

Bleeding Complications Drive In-Hospital Mortality of Patients with Atrial Fibrillation after Transcatheter Aortic Valve Replacement.

Thromb Haemost 2020 Nov 22;120(11):1580-1586. Epub 2020 Aug 22.

Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Background:  Atrial fibrillation (AF) is a risk factor for poor postoperative outcome after transfemoral transcatheter aortic valve replacement (TF-TAVR). The present study analyses the outcomes after TF-TAVR in patients with or without AF and identifies independent predictors for in-hospital mortality in clinical practice.

Methods And Results:  Among all 57,050 patients undergoing isolated TF-TAVR between 2008 and 2016 in Germany, 44.2% of patients ( = 25,309) had AF. Patients with AF were at higher risk for unfavorable in-hospital outcome after TAVR. Including all baseline characteristics for a risk-adjusted comparison, AF was an independent risk factor for in-hospital mortality after TAVR. Among patients with AF, EuroSCORE, New York Heart Association classification class, or renal disease had only moderate effects on mortality, while the occurrence of postprocedural stroke or moderate to major bleeding substantially increased in-hospital mortality (odds ratio [OR] 3.35, 95% confidence interval [CI] 2.61-4.30,  < 0.001 and OR 3.12, 95% CI 2.68-3.62,  < 0.001). However, the strongest independent predictor for in-hospital mortality among patients with AF was severe bleeding (OR 18.00, 95% CI 15.22-21.30,  < 0.001).

Conclusion:  The present study demonstrates that the incidence of bleeding defines the in-hospital outcome of patients with AF after TF-TAVR. Thus, the periprocedural phase demands particular care in bleeding prevention.
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http://dx.doi.org/10.1055/s-0040-1715833DOI Listing
November 2020

Pathogenic Autoimmunity in Atherosclerosis Evolves From Initially Protective Apolipoprotein B-Reactive CD4 T-Regulatory Cells.

Circulation 2020 Sep 24;142(13):1279-1293. Epub 2020 Jul 24.

Division of Vaccine Discovery (B.P., A.S.), La Jolla Institute for Immunology, CA.

Background: Throughout the inflammatory response that accompanies atherosclerosis, autoreactive CD4 T-helper cells accumulate in the atherosclerotic plaque. Apolipoprotein B (apoB), the core protein of low-density lipoprotein, is an autoantigen that drives the generation of pathogenic T-helper type 1 (T1) cells with proinflammatory cytokine secretion. Clinical data suggest the existence of apoB-specific CD4 T cells with an atheroprotective, regulatory T cell (T) phenotype in healthy individuals. Yet, the function of apoB-reactive T and their relationship with pathogenic T1 cells remain unknown.

Methods: To interrogate the function of autoreactive CD4 T cells in atherosclerosis, we used a novel tetramer of major histocompatibility complex II to track T cells reactive to the mouse self-peptide apo B (apoB) at the single-cell level.

Results: We found that apoB T cells build an oligoclonal population in lymph nodes of healthy mice that exhibit a T-like transcriptome, although only 21% of all apoB T cells expressed the T transcription factor FoxP3 (Forkhead Box P3) protein as detected by flow cytometry. In single-cell RNA sequencing, apoB T cells formed several clusters with mixed T signatures that suggested overlapping multilineage phenotypes with pro- and anti-inflammatory transcripts of T1, T helper cell type 2 (T2), and T helper cell type 17 (T17), and of follicular-helper T cells. ApoB T cells were increased in mice and humans with atherosclerosis and progressively converted into pathogenic T1/T17-like cells with proinflammatory properties and only a residual T transcriptome. Plaque T cells that expanded during progression of atherosclerosis consistently showed a mixed T1/T17 phenotype in single-cell RNA sequencing. In addition, we observed a loss of FoxP3 in a fraction of apoB T in lineage tracing of hyperlipidemic mice. In adoptive transfer experiments, converting apoB T failed to protect from atherosclerosis.

Conclusions: Our results demonstrate an unexpected mixed phenotype of apoB-reactive autoimmune T cells in atherosclerosis and suggest an initially protective autoimmune response against apoB with a progressive derangement in clinical disease. These findings identify apoB autoreactive T as a novel cellular target in atherosclerosis.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.042863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515473PMC
September 2020

Meta-Analysis of Leukocyte Diversity in Atherosclerotic Mouse Aortas.

Circ Res 2020 07 16;127(3):402-426. Epub 2020 Jul 16.

La Jolla Institute for Immunology, CA (C.C.H., Y.G., H.Q.D., K.L.).

The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single-cell RNA sequencing and 2 mass cytometry studies. In a comprehensive meta-analysis, we confirm 4 known macrophage subsets-resident, inflammatory, interferon-inducible cell, and Trem2 (triggering receptor expressed on myeloid cells-2) foamy macrophages-and identify a new macrophage subset resembling cavity macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2, and CD (cluster of differentiation)-8 T cells form prominent and separate immune cell populations in atherosclerotic aortas. Many CD4 T cells express IL (interleukin)-17 and the chemokine receptor CXCR (C-X-C chemokine receptor)-6. A small number of regulatory T cells and T helper 1 cells is also identified. Immature and naive T cells are present in both healthy and atherosclerotic aortas. Our meta-analysis overcomes limitations of individual studies that, because of their experimental approach, over- or underrepresent certain cell populations. Mass cytometry studies demonstrate that cell surface phenotype provides valuable information beyond the cell transcriptomes. The present analysis helps resolve some long-standing controversies in the field. First, Trem2 foamy macrophages are not proinflammatory but interferon-inducible cell and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, , which had been considered specific for platelets and megakaryocytes, is also prominently expressed in the main population of resident vascular macrophages. Fourth, a new type of resident macrophage shares transcripts with cavity macrophages. Finally, the discovery of a prominent innate lymphoid cell-2 cluster links the single-cell RNA sequencing work to recent flow cytometry data suggesting a strong atheroprotective role of innate lymphoid cells-2. This resolves apparent discrepancies regarding the role of T helper 2 cells in atherosclerosis based on studies that predated the discovery of innate lymphoid cells-2 cells.
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http://dx.doi.org/10.1161/CIRCRESAHA.120.316903DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371244PMC
July 2020

Pathogenic Role of Air Pollution Particulate Matter in Cardiometabolic Disease: Evidence from Mice and Humans.

Antioxid Redox Signal 2020 08 15;33(4):263-279. Epub 2020 Jun 15.

Department of Cardiology and Angiology I, University Heart Center Freiburg, Freiburg, Germany.

Air pollution is a considerable global threat to human health that dramatically increases the risk for cardiovascular pathologies, such as atherosclerosis, myocardial infarction, and stroke. An estimated 4.2 million cases of premature deaths worldwide are attributable to outdoor air pollution. Among multiple other components, airborne particulate matter (PM) has been identified as the major bioactive constituent in polluted air. While PM-related illness was historically thought to be confined to diseases of the respiratory system, overwhelming clinical and experimental data have now established that acute and chronic exposure to PM causes a systemic inflammatory and oxidative stress response that promotes cardiovascular disease. A large body of evidence has identified an impairment of redox metabolism and the generation of oxidatively modified lipids and proteins in the lung as initial tissue response to PM. In addition, the pathogenicity of PM is mediated by an inflammatory response that involves PM uptake by tissue-resident immune cells, the activation of proinflammatory pathways in various cell types and organs, and the release of proinflammatory cytokines as locally produced tissue response signals that have the ability to affect organ function in a remote manner. In the present review, we summarize and discuss the functional participation of PM in cardiovascular pathologies and its risk factors with an emphasis on how oxidative stress, inflammation, and immunity interact and synergize as a response to PM. The impact of PM constituents, doses, and novel anti-inflammatory therapies against PM-related illness is also discussed.
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http://dx.doi.org/10.1089/ars.2020.8096DOI Listing
August 2020

Outcomes of transcatheter aortic valve implantations in high-volume or low-volume centres in Germany.

Heart 2020 Oct 18;106(20):1604-1608. Epub 2020 Feb 18.

Department of Cardiology and Angiology I, University Heart Center Freiburg, Medical Faculty, University of Freiburg, Freiburg, Germany

Objective: Transcatheter aortic valve implantation (TAVI) is the most common aortic valve replacement in Germany. Since 2015, to ensure high-quality procedures, hospitals in Germany and other countries that meet the minimum requirement of 50 interventions per centre are being certified to perform TAVI. This study analyses the impact of these requirements on case number and in-hospital outcomes.

Methods: All isolated TAVI procedures and in-hospital outcomes between 2008 and 2016 were identified by International Classification of Diseases (ICD) and the German Operation and Procedure Classification codes.

Results: 73 467 isolated transfemoral and transapical TAVI procedures were performed in Germany between 2008 and 2016. During this period, the number of TAVI procedures per year rose steeply, whereas the overall rates of hospital mortality and complications declined. In 2008, the majority of procedures were performed in hospitals with fewer than 50 cases per year (54.63%). Until 2014, the share of patients treated in low-volume centres constantly decreased to 5.35%. After the revision of recommendations, it further declined to 1.99%. In the 2 years after the introduction of the minimum requirements on case numbers, patients were at decreased risk for in-hospital mortality when treated in a high-volume centre (risk-adjusted OR 0.62, p=0.012). The risk for other in-hospital outcomes (stroke, permanent pacemaker implantation and bleeding events) did not differ after risk adjustment (p=0.346, p=0.142 and p=0.633).

Conclusion: A minimum volume of 50 procedures per centre and year appears suitable to allow for sufficient routine and thus better in-hospital outcomes, while ensuring nationwide coverage of TAVI procedures.
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http://dx.doi.org/10.1136/heartjnl-2019-316058DOI Listing
October 2020

Dysregulation of the Mitochondrial Proteome Occurs in Mice Lacking Adiponectin Receptor 1.

Front Endocrinol (Lausanne) 2019 13;10:872. Epub 2019 Dec 13.

Division of Cardiology and Angiology I, Heart Center Freiburg University, Freiburg, Germany.

Decreased serum adiponectin levels in type 2 diabetes has been linked to the onset of mitochondrial dysfunction in diabetic complications by impairing AMPK-SIRT1-PGC-1α signaling via impaired adiponectin receptor 1 (AdipoR1) signaling. Here, we aimed to characterize the previously undefined role of disrupted AdipoR1 signaling on the mitochondrial protein composition of cardiac, renal, and hepatic tissues as three organs principally associated with diabetic complications. Comparative proteomics were performed in mitochondria isolated from the heart, kidneys and liver of mice. A total of 790, 1,573, and 1,833 proteins were identified in cardiac, renal and hepatic mitochondria, respectively. While 121, 98, and 78 proteins were differentially regulated in cardiac, renal, and hepatic tissue of 1 mice, respectively; only 15 proteins were regulated in the same direction across all investigated tissues. Enrichment analysis of differentially expressed proteins revealed disproportionate representation of proteins involved in oxidative phosphorylation conserved across tissue types. Curated pathway analysis identified HNF4, NRF1, LONP, RICTOR, SURF1, insulin receptor, and PGC-1α as candidate upstream regulators. In high fat-fed non-transgenic mice with obesity and insulin resistance, AdipoR1 gene expression was markedly reduced in heart (-70%), kidney (-80%), and liver (-90%) (all < 0.05) as compared to low fat-fed mice. NRF1 was the only upstream regulator downregulated both in 1 mice and in high fat-fed mice, suggesting common mechanisms of regulation. Thus, AdipoR1 signaling regulates mitochondrial protein composition across all investigated tissues in a functionally conserved, yet molecularly distinct, manner. The biological significance and potential implications of impaired AdipoR1 signaling are discussed.
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http://dx.doi.org/10.3389/fendo.2019.00872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6923683PMC
December 2019

Glucose lowering by SGLT2-inhibitor empagliflozin accelerates atherosclerosis regression in hyperglycemic STZ-diabetic mice.

Sci Rep 2019 11 29;9(1):17937. Epub 2019 Nov 29.

University Heart Center Freiburg-Bad Krozingen, Cardiology and Angiology I, University of Freiburg, Freiburg, Germany.

Diabetes worsens atherosclerosis progression and leads to a defect in repair of arteries after cholesterol reduction, a process termed regression. Empagliflozin reduces blood glucose levels via inhibition of the sodium glucose cotransporter 2 (SGLT-2) in the kidney and has been shown to lead to a marked reduction in cardiovascular events in humans. To determine whether glucose lowering by empagliflozin accelerates atherosclerosis regression in a mouse model, male C57BL/6J mice were treated intraperitoneally with LDLR- and SRB1- antisense oligonucleotides and fed a high cholesterol diet for 16 weeks to induce severe hypercholesterolemia and atherosclerosis progression. At week 14 all mice were rendered diabetic by streptozotocin (STZ) injections. At week 16 a baseline group was sacrificed and displayed substantial atherosclerosis of the aortic root. In the remaining mice, plasma cholesterol was lowered by switching to chow diet and treatment with LDLR sense oligonucleotides to induce atherosclerosis regression. These mice then received either empagliflozin or vehicle for three weeks. Atherosclerotic plaques in the empagliflozin treated mice were significantly smaller, showed decreased lipid and CD68 macrophage content, as well as greater collagen content. Proliferation of plaque resident macrophages and leukocyte adhesion to the vascular wall were significantly decreased in empagliflozin-treated mice. In summary, plasma glucose lowering by empagliflozin improves plaque regression in diabetic mice.
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http://dx.doi.org/10.1038/s41598-019-54224-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6884628PMC
November 2019

Platelet CD40 ligand and bleeding during P2Y12 inhibitor treatment in acute coronary syndrome.

Res Pract Thromb Haemost 2019 Oct 26;3(4):684-694. Epub 2019 Jul 26.

Aix Marseille University INSERM, INRA, C2VN Marseille France.

Antiplatelet therapy through inhibition of the adenosine diphosphate (ADP)/P2Y12 pathway is commonly used in the treatment of acute coronary syndrome (ACS). Although efficient in preventing platelet activation and thrombus formation, it increases the risk of bleeding complications. In patients with ACS receiving platelet aggregation inhibitors, that is, P2Y12 blockers (n = 923), we investigated the relationship between plasma and platelet-associated CD40L levels and bleeding events (n = 71). Treatment with P2Y12 inhibitors in patients with ACS did not affect plasma-soluble CD40L levels, but decreased platelet CD40L surface expression (pCD40L) and platelet-released CD40L (rCD40L) levels in response to stimulation as compared to healthy controls. In vitro inhibition of the ADP pathway in healthy control platelets reduced both pCD40L and rCD40L levels. In a multivariable analysis, the reduced pCD40L level observed in ACS patients was significantly associated with the risk of bleeding occurrence (adjusted odds ratio = 0.15; 95% confidence interval = 0.034-0.67). P2Y12 inhibitor-treated (ticagrelor) mice exhibited a 2.5-fold increase in tail bleeding duration compared with controls. A significant reduction in bleeding duration was observed on CD40L but not CD40L platelet infusion. In addition, CD40L blockade in P2Y12 inhibitor-treated blood samples from a healthy human reduced thrombus growth over immobilized collagen under arterial flow. In conclusion, measurement of pCD40L may offer a novel approach to assessing bleeding risk in patients with ACS who are being treated with P2Y12 inhibitors.
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http://dx.doi.org/10.1002/rth2.12244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6781928PMC
October 2019

Migratory and Dancing Macrophage Subsets in Atherosclerotic Lesions.

Circ Res 2019 12 9;125(12):1038-1051. Epub 2019 Oct 9.

Division of Inflammation Biology (S.M., K.B., Y.G., A.B.P., J.M., H.W., D.W., K.L.), La Jolla Institute for Immunology, San Diego, CA.

Rationale: Macrophages are essential regulators of atherosclerosis. They secrete cytokines, process lipoproteins and cholesterol, and take up apoptotic cells. Multiple subsets of plaque macrophages exist and their differential roles are emerging.

Objective: Here, we explore macrophage heterogeneity in atherosclerosis plaques using transgenic fluorescent mice in which subsets of macrophages are labeled by GFP (green fluorescent protein), YFP (yellow fluorescent protein), neither, or both. The objective was to define migration patterns of the visible subsets and relate them to their phenotypes and transcriptomes.

Methods And Results: mice have 4 groups of macrophages in their aortas. The 3 visible subsets show varying movement characteristics. GFP and GFP+YFP+ macrophages extend and retract dendritic processes, dancing on the spot with little net movement while YFP macrophages have a more rounded shape and migrate along the arteries. RNA sequencing of sorted cells revealed significant differences in the gene expression patterns of the 4 subsets defined by GFP and YFP expression, especially concerning chemokine and cytokine expression, matrix remodeling, and cell shape dynamics. Gene set enrichment analysis showed that GFP+ cells have similar transcriptomes to cells found in arteries with tertiary lymphoid organs and regressing plaques while YFP+ cells were associated with progressing and stable plaques.

Conclusions: The combination of quantitative intravital imaging with deep transcriptomes identified 4 subsets of vascular macrophages in atherosclerosis that have unique transcriptomic profiles. Our data link vascular macrophage transcriptomes to their in vivo migratory function. Future work on the functional significance of the change in gene expression and motility characteristics will be needed to fully understand how these subsets contribute to disease progression.
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http://dx.doi.org/10.1161/CIRCRESAHA.119.315175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201888PMC
December 2019

Tumor Necrosis Factor Receptor-Associated Factor 5 Promotes Arterial Neointima Formation through Smooth Muscle Cell Proliferation.

J Vasc Res 2019 22;56(6):308-319. Epub 2019 Aug 22.

Department of Cardiology and Angiology I, Heart Center Freiburg University, Faculty of Medicine, University of Freiburg, Freiburg, Germany.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are cytoplasmic adaptor proteins of the TNF/interleukin (IL)-1/Toll-like receptor superfamily. Ligands of this family such as TNFα, CD40L, and IL-1β promote chronic inflammatory processes such as atherosclerosis and restenosis, the latter being a common adverse reaction after vascular interventions. We previously reported overexpression of TRAF5 in murine and human atheromata and TRAF5-dependent proinflammatory functions in vitro. However, the role of TRAF5 in restenosis remains unsettled. To evaluate whether TRAF5 affects neointima formation, TRAF5-/-LDLR-/- and TRAF5+/+LDLR-/- mice consuming a high cholesterol diet (HCD) received wire-induced injury of the carotid artery. After 28 days, TRAF5-deficient mice showed a 45% decrease in neointimal area formation compared with TRAF5-compentent mice. Furthermore, neointimal vascular smooth muscle cells (vSMC) and macrophages decreased whereas collagen increased in TRAF5-deficient mice. Mechanistically, the latter expressed lower transcript levels of the matrix metalloproteinases 2 and 9, both instrumental in extracellular matrix degradation and vSMC mobilization. Additionally, TRAF5-specific siRNA interference rendered murine vSMC less proliferative upon CD40L stimulation. In accordance with these findings, fewer vSMC isolated from TRAF5-deficient aortas were in a proliferative state as assessed by Ki67 and cyclin B1 expression. In conclusion, TRAF5 deficiency mitigates neointima formation in mice, likely through a TRAF5-dependent decrease in vSMC proliferation.
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http://dx.doi.org/10.1159/000501615DOI Listing
January 2020

Face/On: Multi-Modal Haptic Feedback for Head-Mounted Displays in Virtual Reality.

IEEE Trans Vis Comput Graph 2019 11 12;25(11):3169-3177. Epub 2019 Aug 12.

While the real world provides humans with a huge variety of sensory stimuli, virtual worlds most of all communicate their properties by visual and auditory feedback due to the design of current head mounted displays (HMDs). Since HMDs offer sufficient contact area to integrate additional actuators, prior works utilised a limited amount of haptic actuators to integrate respective information about the virtual world. With the Face/On prototype complex feedback patterns are introduced that combine a high number of vibration motors with additional thermal sources to transport multi-modal and spatial information. A pre-study determining the boundaries of the feedbacks' intensities as well as a user study showing a significant increase of presence and enjoyment validate Face/On's approach.
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http://dx.doi.org/10.1109/TVCG.2019.2932215DOI Listing
November 2019

Residual inflammatory risk in coronary heart disease: incidence of elevated high-sensitive CRP in a real-world cohort.

Clin Res Cardiol 2020 Mar 19;109(3):315-323. Epub 2019 Jul 19.

Department of Cardiology and Angiology I, University Heart Center Freiburg, Medical Faculty, University of Freiburg, Freiburg, Germany.

Background: Inflammation drives atherosclerosis and its complications. Anti-inflammatory therapy with interleukin 1 beta (IL-1β) antibody reduces cardiovascular events in patients with elevated high-sensitive C-reactive protein (hsCRP). This study aims to identify the share of patients with coronary heart disease (CHD) and residual inflammation who may benefit from anti-inflammatory therapy.

Methods: hsCRP and low-density lipoprotein (LDL) levels were determined in 2741 all-comers admitted to the cardiological ward of our tertiary referral hospital between June 2016 and June 2018. Patients without CHD, with acute coronary syndrome, chronic or recurrent systemic infection, use of immunosuppressant or anti-inflammatory agents, chronic inflammatory diseases, chemotherapy, terminal organ failure, traumatic injury and pregnancy were excluded.

Results: 856 patients with stable CHD were included. 42.7% of those had elevated hsCRP ≥ 2 mg/l. Within the group of patients with LDL-cholesterol < 70 mg/dl, 30.9% shared increased hsCRP indicating residual inflammation. After multivariate adjusted backward selection elevated Lipoprotein (a) (OR 1.61, p = 0.048), elevated proBNP (OR 2.57, p < 0.0001), smoking (OR 1.70, p = 0.022), and obesity (OR 2.28, p = 0.007) were associated with elevated hsCRP. In contrast, the use of ezetimibe was associated with normal hsCRP (OR 0.51, p = 0.014). In the subgroup of patients with on-target LDL-cholesterol < 70 mg/dl, backward selection identified elevated proBNP (OR 3.49, p = 0.007) as independent predictor of elevated hsCRP in patients with LDL-cholesterol < 70 mg/dl.

Conclusion: One-third of all-comers patients with CHD showed increased levels of hsCRP despite a LDL-cholesterol < 70 mg/dl potentially qualifying for an anti-inflammatory therapy. Elevated proBNP is an independent risk factor for hsCRP elevation.
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http://dx.doi.org/10.1007/s00392-019-01511-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042185PMC
March 2020

Real-time magnetic resonance imaging - guided coronary intervention in a porcine model.

Sci Rep 2019 06 17;9(1):8663. Epub 2019 Jun 17.

Cardiology and Angiology I, Heart Center Freiburg University and Faculty of Medicine, Freiburg, Germany.

X-ray fluoroscopy is the gold standard for coronary diagnostics and intervention. Magnetic resonance imaging is a radiation-free alternative to x-ray with excellent soft tissue contrast in arbitrary slice orientation. Here, we assessed real-time MRI-guided coronary interventions from femoral access using newly designed MRI technologies. Six Goettingen minipigs were used to investigate coronary intervention using real-time MRI. Catheters were custom-designed and equipped with an active receive tip-coil to improve visibility and navigation capabilities. Using modified standard clinical 5 F catheters, intubation of the left coronary ostium was successful in all animals. For the purpose of MR-guided coronary interventions, a custom-designed 8 F catheter was used. In spite of the large catheter size, and therefore limited steerability, intubation of the left coronary ostium was successful in 3 of 6 animals within seconds. Thereafter, real-time guided implantation of a non-metallic vascular scaffold into coronary arteries was possible. This study demonstrates that real-time MRI-guided coronary catheterization and intervention via femoral access is possible without the use of any contrast agents or radiation, including placement of non-metallic vascular scaffolds into coronary arteries. Further development, especially in catheter and guidewire technology, will be required to drive forward routine MR-guided coronary interventions as an alternative to x-ray fluoroscopy.
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http://dx.doi.org/10.1038/s41598-019-45154-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6572773PMC
June 2019

Impaired SIRT3 activity mediates cardiac dysfunction in endotoxemia by calpain-dependent disruption of ATP synthesis.

J Mol Cell Cardiol 2019 08 13;133:138-147. Epub 2019 Jun 13.

Heart Center Freiburg University, Department of Cardiology and Angiology, Freiburg, Germany; Faculty of Medicine, University of Freiburg, Freiburg, Germany; Division of Cardiology, Medical University of Graz, Graz, Austria. Electronic address:

Background: Sepsis-induced cardiomyopathy contributes to the high mortality of septic shock in critically ill patients. Since the underlying mechanisms are incompletely understood, we hypothesized that sepsis-induced impairment of sirtuin 3 (SIRT3) activity contributes to the development of septic cardiomyopathy.

Methods And Results: Treatment of mice with lipopolysaccharide (LPS) for 6 h resulted in myocardial NAD depletion and increased mitochondrial protein acetylation, indicating impaired myocardial SIRT3 activity due to NAD depletion. LPS treatment also resulted in impaired cardiac output in isolated working hearts, indicating endotoxemia-induced cardiomyopathy. Maintaining normal myocardial NAD levels in LPS-treated mice by Poly(ADP-ribose)polymerase 1 (PARP1) deletion prevented cardiac dysfunction, whereas additional SIRT3 deficiency blunted this beneficial effect, indicating that impaired SIRT3 activity contributes to cardiac dysfunction in endotoxemia. Measurements of mitochondrial ATP synthesis suggest that LPS-induced contractile dysfunction may result from cardiac energy depletion due to impaired SIRT3 activity. Pharmacological inhibition of mitochondrial calpains using MDL28170 normalized LPS-induced cleavage of the ATP5A1 subunit of ATP synthase and normalized contractile dysfunction, suggesting that cardiac energy depletion may result from calpain-mediated cleavage of ATP5A1. These beneficial effects were completely blunted by SIRT3 deficiency. Finally, a gene set enrichment analysis of hearts of patients with septic, ischemic or dilated cardiomyopathy revealed a sepsis-specific suppression of SIRT3 deacetylation targets, including ATP5A1, indicating a functional relevance of SIRT3-dependent pathways in human sepsis.

Conclusions: Impaired SIRT3 activity may mediate cardiac dysfunction in endotoxemia by facilitating calpain-mediated disruption of ATP synthesis, suggesting SIRT3 activation as a potential therapeutic strategy to treat septic cardiomyopathy.
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http://dx.doi.org/10.1016/j.yjmcc.2019.06.008DOI Listing
August 2019

The trafficking protein JFC1 regulates Rac1-GTP localization at the uropod controlling neutrophil chemotaxis and in vivo migration.

J Leukoc Biol 2019 06 12;105(6):1209-1224. Epub 2019 Feb 12.

Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California, USA.

Neutrophil chemotaxis is essential in responses to infection and underlies inflammation. In neutrophils, the small GTPase Rac1 has discrete functions at both the leading edge and in the retraction of the trailing structure at the cell's rear (uropod), but how Rac1 is regulated at the uropod is unknown. Here, we identified a mechanism mediated by the trafficking protein synaptotagmin-like 1 (SYTL1 or JFC1) that controls Rac1-GTP recycling from the uropod and promotes directional migration of neutrophils. JFC1-null neutrophils displayed defective polarization and impaired directional migration to N-formyl-methionine-leucyl-phenylalanine in vitro, but chemoattractant-induced actin remodeling, calcium signaling and Erk activation were normal in these cells. Defective chemotaxis was not explained by impaired azurophilic granule exocytosis associated with JFC1 deficiency. Mechanistically, we show that active Rac1 localizes at dynamic vesicles where endogenous JFC1 colocalizes with Rac1-GTP. Super-resolution microscopy (STORM) analysis shows adjacent distribution of JFC1 and Rac1-GTP, which increases upon activation. JFC1 interacts with Rac1-GTP in a Rab27a-independent manner to regulate Rac1-GTP trafficking. JFC1-null cells exhibited Rac1-GTP accumulation at the uropod and increased tail length, and Rac1-GTP uropod accumulation was recapitulated by inhibition of ROCK or by interference with microtubule remodeling. In vivo, neutrophil dynamic studies in mixed bone marrow chimeric mice show that JFC1 neutrophils are unable to move directionally toward the source of the chemoattractant, supporting the notion that JFC1 deficiency results in defective neutrophil migration. Our results suggest that defective Rac1-GTP recycling from the uropod affects directionality and highlight JFC1-mediated Rac1 trafficking as a potential target to regulate chemotaxis in inflammation and immunity.
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http://dx.doi.org/10.1002/JLB.1VMA0818-320RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543538PMC
June 2019

Immunity and Inflammation in Atherosclerosis.

Circ Res 2019 01;124(2):315-327

Division of Inflammation Biology, La Jolla Institute for Immunology, CA (K.L.).

There is now overwhelming experimental and clinical evidence that atherosclerosis is a chronic inflammatory disease. Lessons from genome-wide association studies, advanced in vivo imaging techniques, transgenic lineage tracing mice, and clinical interventional studies have shown that both innate and adaptive immune mechanisms can accelerate or curb atherosclerosis. Here, we summarize and discuss the pathogenesis of atherosclerosis with a focus on adaptive immunity. We discuss some limitations of animal models and the need for models that are tailored to better translate to human atherosclerosis and ultimately progress in prevention and treatment.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.313591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6342482PMC
January 2019

Platelet Serotonin Aggravates Myocardial Ischemia/Reperfusion Injury via Neutrophil Degranulation.

Circulation 2019 02;139(7):918-931

Faculty of Medicine (M.M., N.H., C.S., T.W., T.M., C.H., C.K., M.S., L.D., C.W., D.S., C.N., H.B., D.W., C.B., I.H., D.D.), University of Freiburg, Germany.

Background: Platelets store large amounts of serotonin that they release during thrombus formation or acute inflammation. This facilitates hemostasis and modulates the inflammatory response.

Methods: Infarct size, heart function, and inflammatory cell composition were analyzed in mouse models of myocardial reperfusion injury with genetic and pharmacological depletion of platelet serotonin. These studies were complemented by in vitro serotonin stimulation assays of platelets and leukocytes in mice and men, and by measuring plasma serotonin levels and leukocyte activation in patients with acute coronary syndrome.

Results: Platelet-derived serotonin induced neutrophil degranulation with release of myeloperoxidase and hydrogen peroxide (HO) and increased expression of membrane-bound leukocyte adhesion molecule CD11b, leading to enhanced inflammation in the infarct area and reduced myocardial salvage. In patients hospitalized with acute coronary syndrome, plasmatic serotonin levels correlated with CD11b expression on neutrophils and myeloperoxidase plasma levels. Long-term serotonin reuptake inhibition-reported to protect patients with depression from cardiovascular events-resulted in the depletion of platelet serotonin stores in mice. These mice displayed a reduction in neutrophil degranulation and preserved cardiac function. In line, patients with depression using serotonin reuptake inhibition, presented with suppressed levels of CD11b surface expression on neutrophils and lower myeloperoxidase levels in blood.

Conclusions: Taken together, we identify serotonin as a potent therapeutic target in neutrophil-dependent thromboinflammation during myocardial reperfusion injury.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.033942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370531PMC
February 2019

Purinergic receptor Y (P2Y)- dependent VCAM-1 expression promotes immune cell infiltration in metabolic syndrome.

Basic Res Cardiol 2018 10 18;113(6):45. Epub 2018 Oct 18.

Department of Cardiology and Angiology I, University Heart Center Freiburg, Faculty of Medicine, University of Freiburg, Hugstetter Str. 55, 79106, Freiburg, Germany.

Sterile inflammation of visceral fat, provoked by dying adipocytes, links the metabolic syndrome to cardiovascular disease. Danger-associated molecular patterns, such as adenosine triphosphate (ATP), are released by activated or dying cells and orchestrate leukocyte infiltration and inflammation via the purinergic receptor P2Y. The gene expression of ATP receptor P2Y did not change in several tissues in the course of obesity, but was increased within epididymal fat. Adipose tissue from P2Y mice consuming high-fat diet (HFD) contained less crown-like structures with a reduced frequency of adipose tissue macrophages (ATMs). This was likely due to decreased leukocyte migration because of missing VCAM-1 exposition on P2Y deficient hypertrophic adipose tissue endothelial cells. Accordingly, P2Y mice showed blunted traits of the metabolic syndrome: they gained less weight compared to P2Y controls, while intake of food and movement behaviour remained unchanged. Liver and adipose tissue were smaller in P2Y animals. Insulin tolerance testing (ITT) performed in obese P2Y mice revealed a better insulin sensitivity as well as lower plasma C-peptide and cholesterol levels. We demonstrate that interfering with somatic P2Y signalling prevents excessive immune cell deposition in diet-induced obesity (DIO), both attenuating adipose tissue inflammation and ameliorating the metabolic phenotype. Thus, blocking the P2Y cascade may be a promising strategy to limit metabolic disease and its sequelae.
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http://dx.doi.org/10.1007/s00395-018-0702-1DOI Listing
October 2018

Atherosclerosis in the single-cell era.

Curr Opin Lipidol 2018 10;29(5):389-396

Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA.

Purpose Of Review: The immune system plays a critical role in the development and modulation of atherosclerosis. New high-parameter technologies, including mass cytometry (CyTOF) and single-cell RNA sequencing (scRNAseq), allow for an encompassing analysis of immune cells. Unexplored marker combinations and transcriptomes can define new immune cell subsets and suggest their functions. Here, we review recent advances describing the immune cells in the artery wall of mice with and without atherosclerosis. We compare technologies and discuss limitations and advantages.

Recent Findings: Both CyTOF and scRNAseq on leukocytes from digested aortae show 10-30 immune cell subsets. Myeloid, T, B and natural killer cells were confirmed. Although cellular functions can be inferred from RNA-Seq data, some subsets cannot be identified based on current knowledge, suggesting they may be new cell types. CyTOF and scRNAseq each identified four B-cell subsets and three macrophage subsets in the atherosclerotic aorta. Limitations include cell death caused by enzymatic digestion and the limited depth of the scRNAseq transcriptomes.

Summary: High-parameter methods are powerful tools for uncovering leukocyte diversity. CyTOF is currently more powerful at discerning leukocyte subsets in the atherosclerotic aorta, whereas scRNAseq provides more insight into their likely functions.
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http://dx.doi.org/10.1097/MOL.0000000000000537DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314310PMC
October 2018

A clinically applicable adjuvant for an atherosclerosis vaccine in mice.

Eur J Immunol 2018 09 12;48(9):1580-1587. Epub 2018 Aug 12.

Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA.

Vaccination with MHC-II-restricted peptides from Apolipoprotein B (ApoB) with complete and incomplete Freund's adjuvant (CFA/IFA) is known to protect mice from atherosclerosis. This vaccination induces antigen-specific IgG1 and IgG2c antibody responses and a robust CD4 T cell response in lymph nodes. However, CFA/IFA cannot be used in humans. To find a clinically applicable adjuvant, we tested the effect of vaccinating Apoe-deficient mice with ApoB peptide P6 (TGAYSNASSTESASY). In a broad screening experiment, Addavax, a squalene-based oil-in-water adjuvant similar to MF59, was the only adjuvant that showed similar efficacy as CFA/IFA. This was confirmed in a confirmation experiment for both the aortic arch and whole aorta analyzed by en face analysis after atherosclerotic lesion staining. Mechanistically, restimulated peritoneal cells from mice immunized with P6 in Addavax released significant amounts of IL-10. Unlike P6 in CFA/IFA, vaccination with P6 in Addavax did not induce any detectable IgG1 or IgG2c antibodies to P6. These data suggest that squalene-based adjuvants such as MF59 are good candidate adjuvants for developing a clinically effective atherosclerosis vaccine.
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http://dx.doi.org/10.1002/eji.201847584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6281392PMC
September 2018

Regulatory CD4 T Cells Recognize Major Histocompatibility Complex Class II Molecule-Restricted Peptide Epitopes of Apolipoprotein B.

Circulation 2018 09;138(11):1130-1143

Department of Bioengineering, University of California, San Diego, La Jolla (K.L.).

Background: CD4 T cells play an important role in atherosclerosis, but their antigen specificity is poorly understood. Immunization with apolipoprotein B (ApoB, core protein of low density lipoprotein) is known to be atheroprotective in animal models. Here, we report on a human APOB peptide, p18, that is sequence-identical in mouse ApoB and binds to both mouse and human major histocompatibility complex class II molecules.

Methods: We constructed p18 tetramers to detect human and mouse APOB-specific T cells and assayed their phenotype by flow cytometry including CD4 lineage transcription factors, intracellular cytokines, and T cell receptor activation. Apolipoprotein E-deficient ( Apoe) mice were vaccinated with p18 peptide or adjuvants alone, and atherosclerotic burden in the aorta was determined.

Results: In human peripheral blood mononuclear cells from donors without cardiovascular disease, p18 specific CD4 T cells detected by a new human leukocyte antigen-antigen D related-p18 tetramers were mostly Foxp3 regulatory T cells (Tregs). Donors with subclinical cardiovascular disease as detected by carotid artery ultrasound had Tregs coexpressing retinoic acid-related orphan receptor gamma t or T-bet, which were both almost absent in donors without cardiovascular disease. In Apoe mice, immunization with p18 induced Tregs and reduced atherosclerotic lesions. After peptide restimulation, responding CD4 T cells identified by Nur77-GFP (green fluorescent protein) were highly enriched in Tregs. A new mouse I-A-p18 tetramer identified the expansion of p18-specific CD4 T cells on vaccination, which were enriched for interleukin-10-producing Tregs.

Conclusions: These findings show that APOB p18-specific CD4 T cells are mainly Tregs in healthy donors, but coexpress other CD4 lineage transcription factors in donors with subclinical cardiovascular disease. This study identifies ApoB peptide 18 as the first Treg epitope in human and mouse atherosclerosis.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.031420DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6160361PMC
September 2018