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Epigenetic regulation of smooth muscle cell plasticity in human vascular disease

Project leader: Till Althoff
Coworkers: Kerstin Wöltje, Andrea Weller, Malte Pietron

Funding: German Research Foundation (DFG individual research grant AL 179511-1);
Deutsches Zentrum für Herz-Kreislaufforschung e.V. (DZHK, German Center for Cardiovascular Research); Berlin Institute of Health (Clinical Scientist program);
Deutsche Herzstiftung (German Heart Foundation)

External cooperations: Wei Chen PhD, Max-Delbrück-Center for Molecular Medicine, Berlin Buch; Yonggang Zhou, PhD (group leader epigenetics) (proteomics), Max-Planck-Institute for Heart and Lung Research, Bad Nauheim; Prof. Marcus Krüger, Head of Proteomics CECAD Cluster of Excellence, University of Cologne; PD Dr. med. Christoph Knosalla, Klinik für Herz-, Thorax- und Gefäßchirurgie & Biomaterialbank, Deutsches Herzzentrum Berlin (DHZB, German Heart Institute Berlin)

Project description:
Unlike skeletal or cardiac muscle cells which are terminally differentiated, vascular smooth muscle cells are highly plastic. This ability to dedifferentiate and redifferentiate is a prerequisite for vascular remodeling processes that physiologically enable vascular development and repair, as well as adaptation to altered hemodynamics. However, smooth muscle cell plasticity also plays a key role in the pathogenesis of vascular diseases. The differentiation state of VSMCs is under control of the transcription factor serum response factor. We recently discovered that G12/G13- and Gq/G11-mediated signaling pathways regulate VSMC differentiation antagonistically by controlling the recruitment of transcriptional co-factors of the myocardin family and the ternary complex factor family, respectively, by serum response factor (Althoff et al. J. Exp. Med. 2012). Accumulating evidence from in vitro-studies suggests that smooth muscle cell plasticity may be co-regulated by epigenetic mechanisms in terms of chromatin modifications.

This project aims to identify epigenetic mechanisms that control smooth muscle cell plasticity in the context of human vascular diseases. Besides applying murine disease models we are performing a patient study to prospectively acquire and analyse human vascular samples. The ultimate goal will be to translate the findings through to the validation of pharmacological agents in vivo.

Atherogenic mechanotransduction of flow-induced endothelial shear stress

Project leader: Till Althoff
Coworkers: Alexander Degkwitz, Andrea Weller

Funding: Deutsches Zentrum für Herz-Kreislaufforschung e.V. (DZHK, German Center for Cardiovascular Research)

External cooperations: Prof. Dr. med. Stefan Offermanns & Dr. Albarràn Juarez, PhD, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim

Project description:
Atherosclerosis is promoted by a number of systemic risk factors like dyslipidemia, diabetes, smoking or hypertension. Although the entire vasculature is equally exposed to these risk factors, atherosclerotic lesions almost exclusively form at specific regions of the arterial tree, where disturbed flow leads to decreased endothelial shear stress. The molecular mechanism of this atherogenic mechanotransduction linking reduced shear stress to atherosclerotic lesion formation is largely unknown.
This project aims to identify molecular sensors and/or transducers of atherogenic shear stress and to define their role in atherosclerosis. The ultimate goal will be to deduce pharmacological targets for anti-atherosclerotic therapy.

Biased GPCR ligands in cardiovascular pharmacotherapy

Project leader: Till Althoff
Coworkers: Kerstin Wöltje, Andrea Weller

External cooperations: Dr. Jens Peter von Kries, Ph.D., Head of Screening Unit,
Leibniz-Institute for Molecular Pharmacology (FMP), Berlin; PD Dr. rer. nat. Patrick Scheerer, Charité, Institute of Medical Physics and Biophysics (IMPB), Research group leader: Protein X-ray Crystallography; PD Dr. rer. nat. Peter Hildebrand, Charité, Institute of Medical Physics and Biophysics (IMPB), Research Group Leader: Computational modelling; Céline Gales, Ph.D., INSERM Toulouse/ Université Paul Sabatier UMR 1048, Institut des Maladies Métaboliques et Cardiovasculaires (I2MC)

Project description:
Classical Agonists and antagonists of G-protein-coupled receptors (GPCR) constitute more than one third of all marketed drugs, and some of the most effective cardiovascular therapeutics target GPCRs like the Angiotensin II receptor (AT1R)- or the endothelin-1 receptor (ETA). While classical antagonists equally inhibit the full network of downstream signalling cascades – which may include both, detrimental and beneficial pathways (Fig. a), a more selective inhibition of would be desirable. In this respect, work by Brian Kobilka and Robert Lefkowitz, that was rewarded with the Nobel Prize in 2012, has led to the novel concept of ligand-biased signaling. This concept, describing functional selectivity of GPCR-ligands and -conformations with respect to the distinct downstream signaling pathways, is adding a new dimension to receptor-based drug discovery and development. Thus we are combining experimental and virtual screening approaches to identify and develop biased ligands that stabilize receptor conformations, which result in activation or deactivation, only of specific pathways (Fig. b).
Methodically we are using luciferase-gene reporter assays for our high-througput screening and Bioluminescence Resonance Energy transfer assays to establish structure function relationships with respect to G-protein recruitment by the receptor. Candidate compounds will be validated in vitro and in murine models for vascular disease. Moreover, we will collaborate with structural biologists to determine the structural basis of ligand-biased signaling using computational modeling and crystallography, which may pave the way for structure-based drug design and lead optimization in the future.

Impact of iRhom2 on atherosclerosis

Project leader: Bernd Hewing
Coworkers: Karl Stangl, Carmen Hannemann, Alica Brettschneider, Phillip van Dijck, Andrea Weller

Funding: Research Grant; Deutsche Forschungsgemeinschaft, DFG (German Research Foundation);
Bernd Hewing is participant of the Charité Clinical Scientist Program (funded by the Charité́-Universitätsmedizin Berlin and the Berlin Institute of Health, BIH).
Alica Brettschneider: Promotionsstipendium des DZHK (Deutsches Zentrum für Herz-Kreislaufforschung e.V.); Phillip van Dijck: Otto-Hess-Promotionsstipendium der DGK (Deutschen Gesellschaft für Kardiologie)

External cooperations: Edward A. Fisher, M.D., Ph.D., M.P.H. NYU School of Medicine, Division of Cardiology, New York, USA; Carl Blobel, M.D., Ph.D. Hospital for Special Surgery, New York, USA;
Prof. Dr. med. Philipp A. Lang, Klinik für Gastroenterologie, Hepatologie und Infektiologie, Universitätsklinikum Düsseldorf, Düsseldorf, Germany

Project description:
Tumor necrosis factor (TNF)-alpha is a potent inflammatory mediator that plays an important role in the development of atherosclerosis. It is expressed as a precursor transmembrane protein and subsequently converted into its soluble, bioactive form by TNF-alpha converting enzyme (TACE) mediated shedding. Recently discovered inactive rhomboid protein 2 (iRhom2) is essential for maturation of TACE in immune cells. A genetic knock-out or knock-down of iRhom2 results in a loss of TACE activity and, consequently, in markedly reduced shedding of TNF-alpha in cells involved in atherosclerosis, such as macrophages. iRhom2-deficient mice exhibit reduced serum levels of TNF-alpha in response to inflammatory stimuli, survive otherwise lethal doses of LPS and are protected from the development of inflammatory arthritis. These findings strongly suggest that the iRhom2/TACE/TNF-alpha signaling axis may contribute to atherosclerosis. However to date, this hypothesis has not been tested experimentally. Therefore, the proposed project evaluates the impact of iRhom2 on early and advanced atherosclerotic plaque development in an iRhom2-deficient atherosclerosis mouse model. Complementary, phenotypic and functional characterization of iRhom2-deficient macrophages will be performed in vitro. The pathophysiological role of iRhom2 in coronary artery disease (CAD) will be evaluated in patients with stable CAD and in the setting of an acute coronary event. Taken together, the proposed project aims at characterizing the role of iRhom2 in atherosclerosis and thus contributes to better understanding of inflammatory processes in atherosclerosis and the development of novel therapeutic strategies for the treatment of this disease.

Monocyte Subsets and Macrophages in Patients with Aortic Valve Stenosis

Project leader: Bernd Hewing, Karl Stangl
Coworkers: Sebastian Chi-Diep Au, Rena Ellerbroek, Nicole Rösener, Antje Ludwig

Funding: Friede-Springer-Herz-Stiftung; Bernd Hewing is participant of the Charité Clinical Scientist Program (funded by the Charité́-Universitätsmedizin Berlin and the Berlin Institute of Health, BIH).

External cooperations: Dr. med. Carolin Giannini, (Institut für Medizinische Immunologie/ BCRT, Charité-Universitätsmedizin Berlin)

Project description:

Aortic valve stenosis (AS) represents the most prevalent valvular disease in the elderly. The pathogenesis of AS involves systemic inflammatory processes that share profound characteristic similarities with atherosclerotic plaque progression in the arterial vessel wall including the infiltration of immune cells; however the exact mechanisms of AS progression are not fully understood yet. Therefore, the present study evaluates the distribution of circulating monocyte subsets in the blood and macrophage phenotypes in aortic valve tissue of patients with severe aortic valve stenosis. Furthermore, we aim to dissect differences in the changes of monocyte subsets after interventional (TAVI) or surgical treatment of aortic valve stenosis.

Epigenetic regulation of angiogenesis

Project leader: Henryk Dreger

Project description:
Over the last decade, our group focused on the role of the ubiquitin-proteasome-system (UPS) in cardiovascular pathology. Among other effects, we observed that partial proteasome inhibition protects cardiovascular cells from oxidative stress via an Nrf2-dependent induction of antioxidative enzymes. Some lasting effects of proteasome inhibition led us to analyze a possible link between the UPS and epigenetic regulation. In a recent project, we identified the target genes of the histone methyltransferase Enhancer of zeste homolog 2 (Ezh2), which is downregulated by proteasome inhibition, in human umbilical vein endothelial cells (HUVEC). Interestingly, knock down of Ezh2 altered the angiogenic properties of HUVEC suggesting epigenetic regulation of angiogenesis. Currently, we are focusing on specific target genes of Ezh2 in HUVEC (e.g. iNOS).


The role of EGCG in vascular dilation in vitro and in vivo

Project leader: Mario Lorenz, Verena Stangl
Coworkers: Angelika Vietzke, Franziska Rauhut, Christine Hofer

Funding: Friede Springer Herz Stiftung

External cooperations: Dr. Benno Zimmermann, Institut für Ernährungs- und Lebensmittelwissenschaften, Rheinische Friedrich-Wilhelms-Universität Bonn

Project description:
Consumption of tea has been inversely associated with the development and progression of cardiovascular diseases. A major mechanism for the cardioprotective effects of tea is the improvement of endothelial function by tea polyphenols. However, the contribution of the main green tea catechin epigallocatechin-3-gallate (EGCG) for the vasodilating effects of tea in vivo is unknown. We previously showed stimulation of eNOS activity in endothelial cells and NO-dependent vasodilation in isolated rat aortic rings by EGCG. Using aortic rings of eNOS-/- mice, we demonstrated that EGCG-induced vasodilation strongly relies on functional nitric oxide synthase and stimulation of NO production in vessels. In vivo, green tea improves flow-mediated vasodilation (FMD) in healthy volunteers. To study the impact of EGCG on endothelial function in vivo, the same dose of EGCG is applied in different intervention arms and FMD is measured in healthy subjects. Since EGCG is used already in many human intervention studies, understanding the role of EGCG in vivo represents an important issue for study design.

Sex-specific differences between female and male human umbilical vein endothelial cells (HUVEC)

Project leader: Mario Lorenz, Henryk Dreger, Verena Stangl

Coworkers: Angelika Vietzke, Cornelia Bartsch, Benjamin Blaschke

Funding: DZHK (Deutsches Zentrum für Herz-Kreislaufforschung e.V.)

External cooperations: Prof. Petra Knaus und Dr. Andreas Benn, Institute of Chemistry and Biochemistry, FU Berlin; Prof. Dr. Uwe Völker und Dr. Elke Hammer, Interfaculty Institute of Genetics and Functional Genomics, Ernst-Moritz-Arndt-University Greifswald; Prof. Dr. Ana Zenclussen und Dr. Anne Schumacher, Experimental Obstetrics and Gynecology, Medical Faculty, Otto-von-Guericke University Magdeburg; Prof. Dr. Werner Reutter und Paul Robin Wratil, Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie, Charité – Universitätsmedizin Berlin (CBF)

Project description:
The underlying causes for sexual dimorphisms in cardiovascular diseases are only partially understood. Experimental studies have focused primarily on the role of sex steroids. However, research in recent years revealed that cells themselves show sex-specific differences. We initially compared the whole transcriptome of female and male HUVEC by gene microarray analysis. We found that immune-related genes are higher expressed in female as compared to male endothelial cells. In addition, female HUVEC showed a more pronounced transcriptional response to physiological shear stress than their male counterparts. The results reveal distinct characteristics at the transcriptional level and demonstrate that HUVEC cells indeed show sex-specific properties. Based on the observed transcriptional differences, we are now investigating whether sex-specific transcriptional differences translate into physiological disparities. As a functional approach, cell migration assays and metabolic studies will be performed. We found that female HUVEC show increased tube formation capacity, compared to male cells.
In ongoing experiments we will focus on HUVEC from female and male dizygotic twin pairs. We plan to analyze sex-specific differences in the whole proteome. The proteome analysis will include measurements of cellular protein extracts as well as of the secretome (soluble factors). The results will provide insights on sex differences in protein levels both intracellular as well as of secreted proteins. In parallel, we expect to identify potential candidates to account for our detected functional differences between male and female endothelial cells. The results of the project will contribute to the understanding of physiological differences and help to disclose mechanisms responsible for gender differences in the vascular system.

Superparamagnetic Iron Oxide Nanoparticles for MR-Imaging of Atherosclerosis

Project leader: Antje Ludwig, Wolfram Poller, Verena Stangl
Coworkers: Anke Stach

Funding: DFG-KFO 213, DZHK

External cooperations: Charité Experimentelle Radiologie, Physikalisch Technische Bundesanstalt Berlin, Working Group Biosignals

Project description:
Magnetic resonance imaging (MRI) with contrast agents that target specific inflammatory components of atherosclerotic lesions have the potential to emerge as promising diagnostic modality for detecting unstable plaques. We explore the potential of electrostatically stabilized very small superparamagnetic iron oxide particles (VSOP) to visualize high-risk plaques on the basis of targeting pathologically altered cellular and extracellular components of atherosclerotic lesions. We study in detail uptake and transport mechanisms of VSOP in cells involved in plaque formation (monocytes/macrophages/foam cells, endothelial cells, vascular smooth muscle cells) as well as in atherosclerotic mice. Moreover, we investigate putative toxic effects of these citrate-coated nanoparticles and the impact of their accumulation on the range of cellular functions and signaling pathways.
Magnetic Particle Imaging (MPI) is a new imaging technique that visualizes the distribution of magnetic iron oxide nanoparticles (MNPs) – so called MPI tracers - in three dimensions by their specific magnetic properties. Our aim is to test the potential of this new imaging technique for cardiovascular diagnostic. We use experimental models of atherosclerosis for investigating the MPI imaging capability to visualize the accumulation of potential MPI tracers in inflammatory atherosclerotic plaques.

The Ubiquitin-Proteasome System in Atherosclerosis

Project leader: Antje Ludwig, Karl Stangl
Coworkers: Nicola Wilck, Bernd Hewing, Henryk Dreger, Anke Stach,

Funding: DZHK (Deutsches Zentrum für Herz-Kreislaufforschung e.V.)

External cooperations: PD Dr. Silke Meiners (Comprehensive Pneumology Center München), Prof. Michael Groll (TU München), Prof. Agnes Görlach (TU München)

Project description:
Atherosclerosis is recognized as a chronic inflammatory disease leading to arterial plaque formation and vessel narrowing in different vascular beds. The ubiquitin-proteasome system (UPS) is the major intracellular degradation system in eukaryotic cells. The UPS influences many processes critical to atherosclerosis progression. Besides its essential role in the degradation of dysfunctional and oxidatively damaged proteins, it regulates inflammatory processes, oxidative defense mechanisms as well as cholesterol metabolism. It is our aim to investigate the involvement of the UPS in key processes of atherosclerosis. Moreover, we investigate whether targeting the UPS (e.g. using proteasome inhibitors) represents a feasible anti-atherosclerotic treatment strategy.

Glycosaminoglycans as targets for non-invasive imaging of unstable atherosclerotic plaques

Project leader: Wolfram Poller, Antje Ludwig, Verena Stangl
Coworkers: Anke Stach, Vasileios Karampelas

Funding: Friede Springer Herz Stiftung; Deutsche Gesellschaft für Kardiologie; Charité BIH Clinical Scientist Program 

External cooperations: Physikalisch-Technische Bundesanstalt Berlin, Working Group Biosignals; Institut für Pathologie; Klinik für Allgemein-, Viszeral-, Gefäß- und Thoraxchirurgie; Institut für Laboratoriumsmedizin, Klinische Chemie und Pathobiochemie

Project description:
Atherosclerotic plaque ruptures cause life-threatening complications including myocardial infarction and stroke. Methods to identify unstable plaques prior to rupture are therefore highly desirable. Proteoglycans (PG) and their glycosaminoglycan (GAG) chains are key components of the extracellular matrix in atherosclerotic plaques and are involved in disease progression. It is currently unknown whether plaque instability correlates with a specific PG/GAG pattern. This project aims at the identification of instability-associated PG/GAG and their use as targets for non-invasive imaging. We will comparatively analyze PG/GAG composition, GAG structure and chemical modifications in stable and unstable human atherosclerotic lesions from coronary and carotid arteries. Glycoanalytical techniques (HPLC, CE-LIF, MALDI-Imaging) as well as histological- and expression analyses (RT-PCR, Western Blot, IHC, TEM, FISH) will be applied to identify instability-associated PG/GAG as novel targets for non-invasive imaging of unstable plaques.