Professor Stephan Baldus

Organisation / Institute
Department III of Internal Medicine
Cologne University Heart Center

Address

Prof. Stephan Baldus
Department III of Internal Medicine,
Cologne University Heart Center
Kerpener Str. 62
50937 Köln, Germany

stephan.baldus[at]uk-koeln.de

 

 

 Dr. Anna Klinke

Organisation / Institute
Department III of Internal Medicine
Cologne University Heart Center

Address

Dr. Anna Klinke
Department III of Internal Medicine,
Cologne University Heart Center
Kerpener Str. 62
50937 Köln, Germany

anna.klinke[at]uk-koeln.de

A. Myeloperoxidase (MPO)-mediated microvascular dysfunction in heart failure – impact of MPO-derived macrophage activation

Professor Stephan Baldus/Dr. Anna Klinike

Rationale and Aims

Current concepts for the treatment of heart failure (HF) center around augmenting cardiac contractility. However, progression of HF is critically propagated by dys-function of resistance vessels, thereby impairing coronary perfusion and yielding increased peripheral vascular tone. Inflammation is recognized as a key factor in vascular dysfunction, with macrophages being a major component of vessel wall infiltrates. There is accumulating evidence that – apart from macrophages – activation of polymorphonuclear neutrophils (PMN) also importantly contributes to vascular dysfunction. However, little is known about the interplay of macrophages and PMN in this context as well as about their mechanistic role in the regulation of vascular resistance. The PMN- and macrophage-derived heme enzyme myeloperoxidase (MPO), which catalyzes the generation of reactive pro-inflammatory species, emerges as an important mediator of inflammatory vascular diseases. Given that MPO release is increased in HF patients, that MPO promotes activation and recruitment of leukocytes, and that the enzyme via nitric oxide (NO)-scavening is causally linked to endothelial dysfunction, the current project intends to investi-gate the significance of MPO-induced macrophage activation for vascular dysfunction in HF.

Current state of research and own preliminary work

Figure 1: A. Analysis of mRNA expression in peritoneal macrophages (PM) revealed increased expression of TNF-α in PM from wild type (WT) as compared to MPO-deficient (Mpo-/-) mice. n=4/5. B. Bone marrow derived macrophages (BMDMs) from Mpo-/- mice were treated with MPO for 24 h. Treated cells showed a significantly higher expression of mRNA for inducible NO-synthase (iNOS), TNF-α and Interleukin (IL)-6 and for arginase as compared to untreated cells. n=4/5. C. Peritoneal lavage was performed in Mpo-/- mice 3 days after i.p. injection of NaCl, MPO or thioglycolate (Thio). Quantification of leukocytes in lavage fluid revealed increased number upon MPO and thioglycolate treatment as compared to control. n=15/16/14 D. The percentage of macrophages from peritoneal cells as described in (C) was increased in MPO and thiogycolate treated animals as assessed by flow cytometry. E. Immunohistochemical quantification of macrophages in myocardial sections of WT and Mpo-/- mice after 3 days of myocardial ischemia disclosed reduced macrophage accumulation in Mpo-/- mice. n=4/3.

In a mouse model of dilated cardiomyopathy (DCM) using muscle LIM protein-deficient (MLP-/-) mice, we found that concomitant MPO-deficiency attenuates the increase in systemic vascular resistance (SVR). This was accompanied by improved systolic left ventricular function. Notably, MLP-/- mice exhibited enhanced microvascular MPO deposition as compared to wild type (WT) littermates. Recent findings have revealed that MPO activates macrophages leading to production of pro-inflammatory cytokines and expression of inducible NO-synthase, as detected in peritoneal macrophages from WT vs. MPO-deficient (Mpo-/-) mice and in MPO-treated bone-marrow derived macrophages (Figure 1 A,B). Thus, MPO appears to promote classical macrophage polarization, thereby causing a pro-inflammatory macrophage phenotype. Furthermore, MPO was found to contribute to macrophage recruitment as disclosed upon peritoneal MPO injection and in a model of myocardial ischemia using WT and Mpo-/- mice (Figure 1 C-E).

Experimental approach and work program

First, we will further characterize the underlying molecular and cellular mechanisms of MPO-mediated macrophage activation and polarization in vitro utilizing peritoneal and bone marrow derived macrophages from WT and Mpo-/- mice as well as the cell line THP-1. The cells will be treated with MPO, MPO-derived reactive species, and inactive MPO variants either alone or in combination with factors inducing classical or alternative polarization (IFN-γ, IL-1β, IL-4). QRT PCR and immunoblotting will be performed to investigate the phenotypic subtype (expression of iNOS, TNF-α, IL-6, IL-12, IL-23, MCP-1, CCR2, arginase, IL-10, TGF-β) and the intracellular signaling (Nf-κB activation, signal transducer, and activator of transcription (STAT)-pathway, Krüppel-like factor (KLF), and peroxisome proliferator-activated receptor (PPAR)γ expression).

Second, in vivo studies will use WT littermates, MLP-/- mice, Mpo-/- mice, and MLP x MPO double-knockout (MLPxMpo DKO) mice to investigate mechanisms underlying the low-grade chronic vascular inflammation reflected by microvascular MPO deposition and increased SVR. Importantly, enhanced pro-inflammatory polarization and retention of macrophages, that leads to the amplification and prolongation of inflammation, results from a disturbed resolution of inflammation. Thus, monocyte recruitment will be investigated by intravital microscopy in cremaster muscle and mesenteric microcirculation and the macrophage phenotype will be characterized via immunostaining in cremaster muscle whole mount and hind limb muscle preparations and flow cytometry of mesentery single cell suspensions. Of note, enhanced PMN infiltration and delayed PMN apoptosis are key factors of disturbed inflammatory resolution mechanisms. Both processes have been previously linked to MPO: In detail, leukocyte recruitment is initiated by MPO interaction with the endothelial glycocalyx. Neutrophil apoptosis is substantially delayed via MPO-binding to CD11b/CD18 integrins, which was linked to prolonged inflammation in a lung injury model. Thus, PMN recruitment and apoptosis will be investigated via intravital microscopy and flow cytometry.

Third, to characterize the causal relationship of macrophages and the HF phenotype, WT, MLP-/-, Mpo-/- and MLPxMpo DKO mice will undergo monocyte/ macrophage depletion by repeated intravenous injection of liposome-encapsulated clodronate (35 µg/g BW). In another cohort, recruitment of activated monocytes will be inhibited by intravenous injection of siRNA targeting CCR2. Furthermore, clodronate- and siCCR2-treated or control MLPxMpo DKO mice will receive chronic MPO infusion via the jugular vein using osmotic mini-pumps (0.05 ng/g/min). Inflammation of vascular endothelium will be characterized by immunohistochemistry in myocardial and hind limb muscle sections and cremaster muscle whole mount preparations and microvascular MPO deposition will be quantified. Endothelial function will be assessed ex vivo (isometric force measurements) and in vivo (determination of SVR), and the severity of HF will be evaluated in vivo by assessing the pressure volume relationship of the left ventricle.

Collectively, this project will decipher the mechanistic role of macrophage polarization as a stress response pathway in HF. The significance of MPO as a modulator of the macrophage´s phenotype and as a central disruptive factor for the resolution of inflammation will be explored. Ultimately, results from this project will help to better understand the potential of MPO as a novel therapeutic target in HF.

Potential therapeutic implications

Given the potential proinflammatory, macrophage activating properties of MPO, MPO inhibition, which is in early preclinical development, may alter vascular function by modulating the activation state of macrophages.

Added value through collaborations within the CCRC

This project will enable us to identify the cytokine-like properties of MPO and to potentially characterize novel pathways of and redundancies in macrophage polarization reaching from diabetes to atherosclerosis. Thus, this project will interconnect research interests of this group with the expertise of the Pasparakis and Brüning groups, both of whom have broad research experience with respect to macrophage polarization and inflammation. Immunohistochemical analysis of macrophages and PMN will be performed by the histopathological core facility (A. Quaas / R. Büttner) and vascular MPO distribution will be analyzed by the molecular imaging core facility (B. Neumaier).

General research interest

  • Role of leukocyte-dependent inflammatory processes for cardiac and vascular plasticity: Molecular mechanisms of leukocyte-mediated 1) endothelial and myocardial dysfunction and 2) vascular and myocardial maladaptive structural remodeling
  • Significance of leukocytes for pathogenesis of heart failure, cardiac reperfusion injury, cardiac arrhythmias and pulmonary hypertension
  • Leukocyte-derived peroxidases as novel therapeutic targets in cardiovascular disease  

Prof. Stephan Baldus

Prof. Stephan Baldus attended the Medical School at the University of Ulm and Hamburg and graduated 1996 in Hamburg. His residency in the Department of Cardiology at the University of Hamburg from 1996 – 1998 and at the Kerckhoff Clinic in Bad Nauheim in 1999 was followed by a position as a postdoctoral research fellow in the laboratory of Prof. Bruce Freeman at the Centre for Free Radical Biology at the University of Alabama at Birmingham, USA from 1999 – 2001. Thereafter, he continued his medical training at the University Heart Centre Hamburg and became an independent group leader supervising clinical and experimental research. In 2004 he received the board approval for Internal Medicine and in 2006 he became an assistant professor at the University Heart Centre Hamburg receiving the board approvals for Cardiology and Intensive Care Medicine in 2007 and 2012. In 2010 he obtained a position as associate professor and vice chair at the department of cardiology at the University Heart Centre Hamburg, which he hold until 2012. Today, he is Full Professor at the University of Cologne and Director of the Department of Internal Medicine and Cardiology at the Heart Centre, University of Cologne. In addition, he is Chair of the Cologne Cardiovascular Research Centre and senior supervisor of the Cluster of Inflammation and Redox Signaling at the University Heart Centre. His work has been acknowledged amongst others by the Albert Fraenkel Award of the German Society of Cardiology in 2008 and the Paul Schölmerich Award of the German Society of Internal Medicine in 2010.   

Dr. Anna Klinke

Dr. Anna Klinke finished her studies of Pharmacy at the University of Münster in 2005. She became PhD student in the laboratory of Prof. Baldus at the University Heart Centre Hamburg and passed collaboration and training attendances in the Czech Academy of Science in Brno, Czech Republic and the Walter Brendel Centre of Experimental Medicine of the LMU Munich. After obtaining her doctoral degree in 2009, she became junior Principal Investigator in the Institute of Experimental Cardiology at the University Heart Centre Hamburg and the Cardiovascular Research Centre Hamburg in 2010. In 2012 she moved with Prof. Baldus to Cologne and established the experimental laboratory at the University of Cologne. Today, she is laboratory supervisor leading one of the groups of the Cluster of Inflammation and Redox Signaling at the Heart Centre in Cologne embedded in the Cardiovascular Research Centre.