3.1 About Project A6 (E) 3.1.1 Title: Cyclophilins in the Regulation Of

PROJECT A6 (E) SEIZER, MAY PAGE 39

3.1 About project A6 (E)

3.1.1 Title: Cyclophilins in the Regulation of Remodeling Processes in DCMi

3.1.2 Principal investigators Seizer, Peter, Dr. med., born 22.09.1978, German Medizinische Klinik und Poliklinik Tübingen, Abt. Innere Medizin III - Kardiologie und Kreislauferkran- kungen, Universitätsklinikum Tübingen, Ottfried-Müller-Straße 10, D-72076 Tübingen Phone: +49 7071 2983 160 Email: [email protected]

May, Andreas E., Prof. Dr. med., born 10.11.1967, German Medizinische Klinik und Poliklinik Tübingen, Abt. Innere Medizin III - Kardiologie und Kreislauferkran- kungen, Universitätsklinikum Tübingen, Ottfried-Müller-Straße 10, D-72076 Tübingen Phone: +49 7071 2983 160 Email: [email protected]

3.2 Project history

3.2.1 Report

Summary Chemoattractants play a critical role in inflammation and innate immunity. Extracellular cyclophilins A and B comprise a group of chemoattractants that are highly elevated during inflammatory responses and are released by a variety of activated and apoptotic/necrotic cells. The most abundant form, cyclophilin A (CyPA), stimulates cell proliferation and migration, extracellular matrix reorganisation and fibrosis, and promotes myocyte hypertrophy. Further, CyPA represents a potential target for antiviral therapy. As a novel project starting in the second funding period (2008) we could show that CyPA and its extracellular receptor EMMPRIN (CD147) are crucially involved in (a) myocardial injury upon ischemia and reperfusion, and (b) are required for an adequate inflammatory response and virus elimination in Coxsackie virus B3-induced myocarditis in mice. (c) In endomyocardial biopsies of patients with congestive heart failure, CyPA and CD147 can distinguish inflammatory from non-inflammatory cardiomyopathies and serve as novel diagnostic markers. Enhanced CyPA expression additionally identifies patients at enhanced risk for an adverse clinical outcome. In the following year we evaluated the role of CyPA and CD147 in non-infectious mouse models of DCMi (e.g. troponin I-induced autoimmune myocarditis), which allows us to study the protective potential of cyclophilin inhibition in a setting independent from virus defense. Indeed in a mouse model of troponine induced myocarditis inhibition of extracellular CyPA via a novel cell-impermeable CyPA- inhibitor reduced myocardial inflammation and cardiac fibrosis dramatically. Analysis of a patient collective with DCM revealed an association between CyPA-expression and cardiac fibrosis. Thus, our data provide evidence that CyPA-inhibitors may serve as potentisl therapeutics for the treatment of DCMi. Moreover we evaluated the expression of EMMPRIN on monocyte subsets in patients with inflammatory cardiovascular diseases. FACS-staining was established in patients with stable coronary artery disease, myocardial infarction and aortic stenosis. Surprisingly we found, that EMMPRIN is expressed predominantly on “classical” monocytes and is upregulated in patients with aortic stenosis or myocardial infarction (not published). Now we started measurement of EMMPRIN on monocyte subsets in patients DCMi. Together, this project extended our knowledge on the role of cyclophilins in DCMi focusing on pharmacological strategies using novel drug molecules in mice and on the diagnostic and prognostic relevance in patients. Report PAGE 40 SEIZER, MAY PROJECT A6 (E)

EMMPRIN and its ligand cyclophilin A regulate MT1-MMP, MMP-9 and M-CSF during foam cell formation (Atherosclerosis. 2010 Mar;209(1):51-7)

Upon coincubation with platelets, CD34+ progenitor cells have the potential to differentiate into foam cells, and thereby may promote the progression of atherosclerosis. The exact mechanism of MMPreg- ulation during the cellular differentiation process to foam cells is still unclear. Thus, we investigated the role of EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) during foam cell formation originating from both monocytes/macrophages and CD34+ progenitor cells. Methods and results: Differentiation of CD34+ progenitor to foam cells was analyzed in a coculture model of progenitor cells and platelets. While CD34+ cells did not express EMMPRIN or MT1-MMP, mature foam cells strongly expressed EMMPRIN, which was associated with MT1-MMP expression as well as MMP-9. Gene silencing of EMMPRIN by siRNA during the cell differentiation process hindered not only the upregulation of MMPs (MT1-MMP, MMP-9), but also the secretion of the cytokine M-CSF. During the differentiation process CyPA was substantially released into the supernatant. The presence of the CyPA inhibitor NIM811 significantly reduced MMP-9 secretion during the differentiation process. Similar results were obtained using the classical pathway of foam cell formation by coincubating human macrophages with AcLDL. Additionally, the presence of soluble EMMPRIN ligands (CyPA, recombinant EMMPRIN) further enhanced MMP-9 secretion by mature foam cells. Consistently, CyPA and EMMPRIN were found in atherosclerotic plaques of ApoE-deficient mice by immunohistochemistry. Conclusion: EMMPRIN is upregulated during the differentiation process from CD34+ progenitor cells to foam cells, whereas its ligand, CyPA, is released. The CyPA/EMMPRIN activation pathway may play a relevant role in promoting the vulnerability of atherosclerotic plaques.

Disrupting the EMMPRIN (CD147)–Cyclophilin A Interaction Reduces Infarct Size and Preserves Systolic Function After Myocardial Ischemia and Reperfusion (Arterioscler Thromb Vasc Biol. 2011 Jun;31(6):1377-86)

Inflammation and proteolysis crucially contribute to myocardial ischemia and reperfusion injury. The extracellular matrix metalloproteinase inducer EMMPRIN (CD147) and its ligand cyclophilin A (CyPA) may be involved in both processes. The aim of the study was to characterize the role of the CD147 and CyPA interplay in myocardial ischemia/reperfusion (I/R) injury. Immunohistochemistry showed enhanced expression of CD147 and CyPA in myocardial sections from human autopsies of patients who had died from acute myocardial infarction and from mice at 24 hours after I/R. At 24 hours and 7 days after I/R, the infarct size was reduced in CD147+/- mice vs CD147+/+ mice (C57Bl/6), in mice (C57Bl/6) treated with monoclonal antibody anti-CD147 vs control monoclonal antibody, and in CyPA-/- mice vs. CyPA+/+ mice (129S6/SvEv), all of which are associated with reduced monocyte and neutrophil recruitment at 24 hours and with a preserved systolic function at 7 days. The combination of CyPA-/- mice with anti-CD147 treatment did not yield further protection compared with either inhibition strategy alone. In vitro, treatment with CyPA induced monocyte chemotaxis in a CD147- and phosphatidylinositol 3-kinase– dependent manner and induced monocyte rolling and adhesion to endothelium (human umbilical vein endothelial cells) under flow in a CD147-dependent manner. CD147 and its ligand CyPA are inflammatory mediators after myocardial ischemia and reperfusion and represent potential targets to prevent myocardial I/R injury.

PROJECT A6 (E) SEIZER, MAY PAGE 41

Figure 1. CyPA-/- mice are protected from myocardial I/R injury. I/R injury was initiated in CyPA+/+ and CyPA-/- mice as described in methods. After 1 day (d1) or 7 days (d7) mice were sacrificed and analyzed for area at risk and infarct size A) Evans Blue staining showed a comparable area at risk (related to the left ventricle in %) after I/R injury at day 1 (d1). B/C) Quantitative analysis of infarct size (related to area at risk in %) at day 1 (d1, B) and day 7 (d7, C) (n=7). D) Echocardiographic analysis of fractional area shortening as an indicator for left ventricular systolic function after 7 days (d7) (n=5). * indicates p<0.05 between groups. (from Seizer et al., ATVB 2011)

EMMPRIN and its ligand Cyclophilin A as novel diagnostic markers in inflammatory cardiomyopathy (Int J Cardiol. 2013 Mar 10;163(3):299-304)

During inflammatory cardiomyopathy matrix metalloproteinases are crucially involved in cardiac remodeling. The aim of the present study was to investigate whether the "extracellular matrix metalloproteinase inducer" EMMPRIN (CD147) and its ligand Cyclophilin A (CyPA) are upregulated in inflammatory cardiomyopathy and may serve as diagnostic markers. Therefore, a series of 102 human endomyocardial biopsies were analyzed for the expression of EMMPRIN and CyPA and correlated with histological and immunohistological findings. Endomyocardial biopsies were stained for EMMPRIN and CyPA in addition to standard histology (HE, Trichrom) and immunohistological stainings (MHC-II, CD68, CD3). 39 (38.2%) biopsies met the immunohistological criteria of an inflammatory cardiomyopathy. EMMPRIN, which was predominantly expressed on cardiomyocytes, was slightly (but significantly) upregulated in non inflammatory cardiomyopathies compared to normal histopathological findings and highly upregulated in inflammatory cardiomyopathy compared to both non inflammatory cardiomyopathy and normal histopathology. In contrast, CyPA reveals no enhanced expression in non inflammatory cardiomyopathies and a highly enhanced expression in inflammatory cardiomyopathy, where it is closely associated with leucocytes infiltrates. We found a strong correlation between both EMMPRIN and CyPA with the expression of MHC-II molecules (correlation coefficient 0.475 and 0.527, p<0.05). Moreover, we found a correlation for both EMMPRIN and CyPA with CD68 (correlation coefficient 0.393 and 0.387, p<0.05) and CD3 (correlation coefficient 0.360 and 0.235, p<0.05). EMMPRIN is enhanced in both inflammatory and non inflammatory cardiomyopathies and can serve as a marker of myocardial remodeling. CyPA may represent a novel and specific marker for cardiac inflammation.

Figure 2. CyPA and EMMPRIN (CD147) in cardiomyopathy. Myocardial tissue sections were stained with Mas- son’s trichrome and mAbs anti-CD3, anti-CD68, anti-CyPA and anti-EMMPRIN (from Seizer et al., Int J Cardiol 2011) PAGE 42 SEIZER, MAY PROJECT A6 (E)

Cyclophilin A affects inflammation, virus elimination and myocardial fibrosis in coxsackievirus B3-induced myocarditis (J Mol Cell Cardiol. 2012 Jul;53(1):6-14) Extracellular cyclophilin A (CyPA) and its receptor Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147) modulate inflammatory processes beyond metalloproteinase (MMP) activity. Re- cently, we have shown that CyPA and CD147 are upregulated in patients with inflammatory cardiomyopathy. Here we investigate the role of CyPA and CD147 in murine coxsackievirus B3 (CVB3)-induced myocarditis. CVB3-infected CyPA−/− mice (129S6/SvEv) revealed a significantly reduced T-cell and macrophage recruitment at 8 days p.i. compared to wild-type mice. In A.BY/SnJ mice, treatment with the cyclophilin-inhibitor NIM811 was associated with a reduction of inflammatory lesions and MMP-9 expression but with enhanced virus replication 8 days p.i. At 28 days p.i. the extent of lesion areas was not affected bei NIM811, whereas the collagen content was reduced. Initiation of NIM811- treatment on day 12 (after an effective virus defense) resulted in an even more pronounced reduction of myocardial fibrosis. In conclusion, in CVB3-induced myocarditis CyPA is important for macrophage and T cell recruitment and effective virus defense and may represent a pharmacological target to modulate myocardial remodeling in myocarditis.

Figure 3. Cyclophilin inhibitor NIM811 attenuates macrophage and T cell recruitment. A.BY/SnJ mice were infected with CVB3 and treated with NIM811 or placebo as described in methods. 8 days p.i. mice were sacrificed and cardiac tissue was analyzed for (A,B) macrophages (Mac-3) and T cells (CD3) (n≥5). (from Seizer et al., JMCC 2012)

Cyclophilin A predicts clinical outcome in patients with congestive heart failure undergoing endomyocardial biopsy (Eur J Heart Fail. 2013 Feb;15(2):176-84) Cyclophilin A (CyPA) represents a ubiquitous intracellular protein, which is secreted by inflammatory and by dying/necrotic cells. The aim of this study was to evaluate the prognostic relevance of CyPA expression in endomyocardial biopsies of consecutive patients with congestive heart failure. A total of 227 unselected patients (age 53.9 ± 15 years) with congestive heart failure undergoing endomyocardial biopsy for diagnostic reasons were enrolled. Biopsies were analysed using established histopathological and immunohistological criteria together with CyPA staining. Virus genome was studied by polymerase chain reaction. CyPA was significantly enhanced in patients with inflammatory cardiomyopathy (n = 127) as compared with patients with non-inflammatory cardiomyopathy (n = 100, P < 0.0001). During a mean follow-up of 16.3 months, 60 patients (26.4%) reached the primary endpoint, a composite of all-cause death, heart transplantation, malignant arrhythmia, and heart failure-related rehospitalization. Of all clinical (ejection fraction, New York Heart Association functional class), labora- tory (brain natriuretic peptide), and immunohistological parameters (CyPA, extracellular matrix metalloproteinase inducer, CD68, CD3, major hisocompatibility complex II, and virus genome) tested, only CyPA was identified as an independent predictor for the composite endpoint [hazard ratio (HR) 2.4; 95% confidence interval (CI) 1.2-5.2; P = 0.019] as well as for all-cause death and heart transplantation alone (HR 4.7; 95% CI 1.1-19.8; P = 0.036). Subgroup analysis revealed CyPA as a predictor in patients with non-inflammatory cardiomyopathy for the composite endpoint (HR 3.0; 95% CI 1.3-6.6; P PROJECT A6 (E) SEIZER, MAY PAGE 43

= 0.007) as well as all-cause death or heart transplantation alone (HR 6.4; 95% CI 1.4-28.1; P = 0.014). CyPA is an independent predictor of clinical outcome in patients with congestive heart failure undergoing endomyocardial biopsy.

Figure 4. Cumulative Kaplan-Meier estimates of the rates of the composite study endpoint in patients with congestive heart failure undergoing enomyocardial biopsy. A. The figure shows data for the composite study point (all-cause death, heart transplantation, arrhythmic events, and heart-failure related rehospitalization) stratified by CyPA. B. Hazard ratio for combined endpoint. (Zürn et al., Eur J Heart Failure 2012)

Electrophysiological characterization of scars detected by contrast enhanced magnetic resonance imaging in patients with non-ischemic cardiomyopathy (Int J Cardiol. 2013 Aug 10;167(3):1070-2) In patients with non-ischemic cardiomyopathy (NICMP) several studies have recently shown that the presence of myocardial fibrosis identified by cardiac magnetic resonance imaging (MRI) is associated with cardiovascular morbidity, overall mortality and likelihood of adequate ICD-therapy. There are clear hints that presence of myocardial fibrosis is associated with ventricular tachycardia. However, the exact mechanismfor pathogenesis of ventricular arrhythmias and its association with late gadolinium enhancement (LGE) in patients with NICMP are unclear. Moreover an electroanatomical characterization of LGE has not been performed so far in a patient collective with LVEF ≤50% and no history of ventricular tachycardias. We have therefore hypothesized that the presence and localization of LGE on MRI correlate with functional relevant findings in electroanatomical voltage mapping (EAVM) and predict higher ventricular tachycardia inducibility in programmed stimulation in patients with non- ischemic congestive heart failure due to reduced left ventricular function. The current findings show that the presence of late gadolinium enhancement (LGE) in contrast enhanced MRI correlates with endomyocardial bipolar electrogram abnormalities. The absence of LGE in patients with NICMP pre- cludes inducibility of ventricular arrhythmiaswith a high sensitivity. Our findings suggest that the presence of LGE identifies patients at risk for sudden cardiac death suffering from NICMP and may be a valuable diagnostic tool for risk stratification in these patients.

The novel extracellular CyPA-inhibitor MM284 reduces myocardial inflammation and remodel- ling in a mouse model of Troponin I-induced myocarditis (Heinzmann D. et al., not published) Cyclophilins (CyPs) are a group of highly conserved cytosolic enzymes that have a peptidylprolyli- somerase activity. This study analyzed the effect of the Cyclosporin A derivative MM284 in a mouse model of autoimmune myocarditis. Due to the modification of Cyclosporin A with a cell-impermeable moiety, MM284 strictly blocks extracellular activities of Cyclophilins such as CyPA. A/J mice were immunized with murine cardiac troponin I (mcTnI) at day 0,7, & 14. Mice were treated with MM284 or diluent every second day starting at day 0. After 28 days the hearts were stained with Hematoxylin& Eosin (H&E), anti-CD3, anti-Mac-3, and Masson’s Trichromestaining procedures. The evaluation of the sections was based on a scoring system. The results showed a considerable reduction of the infiltration of cells in H&E (p<0.05), and a reduced expression of CD3 (p<0.05) and Mac-3 (p<0.01) when treated with MM284. In parallel, the evaluation of Masson’s Trichrome staining also showed a reduction of the cardiac fibrosis (p<0.05), when treated. In addition expression of MMP-9 was reduced significantly. In vitro migration of human monocytes was assessed using a modified Boyden chamber. In the presence of MM284, migration towards extracellular CyPA was almost com- PAGE 44 SEIZER, MAY PROJECT A6 (E)

pletely abolished (p<0.01). Finally analysis of bioptic samples of patients with non ischemic cardiomyopathy revealed that severe cardiac fibrosis was associated with the expression of CyPA.

Figure 5. Treatment with MM284 reduces inflammation in troponin-induced myocarditis. Infiltration of T- cells and macrophages were assessed using anti-CD3 and anti-Mac-3 staining at day 28 after immunization.

Extracellular CyPs, especially CyPA play an important role in inflammatory and profibrotic processes. In the current study we found that the extracellular inhibition of CyPs via MM284 is a successful and potent pharmacological approach for the treatment of myocardial inflammation and reduction of cardiac fibrosis.

Figure 6. Treatment with MM284 reduces myocardial fibrosis in troponine induced myocarditis.

PROJECT A6 (E) SEIZER, MAY PAGE 45

Extracellular Cyclophilin A activates human platelets via CD147 and PI3K/Akt signaling, which promotes platelet adhesion and thrombus formation in vitro and in vivo (Seizer et al., not published) Cyclophilin A (CyPA) is secreted under inflammatory conditions by various cell types including platelets, leukocytes and smooth muscle cells. CyPA interacts with its extracellular receptor CD147 (EMMPRIN) and modifies several cellular functions. Here, we investigated the effect of extracellular CyPA on platelet function.Inhibition of extracellular CyPA through a novel specific inhibitor MM284, that does not pass the plasma membrane, significantly reduced thrombus formation in mesenteric arterioles after FeCl3-induced injury in mice. Treatment of isolated platelets with recombinant CyPA resulted in platelet degranulation in a time and dose dependent manner. CyPA-induced platelet degranulation was attenuated both by MM284 and a permeable CyPA inhibitor NIM811 to a similar degree. Further, blocking of the surface expressed CyPA receptor CD147, by a blocking anti-CD147 mAb significantly reduced CyPA-dependent platelet degranulation. Under arterial shear conditions in vitro, CyPA-stimulated platelets showed an enhanced adhesion on immobilized collagen compared to unstimulated platelets. Further, pretreatment of platelets with CyPA enhanced their recruitment to mouse carotid arteries after arterial injury, which could be inhibited by anti-CD147 mAb (intravital mi- croscopy). Stimulation of platelets with CyPA induced phosphorylation of Akt, which in turn could be inhibited in the presence of PI3K inhibitors. Platelets treated with Akt-inhibitors or derived from Akt-1-/- -mice revealed a markedly decreased degranulation upon CyPA stimulation compared to wild type platelets.Extracellular CyPA activates platelets via CD147-mediated PI3K/Akt-signaling leading to enhanced adhesion and thrombus formation. Targeting extracellular CyPA via a non-permeable specific CyPA inhibitor may be a promising strategy for platelet inhibition without affecting critical functions of intracellular CyPA.

REGULATION OF EMMPRIN (CD147) ON MONOCYTE SUBSETS IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION AND SEVERE AORTIC STENOSIS (Seizer et al., not published) The role of individual monocyte subsets in cardiovascular inflammatory diseases like myocardial infarction or aortic stenosis is insufficiently understood. The Extracellular Matrix Metalloproteinase In- ducer EMMPRIN regulates MMP-release and inflammatory processes on monocytes. To our best of knowledge expression and regulation of EMMPRIN on monocyte subsets have never been character- ized. Thus, the aim of the present study was to characterize the expression of EMMPRIN on monocyte subsets in patients with acute myocardial infarction and aortic stenosis. Blood was obtained from 184 patients (80 with stable CAD, 49 with acute myocardial infarction (AMI), 55 with severe aortic stenosis). Monocytes were divided into 3 subsets according to flow cytometry: CD14++CD16- (low), CD14++CD16+ (intermediate), CD14+CD16++ (high). In addition surface expression of EMMPRIN, CD36 (predominatly expressed on CD16+ monocytes) and CD47 (predominantly expressed on CD16++) was measured. In patients with stable CAD EMMPRIN expression was significantly different on all monocyte subsets with the highest expression on “classical” CD14++/CD16- monocytes. EMMPRIN was upregulated on all monocyte subsets in patients with AMI or aortic stenosis compared to patients with stable CAD. In all patient groups classical CD16- monocytes revealed the highest EMMPRIN expression. EMMPRIN is preferentially expressed on CD16-monocytes and is upregulated in patients with acute myocardial infarction and aortic stenosis.

Discussion and conclusion The project A6 was a new research aspect added in the period of 2008 to 2013 to the research con- sortium. The project focused on the EMMPRIN and Cyclophilin A in inflammatory cardiomyopathy. We found that EMMPRIN as wells as Cyclophilin A are important mediators of cardiac inflammation. Moreover we applied novel extracellular CyP-inhibitors in a mouse model of inflammatory cardiomyopathy and thus etasblished these two proteins as novel targets for the treatment of myocardial inflammation. We established Cyclophilin A as a promising target for diagnostic and therapeutic strategies to control the disease of inflammatory cardiomyopathy an an individualized basis.

3.2.2 Project-related publications of the investigators Seizer P, Gawaz M, May AE. Cyclophilin A and EMMPRIN (CD147) in cardiovascular diseases. Car- diovasc Res 2014, in press. Schmohl JU, Daub K, von Ungern-Sternberg SN, Lindemann S, Schönberger T, Geisler T, Gawaz M, Seizer P. Differential MMP-9 activity in CD34+progenitor cell-derived foam cells from diabetic and normoglycemic patients. Herz 2013, EPub Dec 5. PAGE 46 SEIZER, MAY PROJECT A6 (E)

Seizer P, Klingel K, Stickel J, Dorn C, Horger M, Kandolf R, Bigalke B, May AE, Gawaz M, Schreieck J. Left ventricular site-directed biopsy guided by left ventricular voltage mapping: a proof of princi- ple. Int J Cardiol 2013; 168: 3113-4 Zuern CS, Müller KA, Seizer P, Geisler T, Banya W, Klingel K, Kandolf R, Bauer A, Gawaz M, May AE Cyclophilin A predicts clinical outcome in patients with congestive heart failure undergoing endomyocardial biopsy.. Eur J Heart Fail 2013; 15: 176-84. Seizer P, Zuern CS, Maier J, Kerst G, Weig HJ, Gansser L, Kramer U, May AE, Parade U, Bigalke B, Bauer A, Gawaz M, Geisler T, Schreieck J. Electrophysiological characterization of scars detected by contrast enhanced magnetic resonance imaging in patients with non-ischemic cardiomyopathy. Int J Cardiol 2013; 167: 1070-2 Elvers M, Herrmann A, Seizer P, Münzer P, Beck S, Schönberger T, Borst O, Martin-Romero FJ, Lang F, May AE, Gawaz M Intracellular cyclophilin A is an important Ca(2+) regulator in platelets and critically involved in arterial thrombus formation. Blood 2012; 120: 1317-26. Seizer P, Klingel K, Sauter M, Westermann D, Ochmann C, Schönberger T, Schleicher R, Stellos K, Schmidt EM, Borst O, Bigalke B, Kandolf R, Langer H, Gawaz M, May AE. Cyclophilin A affects inflammation, virus elimination and myocardial fibrosis in coxsackievirus B3-induced myocarditis. J Mol Cell Cardiol 2012; 53: 6-14. Seizer P, Geisler T, Bigalke B, Schneider M, Klingel K, Kandolf R, Stellos K, Schreieck J, Gawaz M, May AE . EMMPRIN and its ligand cyclophilin A as novel diagnostic markers in inflammatory cardiomyopathy. Int J Cardiol 2013; 163: 299-304. Seizer P, Ochmann C, Schönberger T, Zach S, Rose M, Borst O, Klingel K, Kandolf R, MacDonald HR, Nowak RA, Engelhardt S, Lang F, Gawaz M, May AE . Disrupting the EMMPRIN (CD147)- cyclophilin A interaction reduces infarct size and preserves systolic function after myocardial ischemia and reperfusion. Arterioscler Thromb Vasc Biol 2011; 31: 1377-86. Seizer P, Schonberger T, Schott M, Lang MR, Langer HF, Bigalke B … May AE . EMMPRIN and its ligand cyclophilin A regulate MT1-MMP, MMP-9 and M-CSF during foam cell formation. Athero- sclerosis 2010; 209: 51-7 Seizer P, Borst O, Langer HF, Bultmann A, Munch G, Herouy Y … May AE . EMMPRIN (CD147) is a novel receptor for platelet GPVI and mediates platelet rolling via GPVI-EMMPRIN interaction. Thromb Haemost 2009; 101: 682-6

3.3 Funding Funding of the project within the Collaborative Research Centre started July 2008. Funding of the project ended December 2013. The project will be completed by the end of the current funding period.

3.3.1 Project staff in the ending funding period No. Name, aca- Field of Department of Commit- Cate- Funded demic degree, research university or non- ment in gory through position university institu- hours/we : tion ek Available Research staff 1. Andreas E. Inflamma- Internal Medicine, 6 UKT*) May, Prof. Dr. tion and Cardiology & med. MMP- circulatory dis- regulation eases 2. Peter Seizer, Inflamma- Internal Medicine, 12 UKT Dr. med. tion and Cardiology & MMP- circulatory dis- regulation eases 3. Saskia von Treatment Internal Medicine, 8 UKT Ungern- strategies Cardiology & Sternberg, in inflam- circulatory dis- M.Sc. matory eases diseases PROJECT A6 (E) SEIZER, MAY PAGE 47

4. Tanja Schön- Small Internal Medicine, 8 UKT berger, animal Cardiology & Dr. med. analysis circulatory diseases 5. Henrik Sturhan Analysis of Internal Medicine, 15 UKT circulating Cardiology & inflamma- circulatory dis- tory cells eases 6. David, Heinz- Inflamma- Internal Medicine, 20 UKT mann tory cardi- Cardiology & omyopathy circulatory diseases Non-research 7. Klaudia Internal Medicine, 30 UKT staff Posavec Cardiology & circulatory diseases Requested Research staff Dr. Chatterjee, XXXXXX Internal Medicine, XXXXXX Postdoc Madhumita, Cardiology & Postdoc circulatory diseases Christos Kas- XXXXXX Internal Medicine, XXXXXX Doctoral sos, Doctoral Cardiology & student student circulatory diseases

UKT*) University Hospital Tübingen

Job description of staff (supported through available funds):

1. Prof. Dr. Andreas E. May Principal Investigator. The PI was responsible for the design and accomplishment of the present project. He will be in authority for the guidance of the scientific staff (postdocs and PhD students) and design complex experimental tests including the research work with mouse models. Furthermore he coordinates the examination and discussion of the results of the experiments. Prof. May is responsible for publishing the results. 2. Dr. med. Peter Seizer Principal Investigator. Dr. Peter Seizer was involved in the design and organisation of the present project. He was responsible for the guidance of the non-scentific staff and the doctoral students. Dr. Seizer will coordinate the in vitro studies and supervise the FACS-analysis of CD16+ monocytes. Moreover he coordinates and administrates the in vivo studies with the various mouse models. He was involved in the discussion of the results of the experiments as well. 3. Saskia von Unger-Sternberg, M.Sc. She does her PhD-thesis in the lab of the applicants. She is involved in development of EMMPRIN and CyPA-based inhibitors. 4. Dr. med. vet. Tanja Schönberger She leads the core facility for animal experiments including small animal echocardiography and has a great expertise in various in vivo mouse models. 5. Cand. med. Henrik Sturhan He does his MD-thesis in the lab of the applicants. He is working on CD16+/- monocytes in vitro. 6. Cand. med. David Heinzmann He does his MD-thesis in the lab of the applicants. He is experienced in zymography and migration of monocytes and monocytic cell lines. 7. Klaudia Posavec She has been working the lab of the applicants since 01.11.2005. She is expert in FACS-analysis and cell culturing and has profound knowledge in immunohistochemistry and immunofluorescence staining.

Job description of staff (requested):

1. N.N., PhD Student (E13, 65%) PAGE 48 SEIZER, MAY PROJECT A6 (E)

This candidate will perform the analysis of the animal studies and will characterize the mouse models concerning myocardial function, morphology, inflammation, and remodeling. In cooperation with Dr. T. Schönberger (animal core facility) he/she will support her with the animal experiments and learn and perform echocardiographic analyses in mice and will perform the work-up of the animals (immunostainings, ELISA, zymographies, PCR etc.) in both models (autoimmune myocarditis, titin-based ATII-induced DCM).

2. N.N., PhD Student (E13, 65%) This candidate will perform the analyses of the human myocardial biopsies (immunostainings, ELISA, zymographies, PCR etc.) and will correlate with the results with clinical and haemodynamic data including the patient follow-up (prognosis). In addition, he/she will perform the in vitro analyses using cardiomyocytes and (myo)fibroblasts.