Physical Exercise Reduces Transplant Arteriosclerosis in a Fully Allogeneic Mouse Aorta Transplantation Model

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The Journal of Heart and Lung Transplantation, Vol 32, No 4S, April 2014

sphingolipid pathway depends on GJ activation by W6/32. In vivo, a tendency to a reduction of the graft lumen obliteration was observed in mice receiving carbenoxolone, compared to the control group (25 % vs. 40%, respectively). Conclusion: These data highlight the involvement of GJ in the mechanism of transplant vasculopathy, particularly in SMC migration and proliferation, via a regulation of the sphingolipid pathway. Additional in vivo studies (ongoing tests) will help at confirming the efficacy of GJ inhibition in the prevention of chronic rejection. 4( 32) Discovering Novel Inflammatory Triggers after Heart Transplantation Via Proteomics H. Shen ,1 C. Colangelo,2 L. Chung,2 C. Bruce,2 C. Booth,3 D. Kreisel,4 D.R. Goldstein.1  1Internal Medicine and Immunobiology, Yale School of Medicine, New Haven, CT; 2Keck Facility, Yale School of Medicine, New Haven, CT; 3Comparative Medicine, Yale School of Medicine, New Haven, CT; 4Surgery and Immunology, Washington University, St Louis, MO. Purpose: Inflammation has detrimental effects after organ transplantation, yet the identity of inflammatory triggers after transplantation remains largely unknown. In this study, we developed an experimental model and applied non-biased approaches to discover novel inflammatory triggers after heart transplantation. Methods: We employed the following: i) a murine heterotopic heart transplant model and measured intra-graft inflammatory mediators (ELISA); ii) an in vitro assay in which lysates of cardiac transplants are cultured with dendritic cells (DCs) to determine if transplantation increases DC inflammatory responses (ELISA); iii) comparison by mass spectrometry of nontransplanted and transplanted heart proteomes; iv) immune histochemical staining of human endomyocardial biopsies. Results: At 24 hours after transplantation in mice, intragraft levels of IL-12p40, TNFα , MIP1α and MCP1 were elevated with histological evidence of neutrophil infiltration. Importantly, this early inflammatory response was similar between allografts and syngeneic grafts. Cardiac transplantation enhanced DC inflammatory responses, which was dependent on proteins. Comparative proteomics discovered that several proteins were upregulated at 24h post transplantation including those involved in cytoskeletal function, cell adhesion and immune response. Haptoglobin (Hp) was one of the most upregulated proteins and we independently validated its’ upregulation via ELISA. We functionally validated the role of Hp by showing that Hp deficiency diminished inflammation (Figure A) in our syngeneic cardiac transplant model and increased the ability of CTL4 Ig to enhance graft survival in a fully MHC mismatched murine cardiac transplant model (Figure B). We also found that Hp is present in human heart transplants with acute graft rejection but not in human heart transplants without rejection. Conclusion: Proteomics is a powerful strategy to identify novel inflammatory triggers, including Hp, after heart transplantation.

4( 33) Myeloperoxidase is Critically Linked to the Development of Diastolic Heart Failure Following Pressure Overload F.G. Deuschl ,1 A. Klinke,2 K. Friedrichs,2 D. Knappe,1 F. Weinberger,3 K. Müllerleile,1 D. Westermann,1 H. Reichenspurner,4 S. Blankenberg,1 S. Baldus.2  1General and Interventional Cardiology, University Heart

Center Hamburg, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 2Experimental Cardiology, Clinic for Internal Medicine III, Heart Center University Hospital Cologne, Cologne, Germany; 3Department of Experimental Pharmacology and Toxicology, University Hospital Hamburg-Eppendorf, Hamburg, Germany; 4Department for Cardiovascular Surgery, University Heart Center Hamburg, University Hospital Hamburg-Eppendorf, Hamburg, Germany. Purpose: Plasma levels of the leukocyte derived enzyme myeloperoxidase (MPO) are elevated in patients with heart failure (HF). We have shown previously that MPO promotes vasoconstriction, leukocyte recruitment and myocardial fibrosis - suggesting that MPO may be mechanistically linked to the pathophysiology of HF. Here we assessed whether MPO is modulating systolic and diastolic myocardial performance in a pressure overload model. Methods: C57Bl/6-Wild-type (WT) and MPO-deficient (Mpo-/-) mice were subjected to mild transverse aortic constriction (TAC, Δ p max: 50 ±10 mmHg). After 6 weeks echocardiographic as well as invasive hemodynamic measurements were performed and organs were histologically analyzed. Results: After 6 weeks, echocardiography revealed significantly reduced left ventricular (LV) ejection fraction (EF) in TAC-treated animals as compared to sham-operated controls. In vivo hemodynamic assessment of left ventricular function using pressure-volume loops revealed an upward shift of end diastolic pressure volume relation (EDPVR) in both TAC groups as compared to sham animals (p < 0.001). Of note WT-TAC revealed a significantly higher upward shift than Mpo-/- TAC (3.18 ±0.74 mmHg/µl vs. 1.17 ±0.17 mmHg/µl; n = 8; p = 0.02). End diastolic pressure was higher in WT-TAC as compared to Mpo-/- TAC (6.23 ± 1.88 mmHg vs. 2.82 ±0.33 mmHg; n = 8; p = 0.05). In WT-TAC lung weight normalized on tibia length was significantly increased as compared to Mpo-/- TAC mice (20.15 ±2.65 mg/mm vs. 10.78 ±1.00 mg/mm; n = 10; p = 0.006). Immunohistochemical staining of left ventricular myocardium revealed significantly increased collagen I in WT- compared to Mpo-/- TAC mice (5.06 ±0.69 vs. 2.76 ±0.62% area fraction; n = 7; p =  0.001), resembling a pathophysiological mechanism for increased chamber stiffness. Conclusion: The current data reveal that MPO is critically linked to impaired diastolic compliance during pressure overload. This suggests that leukocyte activation in general and MPO release in particular is not only a prognostic marker in heart failure but evolves as an important pathophysiological link and potential pharmacological target in this disease. 4( 34) Physical Exercise Reduces Transplant Arteriosclerosis in a Fully Allogeneic Mouse Aorta Transplantation Model A.K. Knöfel , W. Sommer, N. Madrahimov, K. Jansson, M. Avsar, G. Warnecke, A. Haverich.  Medical School Hanover, Hannover, Germany. Purpose: Transplant arteriosclerosis (TA) is the main factor limiting longterm outcome after heart transplantation. The underlying mechanism of TA is alloreactivity, but it is influenced by various non-immunological co-factors. Physical exercise has positive effects on the development of native arteriosclerosis (NA). Since other cardiovascular risk factors that are relevant for NA also have an impact on TA, we hypothesized that a physical exercise program would reduce the development of TA in our fully allogeneic mouse aorta transplantation model. Methods: Segments of the thoracic aorta from C57.Bl6 mice (H2b) were transplanted into the abdominal aorta of CBA.Ca mice (H2k). In this model untreated control mice (n= 10) developed severe transplant arteriosclerosis within 28 days. Three days following surgery, mice in the intervention group started with a physical exercise protocol (n= 10) consisting of 2x45min treadmill training per day. Transplant arteriosclerosis was assessed on POD 28 after vessel explant. Quantification of intimal hyperplasia (% occlusion of the lumen) was performed by measuring intima to media ratios in EVG-stained sections of the graft. Results: Both untreated control and intervention groups developed transplant arteriosclerosis in their grafts within 28 days. Remarkably, we observed a significant reduction in the luminal occlusion in mice subjected to physical exercise (61.36+/-4.94%), compared with untreated control mice (83.89+/4.98%, p< 0.05). CD31+ endothelial cells (EC) were present in significantly higher numbers in the exercise group (15.44+/-6.19% cells/field) as compared to the untreated group (9.1+/-3.62%; p< 0.05) as evaluated by FACS analysis. In line with these results, we found enhanced eNOS expression in immunohistochemistry in the exercise group (17.6+/-1.43 cells/field) as compared to the untreated group (7+/-1.37, p< 0.05).

Abstracts S165 Conclusion: Here, we show that physical exercise has a direct protective effect on transplant arteriosclerosis. Transplant arteriosclerosis was significantly reduced when a physical exercise program was implemented in our transplant mouse model. Based on these findings we propose that the physical activity may induce the recruitment of functional EC in the graft which subsequently may lead to enhanced eNOS expression and correction of blood vessel function. 4( 35) Donor Single-dose Treatment with VEGFR-3 Antibody Reduces Acute Alloimmune Response by Targeting Lymphatic Endothelial Cell Activation A. Dashkevich ,1 S. Syrjälä,1 M. Keränen,1 R. Tuuminen,1 A. Raissadati,1 R. Krebs,1 K. Alitalo,2 A. Nykänen,1 K. Lemström.1  1Transplantation Laboratory, Haartman Institute, University of Helsinki, Helsinki, Finland,; 2Wiihuri Research Institute, Translational Cancer Biology Program, Biomedicum Helsinki, Helsinki, Finland. Purpose: VEGF-C - VEGFR-3 signaling may regulate the activation of lymphatic endothelial cells and the initiation of alloimmunity after transplantation. We investigated the impact of cold ischemic preservation and ischemia-reperfusion (I/R) injury on the activation of lymphatic endothelial cells and evaluated the effect of donor treatment with VEGFR-3 antibody on subsequent development of acute alloimmune response in rat and mouse cardiac allografts. Methods: DA rat donor hearts were preserved in +4°C PBS for 4h. The fully MHC-mismatched allografts were transplanted to WF rat recipients. The hearts were analyzed after cold and warm preservation and the allografts were analyzed 6h after reperfusion to assess I/R injury. In acute rejection model, the BALB/c mice donors were treated intraperitoneally with VEGFR-3 neutralizing antibody or control IgG a day before transplantation. The C57BL mice recipients received low-dose tacrolimus as immunosuppression. Results: Cold ischemia induced myocardial VEGF-C encoding and VEGFR-3 expression in resident dendritic cells in the DA hearts. At 6h after reperfusion, cold ischemia increased the number of LYVE-1+ lymphatic vessels expressing VEGFR-3, ICAM-1 and VCAM-1 in the allograft and induced the myocardial VEGF-C and VEGFR-3 encoding. Donor treatment with a single dose of monoclonal VEGFR-3 antibody one day before transplantation enhanced allograft survival in acute rejection model. Conclusion: Cold ischemic preservation induced the VEGF-C encoding in the heart. I/R injury activated the lymphatic endothelium by upregulating the VEGF-C core receptor VEGFR-3 and endothelial cell adhesion molecules VCAM-1 and ICAM-1, possibly responsible for the initiation of afferent leucocyte traffic. Donor treatment with a single dose of VEGFR-3 monoclonal antibody reduced acute alloimmune response. Our results suggest a novel clinically feasible lymphatic endothelium targeted immunomodulatory approach. 

4( 36) The Ubiquitin Ligase Mule Is Required for the Maintenance of Normal Cardiac Function L. Hauck ,1 D. Grothe,1 T.W. Mak,2 F. Billia.1  1Toronto General Research Institute, Toronto, ON, Canada; 2Campbell Family Cancer Research Institute, Toronto, ON, Canada. Purpose: Heart failure (HF) is the leading cause of morbidity and mortality worldwide. When exposed to injury, the heart compensates for the increased workload by cardiomyocyte (CM) hypertrophy, ultimately leading to maladaptive remodelling and HF. Defining the mechanisms underlying HF and understanding the factors regulating CM growth has immense clinical consequence. Protein ubiquitination is a key mechanism for controlling biological signalling pathways. The X-chromosome linked Mcl-1 ubiquitin ligase E3 (Mule) gene mediates the polyubiquitination and degradation of several proteins including Arf, Mcl-1, c-Myc and p53, all known to be involved in the control of cell growth and apoptosis. Therefore, we reasoned that Mule’s ability to control the Myc/Arf and Arf/p53 pathways is of critical importance to maintain normal cardiac tissue homeostasis. Methods: Transgenic mice containing loxP sequences that flanked Mule, Myc, Arf and/or p53 genes, were bred with transgenic mice carrying tamoxifen (Tam) -inducible CM-specific Cre recombinase (mcm) to generate cardiac specific knockouts, in vivo. Methods that were employed in the analysis of these mice included echocardiography, Western blotting, qRT-PCR and immunofluorescence microscopy. Results: At 2 weeks (w) post-Tam, Mulef/y;mcm mice developed spontaneous cardiac hypertrophy with an average increase of 54% (P< 0.01) in heart weight/body weight (HBW) accompanied by impaired fractional shortening (FS; 21%±1.9 versus 44%±1.8; P< 0.01). Reduced contractile function, pathological remodeling and significant increases in CM apoptosis was associated with 40% mortality (P< 0.01; n= 34) of Mulef/y; mcm mice at 3 months post-Tam. In contrast, at 4 weeks post-Tam, indices of HBW and FS were normal in Arff/f; mcm, Mycf/f; mcm and p53f/f; mcm mice. The Mulef/y; p53f/f; mcm and Mulef/y; Arff/f mcm mice developed cardiac hypertrophy and worsening of cardiac function while the phenoytype observed in the Mulef/y mice was completely rescued in Mulef/y; Mycf/f;mcm mice. Conclusion: Collectively, our observations provide strong genetic evidence that c-Myc is a physiological target for Mule in the heart. Moreoever, the control of c-Myc and thus Arf/p53 by Mule, is of critical importance in maintaining cardiac tissue homeostasis, in vivo. 4( 37) In Vivo Evaluation of the Cleveland Clinic Continuous-Flow Total Artificial Heart in Calves J.H. Karimov ,1 K. Fukamachi,1 N. Moazami,2 M. Kobayashi,1 S. Sale,3 N. Mielke,1 G. Sunagawa,1 D. Horvath,1 S. Gao,1 L.A. Golding.1  1Biomedical Engineering, Lerner Research Institute, The Cleveland Clinic, Cleveland, OH; 2Thoracic and Cardiovascular Surgery, Kaufman Center for Heart Failure, Heart and Vascular Institute, The Cleveland Clinic, Cleveland, OH; 3Cardiothoracic Anesthesiology, Anesthesiology Institute, The Cleveland Clinic, Cleveland, OH. Purpose: The Cleveland Clinic continuous-flow total artificial heart (CFTAH) is a unique single-piece, valveless, pulsatile, total artificial heart designed to provide a self-balance of the left and right circulation passively without sensors. The purpose of this study was to evaluate the in vivo pump performance, pump-related physiologic and hemodynamic parameters, and biocompatibility of CFTAH in calves. Methods: The CFTAH was implanted in twelve animals (Jersey calves, weight range 77 - 93.9 kg) through a median sternotomy (n= 5) or a right thoracotomy (n= 8). The native ventricles were removed, and the pump was connected through right and left inflow cuffs and outflow grafts. The hemodynamic parameters (pulmonary and systemic pump performance) were monitored post-implant. Results: In vivo experiments demonstrated good hemodynamic performance (pump flow of 7.5 ± 1.8 L/min; left atrial pressure (LAP) 16 ± 3 mm Hg; right atrial pressure (RAP) 17 ± 4 mm Hg; RAP-LAP difference 1.2 ± 2.4 mm Hg; mean arterial pressure 105 ± 8 mm Hg; arterial pulse pressure 31 ±13 mm Hg; pulmonary arterial pressure 33 ± 4 mm Hg) and biocompatibility. The survival of these animals extended up to 14 days of uninterrupted pumping. Post-explant device evaluation did not reveal any sign of major