C. Heim

Combination of clopidogrel and everolimus dramatically reduced the development of transplant arteriosclerosis in murine aortic allografts

Our group has shown that platelet inhibition with clopidogrel, an antagonist of the P2Y12 adenosine diphosphate receptor on platelets, reduced the formation of transplant arteriosclerosis. The aim of this study was to investigate whether a combination of cyclosporin or everolimus with clopidogrel has a beneficial effect on the development of transplant arteriosclerosis. Fully MHC mismatched C57Bl/6 (H2b) donor aortas were transplanted into CBA.J (H2k) recipients and mice received either clopidogrel alone (1 mg/kg/day) or in combination with cyclosporin (2 mg/kg/day) or everolimus (0.05 mg/kg/day). Grafts were analysed by histology and morphometry on day 30 after transplantation. In mice treated with clopidogrel alone, transplant arteriosclerosis was significantly reduced [intima proliferation 56 ± 11% vs. 81 ± 7% (control)/n = 7]. Daily application of everolimus reduced the development of transplant arteriosclerosis compared with untreated controls [intima proliferation of 29 ± 9% vs. 81 ± 7% (control)/n = 7]. Strikingly, combination of clopidogrel and everolimus almost abolished the formation of transplant arteriosclerosis [intima proliferation: 11 ± 8% vs. 81 ± 7% (control)/n = 7]. By contrast, combination of cyclosporin and clopidogrel compared with clopidogrel alone showed no additive effect. These results demonstrate that combination of platelet‐ and mammalian target of Rapamycin‐inhibition can dramatically reduce the development of transplant arteriosclerosis.

Murine cytomegalovirus infection leads to increased levels of transplant arteriosclerosis in a murine aortic allograft model.

Introduction. Cytomegalovirus infection after heart transplantation is considered as risk factor for the development of transplant arteriosclerosis. Therefore, the aim of this study was to investigate the effect of murine cytomegalovirus (MCMV) as a single risk factor on transplant arteriosclerosis in an experimental aortic allograft model. Methods. Major histocompatibility complex class I-mismatched aortas of C.B10-H2(b)/LilMcdJ donor were transplanted into BALB/c recipients, which were either mock-infected or infected with MCMV (strain Smith) on day 7 and harvested 37 days after transplantation. In one experimental group animals received a daily dose of everolimus to increase the viral load of recipients. Grafts were analyzed by histology, morphometry, and immunofluorescence. Intragraft cytokine mRNA production was analyzed by real-time polymerase chain reaction (PCR), and persistence of cytomegalovirus infection was confirmed by TaqMan PCR. Results. After infection with MCMV, there was significantly more intimal proliferation when compared with uninfected controls (intimal proliferation 83.5%±9.6% [MCMV+] vs. 43.9%±5.1% [MCMV−]), indicating that MCMV plays a role in the development of transplant arteriosclerosis. Even after treatment with everolimus, MCMV infection pronounced significantly more intimal proliferation (intimal proliferation 52.5%±7.3% [MCMV+] vs. 20.2%±1.7% [MCMV−]). Intragraft mRNA expression showed significantly higher production of CD62E, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 after infection with MCMV. Cellular infiltration revealed significantly more CD4+, CD8+, and dendritic cells. We could also confirm the presence of MCMV for the duration of the experimental protocol by PCR within the spleen, liver, salivary glands, lung, and the aortic transplant. Conclusion. These data suggest that MCMV infection plays an important role in the development of transplant arteriosclerosis.

Human Cytomegalovirus Infection Leads to Elevated Levels of Transplant Arteriosclerosis in a Humanized Mouse Aortic Xenograft Model

Recent findings emphasized an important role of human cytomegalovirus (HCMV) infection in the development of transplant arteriosclerosis. Therefore, the aim of this study was to develop a human peripheral blood lymphocyte (hu‐PBL)/Rag‐2–/–γc–/– mouse‐xenograft‐model to investigate both immunological as well as viral effector mechanisms in the progression of transplant arteriosclerosis. For this, sidebranches from the internal mammary artery were recovered during coronary artery bypass graft surgery, tissue‐typed and infected with HCMV. Then, size‐matched sidebranches were implanted into the infrarenal aorta of Rag‐2–/–γc–/– mice. The animals were reconstituted with human peripheral blood mononuclear cells (PBMCs) 7 days after transplantation. HCMV‐infection was confirmed by Taqman‐PCR and immunofluorescence analyses. Arterial grafts were analyzed by histology on day 40 after transplantation. PBMC‐reconstituted Rag‐2–/–γc–/– animals showed splenic chimerism levels ranging from 1–16% human cells. After reconstitution, Rag‐2–/–γc–/– mice developed human leukocyte infiltrates in their grafts and vascular lesions that were significantly elevated after infection. Cellular infiltration revealed significantly increased ICAM‐1 and PDGF‐R‐β expression after HCMV‐infection of the graft. Arterial grafts from unreconstituted Rag‐2–/–γc–/– recipients showed no vascular lesions. These data demonstrate a causative relationship between HCMV‐infection as an isolated risk factor and the development of transplant‐arteriosclerosis in a humanized mouse arterial‐transplant‐model possibly by elevated ICAM‐1 and PDGF‐R‐β expression.

Delayed therapy with clopidogrel and everolimus prevents progression of transplant arteriosclerosis and impairs humoral alloimmunity in murine aortic allografts

OBJECTIVES It was previously shown that the combination of clopidogrel and everolimus reduced the development of transplant arteriosclerosis. The aim of this study was to investigate whether delayed onset of treatment, similar to the clinical situation after heart transplantation, inhibits progression of transplant arteriosclerosis. METHODS Fully allogeneic C57BL/6 (H2-b) donor aortas were transplanted into CBA.J (H2-k) recipients treated with clopidogrel and everolimus alone or in combination starting on Days 1, 7 or 14. Grafts were analysed by histology and alloantibodies were detected by fluorescence activated cell sorting after transplantation. RESULTS Delayed platelet inhibition with clopidogrel reduced the development of transplant arteriosclerosis [neointima formation (Day 14): 50±4 vs 84±9% (control)]. The combination of clopidogrel and everolimus almost abolished formation of transplant arteriosclerosis when therapy was started on Day 1 [neointima formation (Day 1): 14±5 vs 84±9% (control)] and also showed a remarkable reduction in both delayed treatment groups [neointima formation (Day 7): 24±7 vs 84±9% (control); neointima formation (Day 14): 28±11 vs 84±9% (control)]. Platelet inhibition alone and in combination with everolimus resulted in reduced alloantibody production. CONCLUSIONS These results demonstrate that delayed treatment with clopidogrel and everolimus-representative of a clinical setting-prevents the progression of transplant arteriosclerosis and impairs humoral immunity in this experimental model.

Enzymatic lipid oxidation by eosinophils propagates coagulation, hemostasis, and thrombotic disease

Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase–derived hydroxyeicosatetraenoic acid–phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease.

Vascular inflammation and media calcification are already present in early stages of chronic kidney disease

BACKGROUND While patients with chronic kidney disease (CKD) have a high prevalence of classical coronary risk factors, there is increasing evidence that atherosclerosis is different in renal compared to nonrenal patients. Therefore, the present study compares changes in different vessels obtained at cardiac surgery between patients with early and advanced CKD and nonrenal control patients. METHODS AND RESULTS Fifty patients undergoing cardiac bypass surgery were divided into three groups: (i) 24 control patients with creatinine <1.3mg/dl, (ii) 14 patients with early CKD (creatinine 1.3-2.0mg/dl), and (iii) 12 patients with advanced CKD (creatinine >2.0mg/dl). Aorta, arteria mammaria interna, and vena saphena (V. saphena) were analyzed using morphometry, Kossa stain for vascular calcification, and immunohistochemistry for markers of inflammation and proosteogenic differentiation of vascular smooth muscle cells (VSMCs). Thereby, aortic wall thickness and calcification score of aortic intima and of V. saphena were significantly higher in advanced CKD patients than in nonrenal control patients, whereas significant vascular inflammation and proosteogenic dedifferentiation of VSMC and calcification of the aortic media were already present in early CKD. Interestingly, marked calcification of the V. saphena magna was seen in advanced CKD. Of note, calcium-phosphate product correlated well with markers of inflammation, but not with calcification itself. CONCLUSIONS Early stages of CKD are already associated with local up-regulation of proinflammatory and proosteogenic molecules in the vascular wall and calcification of the aortic media. These findings point to the importance of local microinflammation in CKD and may shed new light on the potentially overestimated role of the calcium-phosphate product for vessel calcification.

Prolyl-hydroxylase inhibitor activating hypoxia-inducible transcription factors reduce levels of transplant arteriosclerosis in a murine aortic allograft model

OBJECTIVES The development of transplant arteriosclerosis, the hallmark feature of heart transplant rejection, is associated with a chronic immune response and also influenced by an initial injury to the graft through ischaemia and reperfusion. Hypoxia-inducible transcription factor (HIF)-1 pathway signalling has a protective effect against ischaemia-reperfusion injury and has already been demonstrated to ameliorate allograft nephropathy in previous animal studies. Therefore, the aim of this study was to investigate the effect of stabilization of hypoxia-inducible transcription factors with a prolyl-hydroxylase domain (PHD) inhibitor on transplant arteriosclerosis in an experimental aortic allograft model. METHODS MHC-class I mismatched C.B10-H2(b)/LilMcdJ donor thoracic aortas were heterotopically transplanted into the abdominal aorta of BALB/c mice. Donor animals received a single dose of the PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40 mg/kg) or vehicle i.p. 4 h before transplantation. Intragraft HIF accumulation after ICA treatment was detected by immunohistochemistry before and after cold ischaemia (n = 5). Grafts were harvested 30 days after transplantation and analysed by histology (n = 7) and immunofluorescence (n = 7). In addition, intragraft mRNA expression for cytokines, adhesion molecules and growth factors was determined on Day 14 (n = 7). RESULTS Donor preconditioning with ICA resulted in HIF accumulation in the aorta and induction of the HIF target genes vascular endothelial growth factor and transforming growth factor-beta. Vascular lesions were present in both experimental groups. However, there was significantly reduced intimal proliferation in preconditioned grafts when compared with vehicle controls [intimal proliferation 31.3 ± 8% (ICA) vs 55.3 ± 20% (control), P < 0.01]. In addition, experimental groups revealed a down-regulation of E-selectin (-57%) and MCP1 (-33%) expression after ICA pretreatment compared with controls, going along with decreased T-cell [1.4% CD4+ T-cell infiltration vs 8.4% (control) and 4.9% CD8+ T-cell infiltration vs 10.7% (control)], dendritic cell (0.6% dendritic cells infiltration vs 1.9% infiltration(control)] and macrophage infiltration [4.8% macrophages (ICA) vs 10.9% (control)] within vascular grafts. CONCLUSIONS These data of an animal transplant model show that the pharmaceutical activation of HIF with endogenous up-regulation of protective target genes leads to adaptation of the graft to low oxygen-saturation and hereby attenuates the development of transplant arteriosclerosis and allograft injury. Pharmaceutical inhibition of PHDs appears to be a very attractive strategy for organ preservation that deserves further clinical evaluation.

Microvascular integrity plays an important role for graft survival after experimental skin transplantation

BACKGROUND Every transplanted organ relies on a reliable and sound vascular system. Therefore, our study focused on the investigation if platelet inhibition alone or combined with mTOR-inhibition has a beneficial effect on the microvascular integrity in allogeneic murine skin grafts. METHODS Skin transplantation was performed from fully MHC-mismatched C57BL/6 (H-2b) donors to CBA/J (H-2k) recipient mice. Skin allograft recipients were assigned to several experimental groups and either treated with clopidogrel alone, everolimus alone or a combination of both. Graft survival was evaluated and transplants were harvested after 8 days and analyzed for CD31 and C4d by immunohistochemistry. RESULTS Untreated allografts showed a reduced amount of CD31 on postoperative day 8 as well as an increase in C4d compared to isografts. All treated animals showed a significant improvement regarding CD31 [1577.7 ± 200.4 (clopidogrel)/1702.8 ± 151.1 (clopidogrel + everolimus) vs. 479.7 ± 184.2 (control), n = 8, p b 0.05] and C4d [420.9 ± 70.9 (clopidogrel)/324.5 ± 77.3 (clopidogrel + everolimus) vs. 772.4 ± 159.7 (control), n = 8, p b 0.05]. In addition, skin grafts of animals treated with clopidogrel and everolimus survived significantly longer compared to untreated controls [19.2 ± 4.2 d vs. 12.8 ± 2.4 d, n= 10, p b 0.05]. CONCLUSION In this study we could show that clopidogrel alone and in combination with everolimus substantially improved microvascular integrity and resulted in increased survival time of skin grafts.

A new ex vivo beating heart model to investigate the application of heart valve performance tools with a high-speed camera.

High-speed camera examination of heart valves is an established technique to examine heart valve prosthesis. The aim of this study was to examine the possibility to transmit new tools for high-speed camera examination of heart valve behavior under near-physiological conditions in a porcine ex vivo beating heart model. After explantation of the piglet heart, main coronary arteries were cannulated and the heart was reperfused with the previously collected donor blood. When the heart started beating in sinus rhythm again, the motion of the aortic and mitral valve was recorded using a digital high-speed camera system (recording rate 2,000 frames/sec). The image sequences of the mitral valve were analyzed, and digital kymograms were calculated at different angles for the exact analysis of the different closure phases. The image sequence of the aortic valve was analyzed, and several snakes were performed to analyze the effective orifice area over the time. Both processing tools were successfully applied to examine heart valves in this ex vivo beating heart model. We were able to investigate the exact open and closure time of the mitral valve, as well as the projected effective orifice area of the aortic valve over the time. The high-speed camera investigation in an ex vivo beating heart model of heart valve behavior is feasible and also reasonable because of using processing feature such as kymography for exact analysis. These analytical techniques might help to optimize reconstructive surgery of the mitral valve and the development of heart valve prostheses in future.

Die unterschätzte Rolle von Thrombozyten bei der Herztransplantation

ZusammenfassungDie chronische Abstoßung mit ihrem Hauptmerkmal der Transplantatarteriosklerose ist heutzutage die Hauptkomplikation nach erfolgreicher Herztransplantation, deren Ausbildung nicht mit derzeit üblichen Immunsuppressiva verhindert werden kann. In der vorliegenden Übersichtsarbeit wird zunächst dargestellt, dass Thrombozyten eine Rolle in der Herztransplantation spielen, anschließend werden Mechanismen, über welche aktivierte Thrombozyten transplantierte Organe schädigen können, aufgezeigt und therapeutische Strategien in einem experimentellen Transplantationsmodell getestet. So konnte demonstriert werden, dass eine Monotherapie mit Clopidogrel die Ausbildung der Transplantatarteriosklerose signifikant vermindert. Es zeigten sich nach Behandlung mit Clopidogrel deutlich weniger dendritische Zellen und Makrophagen in den transplantierten Gefäßen; bezüglich der T-Zell-Infiltration konnten jedoch keine relevanten Unterschiede gefunden werden. Deshalb wurde in weiteren Experimenten untersucht, ob eine Kombination von einem mTOR(mammalian target of Rapamycin)-Inhibitor mit Clopidogrel einen additiven Effekt auf die Entwicklung von Transplantatarteriosklerose hat. Bei dieser Kombinationsbehandlung konnte eine Transplantatarteriosklerose beinahe vollständig verhindet werden. Angesichts der hohen klinischen Relevanz der erhobenen Befunde wurde unlängst beschlossen, diese Ergebnisse in Form einer klinischen Multicenter-Studie zu untersuchen, um bei nachgewiesenem Erfolg diese neue Therapieform möglichst bald in die klinische Behandlung übernehmen zu können.AbstractTransplant arteriosclerosis, the hallmark feature of chronic rejection, is still the leading cause of late mortality after heart transplantation. Current immunosuppressive therapies do not prevent the formation of transplant arteriosclerosis. This review focuses on the role of platelets in heart transplantation, in particular on the pathogenetic role of activated platelets in the formation of vascular lesions after heart transplantation. In addition, a therapeutic strategy is introduced in an experimental aortic allograft model. Using this model, it was shown that monotherapy with clopidogrel reduced the formation of transplant arteriosclerosis significantly. Cellular analysis of the aortic transplant revealed fewer numbers of infiltrating dendritic cells (CD205+) and macrophages (F4/80+) following application of clopidogrel, whereas T-cells within the graft were unaltered. In a final set of experiments it was shown that a combination of platelet- and mTOR-inhibition can dramatically reduce the development of transplant arteriosclerosis. As a consequence of these encouraging results a multicenter-study was initiated throughout Germany to investigate the benefit of this new therapeutic strategy in clinical practice.