S. Eckl

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.

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.

Clopidogrel reduces post-transplant obliterative bronchiolitis

Survival after lung transplantation is mainly limited by the development of chronic lung allograft dysfunction (CLAD). The aim of this study was to investigate if platelet inhibition by clopidogrel has an influence on the formation of obliterative bronchiolitis, the histopathological correlate to bronchiolitis obliterans syndrome, present in the majority of patients suffering from CLAD. C57Bl/6(H2b) donor tracheas were orthotopically transplanted into CBA.J(H2k). Mice received different doses of clopidogrel alone or in combination with tacrolimus or everolimus. Grafts were analyzed by histology and immunofluorescence method on postoperative days 15, 30 or 60. Cytokines were analyzed by real‐time polymerase chain reaction on postoperative day 21 and alloantibodies by FACS. Mice treated with 20 mg/kg/day clopidogrel for 30 days showed reduced obliteration [34.40 ± 3.76% (20 mg/kg/day clopidogrel) vs. 49.92 ± 2.11% (control), n = 5, P < 0.05]. Platelet inhibition resulted in significant lower infiltration of T cells and macrophages, and we also found significantly lower expression of IL‐12, IL‐4, IL‐6, TNF‐α, TGF‐β, PDGFβ, MCP1, P‐/E‐selectin, ICAM1 and CD40L after treatment with clopidogrel. Combination of 1 mg/kg/day clopidogrel and 0.05 mg/kg/day everolimus or 12 mg/kg/day tacrolimus revealed a synergistic effect. Humoral immunity as manifested by donor‐specific alloantibody secretion was also impaired after treatment with clopidogrel. Here, we can show that platelet inhibition by clopidogrel as a single treatment and in combination with tacrolimus or everolimus reduced the development of fibrosis and obliteration in tracheal allografts.

Attenuation of transplant arteriosclerosis by oral feeding of major histocompatibility complex encoding chitosan-DNA nanoparticles

One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the alloimmune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Therefore, we performed fully allogeneic mouse abdominal aortic transplants using C57BL/6 (H2(b)) mice as donors and CBA.J (H2(k)) mice as recipients. Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for K(b), one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of K(b) encoding nanoparticles (K(b)-NP) or Balb/c (H2(d)) grafts were used instead of C57BL/6 (H2(b)) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the K(b)-NP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. These results demonstrate the potential of chitosan-DNA nanoparticles to induce K(b)-specific tolerance and to reduce the development of transplant arteriosclerosis.

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.

95 Delayed Therapy with Clopidogrel and Everolimus Substantially Inhibits the Development of Transplant Arteriosclerosis

Purpose Our group has shown that combination of clopidogrel and everolimus dramatically reduced the development of transplant arteriosclerosis (TxA). The aim of this study was to investigate whether a delayed onset of treatment- similar to the clinical setting - could inhibit progression of TxA. Methods and Materials Fully allogeneic C57BL/6 (H2(b)) donor aortas were transplanted into CBA (H2(k)) recipients. Recipients were treated with Clopidogrel (1 mg/kg/d) and Everolimus (0.05 mg/kg/d), alone or in combination, starting on postoperative days 1, 7 or 14. Grafts were analyzed by histology and morphometry on day 30 after transplantation. Results In mice treated with clopidogrel alone, TxA was significantly reduced as compared to untreated controls when therapy was started on day 1 (intima proliferation 56%±11% vs. 81%±7% [control]/n=5). This effect was also seen when clopidogrel therapy was started on postoperative day 14 (intima proliferation 50%±4% vs. 81%±7% [control]/n=5). Combination of clopidogrel and everolimus dramatically reduced the formation of TxA when medication was started on day 1 (intima proliferation: 11% ± 8% vs. 81% ± 9% [control], n = 5) and showed remarkable reduction in both delayed onset groups (on day 7: intima proliferation: 36% ± 10% vs. 81% ± 9% [control], n = 5 and on day 14: intima proliferation: 44% ± 3% vs. 81% ± 9% [control], n = 5). Conclusions These results demonstrate that even delayed onset of treatment with clopidogrel and everolimus – representative for a clinical setting - results in a strong reduction of TxA in this murine aortic allograft model.