Development Of Transplant Arteriosclerosis Research Articles

Direct Impact of Human Platelets on the Development of Transplant Arteriosclerosis.

Platelets play an important role in the pathogenesis of inflammatory and proliferative vascular changes. The aim of this study was to investigate whether human platelets are able to induce transplant arteriosclerosis in a humanized C57/Bl6-Rag2-/-γc-/- mouse xenograft model. Nonactivated and in vitro-activated human platelets were analyzed and phenotyped for surface markers by flow cytometry. Side branches of human mammary arteries were implanted into the infrarenal aorta of recipients, followed by daily application of human platelets and histological analyzed on day 30 after transplantation. Human platelets collected by apheresis had low levels of platelet activation markers. However, after in vitro activation, expression was markedly increased. Sixty minutes after injection in recipient mice, nonactivated human platelets become significantly activated. Increased adhesion of platelets to the vascular endothelium was detected by in vivo fluorescence microscopy. After intravenous injection of nonactivated or activated platelets, human xenografts showed pronounced intimal proliferation. Immunohistological analysis showed that the group treated with activated human platelets exhibited significantly increased intragraft protein expression of intracellular adhesion molecule-1 and platelet-derived growth factor receptor beta and smooth muscle cell migration into the neointima. These data demonstrate that an isolated daily application of both in vivo- and in vitro-activated human platelets results in the development of transplant arteriosclerosis in a humanized mouse transplantation model.

Regulatory T Cells and the Effect of Ex Vivo Preservation on the Development of Transplant Arteriosclerosis in a Humanized Mouse Model

Regulatory T Cells and the Effect of Ex Vivo Preservation on the Development of Transplant Arteriosclerosis in a Humanized Mouse Model

Linking CLAD in Lung Transplant Recipients after 3 Years Follow Up with Transplant Arteriosclerosis in Humanized Mice

Purpose Chronic lung allograft dysfunction (CLAD) is a severe complication of lung transplantation (LTX) limiting long term survival. Here, we studied correlations between CLAD after clinical LTX and leukocyte-mediated development of transplant arteriosclerosis (TA) in a humanized mouse model. Methods The pericardiophrenic artery was procured from surplus tissue of 27 donor lungs transplanted in our clinical program and was implanted into the abdominal aorta of immune deficient mice. The experimental mice were then divided into four groups. Negative control mice received no human leukocyte reconstitution (neg. co). PBMC group mice received 5 × 106 allogeneic human peripheral blood mononuclear cells (PBMC) from the respective lung recipients (LR). A further group of animals was reconstituted with the respective PBMC additionally enriched with autologous CD4+CD25high cells (putative regulatory T cells, Treg). Human leukocyte engraftment was monitored by FACS and development of TA was histologically assessed 28 days after PBMC reconstitution. The 27 LR were divided into two groups at least 3 years post transplantation according to their development of chronic rejection. Nine patients (33.3%) developed CLAD 36.2± 4.16 month after LTX. The remaining eighteen patients (66,6%) did not develop CLAD within 35.9± 5.1 month after LTX. Results The neg. co group showed only mild thickening of the intima (6.38±5.70%). In the PBMC CLAD+ group, intimal thickening obliterating the vessel lumen was significantly more severe than in the PBMC CLAD- group (33.19 ± 7.02% vs. 13.37 ± 2.99%, p= 0.011). Then, intimal thickening was significantly inhibited in the PBMC+Treg CLAD+ group as compared to the PBMC CLAD+ group (0.39 ± 3.69% vs 33.19 ± 7.02%, p=0.012). In the experiments using PBMC from lung recipients without CLAD, enriching Treg also further suppressed the development of transplant arteriosclerosis (13.37 ± 2.99% PBMC CLAD- vs. 0.69 ± 5.88% PBMC+Treg CLAD-, p=0.007). Conclusion Lung transplant recipients, who later developed CLAD, had peripheral leukocytes already at the time of transplant that transfered pro-inflammatory properties leading to TA into a humanized mouse model. TA remained sensitive to inhibition by autologous regulatory T cells, suggesting a cell therapy-based approach for the prevention and treatment of CLAD after LTX.

In Vivo Development of Transplant Arteriosclerosis in Humanized Mice Reflects BOS in Lung Transplant Recipients and Is Controlled by Autologous Regulatory T Cells

In Vivo Development of Transplant Arteriosclerosis in Humanized Mice Reflects BOS in Lung Transplant Recipients and Is Controlled by Autologous Regulatory T Cells

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

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. 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). 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. 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.

T Cell Responses in Lung Transplantion – Role of Alloantigen Priming and Regulation on Development of Transplant Arteriosclerosis in a Humanized Mouse Model

Purpose In animal models, both naive and alloantigen-primed Treg inhibit rejection. In clinical recipients of transplants, however, not much is known with regard to the actual function of those Treg that can be found in vivo. Here, we studied the functional importance of both naive and alloantigen-primed T cell responses in a humanized mouse model, utilizing transplant arteriosclerosis as the experimental readout. Methods and Materials The pericardiophrenic artery was procured from leftover tissue of lung grafts transplanted within our clinical program and was implanted into the abdominal aorta of NRG mice. Group A received no human leukocyte and served as negative controls. Group B received 5x106 allogeneic human PBMC from the respective lung recipient harvested at the time of transplant and group C received the same naive allogeneic PBMC, depleted of CD4+CD25+ putative Treg. Group D received naive allogeneic PBMC enriched of these CD4+CD25+ cells. Group E received PBMC harvested 21 days after transplantation, containing the alloantigen-primed T cells. Human leukocyte engraftment was monitored and transplant arteriosclerosis was histologically assessed 28 days later. Results The control group A showed mild thickening of the intima. In group B intimal thickening resulting in severe obliteration of the vessel lumen was evident. In group C obliteration of the lumen was even more severe. By contrast, in group D intimal thickening was less severe. In group E, reconstituted with alloantigen-primed total PBMC, only very mild transplant arteriosclerosis developed, comparable to group D enriched of naive Treg. Conclusions We show the importance of T cell regulation for controlling alloresponses in vivo. The finding of reduced transplant arteriosclerosis elicited by alloantigen-primed PBMC is intriguing and may indicate enhanced T cell regulation in these clinical transplant recipients. Ongoing studies aim at correlating clinical transplant outcomes to T cell function assessed using humanized mouse.

Attenuation of Transplant Arteriosclerosis by Oral Feeding of MHC Encoding Chitosan-DNA Nanoparticles Is Mediated by Humoral Immunity

Purpose 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 allo-immune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Methods and Materials We performed fully allogeneic mouse abdominal aortic transplantats 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. Results 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. Conclusions These results demonstrate the potential of chitosan-DNA nanoparticles to induce K b -specific tolerance and to reduce the development of transplant arteriosclerosis.

Low-Dose Rapamycin Treatment Increases the Ability of Human Regulatory T Cells to Inhibit Transplant Arteriosclerosis In Vivo

Regulatory T cells (Treg) are currently being tested in clinical trials as a potential therapy in cell and solid organ transplantation. The immunosuppressive drug rapamycin has been shown to preferentially promote Treg expansion. Here, we hypothesized that adjunctive rapamycin therapy might potentiate the ability of ex vivo expanded human Treg to inhibit vascular allograft rejection in a humanized mouse model of arterial transplantation. We studied the influence of combined treatment with low-dose rapamycin and subtherapeutic Treg numbers on the development of transplant arteriosclerosis (TA) in human arterial grafts transplanted into immunodeficient BALB/cRag2−/−Il2rg−/− mice reconstituted with allogeneic human peripheral blood mononuclear cell. In addition, we assessed the effects of the treatment on the proliferation and apoptosis of naïve/effector T cells. The combined therapy efficiently suppressed T-cell proliferation in vivo and in vitro. Neointima formation in the human arterial allografts was potently inhibited compared with each treatment alone. Interestingly, CD4+ but not CD8+ T lymphocytes were sensitive to Treg and rapamycin-induced apoptosis in vitro. Our data support the concept that rapamycin can be used as an adjunctive therapy to improve efficacy of Treg-based immunosuppressive protocols in clinical practice. By inhibiting TA, Treg and rapamycin may prevent chronic transplant dysfunction and improve long-term allograft survival

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.

61 PHD (Prolyl-Hydroxylase)-Inhibitor Activating Hypoxia-Inducible Transcription Factors (HIFs) Reduces Levels of Transplant Arteriosclerosis in a Murine Aortic Allograft Model

The development of transplant arteriosclerosis, the hallmark feature of heart transplant rejection is associated with a chronic immune response and influenced by the initial injury caused by ischemia and reperfusion. As previously shown, donor treatment with a PHD-inhibitor activating Hypoxia-inducible transcription factors (HIFs) prevents graft injury and prolongs survival in an allogenic kidney transplant model. Therefore, the aim of this study was to investigate the effect of HIF activation with a PHD-inhibitor on transplant arteriosclerosis in an experimental aortic allograft model.

A20-Mediated Modulation of Inflammatory and Immune Responses in Aortic Allografts and Development of Transplant Arteriosclerosis

Transplant arteriosclerosis (TA) is the pathognomonic feature of chronic rejection, the primary cause of allograft failure. We have shown that the NF-κB inhibitory protein A20 exerts vasculoprotective effects in endothelial and smooth muscle cells (SMC), and hence is a candidate to prevent TA. We sought direct proof for this hypothesis. Fully mismatched, C57BL/6 (H2) into BALB/c (H2), aorta to carotid allografts were preperfused with saline, recombinant A20 adenovirus (rAd.A20) or rAd.β-galactosidase (β-gal), implanted, harvested 4 weeks after transplantation, and analyzed by histology, immunohistochemistry, and immunofluorescence staining. We measured indoleamine 2,3-dioxygenase, interleukin-6, and transforming growth factor-β mRNA and protein levels in nontransduced, and rAd.A20 or rAd.β-gal-transduced human SMC cultures after cytokine treatment. Vascular overexpression of A20 significantly reduced TA lesions. This correlated with decreased graft inflammation and increased apoptosis of neointimal SMC. Paradoxically, T-cell infiltrates increased in A20-expressing allografts, including the immunoprivileged media, which related to A20 preventing indoleamine 2,3-dioxygenase upregulation in SMC. However, infiltrating T cells were predominantly T-regulatory cells (CD25+/Forkhead Box P3 [FoxP3+]). This agrees with A20 inhibiting interleukin-6 and promoting transforming growth factor-β production by medial SMC and in SMC cultures exposed to cytokines, which favors differentiation of regulatory over pathogenic T cells. In summary, A20 prevents immune-mediated remodeling of vascular allografts, therefore reduces TA lesions by affecting apoptotic and inflammatory signals and modifying the local cytokine milieu to promote an immunoregulatory response within the vessel wall. This highlights a novel function for A20 in local immunosurveillance, which added to its vasculoprotective effects, supports its therapeutic promise in TA.

Adenosine Diphosphate Receptor P2Y12-Mediated Migration of Host Smooth Muscle-Like Cells and Leukocytes in the Development of Transplant Arteriosclerosis

We have recently reported that platelet P2Y12 receptors may play a role in the development of transplant arteriosclerosis (TA). In the present study, we investigated the role of P2Y12 receptors on host-derived smooth muscle-like cells (SMLCs, including bone-marrow-derived SMLCs) and CD45+ leukocytes, both of which are believed to be associated with the development of TA, using P2Y12-deficient (KO) mice. Orthotopic carotid artery transplantation was performed from C3H/He (H-2k) donors into KO or wild-type (WT) recipient mice (129S:C57BL/6, H-2b). Grafts were harvested at 8 weeks after transplantation for histology. Plasma monocyte chemoattractant protein-1 (MCP-1) levels were analyzed with a kit. Cell migration was examined using a Boyden chamber system. The expression of MCP-1 messenger RNA was assessed by real-time polymerase chain reaction. Eight weeks after allotransplantation, KO recipient mice showed a significant reduction of luminal occlusion, host-derived SMLCs, CD45+ leukocytes, MCP-1+ cells in the grafts, and of plasma MCP-1 levels. In addition, the migration of host-derived SMLCs (including bone-marrow-derived SMLCs) and CD45+ leukocytes stimulated with adenosine diphosphate (ADP) or 2-methylthio-ADP (2MeSADP, a stable ADP analog) was significantly decreased in KO mice. There were no significant changes in MCP-1-induced cell migration between WT and KO mice. The low concentration of 2MeSADP plus MCP-1 significantly increased cell migration in WT but not KO mice. Furthermore, 2MeSADP-induced MCP-1 messenger RNA expression was significantly reduced in the cells of KO mice. Thus, the P2Y12-mediated migration of host-derived SMLCs and CD45+ leukocytes may play an important role in the development of TA, partly by MCP-1 pathways.

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.

Cordyceps sinensis Extracts Attenuate Aortic Transplant Arteriosclerosis in Rats

Transplant arteriosclerosis is a hallmark of chronic rejection and is still the major limiting factor affecting the success of long-term organ transplants. Development of transplant arteriosclerosis is refractory to conventional immunosuppressive drugs, and adequate therapy is not yet available. The aim of this study was to determine the role of Cordyceps sinensis extracts in reducing the formation of transplant arteriosclerosis in a rat aortic transplant model. Lewis rat aortic allografts were transplanted into Brown-Norway recipient rats. Recipients received 0.5, 1, 2, and 5 mg/kg of Cordyceps sinensis extracts (or control saline) daily via intragastric injection for 60 d. Grafts were harvested 60 d post-transplantation and intimal thickness determined microscopically following hematoxylin and eosin (H and E) staining and abdominal aorta protein profiles determined by Western blot analysis. Cellular localization was assessed by histology and immunohistochemistry and the serum analyzed for tumor necrosis factor alpha (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) by enzyme-linked immunosorbent assay (ELISA). C. sinensis administration resulted in a significant reduction in neointimal formation (neointimal thickness 8.27 ± 1.95 μm [0.5 mg/kg], 3.69 ± 1.43 μm [1 mg/kg], 3.69 ± 1.43 μm [1 mg/kg], 3.69 ± 1.43 μm [1 mg/kg] versus 11.42 ± 2.67 μm [control]) and in the proliferative activity of vascular smooth muscle cells. In addition, localized expression of TNF-α and ICAM-1 in transplant aortas was characterized by immunohistochemistry and immunoblot analyses demonstrating that C. sinensis treatment significantly reduced TNF-α and ICAM-1 levels compared with levels observed in controls (P < 0.05). Serum TNF-α and ICAM-1 levels were significantly reduced in C. sinensis-treated animals compared with controls (P < 0.05). C. sinensis treatment effectively reduced the formation of transplant arteriosclerosis in a rat aortic transplant model.

Murine Cytomegalovirus Infection Leads to Increased Levels of Transplant Arteriosclerosis in a Murine Aortic Allograft Model

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. 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. 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. These data suggest that MCMV infection plays an important role in the development of transplant arteriosclerosis.

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 (H2(b)) donor aortas were transplanted into CBA.J (H2(k)) 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.

382: Reduced Transplant Arteriosclerosis after Treatment with MMF and MMF in Combination with Ganciclovir in a Mouse Aortic Transplant Model

The aim of this study was to investigate whether Ganciclovir and Mycophenolate Mofetil (MMF), alone and in combination, have a beneficial influence on the development of transplant arteriosclerosis (TxA).

Reduced transplant arteriosclerosis after treatment with MMF and MMF in combination with ganciclovir in a mouse aortic transplant model

Objectives: The aim of this study was to investigate whether Ganciclovir and Mycophenolate Mofetil (MMF), alone and in combination, have a beneficial influence on the development of transplant arteriosclerosis (TxA). Methods: Fully allogeneic C57BL/6 (H2b) donor aortas were transplanted into CBA (H2k) recipients. Recipient mice were treated with a single medication of MMF (100mg/kg or 300mg/kg) or a combination of MMF and Ganciclovir (10mg/kg Ganciclovir and 100mg/kg MMF or 72mg/kg Ganciclovir and 300mg/kg MMF) per day. Grafts were analysed by histology and morphometry on day 30 after transplantation. A potential effect of MMF or Ganciclovir on Tregs was investigated by FACS analysis. Results: Two doses of 150mg/kg MMF per day significantly reduced the development of TxA compared to untreated controls (intima proliferation of 23%±8% vs. 68%±7% [control]). 50mg/kg of MMF did not have any effect on the development of TxA. Strikingly, combination of Ganciclovir and low-dose MMF reduced the formation of TxA (intima proliferation of 27%±2% vs. 68%±7% [control]). The high-dose combination group (300mg MMF/72mg Ganciclovir) showed an intima proliferation of 24%±4%. FACS analysis of the spleen within this group revealed 12.73% CD4+cells with 14.70% CD4+CD25+FoxP3+cells. As compared to 17.5% CD4+cells with 11.1% CD4+CD25+FoxP3+cells in the control group. Single treatment with MMF (300mg) showed 10.55% CD4+ cells and 10.87% CD4+CD25+FoxP3+ cells within the analysed spleens. Conclusion: Our results demonstrate a dose-dependent positive effect of MMF on the development of TxA and a synergistic effect of Ganciclovir in combination with MMF.

Combination of clopidogrel and everolimus dramatically reduced the development of transplant arteriosclerosis

Objectives: Our group has previously shown that platelet inhibition with clopidogrel reduced the formation of transplant arteriosclerosis (TxA). The aim of this study was to investigate whether a combination of cyclosporine, tacrolimus or everolimus with clopidogrel has a beneficial effect on the development of TxA. Methods: Fully allogeneic C57BL/6 (H2b) donor aortas were transplanted into CBA (H2k) recipients. Recipient mice were treated with cyclosporine (2mg/kg/d), tacrolimus (12mg/kg/d) and everolimus (0.05mg/kg/d) alone or in combination with clopidogrel (1mg/kg/d). Grafts were analysed by histology and morphometry on day 30 after transplantation. Results: In mice treated with clopidogrel alone, TxA was reduced as compared to untreated controls (intima proliferation 65%±8% vs. 79%±9% [control]/n=5). Daily application of everolimus significantly reduced the development of TxA (intima proliferation of 48%±19% vs. 79%±9% [control], n=5). Strikingly, the combination of clopidogrel and everolimus almost completely abolished the formation of TxA (intima proliferation: 16%±12% vs. 79%±9% [control], n=5). Tacrolimus alone (intima proliferation: 58%±17% vs. 79%±9% [control], n=5) and in combination with clopidogrel (intima proliferation: 30%±5% vs. 79%±9% [control], n=5) also showed a significant reduction. In contrast, daily application of cyclosporine didn't reduce the development of TxA compared to controls (intima proliferation: 74%±8% vs. 79%±9% [control], n=5). Conclusion: These results demonstrate that combination of clopidogrel and everolimus as well as tacrolimus can dramatically reduce the development of TxA in a mouse aortic allograft model.

The inhibitory effect of astilbin on the arteriosclerosis of murine thoracic aorta transplant

The inhibitory effect of astilbin on transplant arteriosclerosis in murine model of thoracic aorta transplantation was examined. Model of rat thoracic aorta transplantation was established. Ninety rats were divided into three groups. In isograft group, the thoracic aorta of Brown Norway (BN) rat was anastomosed with the abdominal aorta of another BN rat. In allograft group, the thoracic aorta of BN rat was anastomosed with the abdominal aorta of Lewis rat. In astilbin group, the rats receiving allo-transplantation were given astilbin 5 mg/kg per day for a time of 28 days. The donor thoracic aorta and the recipient abdominal aorta were anastomosed by means of a polyethylene cannula (inner diameter: 1.5 mm, length: 3 mm length). The grafts were histologically examined for structural changes. The areas of arterial lumen and endatrium were calculated. Our results showed that, in the allograft group, 28 days after allografting, conspicuous proliferation of smooth muscles and infiltration with a great number of inflammatory cells were found in the tunica intima and tunica media. Astilbin significantly inhibited the proliferation of smooth muscles and ameliorated the infiltration of inflammatory cells thereby prevent against the development of transplant arteriosclerosis. It is concluded that asltilbin can effectively prevent the development of arteriosclerosis in allotransplant by inhibiting the proliferation of smooth muscles and inhibit the proliferation of smooth muscles in tunica of intima and media and reducing infiltration of the inflammatory cells.