Development Of Transplant Arteriosclerosis Research Articles

Involvement of Platelet Activation by P2Y12 Receptor in the Development of Transplant Arteriosclerosis in Mice

Although activated platelets influence inflammation by intraplatelet mediators in transplantation, their mechanism of involvement in the progression of transplant arteriosclerosis has not yet been elucidated. We, therefore, investigated this question using P2Y12 receptor knockout (KO) mice, in which platelets cannot be aggregated by adenosine diphosphate stimulation. Carotid arteries from 129X1 mice were orthotopically transplanted into wild-type or KO mice in a minor antigen(s)-mismatched strain combination. No immunosuppression was used. Grafts were harvested at 7, 14, 28, and 56 days after transplantation for morphometry and immunohistology. Fluorescence-activated cell sorting and quantitative real-time reverse-transcriptase polymerase chain reaction were performed at 7 and 14 days after transplantation. The intima/media ratio of grafts in KO mice was significantly reduced compared with wild-type mice at 14, 28, and 56 days after transplantation. Fluorescence-activated cell sorting analysis showed a significant reduction of platelet CD154 expression and platelet-leukocyte aggregates in KO mice at 14 days after transplantation. Additionally, levels of intercellular adhesion molecule-1 and CD40 mRNA, and numbers of intercellular adhesion molecule-1- or CD40-positive cells in the grafts were lower in KO mice at 7 and 14 days after transplantation. These reductions resulted in a significant attenuation of CD45-positive leukocytes adhering to the graft vessel wall in KO mice at 14 days after transplantation. Diminished platelet function by P2Y12 receptor deficiency attenuates initiation and strongly inhibits progression of transplant arteriosclerosis in mice by diminishing adhesion molecule expression and leukocyte accumulation in the grafts during the early phase after transplantation.

Attenuation of Transplant Arteriosclerosis With Clopidogrel Is Associated With a Reduction of Infiltrating Dendritic Cells and Macrophages in Murine Aortic Allografts

Monotherapy with clopidogrel reduced the formation of transplant arteriosclerosis in a murine aortic allograft model. However, the underlying immunologic mechanisms are still unknown. Fully major histocompatibility complex-mismatched C57BL/6 (H2b) donor aortas were transplanted into CBA.J (H2k) recipients and mice received different doses (1, 10, and 20 mg/kg) of clopidogrel, an antagonist of the P2Y12 ADP receptor on platelets, or control saline for 30 days. Blood was analyzed for changes in adhesion molecule and sCD40L concentrations by ELISA. Grafts were analyzed by histology, morphometry, and immunofluorescence on day 30 after transplantation. Intragraft cytokine mRNA production was analyzed by reverse-transcriptase polymerase chain reaction on day 14 after transplantation. Treatment with clopidogrel resulted in significantly decreased blood concentrations of sCD40L and P-selectin after transplantation. Cellular analysis of the aortic transplant revealed fewer numbers of infiltrating dendritic cells (CD205+) and macrophages (F4/80+) after application of clopidogrel, whereas T-cells within the graft were unaltered. In addition cellular P-/E-selectin, ICAM-1, and platelet-derived-growth-factor (PDGF)-beta surface expression were significantly reduced as compared with untreated controls. Intragraft mRNA expression confirmed these results and showed significant lower production of P-/E-selectin, ICAM-1, and PDGF-beta after treatment with clopidogrel. Antiglycoprotein-Ib and antiglycoprotein VI had no beneficial effect on the development of transplant arteriosclerosis. This report shows that application of clopidogrel after transplantation results in a reduction in adhesion molecule expression within the blood and transplant tissue and is associated with reduced transendothelial migration of dendritic cells and macrophages within the vascular wall.

276: Investigations into the Pathogenesis of Cytomegalovirus-Induced Transplant-Arteriosclerosis Using a Humanized Mouse Model

Recent findings have emphasised an important role of human cytomegalovirus infection (HCMV) in the development of transplant arteriosclerosis. Therefore, the aim of this study was to investigate the immunological mechanisms of HCMV in the progression of transplant arteriosclerosis in a human peripheral blood lymphocyte (hu-PBL)/RAG-2(-/-) gc(-/-) mouse-xenograft-model.

563: Development of Transplant Arteriosclerosis in Porcinized Mice Is Regulated by Allogeneic Porcine CD4+CD25+ T Cells

563: Development of Transplant Arteriosclerosis in Porcinized Mice Is Regulated by Allogeneic Porcine CD4+CD25+ T Cells

Investigations into the pathogenesis of cytomegalovirus-induced transplant-arteriosclerosis using a humanized mouse model

Introduction: Recent findings have emphasised an important role of human cytomegalovirus infection (HCMV) in the development of transplant arteriosclerosis. Therefore, the aim of this study was to develop a human peripheral blood lymphocyte (hu-PBL)/RAG-2(–/–)yc(–/–) mouse-xenograft-model to investigate the immunological mechanisms of HCMV in the progression of transplant arteriosclerosis.

Unaltered levels of transplant arteriosclerosis in the absence of the B cell homing chemokine receptor CXCR5

Chemokine receptors and their ligands are crucial for lymphocyte trafficking under both homeostatic and inflammatory conditions. The chemokine receptor CXCR5 controls B cell migration and the organization of B cell follicles. The aim of this study was to investigate the impact of CXCR5 on the development of transplant arteriosclerosis. Fully MHC mismatched BALB/c (H2 d) donor aortas were transplanted into C57BL/6-CXCR5 −/− (H2 b), C57BL/6-CXCR5 +/− (H2 b) or C57BL/6-CXCR5 +/+ (H2 b) recipients. Grafts were analysed by morphometry and immunofluorescence and intra-graft cytokine mRNA production was analysed by RT-PCR. Transplant arteriosclerosis was evident in CXCR5+/+ and CXCR5+/− mice and only mildly reduced in CXCR5−/− recipients indicating that absence of CXCR5 had no substantial effect on the development of transplant arteriosclerosis. Analysis of the cellular infiltrate of aortic grafts implanted in CXCR5−/− recipients revealed no differences in the number of T-cells, macrophages and B cells as compared to controls. Intra-graft cytokine production showed no significant changes in Th1 (IL-12) and Th2 (IL-4) cytokines as well as in TGF-β and iNOS production. These data suggest that lack of CXCR5 expression by recipient T- and B-cells has little effect on the development of transplant arteriosclerosis.

Impaired Notch4 Activity Elicits Endothelial Cell Activation and Apoptosis

Notch signaling pathway controls key functions in vascular and endothelial cells (EC). However, little is known about the role of Notch in allografted vessels during the development of transplant arteriosclerosis (TA). This study investigated regulation of the Notch pathway on cardiac allograft arteriosclerosis and further examined its implication in EC dysfunction. Here we show that, among Notch receptors, Notch2, -3, and -4 transcript levels were markedly downregulated in TA compared to tolerant and syngeneic allografts. TA correlates with high levels of tumor necrosis factor (TNF), transforming growth factor (TGF)beta, and IL10, which consistently decrease Notch4 expression in transplants and cultured ECs. We found that inhibition of Notch activity, reflected by both a reduced CBF1 activity and Hes1 expression, parallels the downregulation of Notch4 expression mediated by TNF in ECs. Notch4 and Hes1 knockdown enhances vascular cell adhesion molecule-1 expression and promotes EC apoptosis. Silencing Notch4 or Hes1 also drastically inhibits repair of endothelial injury. Overall, our results suggest that Notch4 and basal Notch activity are required to maintain EC quiescence and for optimal survival and repair in response to injury. Together, our findings indicate that impaired Notch4 activity in graft ECs is a key event associated with TA by triggering EC activation and apoptosis.

329: Ex Vivo Expanded Human Treg Inhibit the Development of Transplant Arteriosclerosis in a Humanized In Vivo Model

329: Ex Vivo Expanded Human Treg Inhibit the Development of Transplant Arteriosclerosis in a Humanized In Vivo Model

Investigation into the onset and progression of transplant arteriosclerosis in a mice aortic retransplantation model

Long-term function of vascularized human organ grafts is often limited by transplant arteriosclerosis and can lead to graft failure. Here, we have analyzed the impact of an initial rejection episode on the later development of transplant arteriosclerosis. Following transplantation of allogeneic abdominal aortic segments in mice, aortic grafts were retransplanted into either immunodeficient or syngeneic recipients. Retransplantation of grafts from immunocompetent into immunodeficient mice as early as 2 days after the primary transplant resulted in intimal proliferation and obstruction of the graft lumen 30 days after the primary transplant. In contrast, retransplantation of the grafts into donor syngeneic B10 recipients within 7 days did not result in the development of transplant arteriosclerosis. These data suggest that the adaptive immune system can induce intimal proliferation by an initial lethal hit that is sustained by the innate response. However our data demonstrate that development of chronic rejection can be inhibited, in this case by retransplantation into a syngeneic host.

Investigations into the pathogenesis of cytomegalovirus-induced transplant-arteriosclerosis using a humanized mouse model

Introduction: Recent findings have emphasised an important role of human cytomegalovirus infection (HCMV) in the development of transplant arteriosclerosis. Therefore, the aim of this study was to develop a human peripheral blood lymphocyte (hu-PBL)/RAG-2(–/–)gc(–/–) mouse-xenograft-model to investigate the immunological mechanisms of HCMV in the progression of transplant arteriosclerosis.

Anti-TNF-α (Infliximab®) significantly reduced the development of transplant arteriosclerosis in murine aortic allografts

Anti-TNF-α (Infliximab®) significantly reduced the development of transplant arteriosclerosis in murine aortic allografts

The Angiotensin II Type 1 Receptor Blocker Candesartan Attenuates Graft Vasculopathy

Transplant arteriosclerosis remains the major cause of graft failure after cardiac transplantation, although recent progress in immunosuppressive therapy has dramatically improved short-term survival of recipient. We investigated the effects of the angiotensin II type 1 receptor (AT(1)R) blocker candesartan on the development of transplant arteriosclerosis in a murine model of cardiac transplantation. Hearts from DBA/2 (H-2(d)) mice were heterotopically transplanted into B10.D2 (H-2(d)) mice. Recipients were treated with oral administration of candesartan (1 mg/kg per day) or vehicle. Allografts were analyzed at 14 or 30 days after transplantation. Candesartan significantly reduced the development of coronary arteriosclerosis (intima/media ratio: 0.86 +/- 0.09 versus 0.57 +/- 0.10, P < 0.05), without affecting the degree of parenchymal rejection at 30 days. There was no significant difference in the expression of adhesion molecules and cytokines at 14 days. Candesartan significantly reduced the number of peripheral mononuclear cells that differentiated into smooth muscle-like cells in the presence of basic fibroblast growth factor and platelet-derived growth factor BB (27.1 +/- 3.1 versus 17.3 +/- 1.8 cells/HPF, P < 0.05). Angiotensin II may play a role in the pathogenesis of transplant arteriosclerosis. Blockade of AT(1)R might be effective as a prophylactic therapy for transplant arteriosclerosis along with conventional immunosuppressive drugs.

Cytomegalovirus-enhanced development of transplant arteriosclerosis in the rat; effect of timing of infection and recipient responsiveness

Cytomegalovirus (CMV) is put forward as a risk factor for transplant arteriosclerosis (TA). In this article, we studied CMV-enhanced development of TA in rats in different donor/recipient combinations in relation to the timing of infection. Recipient rats transplanted with an aortic allograft (BN to Lew) were infected with rat CMV (RCMV) at different time-points relative to transplantation. The virus-induced effects on TA development were also determined in other strain combinations (PVG to AO and DA to WF). Finally, transmission of RCMV from aortic grafts and its effect on TA was studied. RCMV infection enhanced TA development only in Lew recipients and only after infection early post-transplantation (days 1-5). Virus transmission to the recipient only occurred from 5 and 10 days infected aortic donor-grafts, however without affecting TA development. These data indicate that the acute alloresponse and acute CMV infection need to occur simultaneously to enhance TA. This effect, however, appears to be strain combination dependent and therefore cannot be generalized.

Roles of CCR7 and CXCR5 in the development of transplant arteriosclerosis

Roles of CCR7 and CXCR5 in the development of transplant arteriosclerosis

The angiotensin II type 1 receptor blocker valsartan attenuates graft vasculopathy

Transplant arteriosclerosis remains the major cause of graft failure after cardiac transplantation. Here, we investigated the effects of the angiotensin II type 1 receptor blocker valsartan on the development of transplant arteriosclerosis in a murine model of cardiac transplantation. Hearts from DBA/2 (H-2(d)) mice were heterotopically transplanted into B10.D2 (H-2(d)) mice. Recipients were treated with oral administration of valsartan (10 mg/kg/day) or vehicle. Morphometrical analysis of the cardiac allografts harvested at 30 days revealed that valsartan significantly reduced the development of coronary atherosclerosis (intima/media ratio: 0.39 +/- 0.05 vs. 0.66 +/- 0.08, P < 0.01). At two weeks after transplantation, there was no significant difference between the two groups in expression of adhesion molecules and cytokines. Valsartan significantly reduced the number of peripheral mononuclear cells that differentiated into smooth muscle-like cells in the presence of basic fibroblast growth factor and platelet-derived growth factor BB (18.0 +/- 1.5 vs. 30.3 +/- 4.4 cells/HPF, P = 0.01). These results suggest that angiotensin II plays a role in the pathogenesis of transplant arteriosclerosis and that blockade of angiotensin II type 1 receptor might be effective as a prophylactic therapy for transplant arteriosclerosis along with conventional immunosuppressive drugs.

Chemokinereceptor 7 (CCR7) plays an important role in the development of transplant arteriosclerosis

Introduction: The chemokine receptor CCR7 has been shown to play a pivotal role in the recruitment of native T cells and antigen-activated dendritic cells (DCs) to secondary lymphoid organs. As antigen presentation and T cell recruitment are both crucial events in allograft rejection, the aim of this study was to investigate the impact of CCR7 on the development of transplant arteriosclerosis, the hallmark feature of chronic rejection.

Increased expression of transforming growth factor-beta and eosinophil infiltration is associated with the development of transplant arteriosclerosis in long-term surviving cardiac allografts.

Transplant arteriosclerosis is a major limiting factor for long-term function of allografts in clinical transplantation. This study investigated the impact of three different protocols capable of inducing long-term allograft survival on the development of transplant arteriosclerosis and immune response in cardiac allografts. CBA.Ca (H2k) recipients of fully allogeneic C57/BL10 (H2b) heart grafts received a short-term course of anti-CD154 antibody or were pretreated with anti-CD4 antibody in combination with donor alloantigen in the form of CBK (H2k+Kb) bone marrow or C57BL/10 donor-specific transfusion (DST). Grafts were analyzed on day 40 or 100 after transplantation for transplant arteriosclerosis and expression of interferon-gamma, interleukin (IL)-2, IL-4, IL-10, IL-12p40, inducible nitric oxide synthase, and transforming growth factor (TGF)-beta1 mRNA. Serum was analyzed for the presence of alloantibodies. Intimal proliferation was 62%+/-11% on day 40 in the anti-CD154 group, progressed from 31%+/-10% on day 40 to 68%+/-8% on day 100 in the CBK-bone marrow group, but remained stable at 39%+/-4% in the DST group. Increased transplant arteriosclerosis on day 100 was associated with high intragraft TGF-beta1 mRNA production and eosinophil infiltration, but not alloantibody production. Progressing transplant arteriosclerosis was associated with increased IL-4 expression. Treatment protocols for the induction of long-term allograft survival can differ substantially in the extent and kinetics of transplant arteriosclerosis. IL-4 and TGF-beta1 may be two potential therapeutic targets to attenuate the development of transplant arteriosclerosis in the long term.

Circulating smooth muscle cell plasticity in the development of transplant arteriosclerosis

Abstract To date, chronic transplant dysfunction (CTD) is recognized as the major cause of long-term transplant loss (>1 year) after transplantation. CTD presents histologically with obliterated intragraft arteries as a result of intimal hyperplasia referred to as transplant arteriosclerosis (TA). Neointimal lesions predominantly consist of vascular smooth muscle cells (VSMCs) intermingled with some inflammatory cells. The pathogenesis of TA is believed to be multifactorial, and many risk factors have been identified. Because the precise pathogenetic mechanisms underlying TA are still largely unknown, adequate prevention and treatment protocols are not available. In this review, we discus the origin (donor vs recipient, bone marrow vs non-bone marrow) of neointimal endothelial cells (ECs) and VSMCs in TA lesions, which were formerly believed to be solely graft-derived. On the basis of the data obtained in both clinical and experimental transplantation, it appears that the process leading to TA is heterogeneous and that neointimal ECs and VSMCs can be recruited from different sources, possibly depending on the severity of vascular damage. These data suggest a significant role of host-derived circulating EC-VSMC progenitor cells, which may be partly bone marrow-derived. These circulating progenitor cells are potential targets for therapeutic intervention to ameliorate TA development or occlusive vascular disease in general.

Syngeneic bone marrow transduced with a recombinant retroviral vector to express endoplasmic reticulum signal-sequence-deleted major histocompatibility complex class-I alloantigen can induce specific immunologic unresponsiveness in vivo.

Long-term survival of fully allogeneic cardiac grafts can be induced in mice through transduction of recipient bone marrow cells (BMCs) with a recombinant retroviral vector encoding a single full-length major histocompatibility complex (MHC) class I alloantigen. This study investigated whether cell surface expression of the transduced MHC antigen was necessary for the induction of specific unresponsiveness. METHOD The signal sequence for translocation into the endoplasmic reticulum was deleted from H-2K (SDELKb). Syngeneic BMCs from CBA.Ca (H2k) recipients were transduced with an MFG retroviral vector encoding either wild-type Kb or the mutant SDELKb and reinfused in conjunction with an anti-CD4 therapy. Four weeks later, the recipients underwent transplantation with a fully allogeneic C57BL/10 cardiac graft. Graft survival and the development of transplant arteriosclerosis were assessed. Expression of both the wild-type Kb or SDELK in recipient CBA mice before transplantation resulted in prolonged survival of C57BL/10 grafts. Grafts from recipients pretreated with SDELKb developed 48%+/-22% intimal proliferation compared with 61%+/-21% in grafts from recipients pretreated with wild-type Kb. However, this difference did not reach statistical significance. CONCLUSION Cell surface expression, and therefore direct recognition, of an MHC class I alloantigen is not required to induce long-term survival of fully allogeneic cardiac grafts after retroviral transduction of recipient BMCs.

Mouse endothelial CD40 expression does not play a role during the development of transplant arteriosclerosis.

Endothelial cells (ECs) are of major interest in allograft rejection. The authors and others have recently shown in different mouse allograft models that, during the development of transplant arteriosclerosis, donor ECs are replaced by recipient ECs. It was the aim of this study to characterize the phenotype of ECs during the development of transplant arteriosclerosis further, with particular interest in their capability to express CD40. Abdominal aortic allografts were transplanted across a full major histocompatibility complex (MHC) barrier using BALB/c (H-2d) mice as donors and C57BL/6 (H-2b) mice as transplant recipients. Aortic allografts were harvested on days 7 and 30 after transplantation and analyzed by double-immunohistochemistry for expression of CD40 and CD31. As the authors have previously shown that ECs in the aortic allograft model are of donor origin on day 7 after transplantation, whereas by day 30 donor ECs have been replaced by ECs of recipient origin, these two time points were chosen for analysis of CD40 expression. Double-immunohistochemistry for CD40 and CD31 revealed no endothelial expression of CD40 at either time point. These initial results obtained with the anti-CD40 monoclonal antibody (mAb) (clone 3/23) were confirmed by two further experiments using two different anti-CD40 mAbs (clone HM40-3 and clone 1/10) that bind to epitopes of the CD40 receptor distinct to that of 3/23. Neither mAb revealed endothelial expression of CD40. Further analysis of the composition of the cellular infiltrate demonstrated that the majority of macrophages were CD40 positive. The data indicate that in this mouse model of aortic transplantation, neither recipient- nor donor-derived ECs express CD40 during the development of transplant arteriosclerosis. Therefore, in contrast to rat or human ECs, CD40 expression by mouse ECs does not seem to play a major role in this form of allograft rejection.