Rat Cardiac Allografts Research Articles

Fingolimod (FTY720) prevents chronic rejection of rodent cardiac allografts through inhibition of the RhoA pathway

The Fingolimod (FTY720, Gilenya) is clinically approved for the treatment of multiple sclerosis (MS). Its therapeutic effect on MS is based on the ability to bind sphingosine 1-phosphate (S1P) receptors and block the exit of immune cells from the lymphoid organs, thus preventing immune cell-dependent injury to the central nervous system (CNS). We showed recently that, besides the S1P-related activity, the FTY720 also down-regulates RhoA, which is a master regulator of the actin cytoskeleton. Our previous studies showed that FTY720 also down-regulates Rictor, which is a signature molecule of mTORC2 complex, which regulates RhoA and dictates actin cytoskeleton specificity. Because, our previous studies showed that chronic rejection correlates with the upregulation of RhoA and mTORC2 components and that the inhibition of RhoA pathway prevents chronic rejection, here we studied the effect of FTY720 on the chronic rejection of rat and mouse cardiac allografts. We show that FTY720 in conjunction with the inhibitors of early T cell response, (CTA4-Ig in mice and Everolimus in rats) blocks macrophage infiltration into the grafts and prevents chronic rejection of rat and mouse cardiac transplants. This indicates that FTY720 may be repurposed from the MS application to the clinical transplantation as an anti-chronic rejection drug.

Novel colchicine derivatives enhance graft survival after transplantation via suppression of T-cell differentiation and activity.

Immunosuppressants are crucial in organ transplantation but their side effects are a trade-off for long-term use. Colchicine has been shown to be effective in various diseases, albeit with many side effects. To enhance the immunosuppressive effects of colchicine, in addition to minimizing its side effects, we attempted to synthesize new colchicine derivatives (KR compounds). In rat cardiac and pancreatic islet allograft, long-term graft survival was identified in KR compound-treated groups. The use of cyclosporine A (CsA) or colchicine inhibited the CD3+ and CD4+ T-cell proliferation, whereas KR compounds inhibited the CD8+ T cells as well as CD4+ T cells. KR compounds reduced the apoptosis, interleukin-2 receptor expression, and signal transducer and activator of transcription 3 phosphorylation more than CsA. These results indicate that KR compounds have a potential therapeutic value as novel agents for prevention of graft deterioration by allograft of rejection.

Coinhibition of mTORC1/mTORC2 and RhoA /ROCK pathways prevents chronic rejection of rat cardiac allografts

Chronic rejection of transplanted organs remains an unresolved issue in clinical organ transplantation. The macrophages play a crucial role in the development of chronic rejection. We showed previously that macrophage-specific deletion of RhoA or RhoA/ROCK inhibition prevent macrophage movement to the cardiac allografts and abrogate chronic rejection in rodent transplantation models. Here we assessed the ability of the mTORC1 and mTORC2 inhibitor everolimus, alone or in conjunction with the RhoA/ROCK inhibitor Y27632, to inhibit chronic rejection of Wistar Furth (WF; RT1.Au) rat cardiac allografts heterotopically transplanted into ACI (RT1.Aa) recipients. The transplanted hearts were analyzed for vessel occlusion and tissue fibrosis. T cell and macrophage subsets infiltration was analyzed by immunostaining with T cell and macrophage molecular markers. We found that a combination of the mTOR inhibitor everolimus and the RhoA/ROCK inhibitor Y27632 prolonged allograft survival; decreased vessel occlusion, collagen deposition, and macrophage infiltration; and inhibited the chronic rejection of rat cardiac allografts. These results indicate that coinhibition of the mTORC1/mTORC2 and RhoA pathways in transplant recipients abrogates chronic rejection of rat cardiac allografts, and will aid in the development of clinically applicable anti-chronic rejection therapies.

AS2553627, a novel JAK inhibitor, prevents chronic rejection in rat cardiac allografts

Janus family kinases (JAKs) are essential molecules for cytokine responses and attractive targets for the treatment of transplant rejection and autoimmune diseases. Several JAK inhibitors have shown demonstrable effects on acute rejection in experimental cardiac transplant models. However, little is known about the potential benefits of JAK inhibitors on chronic rejection outcomes such as vasculopathy and fibrosis. Here, we examined the pharmacological profile of a novel JAK inhibitor, AS2553627, and explored its therapeutic potential in chronic rejection as well as acute rejection in a rat cardiac transplant model. AS2553627 potently inhibited JAK kinases but showed no inhibition of other kinases, including TCR-associated molecules. The compound also suppressed proliferation of IL-2 stimulated human and rat T cells. In a rat cardiac transplant model, oral administration of AS2553627 alone or co-administration with a sub-therapeutic dose of tacrolimus effectively prolonged cardiac allograft survival, suggesting the efficacy in treating acute rejection. To evaluate the effect on chronic rejection, recipient rats were administered a therapeutic dose of tacrolimus for 90 days. In combination with tacrolimus, AS2553627 significantly reduced cardiac allograft vasculopathy and fibrosis that tacrolimus alone did not inhibit. AS2553627 at the effective dose in rat transplantation models did not significantly reduce reticulocyte counts in peripheral whole blood after in vivo erythropoietin administration, indicating a low risk for anemia. These results suggest that AS2553627 may be a therapeutic candidate for the prevention of not only acute but also chronic rejection in cardiac transplantation.

Simvastatin Treatment Upregulates Anti-Fibrotic Bone Morphogenetic Protein-7 Expression at Rat Cardiac Allograft Rejection

Background: Bone morphogenetic protein (BMP)-7 mediates ischemic tolerance and anti-fibrotic effects in various organs such as kidney and heart. Recently, reno- and podocyte-protective effects of a potent HMG-CoA reductase inhibitor, pitavastatin, were accompanied by BMP-7 upregulation. Methods: Here, we investigated the effect of simvastatin treatment on BMP-7 expression in major MHC-mismatched rat cardiac allografts subjected to ischemia-reperfusion injury and adaptive immune activation at 10 days. Results: We localized Smad2 activity and Reca-1⁺ fibroblast specific protein-1⁺ immunoreactivity, suggesting endothelial-to-mesenchymal transition, at fibrotic borderline of cardiac allografts at 10 days. Simvastatin donor and recipient combination treatment significantly upregulated cardiac allograft BMP-7 expression when compared to nontreated controls at 10 days. Conclusion: The beneficial effect of statin treatment on cardiac allograft may in part be mediated through the upregulation of BMP-7.

MicroRNA let 7i regulates DCs maturation targeting IL-10 via JAK1-STAT3 signal pathway subsequently induces prolonged cardiac allograft survival in rats

Abstract Objective In this study, we investigated whether microRNA (MiR) let 7i regulates DCs maturation targeting IL-10 via JAK1-STAT3 signal pathway subsequently prolongs rat cardiac allograft survival. Methods QRT-PCR, ELISA, dual luciferase assay were performed to verify whether IL-10 was the target of let 7i. Tregs were assessed by flow cytometry and immunohistochemical study, and JAK1, STAT3 and pSTAT3 expressions were detected by western bolt. Lewis recipients of DA heart were transfused with PBS, LPS-mDCs, or let 7i inhibitor-mDCs. Allograft survival times were recorded, and histologic study were performed. Results Expression of IL-10 mRNA level and production of IL-10 were increased in let 7i inhibitor-mDCs as compared to LPS-mDCs. Luciferase activity showed that the translational level of the IL-10 luciferase reporter was decreased by let 7i mimic, but increased by let 7i inhibitor. MiR let 7i inhibitor suppressed DC maturation by depressing CD80 and CD86, however， pretreatment of IL-10 SiRNA attenuated the suppression. Expressions of JAK1, STAT3, and pSTAT3 in mDCs were suppressed by let 7i mimic and pretreatment of IL-10 SiRNA, however up regulated by let 7i inhibitor. Lewis recipients transfused with let 7i inhibitor-mDCs significantly prolonged DA cardiac allograft survival. The allografts transfused with let 7i inhibitor-mDCs showed slight cell infiltration and significantly preserved graft structure. Inhibition of let 7i increased Tregs and modulated cytokine profiles both in vivo and in vitro. Conclusion MiR let 7i regulated DCs maturation and function targeting the IL-10 through JAK1-STAT3 pathway. Moreover, transfusion of let 7i inhibitor-mDCs induced prolonged cardiac allograft survival and generated Tregs.

Ribonuclease (RNase) Prolongs Survival of Grafts in Experimental Heart Transplantation.

BackgroundCell damage, tissue and vascular injury are associated with the exposure and release of intracellular components such as RNA, which promote inflammatory reactions and thrombosis. Based on the counteracting anti‐inflammatory and cardioprotective functions of ribonuclease A (RNase A) in this context, its role in an experimental model of heart transplantation in rats was studied.Methods and ResultsInbred BN/OrlRj rat cardiac allografts were heterotopically transplanted into inbred LEW/OrlRj rats. Recipients were intravenously treated every other day with saline or bovine pancreatic RNase A (50 μg/kg). Toxic side effects were not found (macroscopically and histologically). Heart tissue flow cytometry and quantitative morphological analyses of explanted hearts at postoperative day 1 or postoperative day 4 showed reduced leukocyte infiltration, edema, and thrombus formation in RNase A‐treated rats. In allogeneic mixed lymphocyte reactions, RNase A decreased the proliferation of effector T cells. RNase A treatment of rats resulted in prolonged median graft survival up to 10.5 days (interquartile range 1.8) compared to 6.5 days (interquartile range 1.0) in saline treatment (P=0.001). Treatment of rats with a new generated (recombinant) human pancreatic RNase 1 prolonged median graft survival similarly, unlike treatment with (recombinant) inactive human RNase 1 (each 50 μg/kg IV every other day, 11.0 days, interquartile range 0.3, versus 8.0 days, interquartile range 0.5, P=0.007).ConclusionsUpon heart transplantation, RNase administration appears to present a promising and safe drug to counteract ischemia/reperfusion injury and graft rejection. Furthermore, RNase treatment may be considered in situations of critical reperfusion after percutaneous coronary interventions or in cardiac surgery using the heart–lung machine.

Platelet-derived Growth Factor-B Protects Rat Cardiac Allografts From Ischemia-reperfusion Injury.

Microvascular dysfunction and cardiomyocyte injury are hallmarks of ischemia-reperfusion injury (IRI) after heart transplantation. Platelet-derived growth factors (PDGF) have an ambiguous role in this deleterious cascade. On one hand, PDGF may exert vascular stabilizing and antiapoptotic actions through endothelial-pericyte and endothelial-cardiomyocyte crosstalk in the heart; and on the other hand, PDGF signaling mediates neointimal formation and exacerbates chronic rejection in cardiac allografts. The balance between these potentially harmful and beneficial actions determines the final outcome of cardiac allografts. We transplanted cardiac allografts from Dark Agouti rat and Balb mouse donors to fully major histocompatibility complex-mismatched Wistar Furth rat or C57 mouse recipients with a clinically relevant 2-hour cold ischemia and 1-hour warm ischemia. Ex vivo intracoronary delivery of adenovirus-mediated gene transfer of recombinant human PDGF-BB upregulated messenger RNA expression of anti-mesenchymal transition and survival factors BMP-7 and Bcl-2 and preserved capillary density in rat cardiac allografts at day 10. In mouse cardiac allografts PDGF receptor-β, but not -α intragraft messenger RNA levels were reduced and capillary protein localization was lost during IRI. The PDGF receptor tyrosine kinase inhibitor imatinib mesylate and a monoclonal antibody against PDGF receptor-α enhanced myocardial damage evidenced by serum cardiac troponin T release in the rat and mouse cardiac allografts 6 hours after reperfusion, respectively. Moreover, imatinib mesylate enhanced rat cardiac allograft vasculopathy, cardiac fibrosis, and late allograft loss at day 56. Our results suggest that PDGF-B signaling may play a role in endothelial and cardiomyocyte recovery from IRI after heart transplantation.

MicroRNA let-7i regulates dendritic cells maturation targeting interleukin-10 via the Janus kinase 1–signal transducer and activator of transcription 3 signal pathway subsequently induces prolonged cardiac allograft survival in rats

In this study, we investig1ated whether microRNA let-7i regulates dendric cell maturation targeting interleukin-10 (IL-10) via the Janus kinase 1-signal transducer and activator of transcription 3 (JAK1-STAT3) signal pathway subsequently prolongs rat cardiac allograft survival. Quantitative real-time reverse transcriptase polymerase chain reaction, enzyme linked immunosorbent assay, and dual-luciferase assay were performed to verify whether IL-10 was the target of let-7i, and regulatory T cells were assessed by flow cytometry and immunohistochemical study. Western blot was performed to detect JAK1, STAT3, and phosphorylated STAT3 expression. Lewis recipients of Dark Agouti hearts were transfused with phosphate-buffered saline, lipopolysaccharide (LPS)-mature dendritic cells (mDCs), or let-7i-inhibitor-mDCs. Allograft survival times were recorded, and histologic studies were performed. Expression of IL-10 messenger RNA level and production of IL-10 were increased in let-7i-inhibitor-mDCs compared with LPS-mDCs. Luciferase activity showed that the translational level of the IL-10 luciferase reporter was decreased by let-7i mimic but increased by let-7i-inhibitor. MicroRNA let-7i inhibitor suppressed DC maturation; however, pretreatment of IL-10 small interfering RNA attenuated the suppression. Expression of JAK1, STAT3, and phosphorylated STAT3 in mDCs were suppressed by let-7i mimic, and pre-treatment of IL-10 small interfering RNA, however, were upregulated by let-7i inhibitor. Lewis recipients transfused with let-7i-inhibitor-mDCs significantly prolonged Dark Agouti cardiac allograft survival. The allografts transfused with let-7i-inhibitor-mDCs showed slight cell infiltration and significantly preserved graft structure. Inhibition of let-7i increased CD4(+)CD25(+)forkhead box P3(+) regulatory T cells and modulated cytokine profiles in vivo and in vitro. MicroRNA let-7i regulated DC maturation and function targeting IL-10 through the JAK1-STAT3 pathway. Moreover, transfusion of LPS-induced mDCs transfected with let-7i inhibitor induced prolonged cardiac allograft survival and generated regulatory T cells.

IL-34 is a Treg-specific cytokine and mediates transplant tolerance

Cytokines and metabolic pathway-controlling enzymes regulate immune responses and have potential as powerful tools to mediate immune tolerance. Blockade of the interaction between CD40 and CD40L induces long-term cardiac allograft survival in rats through a CD8+CD45RClo Treg potentiation. Here, we have shown that the cytokine IL-34, the immunoregulatory properties of which have not been previously studied in transplantation or T cell biology, is expressed by rodent CD8+CD45RClo Tregs and human FOXP3+CD45RCloCD8+ and CD4+ Tregs. IL-34 was involved in the suppressive function of both CD8+ and CD4+ Tregs and markedly inhibited alloreactive immune responses. Additionally, in a rat cardiac allograft model, IL-34 potently induced transplant tolerance that was associated with a total inhibition of alloantibody production. Treatment of rats with IL-34 promoted allograft tolerance that was mediated by induction of CD8+ and CD4+ Tregs. Moreover, these Tregs were capable of serial tolerance induction through modulation of macrophages that migrate early to the graft. Finally, we demonstrated that human macrophages cultured in the presence of IL-34 greatly expanded CD8+ and CD4+ FOXP3+ Tregs, with a superior suppressive potential of antidonor immune responses compared with non-IL-34-expanded Tregs. In conclusion, we reveal that IL-34 serves as a suppressive Treg-specific cytokine and as a tolerogenic cytokine that efficiently inhibits alloreactive immune responses and mediates transplant tolerance.

Rat Cytomegalovirus Vaccine Prevents Accelerated Chronic Rejection in CMV-Naïve Recipients of Infected Donor Allograft Hearts

Cytomegalovirus accelerates transplant vascular sclerosis (TVS) and chronic rejection (CR) in solid organ transplants; however, the mechanisms involved are unclear. We determined the efficacy of a CMV vaccine in preventing CMV‐accelerated rat cardiac allograft rejection in naïve recipients of CMV+ donor hearts. F344 donor rats were infected with RCMV 5 days prior to heterotopic cardiac transplantation into CMV‐naïve or H2O2‐inactivated RCMV‐vaccinated Lewis recipients. Recipients of RCMV‐infected donor hearts rejected at POD59, whereas vaccinated recipients exhibited a significantly prolonged time to rejection‐POD97, similar to recipients of uninfected donor hearts (POD108). Although all of the donor hearts were preinfected, the vaccinated recipients had lower graft and PBMC viral loads at POD 7 compared to unvaccinated controls. Adoptive T cell and passive antibody transfers from vaccinated Lewis rats into naïve recipients demonstrate that both T‐cell and B‐cell arms of the adaptive immune response provide protection against CMV‐accelerated rejection. Similar findings were obtained when testing three different adjuvants in passive transfer experiments. We have determined that the timing of the vaccine prior to transplantation and the specific adjuvant play critical roles in mediating anti‐viral responses and promoting graft survival. CMV vaccination prior to transplantation may effectively increase graft survival.

PDGF-B is Protective during Ischemia-reperfusion Injury in Rat Cardiac Allografts

PDGF-B is Protective during Ischemia-reperfusion Injury in Rat Cardiac Allografts

Proteasome Inhibitors in Experimental Cardiac Transplantation

Objectives: Proteasome inhibition by bortezomib, a constitutive proteasome inhibitor (PI) approved for the treatment of multiple myeloma, was reported in the field of organ transplantation; despite known role in antigen presentation, the positive effect on graft rejection has not been verified. Since bortezomib is associated with many side effects (pancytopenia, gastrointestinal and transient neurological disorders), specific inhibition of immunoproteasomes might reduce them. The currently unapproved immunospecific PI PR-957 achieved positive results in experimental mice studies in the field of autoimmune diseases. Therefore, we compared the treatment with PR-957 and Bortezomib in a rat model of heterotopic heart transplantation. Methods: Brown Norway rat cardiac allografts were heterotopic abdominal transplanted to Lewis rats using microvascular Langendorff technique. Since postoperative day (POD) 1 recipients were daily treated intravenously with bortezomib (0.1 mg/kg), PR-957 (6 mg/kg) or saline (control, n = 6 in each group). The primary end point of the study was graft survival, determined by daily graft palpation and echocardiography. All explanted hearts were collected for histological analyses. A tolerance study was performed treating Lewis rats daily with bortezomib, PR-957 or saline for 16 days (n = 3 in each group). Blood counts of all treated rats were determined every other day. Results: Mean graft survival was 6.8 ± 0.8d in control group versus 11.5 ± 2.9d (p = 0.002 vs control) in bortezomib, and 11.2 ± 1.3d (p = 0.002 vs control) in PR-957 group. On POD 7 only recipients treated with bortezomib suffered from thrombocytopenia (p = 0.019). No significant differences in red and white blood counts were detected between the treatment groups. The tolerance study showed macroscopic alterations of liver, lymphnodes and thymus in bortezomib treated animals, whereas autopsy of PR-957 or saline treated animals revealed no macroscopic abnormalities. Histological analyses and measurement of cytokines are ongoing. Conclusions: Proteasome inhibitors obviously improved allograft survival. Compared with the constitutive PI bortezomib, the treatment with the immunospecific PI PR-957 suggested being superior in avoidance of side effects. Thus, drug development in the new field of immunoproteasome inhibition could improve the treatment of transplant rejection.

Injection of Cardiac Stem Cells Prolongs the Survival of Cardiac Allograft Rats

Cardiac c-kit+cells isolated from cardiac explant-derived cells modestly improve cardiac functions after myocardial infarction; however, their full potential has not yet been realized. The present study was undertaken to determine the isolation and culture of c-kit+cardiac stem cells (CSCs), and the roles of myocardial injection of CSCs on the survival of rat cardiac allograft. Recipient Sprague–Dawley rats were transplanted with hearts from Wistar rats. in the in vitro experiment, c-kit+cells were isolated from mouse heart fragment culture by magnetic cell sorting. CSCs expressed of cardiomyocyte specific protein cardiac troponin I, α smooth muscle actin and von Willebrand factor in conditioned culture. CsC injection increased graft survival of cardiac allograft rats. The effects of CSCs on increase in graft survival of cardiac allograft rats were blocked by stromal-derived factor-1 (SDF-1) knockdown. The expression of SDF-1 was increased after CSC injection into the cardiac of cardiac allograft rats. These results indicate that CSC injection into the cardiac prolongs graft survival of cardiac allograft rats. SDF-1 plays an important role in the effects of CSCs on the graft survival of cardiac allograft rats.

Tranilast, an Analogue of a Tryptophan Catabolite, Induces Allograft Tolerance By Myeloid-Derived Suppressor Cells (MDSCs).

Indoleamine 2, 3-dioxygenase (IDO) converts tryptophan in various tolerogenic metabolites. One is anthranilic acid and has been shown to induce immune tolerance in different immune-mediated diseases, including organ transplantation. Tranilast is a clinically approved and is have ready clinically used (Rizaben®) in asthma and atopic dermatitis. We examined the effect of tranilast in a rat cardiac allograft model. Lewis 1W rat hearts were grafted in MHC-mismatched Lewis 1A rats. The receiver is orally treated with 650 mg/kg of tranilast or clinically used Rizaben® (Kissei Pharmaceutical Co) daily for 30 days. Total splenocytes and purified spleen cell subtypes were sorted by FACS Aria and transferred to sublethaly irradiated rats by i.v injection the day before transplantation. Graft survival in rats treated with tranilast were significantly prolonged (64,6±42.7 days, n=15) and showed tolerance with > 100 days survival vs control group (8.3±2.3 days, n=6). When Rizaben®, was used, all treated animals (n=8) showed tolerance. Adoptive transfer of total splenocytes from tolerant tranilast-treated rats to naïve rats resulted in tolerance in all animals (n=5). We adoptively transferred tolerance only with CD161+TCR- cells from tolerant rats which resulted in tolerance (130±91.6 days, n=5, 3/5 recipients >100 days) whereas CD161-TCR- cells from the same animals did not (12.7±4.6 days, n=3). In the CD161+TCR- cells, we could distinguish 2 cells populations CD161high(NK cells) and CD161low. CD161low cells were MHCII-, CD172a+, CD11b+ and CD86+ a phenotype already described in the rat as MDSCs. When we transferred CD161high and CD161low from the same animals, we obtained prolongation of allograft survival only with the CD161low MDSCs (59±64days, n=5, 2/5 recipients >100 days) and not with the CD161high (18±14 days, n=3).Moreover, tranilast-treated and cell-transfered recipients with indefinite allograft did not have detectable levels of alloantibodies. This is the first demonstration that tranilast mediates transplantation tolerance. Tolerance was active and transferable by MDSCs. Experiments are under way to better define how MDSCs are induced by tranilast and the mechanisms by which they induce tolerance.

Immunomodulation Induced By a Novel Immunosuppressant, NK026680 Plus Donor Specific Transfusion Permits a Long-Term Cardiac Allograft Survival in Mice.

Background: We have recently demonstrated that a novel triazolopylimidine derivative, NK026680 (NK), inhibits T cell proliferation and prolongs rat cardiac allograft survival. In this study, we examined the immunomodulatory effect of NK in combination with donor specific transfusion (DST) in mice. Methods: A fully MHC incompatible BALB/c-to-C57BL/6 mouse heart transplantation was performed on day 0. DST, 20x106 donor-splenocytes, was given intravenously on day -7. NK (40 mg/kg/day) was orally administered from day -7 to day +6. Recipient-splenocytes were cocultured with irradiated donor-splenocytes, and T cell proliferation (3H-thymidine incorporation), IFN-g production (ELISPOT) and cell phenotypes (FACS) were assessed. Results: Untreated mice promptly rejected cardiac allograft at a graft median survival time (gMST) of 6.5 days. DST or NK monotherapy prolonged gMST to 24.5 and 25.5 days, respectively. In a sharp contrast, NK plus DST treatment (NK+DST) markedly prolonged gMST to 75.5 days. (A). In the NK+DST group, both proliferation and IFN-g production against donorantigens transiently increased on day 0, while they were both significantly suppressed on day 7 (p<0.05, vs. Untreated). Following in vivo antigen-challenge by infusing 20x106 donor-splenocytes on day 0, the splenic Foxp3+CD4+ T cells increased[Figure 1](B); however, CD69+ effector T cells were abolished.Figure: No Caption available.(C and D) in the NK+DST group. Conclusion:NK together with DST induces a potent immunomodulatory effect by shifting immune-balance towards a regulatory milieu, and markedly prolongs cardiac allograft survival in mice. DISCLOSURES:Saiga, K.: Employee, salary.

Angiopoietin-2 Inhibition Prevents Transplant Ischemia-Reperfusion Injury and Chronic Rejection in Rat Cardiac Allografts

Transplant ischemia-reperfusion injury (Tx-IRI) and allograft dysfunction remain as two of the major clinical challenges after heart transplantation. We investigated the role of angiopoietin-2 (Ang2) in Tx-IRI and rejection using fully MHC-mismatched rat cardiac allografts. We report that plasma levels of Ang2 were significantly enhanced in the human and rat recipients of cardiac allografts, but not in the rat recipients of syngrafts, during IRI. Ex vivo intracoronary treatment of rat cardiac allografts with anti-Ang2 antibody before 4-h cold preservation prevented microvascular dysfunction, endothelial cell (EC) adhesion molecule expression and leukocyte infiltration, myocardial injury and the development of cardiac fibrosis and allograft vasculopathy. Recipient preoperative and postoperative treatment with anti-Ang2 antibody produced otherwise similar effects without effecting microvascular dysfunction, and in additional experiments prolonged allograft survival. Recipient preoperative treatment alone failed to produce these effects. Moreover, ex vivo intracoronary treatment of allografts with recombinant Ang2 enhanced Tx-IRI and, in an add-back experiment, abolished the beneficial effect of the antibody. We demonstrate that neutralization of Ang2 prevents EC activation, leukocyte infiltration, Tx-IRI and the development of chronic rejection in rat cardiac allografts. Our results suggest that blocking Ang2 pathway is a novel, clinically feasible, T cell-independent strategy to protect cardiac allografts.

MHC-derived allopeptide activates TCR-biased CD8+ Tregs and suppresses organ rejection

In a rat heart allograft model, preventing T cell costimulation with CD40Ig leads to indefinite allograft survival, which is mediated by the induction of CD8+CD45RClo regulatory T cells (CD8+CD40Ig Tregs) interacting with plasmacytoid dendritic cells (pDCs). The role of TCR-MHC-peptide interaction in regulating Treg activity remains a topic of debate. Here, we identified a donor MHC class II-derived peptide (Du51) that is recognized by TCR-biased CD8+CD40Ig Tregs and activating CD8+CD40Ig Tregs in both its phenotype and suppression of antidonor alloreactive T cell responses. We generated a labeled tetramer (MHC-I RT1.Aa/Du51) to localize and quantify Du51-specific T cells within rat cardiac allografts and spleen. RT1.Aa/Du51-specific CD8+CD40Ig Tregs were the most suppressive subset of the total Treg population, were essential for in vivo tolerance induction, and expressed a biased, restricted Vβ11-TCR repertoire in the spleen and the graft. Finally, we demonstrated that treatment of transplant recipients with the Du51 peptide resulted in indefinite prolongation of allograft survival. These results show that CD8+CD40Ig Tregs recognize a dominant donor antigen, resulting in TCR repertoire alterations in the graft and periphery. Furthermore, this allopeptide has strong therapeutic activity and highlights the importance of TCR-peptide-MHC interaction for Treg generation and function.

(435) - Donor Single-dose Treatment with VEGFR-3 Antibody Reduces Acute Alloimmune Response by Targeting Lymphatic Endothelial Cell Activation

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.

RNase A in (Xeno)Transplantation

BackgroundCell injury, particularly as consequence of ischemia/reperfusion or acute rejection after (xeno)transplantation, leads to release of intracellular components like RNA. Extracellular RNA, (i) causes edema as a result of increasing permeability of blood vessels [1], (ii) is described as a procoagulation factor by activation of the contact phase system leading to thrombus formation and vessel occlusion [2], and (iii) promotes leukocyte recruitment to the vascular system by mobilising proinflammatory cytokines [3], and thus, triggers immune response. In rodent models of stroke and thrombosis, treatment with counteracting RNase A, a ribonuclease that specifically degrades single‐stranded RNA, was shown to result in less edema formation and vessel occlusion [4]. In search of additive drugs to protect (xeno)grafts from dysfunction, we investigated if treatment with RNase A in a rat model of heterotopic heart transplantation results in less edema and less coronary occlusion, and, therefore, in improved graft survival.MethodsBrown Norway rat cardiac allografts were transplanted into the abdomen of Lewis rats after perfusion with Bretschneider cardioplegic solution. Microvascular technique for aorto‐aortic anastomosis and pulmonary artery to inferior vena cava anastomosis (Langendorff) was used. Recipients were intravenously treated directly before transplantation and every other day with RNase A (50 μg/kg) or vehicle (saline, n = 6 in each group). In addition, in six cases the donor graft was perfused with Bretschneider solution containing RNase A (8 μg in 10 ml), and afterwards transplanted into RNase A treated recipients. The primary end point of the study was graft survival, which was determined by daily examination of the graft function by palpation and in case of inconclusiveness by echocardiography. A tolerance study was performed treating animals daily with 50 μg/kg of RNase A, with 1,000 μg/kg of RNase A, or vehicle, respectively, for 28 days (n = 3 in each group). All explanted hearts were collected for histological evaluation.ResultsMean graft survival was 6.5 ± 0.55 day (range 6–7 day) in vehicle treated animals vs. 10.2 ± 1.0 day (range 9–11 day) in RNase A treated animals (P = 0.002). Directly after explantation donor grafts had a mean weight of 0.94 ± 0.03 g (range 0.89–0.98 g). After been rejected, donor graft weight increased to 2.06 ± 0.52 g (range 1.59–2.77 g) in vehicle treated recipients, in contrast to 1.28 ± 0.27 g (1.05–1.61 g) in RNase A treated recipients (P = 0.017). No supplemental benefit was observed by additive using of RNase A in Bretschneider solution (graft survival 9.50 ± 1.52 day, graft weight 1.38 ± 0.15 g) compared to single treatment of the recipient (graft survival P = 0.394, graft weight P = 0.589). Tolerance studies showed neither macroscopic nor histological differences compared to saline treated animals.Outlook: Further histological analyses will help to clarify if RNase A will keep the promise to reduce edema formation even in the field of transplantation, as suggested by the significant less graft weight in RNase A treated recipients. Moreover, we will investigate, if RNase A prevents leukocyte recruitment in the context of rejection reaction. Therefore, grafts of RNase A treated and untreated rats will be explanted on day 4 after transplantation.ConclusionsRNase A significantly improved graft survival. On the basis of these sweeping results, however, we suppose that RNase A could be an important adjuvant drug not only in allotransplantation but even in xenotransplantation.