Heart Transplantation Model Research Articles

Knockdown of miR-146a in regulatory T cells suppresses heart transplantation rejection in mice by increasing autophagy

Clinical trials of regulatory T cells (Tregs) have shown that adoptive transfer of Tregs has great promise for the treatment of rejection. However, strategies to improve Treg function are needed in order to enhance their efficacy and reduce the number of Tregs required for adoptive transfer. Autophagy is a process for degrading intracellular components, and it mediates cell death, lymphocyte homeostasis, and Treg function. Studies have shown that the survival and function of Tregs with disrupted autophagy are defective. We found that the autophagic status of Tregs was compromised during acute rejection, allowing us to enhance Treg autophagy by regulating microRNA-146a (miR-146a), which is highly expressed in Tregs and is implicated in their function and metabolism. MiR-146a antagomir-mediated miR-146a knockdown promoted Treg autophagy, as evaluated by Western blot analysis. Further, we evaluated whether altering autophagy affects Treg function in both an in vitro cell coculture model and a heart transplantation model in mice. An increase in autophagy enhanced the inhibitory effects of Tregs on CD4+ T cells and dendritic cells (DCs) in vivo and in vitro. In addition, adoptive transfer of highly autophagic Tregs treated with miR-146a antagomir significantly alleviated rejection. Collectively, these data provide a new method that uses miR-146a knockdown to increase Treg efficacy by increasing autophagy.

NF45/NF90‐mediated rDNA transcription provides a novel target for immunosuppressant development

Herein, we demonstrate that NFAT, a key regulator of the immune response, translocates from cytoplasm to nucleolus and interacts with NF45/NF90 complex to collaboratively promote rDNA transcription via triggering the directly binding of NF45/NF90 to the ARRE2‐like sequences in rDNA promoter upon T‐cell activation in vitro. The elevated pre‐rRNA level of T cells is also observed in both mouse heart or skin transplantation models and in kidney transplanted patients. Importantly, T‐cell activation can be significantly suppressed by inhibiting NF45/NF90‐dependent rDNA transcription. Amazingly, CX5461, a rDNA transcription‐specific inhibitor, outperformed FK506, the most commonly used immunosuppressant, both in terms of potency and off‐target activity (i.e., toxicity), as demonstrated by a series of skin and heart allograft models. Collectively, this reveals NF45/NF90‐mediated rDNA transcription as a novel signaling pathway essential for T‐cell activation and as a new target for the development of safe and effective immunosuppressants.

Dose- and Time-Dependent Effects of Human Mesenchymal Stromal Cell Infusion on Cardiac Allograft Rejection in Mice.

Heart transplantation is the final life-saving therapeutic strategy for many end-stage heart diseases. Long-term immunosuppressive regimens are needed to prevent allograft rejection. Mesenchymal stromal cells (MSCs) have been shown as immunomodulatory therapy for organ transplantation. However, the effect of dose and timing of MSC treatment on heart transplantation has not yet been examined. In this study, we infused three doses (1 × 106, 2 × 106, or 5 × 106 cells) of human MSCs (hMSCs) to the recipient BALB/c mice before (7 days or 24 h) or after (24 h) receiving C57BL/6 cardiac transplants. We found that infusion of high dose hMSCs (5 × 106) at 24 h post-transplantation significantly prolonged the survival time of cardiac grafts. To delineate the underlying mechanism, grafts, spleens, and draining lymph nodes were harvested for analysis. Dose-dependent effect of hMSC treatment was shown in: (1) alleviation of International Society of Heart and Lung Transplantation (ISHLT) score in grafts; (2) reduction of the population of CD4+ and CD8+ T cells; (3) increase of regulatory T (Treg) cells; (4) and decrease of serum levels of inflammatory cytokines and donor-specific antibodies. Taken together, we showed timing critical and dose-dependent immunomodulatory effects of hMSC treatment against acute allograft rejection in a mouse model of heart transplantation.

Donor heart preservation with hypoxic-conditioned medium-derived from bone marrow mesenchymal stem cells improves cardiac function in a heart transplantation model

BackgroundIn heart transplantation, donor hearts inevitably suffer from ischemia/reperfusion (I/R) injury, which leads to primary graft dysfunction and affects patients’ survival rate. Bone marrow mesenchymal stem cells (BMSCs) have been reported to attenuate myocardial I/R injury via their paracrine effects, which can be enhanced by hypoxic preconditioning. We hypothesized that the donor heart preservation with hypoxic conditioned medium (CdM) derived from BMSCs would improve post-transplant graft function.MethodsNormoxic or hypoxic CdM were isolated from rat BMSCs cultured under normoxic (20% O2) or hypoxic (1% O2) condition. Donor hearts were explanted; stored in cardioplegic solution supplemented with either a medium (vehicle), normoxic CdM (N-CdM), or hypoxic CdM (H-CdM); and then heterotopically transplanted. Antibody arrays were performed to compare the differences between hypoxic and normoxic CdM.ResultsAfter heart transplantation, the donor heart preservation with normoxic CdM was associated with a shorter time to return of spontaneous contraction and left ventricular systolic diameter, lower histopathological scores, higher ejection fraction, and fractional shortening of the transplanted hearts. The cardioprotective effects may be associated with the inhibition of apoptosis and inflammation, as reflected by less TUNEL-positive cells and lower levels of plasma proinflammatory cytokines (interleukin-1β, interleukin-6, tumor necrosis factor-α) and cardiac troponin I in the N-CdM group compared with the vehicle group. These therapeutic effects can be further enhanced by hypoxic preconditioning. Antibody arrays revealed that nine proteins were significantly increased in hypoxic CdM compared with normoxic CdM. Furthermore, compared with vehicle and N-CdM groups, the protein levels of PI3K and p-Akt/Akt ratio in the transplanted hearts significantly increased in the H-CdM group. However, no significant difference was found in the phosphorylation of Smad2 and Smad3 for the donor hearts among the three groups.ConclusionsOur results indicate that the cardioplegic solution-enriched with hypoxic CdM can be a novel and promising preservation solution for donor hearts.

Immunosuppressive effect of PLGA-FK506-NPs in treatment of acute cardiac rejection via topical subcutaneous injection

FK506, a first-line immunosuppressant, is routinely administered orally and intravenously to inhibit activation and proliferation of T cells after heart transplantation (HT). Current administration route is not conducive enough to exert its efficacy in lymphatic system. Herein, we proposed that subcutaneous (SC) administration of FK506-loaded nanoparticles (PLGA-FK506-NPs) would be valuable for treating acute rejection after HT. The biodistribution and pharmacokinetic study revealed that it could effectively deliver FK506 to the lymph nodes (LNs) due to their suitable particle size, especially in inguinal LNs. Subsequently, the therapeutic efficacy of PLGA-FK506-NPs on the HT model was evaluated using intravenous (IV), intragastric (IG), or SC injection. Histopathological analysis revealed that 80% of allografts exhibited only grade 1R rejection with negligible lymphocyte infiltration in the SC group. The IV group exhibited 40% 1R rejection with mild lymphocyte infiltration and 20% grade 3R that require further intervention, and the IG group exhibited grades 40% 3R rejection with more lymphocyte infiltration. Moreover, the infiltration of T cells and the secretion of IL-2 and IFN-γ were significantly reduced in the SC group compared with the IG or IV group. The mean survival time (MST) further revealed that 50% of grafts treated with PLGA-FK506-NPs via SC injection survived longer than IG and IV groups. Moreover, the MST of single-dose SC injection of PLGA-FK506-NPs demonstrated that it would effectively reduce the required dose for a similar therapeutic effect. Overall, these results indicate that SC administration of PLGA-FK506-NPs is a more effective route for chronic FK506 treatment.

Rat model of heterotopic heart transplantation to investigate relevant donor heart harvesting method

Rat model of heterotopic heart transplantation to investigate relevant donor heart harvesting method

Inhibition of S-adenosyl-L-homocysteine hydrolase alleviates alloimmune response by down-regulating CD4+ T-cell activation in a mouse heart transplantation model.

BackgroundTransmethylation reactions play an important role on lymphocyte activation and function. S-adenosyl-L-homocysteine hydrolase (SAHH) inhibitors prevent the feedback of transmethylation reactions by S-adenosyl-L-homocysteine (SAH) accumulation, a competitive antagonist of S-adenosylmethionine (SAM)-dependent methyltransferases. However, the role of SAH in solid organ transplantation is currently unclear.MethodsA murine model of cardiac transplantation (BALB/C to C57B/6) was established to assess allograft survival, histology, and T cell infiltration. The reversible SAHH inhibitor, DZ2002, and irreversible SAHH inhibitor, adenosine dialdehyde (AdOx), were used to assess their immunosuppressive effects in murine cardiac transplantation, compared with mice with DMSO.ResultsBoth SAHH inhibitors prolonged the survival of cardiac allografts and alleviated alloimmune response. Notably, AdOx and DZ2002 both eliminated frequencies of Th1 and Th17 in CD4+ T cells in cardiac transplantation, and reduced the frequency of active CD4+ T cell (CD44+ CD62L−). The irreversible SAHH inhibitor facilitated the differentiation of regulatory T cells (Tregs) and increased Bim expression. Furthermore, both SAHH inhibitors alleviated infiltration of CD4+ T cells in cardiac allografts.ConclusionsThe SAHH inhibitors (AdOx and DZ2002) alleviates allograft rejection in cardiac transplantation by inhibition of CD4+ T alloimmune response. SAHH inhibitors, especially DZ2002, is a promising complementary therapeutic agent in organ transplantation.

Triazolopyrimidine derivative NK026680 and donor-specific transfusion induces CD4+CD25+Foxp3+ T cells and ameliorates allograft rejection in an antigen-specific manner

We have previously demonstrated the unique properties of a new triazolopyrimidine derivative, NK026680, which exerts immunosuppressive effects in rat heart transplant model and confers tolerogeneic properties on ex vivo-conditioned dendritic cells in mice. We herein demonstrate that NK026680 promotes the expansion of regulatory T cells (Tregs) with potent immunoregulatory effects when used in combination with donor-specific transfusion (DST).BALB/c (H-2d) heart graft were transplanted into C57BL/6 (H-2b) mice following intravenous injection of donor splenocytes (DST) and oral administration of NK026680. The NK026680 plus DST treatment markedly prolonged the survival time of the donor-graft, but not that of the 3rd party-graft (C3H; H-2k). Treg cells in the recipient spleen on day 0 expanded when stimulated with donor-antigens in vivo and in vitro. After heart transplantation, Treg cells accumulated into the graft and increased in the spleen. NK026680 plus DST also decreased activated CD8+ T cells in the spleen and inhibited infiltration of CD8+ T cells into the graft. Depletion of CD25+ cells inhibited the graft prolonging effect of the NK026680 plus DST treatment.NK026680 administration together with DST induces potent immunoregulatory effects in an antigen-specific manner, likely due to the in vivo generation of donor-specific Tregs.

Donor HLA−DR Drives the Development of De Novo Autoimmunity Following Lung and Heart Transplantation

Background.Individuals harbor preexisting HLA−DR/DQ−restricted responses to collagen type V (ColV) mediated by Th17 cells under Treg control, both specific to peptides that bind to inherited HLA class II antigens. Yet after transplant, the donor−DR type somehow influences graft outcome. We hypothesized that, long after a lung or heart allograft, the particular HLA−DR type of the mismatched transplant donor transforms the specificity of the “anti−self” response. This could explain why, over long term, certain donor DRs could be more immunogenic than others.Methods.We analyzed 7 HLA−DR15neg patients who had received a lung allograft from a DR15+ donor. To determine the mechanism of acquired specificity in self−reactivity, we analyzed the kinetics of DR1 (host) and DR15 (donor) peptide restriction in a heart transplant model using DR−transgenic mice.Results.Beyond 1.5 years post-lung transplant, all patients tested had acquired DR15−restricted immune responses to ColV peptides. These responses were either unrestrained Th17 type (n = 4) or Th17 controlled by Treg arising early (<5 y) or late (>7 y) after transplant (n = 4). Treg suppression via conventional (transforming growth factor−β [TGF−β]) and extracellular vesicle−associated (IL−35) cytokines correlated with superior outcomes. Naïve DR1 and DR15 transgenic mice had preexisting DR−restricted responses, exclusively to ColV fragments containing DR1− or DR15−binding peptides. When HLA−DR1 transgenic recipients of a HLA−DR15 heart developed ColV reactivity post-transplant, mice that acutely rejected (20–25 d) responded only to the DR1−restricted ColV peptide epitope. In animals whose grafts survived long term, we could detect acquisition of DR from the transplant donor onto the surface of recipient dendritic cells, and immune responses against a donor DR15–restricted ColV peptide.Conclusions.These results might explain how certain donor HLA−DR types redirect host immune responses to novel peptides of critical self−antigens. Unless regulated, such responses may predispose the allograft to chronic rejection.

Exosomes from dendritic cells with Mettl3 gene knockdown prevent immune rejection in a mouse cardiac allograft model.

We have previously demonstrated that Mettl3-silencing dendritic cells (DCs) exhibited immature properties and prolonged allograft survival in a murine heart transplantation model. Exosomes derived from donor DCs (Dex) are involved in the immune rejection of organ transplantation, and blocking Dex transfer may suppress immune rejection. Herein, this study aimed to investigate whether Mettl3 knockdown inhibits the secretion and activity of donor Dex, thereby inhibiting donor Dex-mediated immune rejection. The imDex, mDex, shCtrl-mDex, and shMettl3-mDex were obtained from the culture supernatant of DCs (immature DCs, mature DCs, shCtrl-infected mature DCs, shMettl3-infected mature DCs) derived from donor BALB/c mouse bone marrow and then co-cultured with splenic T cell lymphocyte suspension from recipient C57BL/6 mice in vitro or injected into recipient C57BL/6 mice before the cardiac transplantation. Donor shMettl3-mDex expressed lower concentration of exosomes and lower expression of Mettl3, Dex markers (ICAM-1, MHC-I, MHC-II), as well as lower ability to activate T cell immune response than shCtrl-mDex. Administration of donor shMettl3-mDex attenuated immune rejection after mouse heart transplantation and prolonged the allograft survival. In summary, Mettl3 knockdown inhibits the immune rejection of Dex in a mouse cardiac allograft model.

Graft IL-33 regulates infiltrating macrophages to protect against chronic rejection.

Alarmins, sequestered self-molecules containing damage-associated molecular patterns, are released during tissue injury to drive innate immune cell proinflammatory responses. Whether endogenous negative regulators controlling early immune responses are also released at the site of injury is poorly understood. Herein, we establish that the stromal cell-derived alarmin interleukin 33 (IL-33) is a local factor that directly restricts the proinflammatory capacity of graft-infiltrating macrophages early after transplantation. By assessing heart transplant recipient samples and using a mouse heart transplant model, we establish that IL-33 is upregulated in allografts to limit chronic rejection. Mouse cardiac transplants lacking IL-33 displayed dramatically accelerated vascular occlusion and subsequent fibrosis, which was not due to altered systemic immune responses. Instead, a lack of graft IL-33 caused local augmentation of proinflammatory iNOS+ macrophages that accelerated graft loss. IL-33 facilitated a metabolic program in macrophages associated with reparative and regulatory functions, and local delivery of IL-33 prevented the chronic rejection of IL-33-deficient cardiac transplants. Therefore, IL-33 represents what we believe is a novel regulatory alarmin in transplantation that limits chronic rejection by restraining the local activation of proinflammatory macrophages. The local delivery of IL-33 in extracellular matrix-based materials may be a promising biologic for chronic rejection prophylaxis.

Fingolimod (FTY720) Preserves High Energy Phosphates and Improves Cardiac Function in Heterotopic Heart Transplantation Model.

During heart transplantation, donor heart leads to reduced oxygen supply resulting in low level of high energy phosphate (HEP) reserves in cardiomyocyte. Lower HEP is one of the underlying reasons of cell death due to ischemia. In this study we investigated the role of Fingolimod (FTY720) in heart transplantation ischemia. Eight groups of Sprague-Dawley rats (n = 5 for each subgroup) were made, A1 and C1 were given FTY720 1 mg/kg while B1 and D1 were given normal saline. The hearts were implanted into another set of similar rats after preservation period of 1 h at 4–8 °C. Significantly higher Left ventricular systolic pressure (LVSP), dP/dT maximum (p < 0.05), dP/dT minimum (p < 0.05) were recorded in the FTY720 treated group after 24 h of reperfusion while after 1 h of reperfusion, there were no significant differences in LVSP, maximum and negative dP/dT, and Left ventricular end diastolic pressure (LVEDP) between the control and the FTY720-treated transplant groups. Coronary blood flow (CBF) was enhanced (p < 0.05) in the FTY720 treated group after 1 and 24 h. ATP p < 0.001, p < 0.05 at 1 and 24 h, ADP p < 0.001, p > 0.05 at 1 and 24 h, and phosphocreatine p < 0.05, p > 0.05 at 1 and 24 h were better preserved by FTY720 treatment as compared to control group. The study concluded that pretreatment of grafted hearts with FTY720 improved hemodynamics, CBF, high energy phosphate reserves, reduces the peroxynitrite level and poly (ADP ribose) polymerase (PARP) inhibition that prevents ischemia-reperfusion injury.

An Addition of U0126 Protecting Heart Grafts From Prolonged Cold Ischemia-Reperfusion Injury in Heart Transplantation: A New Preservation Strategy.

Ischemia-reperfusion injury (IRI) is the major cause of primary graft dysfunction in organ transplantation. The mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) signaling pathway plays a crucial role in cell physiological and pathological processes including IRI. This study aims to investigate whether inhibition of ERK signaling with U0126 can prevent prolonged cold IRI in heart transplantation. Rat cardiac cell line H9c2 cells were treated with U0126 before exposure to hypothermic hypoxia/reoxygenation (H/R) conditions. The effect of U0126 on H9c2 cells in response to H/R stress was determined by measuring cell death, reactive oxygen species production, mitochondrial membrane potential, and ERK signaling activation. Mouse syngeneic heterotopic heart transplantation was conducted, where a donor heart was preserved in the University of Wisconsin (UW) solution supplemented with U0126 for 24 hours at 4°C before transplantation. Heart graft function, histopathologic changes, apoptosis, and fibrosis were measured to assess IRI. Phosphorylated ERK was increased in both in vitro H/R-injured H9c2 cells and in vivo heart grafts with IRI. Pretreatment with U0126 inhibited ERK phosphorylation and prevented H9c2 cells from cell death, reactive oxygen species generation, and mitochondrial membrane potential loss in response to H/R. Preservation of donor hearts with U0126-supplemented solution improved graft function and reduced IRI by reductions in cell apoptosis/death, neutrophil infiltration, and fibrosis of the graft. Addition of U0126 to UW solution reduces ERK signal activation and attenuates prolonged cold IRI in a heart transplantation model. ERK inhibition with U0126 may be a useful strategy to minimize IRI in organ transplantation.

Role of syndecan-1 in the interaction between dendritic cells and T cells.

Syndecan-1 (Sdc-1) is a heparan sulfate proteoglycan that can bind cytokines and chemokines via its heparan sulfate side chains, and has immunomodulatory properties in experimental models. Sdc-1 expression has been reported on dendritic cells (DC) and T cells. The potential role of Sdc-1 in DC-T cell interaction has not been investigated yet. We postulate that Sdc-1 is involved in DC-T cell interaction and may influence graft survival in an allogeneic transplant model. Sdc-1 expression on bone marrow-derived DC and T cells was analyzed by flow cytometry. Unstimulated and LPS stimulated Sdc-1 deficient DC were evaluated in vitro for phenotype and stimulatory capacity in mixed lymphocyte reaction. Sdc-1 deficient T cells were evaluated for proliferative capacity and differentiation in a mixed lymphocyte reaction and a proliferation assay. Allograft survival was evaluated in a fully MHC mismatched heterotopic heart transplant model, with either Sdc-1 deficient donors or recipients. Sdc-1 was expressed on the cell surface of unstimulated and LPS matured DC. Sdc-1 deficiency had no effect on expression of co-stimulatory molecules, cytokine production or T cell stimulatory capacity as compared to WT DC. Sdc-1 expression was not detectable on WT T cells, although intracellular Sdc-1 expression could be demonstrated after ConA activation. Sdc-1 deficient T cells showed reduced proliferation upon DC or ConA stimulation and reduced IL-17 production upon ConA stimulation, compared to WT T cells. Sdc-1 deficiency of either allograft or recipient did not prolong allograft survival. In conclusion, Sdc-1 is expressed on the cell surface of DC, where its absence does not affect DC phenotype or T cell stimulatory capacity. Sdc-1 is intracellularly expressed in ConA activated T cells. Sdc-1 deficiency in T cells results in a reduced proliferative response in vitro, as induced by DC and ConA. Sdc-1 deficiency in donor or recipient does not affect allograft survival.

Use of peroxiredoxin for preconditioning of heterotopic heart transplantation in a rat

Peroxiredoxin 6 (Prdx6) is an antioxidant enzyme in the human body that performs a number of important functions in the cell. Prdx6 restores a wide range of peroxide substrates, thus playing a leading role in maintaining redox homeostasis in mammalian cells. In addition to peroxidase activity, Prdx6 has an activity of phospholipase A2, thus taking part in membrane phospholipid metabolism. Due to its peroxidase and phospholipase activity, Prdx6 participates in intracellular and intercellular signal transmission, thereby facilitating the initiation of regenerative processes in the cell, suppression of apoptosis and activation of cell proliferation. Given the functions performed, Prdx6 can effectively deal with oxidative stress caused by various factors, including ischemia-reperfusion injury. On an animal model of rat heterotopic heart transplantation, we showed the cardioprotective potential of exogenous recombinant Prdx6, introduced before transplantation and subsequent reperfusion injury of the heart. It has been demonstrated that exogenous Prdx6 effectively alleviates the severity of ischemia-reperfusion injury of the heart by 2–3 times, providing normalization of its structural and functional state during heterotopic transplantation. The use of recombinant Prdx6 can be an effective approach in preventing/alleviating ischemia-reperfusion injury of the heart, as well as in maintaining an isolated heart during transplantation.

Novel delivery of cellular therapy to reduce ischemia reperfusion injury in kidney transplantation.

Ex vivo normothermic machine perfusion (NMP) of donor kidneys prior to transplantation provides a platform for direct delivery of cellular therapeutics to optimize organ quality prior to transplantation. Multipotent Adult Progenitor Cells (MAPC® ) possess potent immunomodulatory properties that could minimize ischemia reperfusion injury. We investigated the potential capability of MAPC cells in kidney NMP. Pairs (5) of human kidneys, from the same donor, were simultaneously perfused for 7 hours. Kidneys were randomly allocated to receive MAPC treatment or control. Serial samples of perfusate, urine, and tissue biopsies were taken for comparison. MAPC-treated kidneys demonstrated improved urine output (P=.009), decreased expression of injury biomarker NGAL (P=.012), improved microvascular perfusion on contrast-enhanced ultrasound (cortex P=.019, medulla P=.001), downregulation of interleukin (IL)-1β (P=.050), and upregulation of IL-10 (P<.047) and Indolamine-2, 3-dioxygenase (P=.050). A chemotaxis model demonstrated decreased neutrophil recruitment when stimulated with perfusate from MAPC-treated kidneys (P<.001). Immunofluorescence revealed prelabeled MAPC cells in the perivascular space of kidneys during NMP. We report the first successful delivery of cellular therapy to a human kidney during NMP. Kidneys treated with MAPC cells demonstrate improvement in clinically relevant parameters and injury biomarkers. This novel method of cell therapy delivery provides an exciting opportunity to recondition organs prior to transplantation.

Combined abdominal heterotopic heart and aorta transplant model in mice.

Allograft vasculopathy (AV) remains a major obstacle to long-term allograft survival. While the mouse aortic transplantation model has been proven as a useful tool for study of the pathogenesis of AV, simultaneous transplantation of the aorta alongside the transplantation of another organ may reveal more clinically relevant mechanisms that contribute to the pathogenesis of chronic allograft rejection. Therefore, we developed a combined abdominal heart and aorta transplantation model in mice which benefits from reducing animal and drug utilization, while providing an improved model to study the progressive nature of AV. The middle of the infrarenal aorta of the recipient mouse was ligatured between the renal artery and its bifurcation. Proximal and distal aortotomies were performed at this site above and below the ligature, respectively, for the subsequent anastomoses of the donor aorta and heart grafts to the recipient infrarenal aorta in an end-to-side fashion. The distal anastomotic site of the recipient infrarenal aorta was connected with the outlet of the donor aorta. Uniquely, the proximal anastomotic site on the recipient infrarenal aorta was shared to connect with both the inlet of the donor aorta and the inflow tract to the donor heart. The outflow tract from the donor heart was connected to the recipient inferior vena cava (IVC). The median times for harvesting the heart graft, aorta graft, recipient preparation and anastomosis were 11.5, 8.0, 9.0 and 40.5 min, respectively, resulting in a total median ischemic time of 70 min. The surgery survival rate was more than 96% (29/30). Both the syngeneic C57Bl/6 aorta and heart grafts survived more than 90 days in 29 C57Bl/6 recipients. Further, Balb/c to C57Bl/6 allografts treated with anti-CD40L and CTLA4.Ig survived more than 90 days with a 100% (3/3) survival rate. (3/3). This model is presented as a new tool for researchers to investigate transplant immunology and assess immunosuppressive strategies. It is possible to share a common anastomotic stoma on the recipient abdominal aorta to reconstruct both the aorta graft entrance and heart graft inflow tract. This allows for the study of allogeneic effects on both the aorta and heart from the same animal in a single survival surgery.

PARP-1 inhibitor-AG14361 suppresses acute allograft rejection via stabilizing CD4+FoxP3+ regulatory T cells

Acute allograft rejection is the most common complication in organ transplantation leading to organ loss. Treg cells play an important role in preventing acute rejection, but they are unstable and easily lose function. Poly(ADP-ribose) polymerase 1(PARP-1) is involved in the differentiation stabilization of Treg cells, it has been suggested that PARP-1 inhibition could prevent acute rejection and prolong allograft survival. This study investigated AG14361 effects on acute allograft rejection. We used a fully MHC-mismatched murine heart transplantation model to compare the effect of PARP-1 inhibitor-AG14361 on alloimmunity to the control. Mice treated with PARP-1 inhibitors showed a longer median survival time of allografts (MS14 compared with the control group, MST was 8 days, and AG14361 was 6 days, P = 0.019). The combination of sirolimus and AG14361 significantly delayed allograft MST (AG14361 + sirolimus for 30 days, sirolimus for 16 days, P = 0.002). AG14361 markedly augmented the number of the CD25+FoxP3+ Treg cells in the graft and periphery. In addition, it could enhance the suppressive function of Treg cells by upregulating the level of CTLA-4, PD-1 and ICOS. In vivo, the Treg/Th17 ratio increased significantly in the AG14351 group compared to the control. In the combination with sirolimus treatment, AG14361 promoted the long-term allograft survival. Our results highlight novel effects of a PARP-1 inhibitor. PARP-1 inhibitor AG14361 may be a promising agent to attenuate acute allograft rejection as it can maintain the number and function of Treg cells in allografts.

Reduction of Cold Ischemic Injury with the Addition of Compound Glycyrrhizin in HTK Solution in a Mouse Heart Transplantation Model

Cold ischemic injury in heart storage is an important issue pertaining to heart transplantation. This study aims to evaluate the addition of compound glycyrrhizin (CG) in histidine-tryptophan-ketoglutarate (HTK) solution on chronic isograft injury in comparison to traditional HTK solution.Hearts of mouse were stored for 8 h in 4°C cold preservation solution and then transplanted heterotopically into mouse. Five groups were evaluated: HTK, low dose of CG solution (LCG), medium dose of CG solution (MCG), high dose of CG solution (HCG), and hearts without cold ischemia (sham). Survival was assessed. Time to restoration of heartbeat and strength of the heartbeat was measured. Lactate dehydrogenase (LDH) and creatine kinase (CK) levels in the preservation solution were determined. The myocardial damage and interstitial fibrosis of transplanted hearts were evaluated. TGF-β1 expression in the transplanted hearts was assessed.Addition of CG to HTK solution significantly attenuated cold ischemic injury during cold storage, as evidenced by the lower time to restoration of heartbeat, higher strength of the heartbeat, lower LDH, and CK leakage. After transplantation, hearts stored in HTK solution containing CG had decreased the myocardial damage and interstitial fibrosis, compared with those stored without CG. The percentage of TGF-β1-positive cells and TGF-β1 level in the transplanted hearts were also decreased when stored in CG-containing HTK solution.The addition of CG to HTK solution attenuates cold ischemic injury during cold storage.

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats