Rat Heart Transplantation Model Research Articles

Normothermic Ex Vivo Heart Perfusion With Exosomes From Human Umbilical Cord Mesenchymal Stem Cells Improves Graft Function in Donation After Circulatory Death Hearts.

This study aimed to investigate the cardioprotective effect of exosomes derived from human umbilical cord mesenchymal stem cells on donation after circulatory death (DCD) hearts preserved with normothermic ex vivo heart perfusion (EVHP) in a rat heart transplantation model. Thirty-two male Lewis rats were divided into 2 groups: the control group and the exosome group. The donor-heart rats were subjected to the DCD procedure by suffering a 15-min warm ischemia injury, subsequently preserved with EVHP for 90 min, and then transplanted into recipients via abdominal heterotopic heart transplantation. Vehicle or exosome was added into the perfusate of normothermic EVHP in the control or exosome group. We evaluated left ventricular graft function, myocardial inflammation, and myocardial apoptosis of the donor heart 1.5 h after heart transplantation. Furthermore, we investigate the alternation of myocardial gene expression in the donor hearts between both groups by transcriptome sequencing. The treatment with exosome significantly enhanced cardiac function through increasing left ventricular developed pressure, dp/dtmax, and dp/dtmin of DCD hearts at 90 min after heart transplantation compared with the control group. The myocardial cells in the exosome group exhibited an orderly arrangement without obvious edema. Furthermore, exosome added into perfusate in the exosome group significantly attenuated the level of inflammatory response and apoptosis. Transcriptome sequencing and RT-qPCR showed the phosphoinositide 3-kinase/protein kinase B pathway was activated after exosome treatment. Normothermic EVHP combined with exosome can be a promising and novel DCD heart preservation strategy, alleviating myocardial ischemia-reperfusion injury in the DCD heart.

Cirbp suppression compromises DHODH-mediated ferroptosis defense and attenuates hypothermic cardioprotection in an aged donor transplantation model.

Hypothermia is commonly used to protect donor hearts during transplantation. However, patients transplanted with aged donor hearts still have severe myocardial injury and decreased survival rates, but the underlying mechanism remains unknown. Because aged hearts are not considered suitable for donation, the number of patients awaiting heart transplants is increasing. In this study, we examined whether hypothermic cardioprotection was attenuated in aged donor hearts during transplantation and evaluated potential therapeutic targets. Using a rat heart transplantation model, we found that hypothermic cardioprotection was impaired in aged donor hearts but preserved in young donor hearts. RNA-Seq showed that cold-inducible RNA-binding protein (Cirbp) expression was decreased in aged donor hearts, and these hearts showed severe ferroptosis after transplantation. The young donor hearts from Cirbp-KO rats exhibited attenuated hypothermic cardioprotection, but Cirbp overexpression in aged donor hearts ameliorated hypothermic cardioprotection. Cardiac proteomes revealed that dihydroorotate dehydrogenase (DHODH) expression was significantly decreased in Cirbp-KO donor hearts during transplantation. Consequently, DHODH-mediated ubiquinone reduction was compromised, thereby exacerbating cardiac lipid peroxidation and triggering ferroptosis after transplantation. A cardioplegic solution supplemented with CIRBP agonists improved hypothermic cardioprotection in aged donor hearts, indicating that this method has the potential to broaden the indications for using aged donor hearts in transplantation.

Normothermic ex vivo heart perfusion with NLRP3 inflammasome inhibitor Mcc950 treatment improves cardiac function of circulatory death hearts after transplantation.

The utilization of donation after circulatory death (DCD) hearts can enlarge the donor pool. However, DCD hearts suffer from serious ischemia/reperfusion injury (IRI). Recent studies found that the activation of NLRP3 inflammasome could play a significant role in organ IRI. Mcc950, which is a novel inhibitor of the NLRP3 inflammasome, can be applied to treat various kinds of cardiovascular diseases. Therefore, we hypothesized that the treatment of mcc950 could protect DCD hearts preserved with normothermic ex vivo heart perfusion (EVHP) against myocardial IRI via inhibiting NLRP3 inflammasome in a rat heart transplantation model of DCD. Donor-heart rats were randomly divided into four groups: Control group; Vehicle group; MP-mcc950 group; and MP + PO-mcc950 group. Mcc950 was added into the perfusate of normothermic EVHP in the MP-mcc950 and MP + PO-mcc950 groups, and was injected into the left external jugular vein after heart transplantation in the MP + PO-mcc950 group. Cardiac functional assessment was performed. The level of oxidative stress, inflammatory response, apoptosis, and NLRP3 inflammasome-associated protein of donor hearts were evaluated. The treatment with mcc950 significantly increased the developed pressure (DP), dP/dtmax, and dP/dtmin of the left ventricular of DCD hearts at 90 min after heart transplantation in both MP-mcc950 and MP + PO-mcc950 groups. Furthermore, mcc950 added into perfusate and injected after transplantation in both MP-mcc950 and MP + PO-mcc950 groups significantly attenuated the level of oxidative stress, inflammatory response, apoptosis, and NLRP3 inflammasome compared with the vehicle group. Normothermic EVHP combined with mcc950 treatment can be a promising and novel DCD heart preservation strategy, which can alleviate myocardial IRI via inhibiting NLRP3 inflammasome.

Poly(Lactic-Co-Glycolic Acid) (PLGA) Nanoparticles Loaded with FK506 Inhibits Acute Heart Transplantation Rejection via Regulation of Monocyte Dendritic Cells Receptor

FK506-loaded poly(lactic-co-glycolic acid)-nanoparticles (PLGA-NPs) (PLGA-FK506-NPs) have been indicated to exert better curative effect on rejection. Therefore, were herein studied mechanism underlying PLGA-FK506-NPs suppression of heart transplantation rejection. After construction of heterotopic heart transplant model in rats and preparation of composite NPs, the animals were administered with normal saline, FK506 and PLGA-FK506-NPs. With measurement of survival time for transplanted hearts and detection of NPs toxicity, rat tissue sample was collected for Hematoxylin and eosin (H&E) staining observation. T cell infiltration and contents of IL-12 and IL-23 in dendritic cell (DCs) were also detected. In the presence of FK506 or PLGA-FK506-NPs, cell viability did not change significantly (p > 0.05), indicating low toxicity of PLGA-FK506-NPs. Importantly, treatment with FK506 or PLGA-FK506-NPs alleviated CD3+ T cell infiltration and rejection, compared with control group. Of note was that, 40% of the rat hearts in the PLGA-FK506-NPs group had an Acute Rejection (AR) level of 1R, but only 20% in the FK506 group. PLGA-FK506-NPs group had a longer heart transplant survival time than both control and FK506 groups (p <0.001). Over time, FK506 concentration decreased in blood from the rats in the FK506 and PLGA-FK506-NPs groups, indicating that, FK506 was gradually metabolized. Additionally, PLGA-FK506-NPs and FK506 resulted in increased secretion of IL-12 and IL-23, with a higher level in the PLGA-FK506-NPs group. PLGA-FK506-NPs can effectively increase FK506 content in the body, prolonging survival time of heart transplant recipients, relieving AR, and improving secretion of related factors in the mono-DCs recipients.

A Heterotopic Rat Heart Transplantation Model using Circulatory Death Donor Hearts.

The objective of this protocol is to set up a rat heterotopic heart transplantation model with donation after circulatory death (DCD) donor hearts. There are two setups for this protocol: heart donor setup and recipient setup. In the heart donor setup, Sprague Dawley rats are anesthetized, endotracheally intubated, and ventilated. The right carotid artery is cannulated to deliver heparin and the paralytic agent vecuronium-bromide. The DCD process is initiated by terminating the ventilation. After 20 min, the heart is exposed and the aorta distal to the brachiocephalic branch is clamped. At 25 min from terminating the ventilator, ice-cold University of Wisconsin (UW) solution is perfused through the carotid catheter to flush the heart. The heart is procured by dividing the aorta, pulmonary artery, venae cavae, and pulmonary veins and stored in UW solution for implantation. In the recipient setup, the Lewis rat is anesthetized with isoflurane. Slow-release buprenorphine is administered subcutaneously to facilitate a smooth postoperative recovery. Through a midline abdominal incision, the infra-renal aorta and the inferior vena cava are isolated and clamped with an atraumatic vascular clamp. The donor heart aorta and pulmonary artery are sutured to the recipient abdominal aorta and vena cava, respectively, with a running 8-0 Prolene. The vascular clamp is removed to reperfuse the heart. The abdominal wall is closed and the rat is recovered. After a set interval (24 h to 2 weeks), the recipient rat is anesthetized, the transplanted heart is exposed, and a balloon-tip-catheter is inserted into the left ventricle via the apex to record developed pressure and dP/dt using a data acquisition system. The heart tissue is collected for histology, immunology, or molecular analysis. A successful DCD donor rat heart transplantation model will allow further studies on the cardioprotective approaches to improve heart transplantation outcomes from DCD donors.

Spatial Transcriptomic Analysis of Acute Heart Rejection Model

<h3>Purpose</h3> Heart transplantation (HTx) is sometimes the only treatment for end-stage cardiac disease, but survival after HTx is limited. Understanding the molecular mechanisms behind allograft injury, dysfunction, and failure, are critical in improving the patients' outcome. Rapidly evolving single cell and spatial transcriptomic technologies have opened a new field of precision to diagnostics and therapeutics of cardiovascular diseases. Here we studied the cellular mechanisms of acute rejection in a rat heart transplant model with spatial transcriptomics methods. <h3>Methods</h3> We investigated the effects of acute rejection in heterotopic rat heart transplantation model with syngeneic (DA-DA) and major MHC-mismatched allogeneic (DA-WF) recipients. The recipient rats were sacrificed 5 days after transplantation, and graft cross-sections were analyzed with the Visium Spatial Gene Expression platform (10xGenomics). Raw sequencing reads were processed using SpaceRanger. All main data analysis steps were performed using Seurat. Clusters were identified based on the expression of several known cell markers. <h3>Results</h3> From all samples together, a total of 17 clusters of spots were identified. Our analysis suggests that two of the clusters presented mainly cardiomyocytes, one cluster of smooth muscle cells, two clusters of fibroblasts, 11 immune cells and one cluster of erythrocytes. Endothelial cells did not form a unique cluster. Syngeneic HTx alone induced marked transcriptomic changes in 5 clusters based on differential gene expression analysis, mainly increasing the proportion of clusters with certain types of macrophages. Few clusters remained unchanged between the allogenic and the syngeneic groups. Allogenic group had four unique clusters representing mainly different immune cell populations: granulocytes, lymphocytes and a type of macrophages that were not present in other samples. One cluster consisted of erythrocytes from hemorrhagic area. <h3>Conclusion</h3> Acute rejection changes the cellular composition of the heart muscle dramatically and induces transcriptomic changes in the structural cells of the heart. Our spatial transcriptomic analysis provides novel transcriptomic data on cellular changes occurring following acute rejection. This method allows us to study the cells and mechanisms involved in rejection and look for potential targets for drug development and therapeutics.

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.

A modified heterotopic heart transplantation in the rat - as an important model in experimental regeneration and replacement of the failing organ.

The qualification of new knowledge is one of the oldest problems in experimental medicine that provides a link between fundamental discovery, hypothesis, ‘proof of concept’ preclinical studies and development of clinical trials. The biggest challenge in animal models is the proper evaluation of all the aspects that are crucial in specific studied pathologies as well as the prediction of their progression. The aim of this review was to describe and discuss the rat animal model of heart transplant. The rat model of heart transplantation is an excellent yet underestimated method of research of prevention, monitoring and treatment of acute and chronic, immune and nonimmune response to organ transplantation. Despite being a technically and logistically demanding model, it provides a tool for reproducible experiments with longterm animal survival and excellent graft survival.

Indoleamine 2,3-dioxgenase-transfected mesenchymal stem cells suppress heart allograft rejection by increasing the production and activity of dendritic cells and regulatory T cells

Expression of indoleamine 2,3-dioxygenase (IDO) in mesenchymal stem cells (MSC) is thought to contribute to MSC-mediated immunosuppression. A lentiviral-based transgenic system was used to generate bone marrow stem cells (BMSC)...

Effect of Prostaglandin E1 Injected Into Donors in a Heterotopic Heart Transplant Model of Sprague Dawley Rats

IntroductionProstaglandin E1 (PGE1) administered to patients in the immediate post-transplant period has been known to reduce ischemic reperfusion injuries (IRIs), but the effect on IRI of PGE1 administered to the donor is unknown. The purpose of this study was to determine the effect on IRI of PGE1 injected into donor rats during heterotopic heart transplantation. MethodsGenetically identical male Sprague Dawley rats with a body weight of 300–320 g at 8–9 weeks of age were used for the study. Experimental methods were the same in the control (G0, n = 6) and experimental groups (G1, n = 6), but only the donor rats in the experimental group received an intramuscular injection of PGE1 (5 μg/kg) prior to the donor surgery. On day 1 the animals were sacrificed with the removal of the transplanted heart. Histologic analysis was performed in the hematoxylin-eosin-stained slides to assess interstitial edema and neutrophil infiltration by a pathologist. ResultsMedian times of the donor organ procurement, cold ischemia, and warm ischemia were 37, 69, and 35 minutes, respectively, in the G0 group and 38, 76.5, and 33 minutes respectively in G1 group; there were no statistical differences. Heartbeats were observed in the transplanted graft in 2 of the G0 group and 2 of G1 group immediately after heart transplantation, but in all transplanted grafts on day 1 after surgery. Histologic scores for neutrophil infiltration showed significantly lower in the G1 group than in the G0 group. ConclusionPGE1 administration to donors in a rat heart transplantation model may significantly reduce IRI.

Sphingosine-1-Phosphate Receptor Agonist Fingolimod Reduces Myocardial Ischemia-Reperfusion Injury and Apoptosis Increasing Long-Term Left Ventricular Function after Heart Transplantation

Purpose Fingolimod, a sphingosine-1-phosphate receptor agonist, is approved for the treatment of multiple sclerosis and exerts antiapoptotic properties. We hypothesized that sphingosine-1-phosphate receptor activation with fingolimod in the donor hearts could protect against ischemia/reperfusion injury and preserve myocardial function in a heterotopic rat heart transplantation model. Methods Before explantation, donor Wistar rats received finfolimod (1 mg/kg iv, n= 40) or saline (control, n=40). The hearts were excised, stored in cold preservation solution for 1 hour and heterotopically transplanted. Left ventricular function of the graft and myocardial markers of apoptosis, nitro-oxidative stress, inflammation and fibrosis have been assessed after 24 hour (early phase) and 1 month (late phase) from transplantation. Results Fingolimod treatment activated the cardioprotective reperfusion injury salvage kinase Akt and extracellular receptor kinase 1/2 in the early phase after transplantation, leading to decreased cardiomyocyte apoptosis (TUNEL) and reduced myocardial nitro-oxidative stress (malondialdehyde, nitrotyrosine) and inflammation (CCL5/RANTES). In the late phase fingolimod decreased interstitial fibrosis and improved LV systolic and diastolic functions (pressure-volume analysis). Conclusion Sphingosine-1-phosphate receptor activation with fingolimod before heart transplantation activates anti-apoptotic and anti-inflammatory pathway reducing graft ischemia-reperfusion injury. This produces a reduced myocardial fibrosis and improved left ventricular function in a chronic phase. Fingolimod might represent a useful tool for cardioprotection in the clinical setting of heart transplantation surgery soon. Fingolimod, a sphingosine-1-phosphate receptor agonist, is approved for the treatment of multiple sclerosis and exerts antiapoptotic properties. We hypothesized that sphingosine-1-phosphate receptor activation with fingolimod in the donor hearts could protect against ischemia/reperfusion injury and preserve myocardial function in a heterotopic rat heart transplantation model. Before explantation, donor Wistar rats received finfolimod (1 mg/kg iv, n= 40) or saline (control, n=40). The hearts were excised, stored in cold preservation solution for 1 hour and heterotopically transplanted. Left ventricular function of the graft and myocardial markers of apoptosis, nitro-oxidative stress, inflammation and fibrosis have been assessed after 24 hour (early phase) and 1 month (late phase) from transplantation. Fingolimod treatment activated the cardioprotective reperfusion injury salvage kinase Akt and extracellular receptor kinase 1/2 in the early phase after transplantation, leading to decreased cardiomyocyte apoptosis (TUNEL) and reduced myocardial nitro-oxidative stress (malondialdehyde, nitrotyrosine) and inflammation (CCL5/RANTES). In the late phase fingolimod decreased interstitial fibrosis and improved LV systolic and diastolic functions (pressure-volume analysis). Sphingosine-1-phosphate receptor activation with fingolimod before heart transplantation activates anti-apoptotic and anti-inflammatory pathway reducing graft ischemia-reperfusion injury. This produces a reduced myocardial fibrosis and improved left ventricular function in a chronic phase. Fingolimod might represent a useful tool for cardioprotection in the clinical setting of heart transplantation surgery soon.

A novel 2,4-diaminopyrimidine derivative as selective inhibitor of protein kinase C theta prevents allograft rejection in a rat heart transplant model

Protein kinase C theta (PKCθ) plays a critical role in T cell signaling and is an attractive target for the treatment of T cell-mediated diseases such as transplant rejection and autoimmune disease. To identify PKCθ inhibitors, we focused on the 2,6-diamino-3-carbamoyl-5-cyanopyrazine derivative 2, which exhibited moderate PKCθ inhibitory activity. Optimization of 2 identified the 2,4-diamino-5-cyanopyrimidine derivative 16c, which exhibited potent PKCθ inhibitory activity and showed good selectivity against other PKC isozymes. Compound 16c prolonged graft survival in an in vivo rat heterotopic cardiac transplant model.

Depletion of macrophages from Cytomegalovirus (CMV) latently infected donor allografts decreases CMV mediated immunopathology and prevents CMV-accelerated chronic rejection

Abstract Cytomegalovirus (CMV), a ubiquitous β-herpesvirus, is linked to acceleration of solid organ transplant vascular sclerosis (TVS) and chronic rejection (CR). Latent CMV infection increases cardiac-resident macrophages and T cells that may increase inflammation and promote allograft rejection. To investigate the role of cardiac macrophages in CMV mediated TVS/CR, macrophages were depleted from CMV+ donor allografts prior to transplantation using clodronate liposomes (clod) in our rat heart transplant model. Latently rat CMV infected donor F344 rats were treated with clod or PBS or control liposomes 3 days prior to heterotopic cardiac transplant into CMV-naïve Lewis rats and macrophage depletion was validated. Donor hearts were transplanted and monitored daily for rejection. Clod treatment significantly increased graft survival from 61 days post-transplant to 84 days. The severity of graft vessel disease was decreased compared to controls. Clod treatment significant decreased macrophage and T cell infiltration into allograft tissue versus controls. Clod treatment also altered allograft tissue protein and gene expression cytokine/chemokine profiles toward a phenotype that promotes graft survival by reducing the allograft inflammation. Clod treatment did not prevent CMV reactivation since all recipients seroconverted and CMV DNA was detectable in recipient salivary glands. Depletion of donor macrophages prior to transplant represents a novel method to modulate allograft rejection immunity and prevent the negative impact of CMV on allograft recipient survival.

Role of mTOR C1/C2 pathways involve in abrogation of chronic rejection in rat heart transplantation model

Objective To study the expression of the components of the mammalian rapamycin target protein (mTOR) pathway in T lymphocytes in rats with chronic rejection (CR), and to explore the role of mTOR pathway in the inhibition of CR. Methods ACI rat recipients received intraperitoneal ectopic cardiac transplantation with Wistar-Furth rat hearts. In the experimental group, a mutated class I major histocompatibility complex (MHC I) that can eliminate CR was delivered into recipients prior-operation, and a sub-therapeutic cyclosporine A (CsA) (10 mg/kg, 3 d) was also administered. In the experimental control group, the heart allograft recipients were treated with sub-therapeutic CsA (10 mg/kg, 3 d). The blank controls were the untreated recipients. Each group was divided into three subgroups (5 rats in each subgroup) according to the sacrifice time on the postoperative 1st, 3rd and 7th days. The spleen samples were taken for T cell extraction and Western blot analysis. Results Western blot results showed that rat heart allografts with abolished CR exhibited downregulation of the RAPA-sensitive mTORC1 elements including mTOR and Raptor, and down-regulation of the RAPA-insensitive mTORC2 elements including Rictor and Sin1. Conclusions Abrogation of CR in rat model system involves modulation of mTOR C1 and mTOR C2 pathways. The mTOR C1 pathway regulates cellular proliferation and the mTORC2 pathway regulates T-cell motility. Selective targeting of T-cell actin cytoskeletal pathways shows potential for pathway-targeted immunosuppression therapies. Key words: Chronic rejection; Transplantation; Immune tolerance; Actin cytoskeleton

Olaparib protects cardiomyocytes against oxidative stress and improves graft contractility during the early phase after heart transplantation in rats.

Olaparib, rucaparib and niraparib, potent inhibitors of poly(ADP-ribose) polymerase (PARP) are approved as anti-cancer drugs in humans. Considering the previously demonstrated role of PARP in various forms of acute and chronic myocardial injury, we tested the effects of olaparib in in-vitro models of oxidative stress in cardiomyocytes, and in an in vivo model of cardiac transplantation. H9c2-embryonic rat heart-derived myoblasts pretreated with vehicle or olaparib (10μM) were challenged with either hydrogen peroxide (H2 O2 ) or with glucose oxidase (GOx, which generates H2 O2 in the tissue culture medium). Cell viability assays (MTT, lactate dehydrogenase) and Western blotting for PARP and its product, PAR was performed. Heterotopic heart transplantation was performed in Lewis rats; recipients were treated either with vehicle or olaparib (10mg kg-1 ). Left ventricular function of transplanted hearts was monitored via a Millar catheter. Multiple gene expression in the graft was measured by qPCR. Olaparib blocked autoPARylation of PARP1 and attenuated the rapid onset of death in H9c2 cells, induced by H2 O2 , but did not affect cell death following chronic, prolonged oxidative stress induced by GOx. In rats, after transplantation, left ventricular systolic and diastolic function were improved by olaparib. In the transplanted hearts, olaparib also reduced gene expression for c-jun, caspase-12, catalase, and NADPH oxidase-2. Olaparib protected cardiomyocytes against oxidative stress and improved graft contractility in a rat model of heart transplantation. These findings raise the possibility of repurposing this clinically approved oncology drug, to be used in heart transplantation. This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.

Sphingosine 1-Phosphate Receptor Modulator Fingolimod (FTY720) Attenuates Myocardial Fibrosis in Post-heterotopic Heart Transplantation.

Background and Objective: Sphingosine 1-phosphate (S1P), and S1P receptor modulator fingolimod have been suggested to play important cardioprotective role in animal models of myocardial ischemia/reperfusion injuries. To understand the cardioprotective function of S1P and its mechanism in vivo, we analyzed apoptotic, inflammatory biomarkers, and myocardial fibrosis in an in vivo heterotopic rat heart transplantation model.Methods: Heterotopic heart transplantation is performed in 60 Sprague–Dawley (SD) rats (350–400 g). The heart transplant recipients (n = 60) are categorized into Group A (control) and Group B (fingolimod treated 1 mg/kg intravenous). At baseline with 24 h after heart transplantation, blood and myocardial tissue are collected for analysis of myocardial biomarkers, apoptosis, inflammatory markers, oxidative stress, and phosphorylation of Akt/Erk/STAT-3 signaling pathways. Myocardial fibrosis was investigated using Masson’s trichrome staining and L-hydroxyline.Results: Fingolimod treatment activates both Reperfusion Injury Salvage Kinase (RISK) and Survivor Activating Factor Enhancement (SAFE) pathways as evident from activation of anti-apoptotic and anti-inflammatory pathways. Fingolimod treatment caused a reduction in myocardial oxidative stress and hence cardiomyocyte apoptosis resulting in a decrease in myocardial reperfusion injury. Moreover, a significant (p < 0.001) reduction in collagen staining and hydroxyproline content was observed in fingolimod treated animals 30 days after transplantation demonstrating a reduction in cardiac fibrosis.Conclusion: S1P receptor activation with fingolimod activates anti-apoptotic and anti-inflammatory pathways, leading to improved myocardial salvage causing a reduction in cardiac fibrosis.

Immunosuppressive effects of mesenchymal stem cells on tacrolimus therapy in rat heart transplant model

Introduction: Mesenchymal stem cells (MSCs) are used in various clinical settings for effective cell therapy by taking advantage of their immunomodulatory, regenerative, and anti-inflammatory properties. We previously investigated the effects of the regenerative and anti-inflammatory properties of MSCs for consideration of their clinical application in organ transplantation. Since immunosuppressant agents are administered in nearly all clinical cases of organ transplantation, clarification of the immunological interaction between MSCs and the calcineurin inhibitor tacrolimus (TAC) is needed.Aim: Due to potential use of MSCs in organ transplantation, we assessed immunological interactions between TAC and MSCs in vivo.Methods: Adipose tissue-derived MSCs (AT-MSCs) were obtained from wild-type Lewis rats. We then used a rat heart transplantation model to observe the immunosuppressive effects of AT-MSCs, TAC, and those in combination. Following allogeneic heterotopic heart transplantation from ACI rats to Lewis rats, graft survival was assessed in groups treated with AT-MSCs, TAC, both, or neither.Results: Median survival of the allograft was 14.5 days in the TAC-alone group and 11 days in the MSC+TAC group. Although the difference between those groups was not significant, the survival periods of both were significantly longer as compared to that of the control (median survival =5 days, P<0.05) and MSC-alone (median survival =5 days, P<0.05) groups.Conclusions: Administration of AT-MSCs did not decrease the beneficial immunosuppressive effect of TAC on heart graft survival. TAC appears to be compatible and effective for clinical use as an immunosuppressant with AT-MSCs.

Transplant tolerance: a new role for IL-34.

Immune-suppressive cell populations, including Tregs and suppressor monocytes, have been implicated in long-term survival of allografts in both human transplant recipients and animal models. The factors that drive differentiation and function of these cell populations are not completely understood. In this issue, Bézie and colleagues identify IL-34 as an important mediator of allograft tolerance in a rat model of heart transplantation. Their data support a model in which IL-34 production by Tregs promotes a population of suppressive macrophages that in turn promote Treg differentiation. The results of this study support further exploration of the immunosuppressive properties of IL-34.

Release mechanisms of tacrolimus-loaded PLGA and PLA microspheres and immunosuppressive effects of the microspheres in a rat heart transplantation model

The objective of this study was to elucidate the release and absorption mechanisms of tacrolimus loaded into microspheres composed of poly(lactic-co-glycolic acid) (PLGA) and/or polylactic acid (PLA). Tacrolimus-loaded microspheres were prepared by the o/w emulsion solvent evaporation method. The entrapment efficiency correlated with the molecular weight of PLGA, and the glass transition temperature of PLGA microspheres was not decreased by the addition of tacrolimus. These results indicate that intermolecular interaction between tacrolimus and the polymer would affect the entrapment of tacrolimus in the microspheres. Tacrolimus was released with weight loss of the microspheres, and the dominant release mechanism of tacrolimus was considered to be erosion of the polymer rather than diffusion of the drug. The whole-blood concentration of tacrolimus in rats was maintained for at least 2 weeks after a single subcutaneous administration of the microspheres. The pharmacokinetic profile of tacrolimus following subcutaneous administration was similar to that following intramuscular administration, suggesting that the release and dissolution of tacrolimus, rather than the absorption of the dissolved tacrolimus, were rate-limiting steps. Graft-survival time in a heart transplantation rat model was prolonged by the administration of tacrolimus-loaded microspheres. The microsphere formulation of tacrolimus would be expected to precisely control the blood concentration while maintaining the immunosuppressive effect of the drug.

Effects of soluble guanylate cyclase activation on heart transplantation in a rat model

The nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway is an important key mechanism to protect the heart from ischemia/reperfusion injury. However, this pathway is disrupted in several cardiovascular diseases as a result of decreased NO bioavailability and increased NO-insensitive forms of sGC. Cinaciguat preferentially activates these NO-insensitive, oxidized forms of sGC. We assessed the hypothesis that targeting NO-unresponsive sGC would protect the graft against ischemia/reperfusion injury in a rat heart transplantation model. Before explantation, donor Lewis rats received methylcellulose (1%) vehicle or cinaciguat 10 mg/kg. The hearts were excised, stored in cold preservation solution, and heterotopically transplanted. We evaluated in vivo left ventricular function of the graft. After transplantation, decreased left ventricular systolic pressure (77 ± 3 mm Hg vs 123 ± 13 mm Hg, p < 0.05), dP/dt(max) (1,703 ± 162 mm Hg vs 3,350 ± 444 mm Hg, p < 0.05), and dP/dt(min) (995 ± 110 mm Hg vs 1,925 ± 332 mm Hg, p < 0.05) were significantly increased by cinaciguat. Coronary blood flow was significantly higher in the cinaciguat group compared with the control group. Additionally, cinaciguat increased adenosine triphosphate levels (1.9 ± 0.4 µmol/g vs 6.6 ± 0.8 µmol/g, p < 0.05) and improved energy charge potential. After transplantation, increased c-jun messenger RNA expression was downregulated, whereas superoxide dismutase-1 and cytochrome-c oxidase mRNA levels were upregulated by cinaciguat. Cinaciguat also significantly decreased myocardial DNA strand breaks induced by ischemia/reperfusion during transplantation and reduced death of cardiomyocytes in a cellular model of oxidative stress. By interacting with NO-unresponsive sGC, cinaciguat enhances the protective effects of the NO/cGMP pathway at different steps of signal transduction after global myocardial ischemia/reperfusion. Its clinical use as pre-conditioning agent could be a novel approach in cardiac surgery.