Heterotopic Heart Transplantation Model Research Articles

Stimulation of P2Y11 receptor protects human cardiomyocytes against Hypoxia/Reoxygenation injury and involves PKC\u03b5 signaling pathway

Sterile inflammation is a key determinant of myocardial reperfusion injuries. It participates in infarct size determination in acute myocardial infarction and graft rejection following heart transplantation. We previously showed that P2Y11 exerted an immunosuppressive role in human dendritic cells, modulated cardiofibroblasts’ response to ischemia/reperfusion in vitro and delayed graft rejection in an allogeneic heterotopic heart transplantation model. We sought to investigate a possible role of P2Y11 in the cellular response of cardiomyocytes to ischemia/reperfusion. We subjected human AC16 cardiomyocytes to 5 h hypoxia/1 h reoxygenation (H/R). P2Y11R (P2Y11 receptor) selective agonist NF546 and/or antagonist NF340 were added at the onset of reoxygenation. Cellular damages were assessed by LDH release, MTT assay and intracellular ATP level; intracellular signaling pathways were explored. The role of P2Y11R in mitochondria-derived ROS production and mitochondrial respiration was investigated. In vitro H/R injuries were significantly reduced by P2Y11R stimulation at reoxygenation. This protection was suppressed with P2Y11R antagonism. P2Y11R stimulation following H2O2-induced oxidative stress reduced mitochondria-derived ROS production and damages through PKCε signaling pathway activation. Our results suggest a novel protective role of P2Y11 in cardiomyocytes against reperfusion injuries. Pharmacological post-conditioning targeting P2Y11R could therefore contribute to improve myocardial ischemia/reperfusion outcomes in acute myocardial infarction and cardiac transplantation.

ASP1126, a Novel Sphingosine-1-Phosphate–Selective Agonist With a Favorable Safety Profile, Prolongs Allograft Survival in Rats and Nonhuman Primates in Combination With Tacrolimus With a Broad Safety Margin for Bradycardia

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that acts through the members of a family of 5 G protein–coupled receptors (S1P1 to S1P5). Among these, S1P1 is a major regulator of lymphocyte trafficking. Fingolimod, whose active metabolite, fingolimod phosphate, acts as a nonselective S1P-receptor agonist, exerts its immunomodulatory effect, at least in part, by regulating lymphocyte trafficking via downregulation of S1P1 expression on lymphocytes. Here, we describe the pharmacologic profile of a novel S1P1 agonist, ASP1126. ASP1126 preferentially activated S1P1 compared to S1P3 in rat and human guanosine-5'-(γ-thio)-triphosphate (GTPγS) assays. Oral single administration of ASP1126 decreased the number of peripheral lymphocytes and repeated dosing showed a cumulative effect on lymphopenia in both rats and monkeys. ASP1126 prolonged allograft survival in a rat heterotopic heart transplantation model in combination with a subtherapeutic dose of tacrolimus that was independent of drug-drug interactions. In addition, in nonhuman primate (NHP) renal transplantation, pretreatment with ASP1126 reduced not only the number of naive T cells and central memory T cells but also effector memory T cells in the peripheral blood, all of which could contribute to acute graft rejection and prolonged allograft survival in combination with tacrolimus. Further, we confirmed that ASP1126 has a broad ranging safety margin with respect to its effect on lung weight in rats and bradycardia in NHPs, which were the adverse events found in clinical studies of fingolimod. ASP1126 with improved safety profile has the potential to be an adjunct therapy in combination with tacrolimus in clinical transplantation.

Comparison of hemodynamic and infarct-limiting effects of preservation solution based on Krebs–Henseleit buffer and HTK solution in the rat model of heterotopic heart transplantation

Objective. To compare the cardioprotective effcacy of a preservation solution based on Krebs–Henseleit buffer and HTK solution in the model of heterotopic heart transplantation in rat.Design and methods. A study was conducted on 12 Wistar rats. The animals were divided into groups depending on the preservation solution used: 1) Krebs–Henseleit buffer-based solution (n = 7), 2) HTK (n = 5). Each experiment consisted of collecting donor heart, preserving it with an appropriate cardioplegic solution, heterotopic transplantation into a recipient rat followed by explantation and evaluation of left ventricular contractility using the Langendorff model and histochemical assessment of the irreversible myocardial damage. Coronary blood flow in the donor heart was assessed in vivo using ultrasound doppler flowmetry. After 3 hours, the donor heart was explanted and connected to the Langendorff apparatus to assess left ventricular contractility, and the myocardium was subjected to histochemical staining with 1 % triphenyltetrazolium chloride for the assessment of the irreversible myocardial damage.Results. In the group of Krebs–Henseleit buffer-based cardioplegic solution, 7 experiments were performed. Myocardial infarct size was 3,5 ± 1,2%, the coronary flow rate was 4,5 ± 1,3 ml/min, and the developed left ventricular pressure of the donor heart was 70 ± 6,3 mmHg at diastolic left ventricular pressure of 10 mmHg. In the HTK solution group (n = 5), in all of the experiments after the start of blood flow, the transplanted heart did not begin to contract, and all 5 hearts remained in an asystole state. Therefore, after the end of the 3-hour reperfusion period, the assessment of the contractility of the left ventricle using the Langendorff apparatus was not carried out. Coronary flow rate was only 0,4 ± 0,1 ml/min, probably resulting from inadequate cardioprotection with HTK solution. Due to the lack of adequate reperfusion, the accurate assessment of the irreversible myocardial damage was impossible in the HTK solution group.Conclusions. The technique of heterotopic heart transplantation in rats is adequate and informative for the study of ischemia-reperfusion myocardial damage as well as for the study of the effectiveness of cardioplegic and cardiac preservation solutions. Cardioplegic solution based on Krebs– Henseleit buffer demonstrated greater cardioprotective effectiveness in our model compared to HTK solution.

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.

Circulating mitochondria in organ donors promote allograft rejection.

The innate immune system is a critical regulator of the adaptive immune responses that lead to allograft rejection. It is increasingly recognized that endogenous molecules released from tissue injury and cell death are potent activators of innate immunity. Mitochondria, ancestrally related to bacteria, possess an array of endogenous innate immune-activating molecules. We have recently demonstrated that extracellular mitochondria are abundant in the circulation of deceased organ donors and that their presence correlates with early allograft dysfunction. Here we demonstrate the ability of mitochondria to activate endothelial cells (ECs), the initial barrier between a solid organ allograft and its host. We find that mitochondria exposure leads to the upregulation of EC adhesion molecules and their production of inflammatory cytokines and chemokines. Additionally, mitochondrial exposure causes dendritic cells to upregulate costimulatory molecules. Infusion of isolated mitochondria into heart donors leads to significant increase in allograft rejection in a murine heterotopic heart transplantation model. Finally, co-incubation of human peripheral blood mononuclear cells with mitochondria-treated ECs results in increased numbers of effector (IFN-γ+ , TNF-α+ ) CD8+ T cells. These data indicate that circulating extracellular mitochondria in deceased organ donors may directly activate allograft ECs and promote graft rejection in transplant recipients.

Inhibition of cardiac allograft rejection in mice using interleukin-35-modified mesenchymal stem cells.

Interleukin-35 (IL-35) is a cytokine recently discovered to play a potent immunosuppressive role by intensifying the functions of regulatory T cells and inhibiting the proliferation and functions of T helper 1 and T helper 17 cells. Mesenchymal stem cells (MSCs) have recently emerged as promising candidates for cell-based immune therapy, and our previous study showed that IL-35 gene modification can effectively enhance the therapeutic effect of MSCs in vitro. In this study, we isolated adipose tissue-derived MSCs in vitro and infected them with lentiviral vectors overexpressing the IL-35 gene, thereby creating IL-35-MSCs. Subsequently, IL-35-MSCs were then injected into mice of the allogeneic heterotopic abdominal heart transplant model to determine their effect on allograft rejection. The results showed that IL-35-MSCs could continuously secrete IL-35 in vivo and in vitro, successfully alleviate allograft rejection and prolong graft survival. In addition, compared to MSCs, IL-35-MSCs showed a stronger immunosuppressive ability and further reduced the percentage of Th17 cells, increased the proportion of CD4+ Foxp3+ T cells, and regulated Th1/Th2 balance in heart transplant mice. These findings suggest that IL-35-MSCs have more advantages than MSCs in inhibiting graft rejection and may thus provide a new approach for inducing immune tolerance during transplantation.

Intracardiac hemodynamics, infarct-limiting effects and myocardial expression of microRNA 223 after necroptosis inhibition in a rat model of heterotopic allogeneic heart transplantation

Objective.To investigate the effect of necroptosis inhibition on the morphofunctional state of the myocardium and the expression of microRNA 223 after heterotopic allogeneic heart transplantation in rat.Design and methods. Twenty Wistar rats were examined in the study. Animals were divided into the following groups: 1) control (n = 7), 2) dimethyl sulfoxide (DMSO) (n = 6), 3) necrostatin-1s (n = 7). Necrostatin-1s was used as an inhibitor of necroptosis, which was administered intraperitoneally in DMSO solution 1 hour before the start of the experiment at a dose of 1,65 mg/kg. HTK solution cooled to 4 °C was used as a preservation solution. Two hours after heart arrest, the heart was heterotopically transplanted in the abdominal cavity of recipient rat using the scheme “aorta-aorta, pulmonary trunc-posterior vena cava”. Three hours later, intracardiac hemodynamics was assessed by recording the pressure in the left ventricle, heart rate and coronary flow rate in Langendorff-perfused heart. The expression level of microRNA 223–5p and –3p in left ventricular myocardium was assessed using real-time polymerase chain reaction. The plasma levels of troponin I were assessed by enzyme immunoassay. Myocardial infarct size was measured planimetrically at the end of the experiment by staining myocardium with triphenyltetrazolium chloride.Results. Inhibition of necroptosis significantly improved the morphofunctional state of the myocardium, which manifested in a decrease of myocardial infarct size in the necrostatin-1s group compared with the control group and DMSO group. Thus, in the necrostatin-1s group, myocardial infarct size was 25 ± 8,7 %, which was smaller than in the control and DMSO groups (56 ± 9,5 and 57 ± 8,7 %, respectively; p < 0,05). Also in the necrostatin-1s group, lower diastolic intraventricular pressure was recorded, as well as higher values of pulse intraventricular pressure and coronary flow rate than in control group and DMSO group (p < 0,05). Left ventricular myocardium in the necrostatin-1s group demonstrated higher expression of the antinecroptotic miRNA 223–5p and –3p as compared with the control and DMSO groups, as well as lower plasma levels of troponin I (p < 0,05).Conclusions.Pharmacological inhibition of necroptosis in heterotopically transplanted donor heart is accompanied by marked cardioprotective effects and increases the expression of antinecroptotic microRNA 223–5p and –3p.

Role of MicroRNA 146a in Regulating Regulatory T Cell Function to Ameliorate Acute Cardiac Rejection in Mice

BackgroundMiR-146a plays a critical regulatory role in the homeostasis and function of regulatory T cells (Treg cells). The purpose of this study was to investigate the role of miR-146 in regulating Treg function to ameliorate acute cardiac rejection in mice. MethodsWe downregulated the miR-146a expression of Treg cells in recipients and constructed murine heterotopic heart transplantation models. Flow cytometry was used to analyze phenotypes of T cells. Graft rejection and cytokine changes during allograft rejection were detected. The changes of miR-146a target genes in Treg cells were detected by quantitative real-time polymerase chain reaction and western blotting. ResultsOur results indicated the miR-146a antagomir increased the Treg proportion in splenocytes and blood cells in the normal immune and inflammatory conditions, but impaired the ability of Treg cells to inhibit Th1 in the inflammatory condition. During the rejection process of murine heterotopic heart transplantation, miR-146a was mainly involved in regulating Treg cells through the STAT1/IFN-γ signaling pathway and the target gene was STAT1. Reducing the miR-146a of Treg and simultaneously collaboratively applying IFN-γ neutralizing antibodies can inhibit the proliferation of CD4+ T cell, alleviate the rejection process, and promote the survival of the donor's heart. ConclusionIt is possible to enhance the function of Treg cells in vivo of recipient by regulating a single miRNA, miR-146a, to achieve the purpose of inhibiting graft rejection. Collaboratively applying immunosuppressive drugs may effectively control the immune rejection and allow for the dosage and side effects of drugs to be reduced, a process that needs further study.

Heart Failure Stimulates Tumor Growth by Circulating Factors.

Heart failure (HF) survival has improved, and nowadays, many patients with HF die of noncardiac causes, including cancer. Our aim was to investigate whether a causal relationship exists between HF and the development of cancer. HF was induced by inflicting large anterior myocardial infarction in APCmin mice, which are prone to developing precancerous intestinal tumors, and tumor growth was measured. In addition, to rule out hemodynamic impairment, a heterotopic heart transplantation model was used in which an infarcted or sham-operated heart was transplanted into a recipient mouse while the native heart was left in situ. After 6 weeks, tumor number, volume, and proliferation were quantified. Candidate secreted proteins were selected because they were previously associated both with (colon) tumor growth and with myocardial production in post-myocardial infarction proteomic studies. Myocardial gene expression levels of these selected candidates were analyzed, as well as their proliferative effects on HT-29 (colon cancer) cells. We validated these candidates by measuring them in plasma of healthy subjects and patients with HF. Finally, we associated the relation between cardiac specific and inflammatory biomarkers and new-onset cancer in a large, prospective general population cohort. The presence of failing hearts, both native and heterotopically transplanted, resulted in significantly increased intestinal tumor load of 2.4-fold in APCmin mice (all P<0.0001). The severity of left ventricular dysfunction and fibrotic scar strongly correlated with tumor growth ( P=0.002 and P=0.016, respectively). We identified several proteins (including serpinA3 and A1, fibronectin, ceruloplasmin, and paraoxonase 1) that were elevated in human patients with chronic HF (n=101) compared with healthy subjects (n=180; P<0.001). Functionally, serpinA3 resulted in marked proliferation effects in human colon cancer (HT-29) cells, associated with Akt-S6 phosphorylation. Finally, elevated cardiac and inflammation biomarkers in apparently healthy humans (n=8319) were predictive of new-onset cancer (n=1124) independently of risk factors for cancer (age, smoking status, and body mass index). We demonstrate that the presence of HF is associated with enhanced tumor growth and that this is independent of hemodynamic impairment and could be caused by cardiac excreted factors. A diagnosis of HF may therefore be considered a risk factor for incident cancer.

Overcoming Coagulation Dysregulation in Pig Solid Organ Transplantation in Nonhuman Primates

There has recently been considerable progress in the results of pig organ transplantation in nonhuman primates, largely associated with the availability of (i) pigs genetically engineered to overcome coagulation dysregulation, and (ii) novel immunosuppressive agents. The barriers of thrombotic microangiopathy and/or consumptive coagulation were believed to be associated with (i) activation of the graft vascular endothelial cells by a low level of antipig antibody binding and/or complement deposition and/or innate immune cell activity, and (ii) molecular incompatibilities between the nonhuman primate and pig coagulation-anticoagulation systems. The introduction of a human coagulation-regulatory transgene, for example, thrombomodulin, endothelial protein C receptor, into the pig vascular endothelial cells has contributed to preventing a procoagulant state from developing, resulting in a considerable increase in graft survival. In the heterotopic (non-life-supporting) heart transplant model, graft survival has increased from a maximum of 179 days in 2005 to 945 days. After life-supporting kidney transplantation, survival has been extended from 90 days in 2004 to 499 days. In view of the more complex coagulation dysfunction seen after pig liver and, particularly, lung transplantation, progress has been less dramatic, but the maximum survival of a pig liver has been increased from 7 days in 2010 to 29 days, and of a pig lung from 4 days in 2007 to 9 days. There is a realistic prospect that the transplantation of a kidney or heart, in combination with a conventional immunosuppressive regimen, will enable long-term recipient survival.

T2 Mapping for Noninvasive Assessment of Interstitial Edema in Acute Cardiac Allograft Rejection in a Mouse Model of Heterotopic Heart Transplantation.

Heart transplantation (HTX) in mice is used to characterize gene-deficient mice and to test new treatment strategies. The purpose was to establish noninvasive magnetic resonance imaging techniques in mice to monitor pathophysiological changes of the allograft during rejection. Magnetic resonance imaging was performed at baseline and days 1 and 6 after isogenic (n = 10, C57BL/6) and allogenic (n = 12, C57BL/6 to BALB/c) heterotopic HTX on a 7 T small animal scanner. Respiratory- and electrocardiogram-gated multislice multi-echo spin echo sequences were acquired, and parameter maps of T2 relaxation time were generated. T2 times in septal, anterior, lateral, and posterior myocardial segments as well as global T2 times were calculated and compared between groups. At day 7 animals were sacrificed and graft pathology was assessed by semiquantitative regional analysis and correlated with magnetic resonance imaging results. Myocardial T2 relaxation time was significantly increased in allogenic (33.4 ± 0.1 ms) and isogenic cardiac grafts (31.8 ± 1.8 ms) on day 1 after HTX compared with healthy donor hearts at baseline (23.1 ± 0.3 ms, P < 0.001). Until day 6 after HTX, myocardial T2 further increased markedly in allografts but not in isografts (43.4 ± 1.9 vs 31.2 ± 1.1 ms, P < 0.001). Mean segmental T2 values as well as mean global T2 values in allogenic compared with isogenic cardiac grafts on day 6 were significantly higher (P < 0.01). Histologically, isogenic grafts were almost normal besides small focal leukocyte infiltrates and signs of interstitial edema, most likely due to ischemia reperfusion injury (histological sum score, 0.9 ± 0.4). In allogenic HTX, histology revealed severe inflammation and tissue edema representing allograft rejection with increased histological scores (5.3 ± 0.7, P < 0.001). Higher histological scores of rejection were significantly associated with increased T2 times on a segmental and a global level. We could show that T2 mapping is a suitable noninvasive imaging method to monitor global and regional HTX pathologies in experimental heart transplantation in mice. Progressive prolongation of T2 time was significantly associated with pathological signs of rejection.

Inhibitory effect of wild-type P53 gene transfer on graft coronary artery disease

ObjectiveThe aim of the present study was to investigate the inhibitory effect of wild-type P53 gene transfer on graft coronary artery disease (GCAD) after heart transplantation and the underlying mechanisms. MethodsA rat model of heterotopic heart transplantation was established using Wistar rats as donors and Sprague-Dawley (SD) rats as recipients. The donor hearts were collected and perfused, via the coronary artery, with 800 μl of recombinant adenovirus carrying the P53 gene (Ad-P53). Thirty minutes later, heart transplant was performed. At 5 d after the transplant surgery, the expression of the exogenous P53 gene and protein in the coronary artery tissues of the donor hearts was examined. At 28 d after the transplant surgery, tissues were collected from the transplanted hearts. The degree of coronary artery stenosis was examined, and apoptosis of the coronary artery smooth muscle cells in the donor hearts was analysed. In addition, histological changes in the vital organs of the recipient rats and the levels of serum biochemical indicators in the rats were also examined. ResultsThe exogenous gene was successfully transferred into donor heart tissues and the coronary artery and was highly expressed. At 28 d after the transplant surgery, the ratio of tunica intima thickness to tunica media thickness (I/M) and the ratio of wall thickness to the lumen diameter of the coronary artery were decreased in the Ad-P53 group compared to those in the Ad-LacZ group and the control group (P < 0.05). A terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay revealed that the percentage of apoptotic coronary artery smooth muscle cells in the donor hearts was significantly increased in the Ad-P53 group compared to that in the Ad-LacZ group and the control group (P < 0.01). The wild-type P53 gene had no effect on the morphology and functions of the vital organs of the recipient rats. ConclusionsP53 gene transfer inhibits coronary artery intimal hyperplasia and reduces the degree of luminal stenosis in transplanted hearts. The inhibitory effect may be related to the wild-type P53 gene-induced apoptosis of vascular smooth muscle cells and inhibition of vascular smooth muscle cell proliferation. This approach is effective and safe and may have good prospects for clinical application.

Donor tissue-specific exosome profiling enables noninvasive monitoring of acute rejection in mouse allogeneic heart transplantation

ObjectiveIn heart transplantation, there is a critical need for development of biomarkers to noninvasively monitor cardiac allografts for immunologic rejection or injury. Exosomes are tissue-specific nanovesicles released into circulation by many cell types. Their profiles are dynamic, reflecting conditional changes imposed on their tissue counterparts. We proposed that a transplanted heart releases donor-specific exosomes into the recipient's circulation that are conditionally altered during immunologic rejection. We investigated this novel concept in a rodent heterotopic heart transplantation model. Materials and MethodsFull major histocompatibility mismatch (BALB/c [H2-Kd] into C57BL/6 [H2-Kb]) heterotopic heart transplantation was performed in 2 study arms: Rejection (n = 64) and Maintenance (n = 28). In the Rejection arm, immunocompetent recipients fully rejected the donor heart, whereas in the Maintenance arm, immunodeficient recipients (C57BL/6 PrkdcSCID) accepted the allograft. Recipient plasma exosomes were isolated and a donor heart-specific exosome signal was characterized on the nanoparticle detector for time-specific profile changes using anti-H2-Kd antibody quantum dot. ResultsIn the Maintenance arm, allografts were viable throughout follow-up of 30 days, with histology confirming absence of rejection or injury. Time course analysis (days 1, 2, 3, 4, 5, 7, 9, 11, 15, and 30) showed that total plasma exosome concentration (P = .157) and donor heart exosome signal (P = .538) was similar between time points. In the Rejection arm, allografts were universally rejected (median, day 11). Total plasma exosome quantity and size distribution were similar between follow-up time points (P = .278). Donor heart exosome signals peaked on day 1, but significantly decreased by day 2 (P = 2 × 10−4) and day 3 (P = 3.3 × 10−6), when histology showed grade 0R rejection. The receiver operating characteristic curve for a binary separation of the 2 study arms (Maintenance vs Rejection) demonstrated that a donor heart exosome signal threshold < 0.3146 was 91.4% sensitive and 95.8% specific for diagnosis of early acute rejection. ConclusionsTransplant heart exosome profiling enables noninvasive monitoring of early acute rejection with high accuracy. Translation of this concept to clinical settings might enable development of a novel biomarker platform for allograft monitoring in transplantation diagnostics.

Graft vasculopathy in a Wistar rat model of heterotopic heart transplantation depending on gender matching between donors and recipients.

IntroductionHeart transplant is an accepted treatment modality in end-stage heart failure. The graft coronary artery vasculopathy is a main concern to explain the heterogeneity of the rejection process according to the gender of the donor and recipient.AimTo assess the severity and type of mechanisms leading to failure of the graft.Material and methodsExperimental allogenic heart transplantation in the abdomen was performed on Wistar rats depending on the gender of the donor and recipient (F – female; M – male) in four groups (FF, FM, MM MF). The donor heart was implanted in the abdominal cavity of the recipient. Complete time of observation was 10 weeks. Bromodeoxyuridine was administered intraperitoneally to detect proliferating cells.ResultsThere was 42.5% graft survival in all experiments. The mean time of graft survival was 60 ±18, 54 ±29, 58 ±23 and 64 ±18 days (FF, FM, MM and MF) and no significant difference was found in graft survival time among the four experimental groups (p = 0.73). None of the heart weight changes reached statistical significance.ConclusionsThe use of an animal experimental model helps to understand the mechanisms leading to graft failure and to compare the changes that occur in rats to human hearts. The gender matching affects the survival of the transplanted heart and severity of the graft vasculopathy.

Sirtinol regulates the balance of Th17/Treg to prevent allograft rejection

BackgroundCurrent immunosuppressive medications used after transplantation induce significant toxicity , and a new medication regimen is needed. Based on recent research, Sirt1 exerts a proinflammatory effect on the immune response. Sirtinol is a Sirt1 inhibitor, but its impact on allograft rejection and its molecular mechanisms of action have not yet been reported.ReslutsIn this study, we examined the effect of sirtinol on prolonging allograft survival in a mouse cervical heterotopic heart transplantation model. Based on an examination of the allograft, allografts from sirtinol-treated recipients show significantly lower levels of IL-17A expression and higher levels of Foxp3 expression. In vivo, sirtinol reduces the proportion of Th17 cells and increases the proportion of Treg cells in splenocytes from recipients. In vitro, sirtinol reduces the proportion of Th17 cells and decreases the expression of IL-17A and RORγt in an isolated CD4+ T cell population. Moreover, we identified synergistic effects of sirtinol and FK506 on prolonging allograft survival, and sirtinol synergizes with FK506 to promote Foxp3 expression.ConclusionSirtinol, a Sirt1 inhibitor, may be a promising immunosuppressive drug to prevent the rejection reaction in combination with FK506.

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.

A highly reproducible cervical cuff technique for rat-to-mouse heterotopic heart xenotransplantation.

Xenotransplantation is an effective way to solve the problem of donor shortage in clinical transplantation. However, clinical use of xenotransplantation is currently limited due to immunological challenges such as acute vascular rejection and cell-mediated rejection. To finally surpass this immunological barrier, more preclinical research is needed into the molecular mechanisms of rejection and the possible effects of new immunosuppressants. Our aim was to create a refined, highly reproducible protocol to establish the most suitable rat-to-mouse heterotopic heart transplantation model using the cuff technique.

ADIPOSE DERIVED STROMAL CELLS CULTURED AS SPHEROIDS IMPROVES CARDIAC ALLOGRAFT VASCULOPATHY DEVELOPMENT IN A HEART TRANSPLANTATION MODEL

Cardiac transplantation remains the definitive therapy for eligible patients with end-stage heart failure. Beyond the first year post-transplant period, cardiac allograft vasculopathy (CAV) represents one of the major causes of death which implies immunologic and non-immunologic factors. Adipose tissue is a rich and very convenient source of cells for regenerative medicine therapeutic approaches. Adipose-derived stromal cell (ASCs) treatment is a potentially effective cell therapy for cardiac disease. We investigated the effects of epicardial delivery of ASC matrix in a heterotopic heart transplantation (HHT) model associated with CAV.

Erythroblast transformation-specific 2 correlates with vascular smooth muscle cell apoptosis in rat heterotopic heart transplantation model.

Cardiac allograft vasculopathy (CAV) decreases the long-term survival of heart transplantation recipients. Vascular smooth muscle cell (VSMC) apoptosis is an important pathological feature of CAV. Erythroblast transformation-specific 2 (Ets-2), as a transcription factor, participates in cell apoptosis and plays an important role in organ transplantation. Hearts from Wistar-Furth (WF:RT1u) rats were heterotopically transplanted into Lewis (Lew:RT1(l)) rats without immunosuppression. Additional syngeneic heterotopic cardiac transplantations were performed in Lewis rats. HE staining was used to identify CAV. Ets-2 expression was examined by western blot. Ets-2 tissue location was examined by immunohistochemical assay and double immunostaining. Cleaved caspase 3 expression was detected by western blot. Co-localization of Ets-2 and cleaved caspase 3 was detected by double immunostaining. Ets-2, p53, cleaved caspase 3 and Bcl-xl expression in rat VSMC line A7R5 was examined after Ets-2 siRNA transfection. TUNEL assay was applied to detect A7R5 apoptosis with or without ETS-2 siRNA transfection. Immunoprecipitation was performed to explore the interaction between Ets-2 and p53. Ets-2 expression decreased in the allograft group but had no obvious change in the isograft group. Meanwhile, the phenomenon of CAV was observed in the allograft group and there is neointima formation in the isograft group which is not obvious compared with allograft group. Additionally, Ets-2 expression was opposite to VSMC apoptosis in the allograft group. In vitro, Ets-2 siRNA transfection in A7R5cells resulted in enhanced cell apoptosis. Finally, Ets-2 interacted with p53. Ets-2 might inhibit VSMC apoptosis via p53 pathway. The results further elucidate the molecular mechanism of VSMC apoptosis after heart transplantation during CAV and provide theoretical basis for seeking new specific drug targets for CAV prevention and treatment.

Knockout of microRNA-155 ameliorates the Th1/Th17 immune response and tissue injury in chronic rejection

MicroRNAs (miRNAs) are integral for maintaining immune homeostasis and self-tolerance. The influence of miRNAs on T-cell differentiation and plasticity are critical in the development of chronic rejection of transplanted hearts. In this study, we sought to determine whether the knockout of miR-155 affects the development of cardiac allograft vasculopathy (CAV) in a murine model. miRNA microarray and quantitative polymerase chain reaction (qPCR) analyses were performed for allograft neointimal lesion samples in chronic rejection. A model of heterotopic murine heart transplantation (bm12 to miR-155+/+ or miR-155-/- mice) was then used to analyze allograft survival, histology, mRNA expression and T-cell sub-populations in spleens. The accelerated experiments were performed by intraperitoneal injection of either recombinant interleukin-17A or phosphate-buffered saline (PBS) after heart transplantation. For the competitive transfer experiments, CD4+ splenocytes from wild-type (WT) or miR-155-/- mice were mixed and injected into Rag1-/- mice, and cardiac transplantation was performed after 24 hours. The differentiation of T-helper subsets (Th1/Th17/iTreg) was investigated in vitro. miR-155-/- mice showed resistance to cardiac rejection along with weakened T-cell-mediated inflammation, especially for Th17 cells. Recombinant IL-17A could restore this relieved injury. The competitive experiments implied that miR-155 plays a vital role in the stability of the Th17 phenotype. In vitro, we also demonstrated that miR-155-/- mice exhibit a defect in Th17 differentiation. miR-155 regulates Th1/Th17-related inflammation in chronic cardiac rejection and may be a potential therapeutic target to attenuate cardiac allograft rejection. Despite advancements in immunosuppressive therapy, the immunologic mechanisms responsible for allograft rejection remain an important issue for both clinicians and researchers. Allograft rejection is a T-cell-dependent phenomenon and is critically dependent on inflammation mediated by CD4+ Th subsets, including Th1, Th2, Th17, Th9 and regulatory T (Treg) cells.