Heterotopic Heart Transplantation Research Articles

Electron Transport Chain Inhibition to Decrease Injury in Transplanted Donation After Circulatory Death Rat Hearts.

Donation after circulatory death (DCD) donor hearts sustain ischemic damage and are not routinely used for heart transplantation. DCD heart injury, particularly reperfusion injury, is primarily mediated by releasing reactive oxygen species from the damaged mitochondria (complex I of the electron transport chain). Amobarbital (AMO) is a transient inhibitor of complex I and is known to reduce releasing reactive oxygen species generation. We studied the beneficial effects of AMO in transplanted DCD hearts. Sprague-Dawley rats were assigned to 4 groups-DCD or DCD + AMO donors and control beating-heart donors (CBD) or CBD + AMO donors (n = 6-8 each). Anesthetized rats were connected to a ventilator. The right carotid artery was cannulated, heparin and vecuronium were administered. The DCD process started by disconnecting the ventilator. DCD hearts were procured after 25 minutes of in-vivo ischemia, whereas CBD hearts were procured without ischemia. At procurement, all donor hearts received 10 mL of University of Wisconsin cardioplegia solution. The CBD + AMO and DCD + AMO groups received AMO (2 mM) dissolved in cardioplegia. Heterotopic heart transplantation was performed by anastomosing the donor aorta and pulmonary artery to the recipient's abdominal aorta and inferior vena cava. After 14 days, transplanted heart function was measured with a balloon tip catheter placed in the left ventricle. Compared with CBD hearts, DCD hearts had significantly lower developed pressure. AMO treatment significantly improved cardiac function in DCD hearts. Treatment of DCD hearts at the time of reperfusion with AMO resulted in an improvement of transplanted heart function that was comparable with the CBD hearts.

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.

Mst1 attenuates myocardial ischemia/reperfusion injury following heterotopic heart transplantation in mice through regulating Keap1/Nrf2 axis

Ischemia reperfusion (I/R) injury remains a frequent adverse event that accompanies heart transplantation. Oxidative stress and aberrant production of free radicals were regarded as the culprit of cell death and tissue damage in post-transplant IR injury. Mst1 has been identified as a mediator of oxidative stress and Nrf2 regulates anti-oxidative enzymes, however, the interaction between Mst1 and Nrf2 anti-oxidative stress pathway remains to be clarified in the event of cardiac IR injury. Herein, the model of ischemia-reperfusion injury in heterotopic heart transplantation mice was firstly established.. We observed that cardiac IR induced upregulation of Mst1 and activation of Nrf2/HO-1pathway in mice receiving heterotopic heart transplantation. Further Cobalt dichloride-induced oxidative stress model of RAW264.7 macrophage cells were then established to mimic cardiac I/R injury, results showed that exposure to CoCl2 induced the upregulation of Mst1 and activation of Keap1/Nrf2 pathway, and genetic ablation of Mst-1 and inhibition of Keap1/Nrf2 pathway aggravated oxidative damage in those cells. Additional in vivo study showed that transfection of Mst1 shRNA spurred ROS generation and worsened cardiac damage in IR mice. Meanwhile, Mst1-KD mice receiving heart transplantation showed markedly downregulation of Nrf2, HO-1 yet upregulation of Keap1, indicating diminished protective effect against tissue damage caused by IR probably owing to the frustration of Keap1/Nrf2 pathway. Taken together, our findings demonstrated the protective effect of Mst1 from cardiac IR injury via triggering Keap1/Nrf2 axis and suppressing ROS generation, which shed light on the promising role of Mst1 in transitional management of IR injury resulted from cardiac transplantation.

An efficient combination immunotherapy for antitumor immunity without accelerating cardiac allograft rejection.

Immunotherapy with immune checkpoint inhibitors (ICIs), including antibodies against programmed cell death protein-1 (PD-1) and its receptor programmed cell death ligand-1 (PD-L1), represents a promising systematic treatment for advanced human malignancies. Transplantation remains the ultimate therapy for end-stage organ diseases. However, the efficacy of ICI treatment in solid organ transplant (SOT) recipients remains controversial. We established a transgenic primary liver cancer mouse model and performed allogeneic heterotopic heart transplantation. Different treatments were performed and survival curves were calculated. Graft samples were collected, and immune cells and the cell surface expression of PD-L1 were analysed by flow cytometry. Inflammatory cytokine levels in the serum were measured by an inflammatory array. The specificity of the histochemical techniques was tested by staining sections. A combination immunotherapy comprising a BET protein inhibitor (JQ1) and an immune checkpoint inhibitor (anti-PD-L1 antibody) was administered to primary liver cancer model mice bearing cardiac allografts. Interestingly, the combination immunotherapy effectively suppressed the progression of primary liver cancer but did not accelerate allograft rejection. In accordance with our previous findings, BET protein inhibition enhances the expression of a putative membrane transporter (Rab8A), which upregulates the expression of PD-L1 on the plasma membrane in a transgenic primary liver cancer mouse model. This may be a crucial mechanism of tumour progression arrest. Our data showed that heart transplantation upregulated the expression of the proinflammatory factor IFN-γ and suggested that BET protein inhibition (with JQ1) decreased PD-L1 expression in heart tissues after cardiac transplantation. This phenomenon was accompanied by enhanced infiltration of inflammatory IFN-γ. Our study provides a novel and efficient therapeutic strategy for SOT recipients.

Comparison of hypothermic static and normothermic ex situ donor heart preservation in heterotopic heart transplantation with the murine model

Comparison of hypothermic static and normothermic ex situ donor heart preservation in heterotopic heart transplantation with the murine model

Abstract 15042: Cardiac-Enhanced Adeno-Associated Viral Vector Achieves Long-Term Transgene Expression in Transplanted Porcine Hearts When Administered During Ex Vivo Perfusion

Introduction: Use of gene therapy for cardiac transplantation is limited by the lack of an effective recombinant Adeno-associated viral vector (rAAV) gene delivery strategy. Ex vivo normothermic perfusion has been increasingly used in clinical practice and is an effective platform for achieving robust global gene delivery to a donor heart. Here we utilized a rationally designed cardiac-enhanced AAV3b variant, SASTG, to deliver the gene firefly luciferase to porcine hearts and evaluated transgene expression 30-days post-transplantation. Methods: Heterotopic heart transplantation was performed using blood-type and MHC matched Yucatan pigs (n=4). Donor hearts underwent ex vivo perfusion for 2-hours with perfusate containing the rAAV SASTG-Luciferase. Three different doses of rAAV were used: 2x10 13 , 8x10 13 , 1x10 14 viral genome copies (VGC). Recipients were survived for 30 days at which time they were euthanized and donor and recipient hearts were examined for overall levels of luciferase activity, luciferase DNA copy number, and histology for luciferase expression on immunostaining and evidence of inflammation. Results: There was a dose-dependent response in luciferase activity observed in the donor heart ventricles with the lowest activity detected at the 2x10 13 VGC dose (1.5-9.9x10 3 RLU/mg) mid-level activity at the 8x10 13 VGC dose (32.7-71.2x10 3 RLU/mg) and highest activity at the 1x10 14 VGC dose (27.6-278.1x10 3 RLU/mg). Luciferase DNA was detectable in a dose-dependent manner in the donor heart ventricles (0.02-131.1x10 3 VGC/pg of DNA), but undetectable in all native hearts. Finally, immunostaining demonstrated diffuse luciferase expression in the donor heart but none in the recipient hearts. There was also no evidence of inflammation in any of the donor hearts. Conclusions: The cardiac-enhanced AAV3b variant, SASTG, is associated with robust, global transgene expression in porcine cardiac allografts when administered during ex vivo perfusion.

Abstract 12689: Electron Transport Chain Inhibition to Decrease Reperfusion Injury in Transplanted Donation After Circulatory Death Rat Hearts

Introduction: Intrinsic to the donation after circulatory death (DCD) donation is ischemia and reperfusion injury, primarily mediated through release of reactive oxygen species (ROS) from the damaged mitochondria. Amobarbital (AMO) is a transient, reversible inhibitor of complex I known to decrease ROS generation. We studied whether AMO treatment during reperfusion can improve function in transplanted DCD hearts. Methods: Sprague Dawley rats, were assigned to four heart donor groups- DCD or DCD+AMO and control beating-heart donor (CBD) or CBD+AMO (n=6-8 each). Donor rats were anesthetized using ketamine/xylazine, intubated and connected to a ventilator. The carotid access was used to deliver heparin (1000 U/kg) and vecuronium bromide (4 mg/kg) to paralyze diaphragmatic muscles. The DCD process was initiated by disconnecting the ventilator. Donor hearts were procured following 25 minutes of in-vivo ischemia in the DCD groups, while CBD hearts were procured without terminating the ventilation. At procurement, after clamping the aortic arch, all donor hearts received 10 ml of University of Wisconsin cardioplegia solution delivered through the carotid cannula. The CBD+AMO and DCD+AMO groups received AMO (2 mM), dissolved in the cardioplegia. Heterotopic heart transplantation was performed in recipient rat’s abdomen. After 14 days the transplanted heart function was measured with a balloon tip catheter. Results: Compared to CBD hearts, DCD hearts had significantly lower developed pressure, the rate pressure product (developed pressure times heart rate), and negative dP/dt indicating decreased contractile and impaired diastolic function (Figure). AMO treatment of the DCD hearts resulted in significantly improved cardiac function compared to the untreated DCD hearts. Conclusions: A single treatment with AMO during the heart procurement improves the transplanted DCD heart function which was comparable to the control CBD hearts.

Transvenous Endomyocardial Biopsy Technique for Intra-abdominal Heterotopic Cardiac Grafts.

The porcine intra-abdominal heterotopic heart transplantation model allows for the assessment of immunologic effects on cardiac transplantation without relying on the allograft to maintain hemodynamic support for the animal. Historically, allograft function and histology is monitored by physical exam, echocardiogram evaluation, percutaneous core biopsy, and open biopsy. We performed transvenous endomyocardial biopsies in three pigs that had undergone heterotopic heart implantation. We describe the procedure to be feasible and reproducible, and that histologic results from these biopsies correlated with those from corresponding tissue collected by surgical dissection at the time of allograft explantation. The ability to perform endomyocardial biopsies in the heterotopic heart transplantation model allows for serial non-invasive monitoring of allograft histology.

Antagonist of sphingosine 1-phosphate receptor 3 reduces cold injury of rat donor hearts for transplantation

Cold storage is widely used to preserve an organ for transplantation; however, a long duration of cold storage negatively impacts graft function. Unfortunately, the mechanisms underlying cold exposure remain unclear. Based on the sphingosine-1-phosphate (S1P) signal involved in cold tolerance in hibernating mammals, we hypothesized that S1P signal blockage reduces damage from cold storage. We used an in vitro cold storage and rewarming model to evaluate cold injury and investigated the relationship between cold injury and S1P signal. Compounds affecting S1P receptors (S1PR) were screened for their protective effect in this model and its inhibitory effect on S1PRs was measured using the NanoLuc Binary Technology (NanoBiT)-β-arrestin recruitment assays. The effects of a potent antagonist were examined via heterotopic abdominal rat heart transplantation. The heart grafts were transplanted after 24-hour preservation and evaluated on day 7 after transplantation. Cold injury increased depending on the cold storage time and was induced by S1P. The most potent antagonist strongly suppressed cold injury consistent with the effect of S1P deprivation in vitro. In vivo, this antagonist enabled 24-hour preservation, and drastically improved the beating score, cardiac size, and serological markers. Pathological analysis revealed that it suppressed the interstitial edema, inflammatory cell infiltration, myocyte lesion, TUNEL-positive cell death, and fibrosis. In conclusion, S1PR3 antagonist reduced cold injury, extended the cold preservation time, and improved graft viability. Cold preservation strategies via S1P signaling may have clinical applications in organ preservation for transplantation and contribute to an increase in the donor pool.

Ghrelin May Inhibit Inflammatory Response and Apoptosis During Ischemia-Reperfusion Injury

Ghrelin, a novel growth hormone-releasing peptide, has both anti-inflammatory and anti-apoptotic effects on human endothelial cells. We evaluated the protective effects of ghrelin against ischemia-reperfusion injury (IRI) in a murine heterotopic cervical heart transplantation model. Donor hearts from C57BL/6J wild-type mice, which were kept in cold saline for 60 minutes, were heterotopically transplanted into C57BL/6J wild-type recipients. A day prior to heterotopic cervical heart transplantation, donor animals received either ghrelin (300 nmol/kg) or saline (0.3 mL) intraperitoneally. Upon reperfusion and postoperative day 1, ghrelin or saline was administered to the recipients. Donor hearts were procured on day 2. Ghrelin injection did not result in any adverse effects in donors or recipients. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were significantly decreased in the ghrelin group (0.38% ± 0.21% vs 5.74% ± 3.68%; P < .001). Both cleaved caspase-3 activity and Bcl-2/Bax ratio from the ghrelin group were significantly reduced compared to those in the control. Furthermore, the phosphorylated Akt/Akt ratio was higher in the ghrelin group (0.44 ± 0.21 vs 0.14 ± 0.03; P=.043). Nuclear factor-kappa B p65 nuclear translocation was reduced in the ghrelin hearts compared to the controls (3.17% ± 1.84% vs 19.28% ± 13.14%; P=.009). Vascular cell adhesion molecule-1, intracellular adhesion molecule-1, nuclear factor-kappa B, and tumor necrosis factor alpha levels were also significantly reduced in the ghrelin-treated group. No significant difference was observed in 8-isoprostane production between groups. Ghrelin inhibits the inflammatory response and apoptosis during transplant-related IRI. This study demonstrates the protective effects of ghrelin against IRI.

PHENOMENON OF DEMIKHOV. Heart transplantation in experiment and clinical cases in the USSR and abroad (1968–1972). The Second in the USSR heart transplantation in a Clinic (Solovyev G.M., June 10, 1971)

The article presents materials of Soviet and foreign medical literature of 1968–1972 devoted to heart transplantation in experiment and clinical practice. It is shown that in the USSR after unsuccessful heart transplantation performed by A.A. Vishnevsky on November 4, 1968, experimental studies on isolated heart preservation in order to preserve its viability were conducted; models of orthotopic and heterotopic heart transplantation on large and small animals were created; morphological, physiological, biochemical and immunological changes in the transplanted heart were studied. The second heart transplantation in this country was performed by G.M. Solovyev on June 10, 1971, but it was also unsuccessful. It’s remarkable that the 20-year experience of V.P. Demikhov in experimental heart transplantation was used only partially: a number of surgeons applied the cardiopulmonary complex isolated according to V.P. Demikhov's technique for biological heart preservation in experimental animals. At the same time after the successful heart transplantation performed by C. Barnard on December 3, 1967, the world boom of clinical transplantation began. In 1969, 101 such operations were performed. For example, D. Cooley performed them on 21 patients and on another one performed two surgeries. There were observations that patients operated on in 1968 lived 800–900 days or more. The best results were shown by R. Lower, M. DeBakey and N. Shumway. However, the vast majority of heart transplants resulted in lethal outcomes in the immediate or distant postoperative period. This led to the fact that the euphoria of successful transplants gradually began to diminish: in 1969 only 47 operations were performed, and in 1970–1971 only 17 surgeries per year.

337.2: Comparison of Hypothermic Static and Normothermic Ex-situ Donor Heart Preservation in Heterotopic Heart Transplantation With the Murine Model

Introduction and Background: Hypothermic static and Normothermic ex-situ preservation methods are the most widely used preservation technique worldwide. While cold static storage is associated with subsequent ischemic injury, Normothermic perfusion preservation has the potential to elongate preservation time with less ischemia. The current study compares the hypothermic and normothermic preservation methods in terms of graft performance, morphologic changes, and acute immune response in an experimental model. Materials and Method: Twenty-five rats underwent heterotopic abdominal heart transplantation after two hours of donor heart hypothermic storage (HT group; n=10) or normothermic ex-situ preservation (NT group; n=15). Blood samples were obtained just before and after 4-hours of implantation to analyze surface markers of immune cells (CD4, CD8, CD161, CD45R). ECG and Echocardiography were performed before harvesting and after implantation. Hearts were extracted after four hours of implantation for Hematoxylin-Eosin (HE) and TUNEL-staining, and recipient rats were euthanized. Results: 19 (76%) rats successfully survived after implantation (HT group: 100%, NT group: 60%). The mean ischemic time of the donor heart was 162.7±8.34 minutes in the HT group and 45±3.7 minutes in the NT group. The NT group showed no significant change in the heart rate before and after implantation but significantly decreased in the first hour of implantation in the HT group (259.5±24.3rpm) and normalized at 3 hours (338.3±34.1rpm). Ejection fraction and fractional shortening significantly decreased after implantation in both groups but were less significant in the NT group (P=0.001). Granulocyte was less significantly increased in the NT group compared to the HT group after the experiment (p=0.037). Although the gross structure was well preserved in both groups in HE-staining, the number of TUNEL positive cells was significantly higher in the HT group. Conclusions: Our findings suggest that normothermic ex-situ preservation is associated with well-preserved donor hearts but similar recipient immune response in comparison with hypothermic static preservation.

Anti-HLA-A2-CAR Tregs prolong vascularized mouse heterotopic heart allograft survival

Alloantigen-specific regulatory T cell (Treg) therapy is a promising approach for suppressing alloimmune responses and minimizing immunosuppression after solid organ transplantation. Chimeric antigen receptor (CAR) targeting donor alloantigens can confer donor reactivity to Tregs. However, CAR Treg therapy has not been evaluated in vascularized transplant or multi-MHC mismatched models. Here, we evaluated the ability of CAR Tregs targeting HLA-A2 (A2-CAR) to prolong the survival of heterotopic heart transplants in mice. After verifying the in vitro activation, proliferation, and enhanced suppressive function of A2-CAR Tregs in the presence of A2-antigen, we analyzed the in vivo function of Tregs in C57BL/6 (B6) mice receiving A2-expressing heart allografts. A2-CAR Treg infusion increased the median survival of grafts from B6.HLA-A2 transgenic donors from 23 to 99days, whereas median survival with polyclonal Treg infusion was 35days. In a more stringent model of haplo-mismatched hearts from BALB/cxB6.HLA-A2 F1 donors, A2-CAR Tregs slightly increased median graft survival from 11 to 14days, which was further extended to >100days when combined with a 9-day course of rapamycin treatment. These findings demonstrate the efficacy of CAR Tregs, alone or in combination with immunosuppressive agents, toward protecting vascularized grafts in fully immunocompetent recipients.

Abstract P3069: The Impact Of Lymphatic Vasculature Post-Cardiac Transplantation On Allograft Function, Fibrotic Remodeling, And Lymphatic Drainage

Lymphatic drainage inherently modulates cardiac function by maintaining the immune response and tissue-fluid homeostasis. During cardiac transplantation, the lymphatic collecting vessels are severed at the time of donor heart excision and not surgically reconstructed in the recipient. We hypothesize severed cardiac lymphatics contribute to transplant rejection and reduced cardiac function by influencing myocardial edema and immune cell transit. Methods: A heterotopic abdominal heart transplant (HAHT) rodent model permitted the immunologic evaluation of transplant rejection as supported in past literature. Echocardiography confirmed allograft survival via active contraction, while histologic quantification of lymphatic vessels and fibrosis were obtained for native and transplanted rodent hearts (n=3). Lymphatic drainage was assessed via intramyocardial injection of fluorescein isothiocyanate (FITC)-dextran. Results: Electrocardiographic impulses detected during the echocardiograms revealed additional QRS complexes attributed to contractions of the transplanted hearts. Histologic analysis of fibrosis in rodent allografts at an early timepoint exhibited increases in total tissue accumulation (1.48 – 5.15 fold) and significant increases in various focal regions (left ventricle, ***p &lt; 0.001; interventricular septum, ****p &lt; 0.0001) compared to the native hearts. Furthermore, large amounts of cellular infiltrate and marginal increases in cardiac diameter (1.10 – 1.42 fold) were observed in the transplants. While the scarcity of immunofluorescence signal for lymphatic markers has caused cardiac lymphatic quantification to be inconclusive, evaluation of lymphatic transport revealed fluctuating time-dependent circulating FITC levels (0.002 - 0.010 mg/mL; *p &lt; 0.05) suggesting multi-teared compartmental release properties between the microcirculatory system, lymphatic system, and cardiac tissue. Conclusion: The HAHT rodent model provides a promising modality to assess both lymphatic transport and transplant rejection. Moreover, these studies will progress understanding of transplant rejection’s pathological intricacies and provide the groundwork for lymphangiogenic-based therapeutic intervention.

Abstract P1003: Mechanical Loading Controls Cardiomyocyte Proliferation

Introduction: Mammal cardiomyocytes (CMs) lose their proliferative capacity early after birth and switch to an adult phenotype, characterized by organized sarcomeres. The mechanisms that control this switch remain largely elusive. Among the changes experienced by the heart at birth is a sudden increase in mechanical load. Hypothesis: This study tests the hypothesis that variations in mechanical loading may regulate both the proliferation and the maturation of cardiomyocytes. Methods: Engineered heart tissues (EHTs) were generated using neonatal rat heart cells, with a modified protocol allowing to alter mechanical load. Unloading is obtained by reducing the distance between the silicon posts that anchor the extremities of the EHT, while overloading is induced by changing Young’s module of posts using metal braces. Adult heart unloading in vivo was achieved by heterotopically transplanting a mouse heart into the neck of syngeneic recipient mice, which were injected with EdU twice per week. Both hearts, native and transplanted, were harvested at day 30. Results: In line with our hypothesis, mechanical unloading of developed EHTs significantly increased the percentage of EdU-, Ki67- and pH3-positive CMs at 72 hours. In contrast, increasing afterload induced CM maturation, characterized by increased cross-sectional area and force of contraction, associated with reduced proliferation. Consistently, heterotopically transplanted hearts showed a significant increase in the the number of proliferating CMs in both ventricles, which were almost absent in native hearts. Conclusions: Our data indicate that mechanical unloading induce CM proliferation in both rat EHTs and an in vivo in a mouse model of heterotopic heart transplantation. These results support our initial hypothesis and indicate that mechanical loading is a master regulator of cardiomyocytes proliferation and maturation. Ongoing experiments aim at identifying the molecular mechanisms that regulate this process.

Lactobacillus rhamnosus HN001 Ameliorates BEZ235-Induced Intestinal Dysbiosis and Prolongs Cardiac Transplant Survival.

ABSTRACTCardiac allograft rejection remains a major factor limiting long-term engraftment after transplantation. A novel phosphoinositide 3-kinase (PI3K)/mTOR dual inhibitor, BEZ235, prolonged cardiac allograft survival by effectively suppressing activation of the PI3K/serine/threonine kinase (AKT)/mTOR pathway. However, long-term usage of pharmacological immunosuppressant drugs can cause intestinal microbiota dysbiosis. We established mouse models of allogeneic heterotopic heart transplantation with different treatments. Fecal samples were collected and subjected to 16S rRNA sequencing and targeted fecal metabolomic analysis. Graft samples were taken for immune cell detection by flow cytometry. Inflammatory cytokines in serum were quantified by enzyme-linked immunosorbent assay (ELISA). Compared to single-target approaches (IC-87114 and rapamycin), BEZ235 more efficiently prolongs cardiac transplant survival. Interestingly, BEZ235 reduces the diversity and abundance of the intestinal microbiota community. We demonstrated that Lactobacillus rhamnosus HN001 rescues the intestinal microbiota imbalance induced by BEZ235.IMPORTANCE Our data confirmed that the combination of BEZ235 and Lactobacillus rhamnosus HN001 significantly prolongs cardiac transplant survival. A main metabolic product of Lactobacillus rhamnosus HN001, propionic acid (PA), enriches regulatory T (Treg) cells and serves as a potent immunomodulatory supplement to BEZ235. Our study provides a novel and efficient therapeutic strategy for transplant recipients.

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs.

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Linking erythropoietin to Treg-dependent allograft survival through myeloid cells.

Erythropoietin (EPO) has multiple nonerythropoietic functions, including immune modulation, but EPO’s effects in transplantation remain incompletely understood. We tested the mechanisms linking EPO administration to prolongation of murine heterotopic heart transplantation using WT and conditional EPO receptor–knockout (EPOR-knockout) mice as recipients. In WT controls, peritransplant administration of EPO synergized with CTLA4-Ig to prolong allograft survival (P < 0.001), reduce frequencies of donor-reactive effector CD8+ T cells in the spleen (P < 0.001) and in the graft (P < 0.05), and increase frequencies and total numbers of donor-reactive Tregs (P < 0.01 for each) versus CTLA4-Ig alone. Studies performed in conditional EPOR-knockout recipients showed that each of these differences required EPOR expression in myeloid cells but not in T cells. Analysis of mRNA isolated from spleen monocytes showed that EPO/EPOR ligation upregulated macrophage-expressed, antiinflammatory, regulatory, and pro-efferocytosis genes and downregulated selected proinflammatory genes. Taken together, the data support the conclusion that EPO promotes Treg-dependent murine cardiac allograft survival by crucially altering the phenotype and function of macrophages. Coupled with our previous documentation that EPO promotes Treg expansion in humans, the data support the need for testing the addition of EPO to costimulatory blockade-containing immunosuppression regimens in an effort to prolong human transplant survival.

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.

Gluconate-Lactobionate-Dextran Perfusion Solutions Attenuate Ischemic Injury and Improve Function in a Murine Cardiac Transplant Model.

Static cold storage is the cheapest and easiest method and current gold standard to store and preserve donor organs. This study aimed to compare the preservative capacity of gluconate-lactobionate-dextran (Unisol) solutions to histidine-tryptophan-ketoglutarate (HTK) solution. Murine syngeneic heterotopic heart transplantations (Balb/c-Balb/c) were carried out after 18 h of static cold storage. Cardiac grafts were either flushed and stored with Unisol-based solutions with high-(UHK) and low-potassium (ULK) ± glutathione, or HTK. Cardiac grafts were assessed for rebeating and functionality, histomorphologic alterations, and cytokine expression. Unisol-based solutions demonstrated a faster rebeating time (UHK 56 s, UHK + Glut 44 s, ULK 45 s, ULK + Glut 47 s) compared to HTK (119.5 s) along with a better contractility early after reperfusion and at the endpoint on POD 3. Ischemic injury led to a significantly increased leukocyte recruitment, with similar degrees of tissue damage and inflammatory infiltrate in all groups, yet the number of apoptotic cells tended to be lower in ULK compared to HTK. In UHK- and ULK-treated animals, a trend toward decreased expression of proinflammatory markers was seen when compared to HTK. Unisol-based solutions showed an improved preservative capacity compared with the gold standard HTK early after cardiac transplantation. Supplemented glutathione did not further improve tissue-protective properties.