Heterotopic HTX Research Articles

Preservation solution Custodiol containing human alpha-1-antitrypsin improves graft recovery after prolonged cold ischemic storage in a rat model of heart transplantation.

The shortage of available donor hearts and the risk of ischemia/reperfusion injury restrict heart transplantation (HTX). Alpha-1-antitrypsin (AAT), a well-characterized inhibitor of neutrophil serine protease, is used in augmentation therapy to treat emphysema due to severe AAT deficiency. Evidence demonstrates its additional anti-inflammatory and tissue-protective effects. We hypothesized that adding human AAT in a preservation solution reduces graft dysfunction in a rat model of HTX following extended cold ischemic storage. The hearts from isogenic Lewis donor rats were explanted, stored for either 1h or 5h in cold Custodiol supplemented with either vehicle (1h ischemia, n=7 or 5h ischemia, n=7 groups) or 1 mg/ml AAT (1h ischemia+AAT, n=7 or 5h ischemia+AAT, n=9 groups) before heterotopic HTX. Left-ventricular (LV) graft function was evaluated in vivo 1.5h after HTX. Immunohistochemical detection of myeloperoxydase (MPO) was performed in myocardial tissue and expression of 88 gene quantified with PCR was analyzed both statistical and with machine-learning methods. After HTX, LV systolic function (dP/dtmax 1h ischemia+AAT 4197 ± 256 vs 1h ischemia 3123 ± 110; 5h ischemia+AAT 2858 ± 154 vs 5h ischemia 1843 ± 104mmHg/s, p<0.05) and diastolic function (dP/dtmin 5h ischemia+AAT 1516 ± 68 vs 5h ischemia 1095 ± 67mmHg/s, p<0.05) at an intraventricular volume of 90µl were improved in the AAT groups compared with the corresponding vehicle groups. In addition, the rate pressure product (1h ischemia+AAT 53 ± 4 vs 1h ischemia 26 ± 1; 5h ischemia+AAT 37 ± 3 vs 5h ischemia 21 ± 1mmHg*beats/min at an intraventricular volume of 90µl; p<0.05) was increased in the AAT groups compared with the corresponding vehicle groups. Moreover, the 5h ischemia+AAT hearts exhibited a significant reduction in MPO-positive cell infiltration in comparison to the 5h ischemia group. Our computational analysis shows that ischemia+AAT network displays higher homogeneity, more positive and fewer negative gene correlations than the ischemia+placebo network. We provided experimental evidence that AAT protects cardiac grafts from prolonged cold ischemia during HTX in rats.

Dysregulated regulatory T cell responses target fibroblasts and lead to chronic rejection after heart transplantation

Abstract Suppressive and reparative regulatory T cell (Treg) functions are described. However, in transplantation (Tx), recipient immune responses to persistent MHC differences will sustain injury and impact repair. The development of immunosuppressant-resistant graft vasculopathy and fibrosis, or chronic rejection (CR) remains a leading cause of death post-heart Tx (HTx) and may reflect failed tissue repair. We tested the hypothesis that Treg repair responses become dysregulated due to sustained alloreactive immune responses. Our studies utilized IL-33 as a model injury signal, or damage-associated molecular pattern (DAMP) and B6 (H-2b) CD45.1+ mice received heterotopic HTx from MHCII-mismatched Bm12 (H2-I-Abm12) donors that were IL-33-deficient or competent. On day (d)14, scRNAseq and CITE-seq were used to compare CD45.1+ cells from HTx digests (n=4). IL-33 modulated &amp;gt;800 genes in HTx-infiltrating Treg, including the growth factor amphiregulin (Areg; 2.25x; P=0.0241). Areg is implicated in tissue repair, yet when B6 Foxp3YFP-CrexAregfl/fl and Foxp3YFP-Cre mice receiving Bm12 HTx were assessed at d100 (n=6–10), the deletion of Areg in Treg protected against CR. Using IncuCyte Live-Cell imaging, it was clear that Areg from Treg stimulated fibroblast proliferation and migration. Tregs stimulated with IL-2 alone or with IL-33 were assessed at d14 or d21 for CpG methylation of Treg-associated genes. These data revealed that sustained exposure to IL-33 increased methylation of Foxp3, suggesting that sustained DAMP stimulation makes Treg unstable. In total, our studies identify how a sustained and dysregulated repair response involving interactions between fibroblasts and recipient Treg contributes to the progression of HTx CR. Supported by grants from NIH (F30AI147437, T32 CA082084) (GKD), NIH (R01HL122489, R01AR073527) (HRT)

Targeting fatty acid β-oxidation impairs monocyte differentiation and prolongs heart allograft survival.

Monocytes play an important role in the regulation of alloimmune responses after heart transplantation (HTx). Recent studies have highlighted the importance of immunometabolism in the differentiation and function of myeloid cells. While the importance of glucose metabolism in monocyte differentiation and function has been reported, a role for fatty acid β-oxidation (FAO) has not been explored. Heterotopic HTx was performed using hearts from BALB/c donor mice implanted into C57BL/6 recipient mice and treated with etomoxir (eto), an irreversible inhibitor of carnitine palmitoyltransferase 1 (Cpt1), a rate-limiting step of FAO, or vehicle control. FAO inhibition prolonged HTx survival, reduced early T cell infiltration/activation, and reduced DC and macrophage infiltration to heart allografts of eto-treated recipients. ELISPOT demonstrated that splenocytes from eto-treated HTx recipients were less reactive to activated donor antigen-presenting cells. FAO inhibition reduced monocyte-to-DC and monocyte-to-macrophage differentiation in vitro and in vivo. FAO inhibition did not alter the survival of heart allografts when transplanted into Ccr2-deficient recipients, suggesting that the effects of FAO inhibition were dependent on monocyte mobilization. Finally, we confirmed the importance of FAO on monocyte differentiation in vivo using conditional deletion of Cpt1a. Our findings demonstrate that targeting FAO attenuates alloimmunity after HTx, in part through impairing monocyte differentiation.

Abstract 13594: Cyclosporine-A Reduces Myocardial Damage in Donation After Circulatory Death Hearts

Background: Mitochondria contribute to ischemia and reperfusion injury (IRI) in donation after circulatory death (DCD) donor hearts. Cyclosporine A (CyA), an inhibitor of mitochondrial permeability transition pore (MPTP) opening, limits IRI. CyA administration at the onset of reperfusion is essential for therapeutic efficacy. DCD heart transplantation (HTx) is a unique setting for controlled administration of CyA. Hypothesis: CyA administered to DCD hearts during reperfusion is cardioprotective. Methods: Under general anesthesia Sprague Dawley rats underwent simulated clinical DCD setup with a global ischemia of 25 minutes. Control beating-heart donor (CBD) hearts served as controls (n=10). Hearts were reanimated ex-vivo with Krebs-Henseleit buffer (KH) perfusion at 37 o C for 90 minutes. DCD hearts were randomly assigned to receive CyA (0.5mM) or DMSO added to KH buffer for the first 15 minutes of perfusion (n=10 each). The hearts were collected to measure oxidative phosphorylation (OXPHOS) and calcium retention capacity (CRC). Additional CBD (n=5) and DCD (with or without CyA, n=15) hearts underwent heterotopic HTx in recipient rats to measure developed pressure (DP) and rate pressure product (RPP) at 48 hours. Results: Compared to CBD hearts, OXPHOS in DCD hearts was significantly reduced. CyA improved OXPHOS and was similar to CBD hearts. CRC was reduced in DCD hearts compared to CBD hearts (317 vs.578nM/mg) and was increased with CyA treatment (413 vs. 317nM/mg; p&lt;0.05, Figure). Infarct size decreased significantly with CyA treatment (15% vs. 25%). In transplanted DCD hearts, DP and RPP were significantly reduced compared to CBD hearts (80 vs.153mmHg and 17639 vs. 29545mmHg*bpm). CyA treatment improved DP (124vs.80mmHg; p&lt;0.05) and RPP (35491vs.17639mmHg*bpm; p&lt;0.05) which was comparable to CBD hearts. Conclusions: CyA administered at reperfusion reduces mitochondrial damage, limits infarct size, and preserves function in transplanted DCD hearts.

Methane supplementation improves graft function in experimental heart transplantation

Maintenance of cell viability during cold storage is a key issue in organ transplantation. Methane (CH4) bioactivity has recently been recognized in ischemia/reperfusion conditions; we therefore hypothesized that cold storage in CH4-enriched preservation solution can provide an increased defense against organ dysfunction during experimental heart transplantation (HTX). The hearts of donor Lewis rats were stored for 60 minutes in cold histidine-tryptophan-ketoglutarate (Custodiol [CS]) or CH4-saturated CS solution (CS-CH4) (n = 12 each). Standard heterotopic HTX was performed, and 60 minutes later, the left ventricular (LV) pressure-volume relationships LV systolic pressure (LVSP), systolic pressure increment (dP/dtmax), diastolic pressure decrement, and coronary blood flow (CBF) were measured. Tissue samples were taken to detect proinflammatory parameters, structural damage (by light microscopy), endoplasmic reticulum (ER) stress, and apoptosis markers (CCAAT/enhancer binding protein [C/EBP] homologous protein, GRP78, glycogen synthase kinase-3β, very low-density lipoprotein receptor, caspase 3 and 9, B-cell lymphoma 2, and bcl-2-like protein 4), whereas mitochondrial functional changes were analyzed by high-resolution respirometry. LVSP and dP/dtmax increased significantly at the largest pre-load volumes in CS-CH4 grafts as compared with the CS group (114.5 ± 16.6 mmHg vs 82.8 ± 4.6 mmHg and 3,133 ± 430 mmHg/s vs 1,739 ± 169 mmHg/s, respectively); the diastolic function and CBF (2.4 ± 0.4 ml/min/g vs 1.3 ± 0.3 ml/min/g) also improved. Mitochondrial oxidative phosphorylation capacity was more preserved (58.5 ± 9.4 pmol/s/ml vs 27.7 ± 6.6 pmol/s/ml), and cytochrome c release was reduced in CS-CH4 storage. Signs of HTX-caused myocardial damage, level of ER stress, and the transcription of proapoptotic proteins were significantly lower in CS-CH4 grafts. The addition of CH4 during 1 hour of cold storage improved early in vitro graft function and reduced mitochondrial dysfunction and activation of inflammation. Evidence shows that CH4 reduced ER stress-linked proapoptotic signaling.

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.

Complex ventricular arrhythmias in patients after heterotopic heart transplantation

Heart transplantation (HTx) has become a valuable therapy of end-stage heart failure and for some other specific indications [1]. Mostly, the native heart is replaced by the donor heart. Only in rare cases, a heterotopic HTx is performed where the donor heart is inserted auxiliary to the native heart which stays in situ, as a kind of biological biventricular assist device [2]. This kind of transplantation is mostly used if the pulmonary resistance of the recipient is too high and if there is no possibility to transplant heart and lungs.

363: Intraluminal Bone Marrow Cells Inhibit Allograft Fibrosis Via the Indoleamine Dioxygenase Pathway

Purpose: Bilirubin is a final byproduct of the heme oxygenase system and is known to be a powerful antioxidant. In this study, we examined whether recipient hyperbilirubinemia would protect cardiac grafts from ischemia/reperfusion (I/R) injury and chronic rejection in a rat heart transplantation (HTx) model. Methods and Materials: Heterotopic HTx was performed using congenitally hyperbilirubinemic GUNN (j/j) and normobilirubinemic GUNN ( / ) rats. Serum bilirubin levels of j/j rats were 4.8 mg/dl, while / rats had bilirubin levels 0.1 mg/dl. To evaluate I/R injury, syngenic grafts from / were transplanted into / or j/j rats with 18 hrs cold storage in UW solution. To evaluate the effect on allograft rejection, fully allogenic BN heart grafts were transplanted into / or j/j rats under a short course of tacrolimus (0.5 mg/kg, d0-6). Results: The / grafts in j/j rats resulted in a significantly lower serum CPK level (5492 IU/L) with smaller infarct area determined by TTC stain, compared to those in / recipients 3 hrs after HTx (9617 IU/L). Graft function evaluated by Langendorf apparatus revealed higher left ventricular developed pressure than those in / at 3 hrs after HTx. Tissue MDA levels, a marker of lipid peroxidation, were significantly lower in j/j recipients (16.3 M/g) compared to those in / recipients (39.5 M/g). Graft survival in j/j recipients improved to 41.7% from 0% in / rats and was associated with reduction of the levels of inflammatory mediator mRNA (TNF and IL-6) and phosphorylation of ERK. The mean allograft survival in j/j recipients was prolonged to a median survival of 150 days from 84 days in / recipients. Prolongation of allograft survival was associated with less inflammatory infiltrates and decrease of intragraft inflammatory cytokine mRNA including IFN , TNF and IL-6 at d50. In vitro T cell proliferation was significantly inhibited in the presence of bilirubin. Conclusions: Recipient hyperbilirubinemia ameliorated cardiac I/R injury, as well as chronic allograft rejection following HTx via regulation of inflammatory responses or T cell proliferation.

CD26/DPP IV in experimental and clinical organ transplantation.

The T-cell activation-Ag CD26 possesses dipeptidyl peptidase IV (DPP IV) enzymatic activity. Costimulatory efficacy and immunocompetence are associated with the enzymatic activity. In models of experimental cardiac allograft transplantation (HTx), we analyzed the role of CD26/DPP IV during organ rejection. Also, we investigated CD26 enzymatic and cellular expression in human recipients of kidney transplants (Tx). Heterotopic HTx in rats, models of acute and accelerated rejection. Monitoring of DPP IV serum levels and humoral immunity. Propro-diphenyl phosphonate was employed to inhibit DPP IV activity during rejection. In a prospective study, surface expression of CD26, 3, 4, 8, 45, 122 and ADA on PBL and DPP IV serum activity were measured in kidney recipients for 24 months post-transplantation. Acute rejection was . associated with increased serum DPP IV activity (p < 0.005). Specific inhibition abrogated acute (p < 0.0001) and accelerated (p < 0.01) rejection, impairing cytotoxicity and allospecific Ig-synthesis. Kidney recipients displayed a significant drop in CD26 expression on PBL for up to 18 months postoperatively (p < 0.001). CD4, 8, 45, 122 and ADA expression kinetics were only briefly affected. DPP IV enzymic activity stayed depressed for at least 12 months (p < 0.001). CD26/DPP IV is pivotal in T-cell mediated immune responses toward allo-Ag. In clinical transplantation, engraftment/immunosuppression are reflected by CD26 cellular and enzymatic expression posttransplantation and may serve as an indicator for immunomodulation.

The effect of FTY 720 on engraftment in a model of spontaneous allograft acceptance.

Further development in organ transplantation requires the utilization of new immunosuppressive drugs that-in addition to being effective against rejection-do not block tolerance. We previously reported that FTY 720, a drug that alters lymphocyte trafficking, has marked anti-rejection properties. We now investigate how FTY 720 influences tolerance in a model of graft acceptance by donor-specific blood transfusion (DSBT). Two different transplant models--heart transplantation (Htx) and intestinal transplantation (Itx)--were studied. We performed orthotopic Itx and heterotopic Htx using fully mismatched inbred male RA (RT1(p)) and PVG (RT1(c)) rats as donors and recipients. Tolerance was induced by DSBT on pre-transplant day -12. To test the effect of FTY 720 on DSBT-induced tolerance, we administered FTY 720 orally prior to DSBT. Itx: control rats succumbed to rejection at 18+/-4 days. DSBT alone prolonged survival to 101.9+/-18 days (P<0.05 vs untreated). Long-term survivors were tolerant (acceptance of secondary donor-specific Htx). Adjunction of FTY 720 prior to DSBT reduced survival to 55.9+/-44.7 days (P<0.05). However, long-term survivors still accepted secondary donor-specific Htx. Htx: control rats survived 9+/-0.6 days. DSBT alone prolonged survival indefinitely (>120 days) and induced tolerance (acceptance of secondary donor-specific Htx). Unlike in Itx, adjunction of FTY 720 prior to DSBT did not reduce Htx survival. Acceptance of secondary donor-specific Htx was not influenced by FTY 720. In Itx, FTY 720 counteracts the beneficial effect of pre-transplant DSBT and triggers acute rejection of primary, but not secondary grafts. In Htx, however, FTY 720 allows full development of tolerance. The mechanisms by which FTY 720 causes rejection in primary intestinal but not in heart grafts need to be elucidated.

Heart Transplantation: Intraoperative Management, Postoperative Therapy and Complications

Currently, heart transplantation (HTX) is performed as an orthotopic cardiac replacement according to the technique of Lower and Shumway in over 95% of the cases with good results. Survival after heterotopic HTX, by contrast, remain poor (one year survival: 50%). Postoperative therapy compiles primarily prophylactic measures to prevent complications, especially organ rejection and infections. Immunosuppressive prophylaxis generally includes a triple drug therapy consisting of cyclosporin, prednisolone, and azathioprine for maintenance therapy. Initially there is often an additional application of poly- or monoclonal antibodies. The prime measure to prevent infection during the initial hospital stay will be reversed isolation of the recipient. Initial antibiotic prophylaxis resembles that of conservative cardiac surgery, but in addition antiviral and antifungal prophylaxis is applied. The most common postoperative complication following HTX is cardiac rejection, which is detected by routine endomyocardial biopsies. At our institution the incidence of rejection decreases from 3.07 episodes per patient in the first 3 months to 1.97 episodes during the last 6 months of the first year after HTX. In general, acute rejection is treated by methylprednisolone (3 x 500 mg/day) or anti-T-cell-antibodies. Infections often occur following intervals of increased immunosuppression, usually early postoperatively and following therapy of acute rejections. Often, invasive diagnostic measures have to be taken rapidly in order to allow for specific therapy (antibiotics, antimycotic treatment, virostatic agents). Close follow-up of the heart transplant recipient and rapid therapy of possible postoperative complications enable the current one-year survival rates of 80% or more.