Donor plasma VEGF-A as a biomarker for myocardial injury and primary graft dysfunction after heart transplantation.

BACKGROUND: Vascular endothelial growth factor (VEGF)-A is an angiogenic and proinflammatory cytokine with profound effects on microvascular permeability and vasodilation. Several processes may induce VEGF-A expression in brain-dead organ donors. However, it remains unclear whether donor VEGF-A is linked to adverse outcomes after heart transplantation. METHODS: We examined plasma VEGF-A levels from 83 heart transplant donors as well as the clinical data of these donors and their respective recipients operated between 2010 and 2016. The donor plasma was analyzed using Luminex-based Multiplex and confirmed with a single-target ELISA. Based on donor VEGF-A plasma levels, the recipients were divided into 3 equal-sized groups (low VEGF <500 ng/liter, n = 28; moderate VEGF 500-3000 ng/liter, n = 28; and high VEGF >3000 ng/liter, n = 27). Biochemical and clinical parameters of myocardial injury as well as heart transplant and kidney function were followed-up for one year, while rejection episodes, development of cardiac allograft vasculopathy, and mortality were monitored for 5 years. RESULTS: Baseline parameters were comparable between the donor groups, except for age, where median ages of 40, 45, and 50 were observed for low, moderate, and high donor plasma VEGF levels groups, respectively, and therefore donor age was included as a confounding factor. High donor plasma VEGF-A levels were associated with pronounced myocardial injury (TnT and TnI), a higher inotrope score, and a higher incidence of primary graft dysfunction in the recipient after heart transplantation. Furthermore, recipients with allografts from donors with high plasma VEGF-A levels had a longer length of stay in the intensive care unit and the hospital, and an increased likelihood for prolonged renal replacement therapy. CONCLUSIONS: Our findings suggest that elevated donor plasma VEGF-A levels were associated with adverse outcomes in heart transplant recipients, particularly in terms of myocardial injury, primary graft dysfunction, and long-term renal complications. Donor VEGF-A may serve as a potential biomarker for predicting these adverse outcomes and identifying extended donor criteria.

Transcriptomic Landscape of Circulating Extracellular Vesicles in Heart Transplant Ischemia-Reperfusion.

Ischemia-reperfusion injury (IRI) is an inevitable event during heart transplantation, which is known to exacerbate damage to the allograft. However, the precise mechanisms underlying IRI remain incompletely understood. Here, we profiled the whole transcriptome of plasma extracellular vesicles (EVs) by RNA sequencing from 41 heart transplant recipients immediately before and at 12 h after transplant reperfusion. We found that the expression of 1317 protein-coding genes in plasma EVs was changed at 12 h after reperfusion. Upregulated genes of plasma EVs were related to metabolism and immune activation, while downregulated genes were related to cell survival and extracellular matrix organization. In addition, we performed correlation analyses between EV transcriptome and intensity of graft IRI (i.e., cardiomyocyte injury), as well as EV transcriptome and primary graft dysfunction, as well as any biopsy-proven acute rejection after heart transplantation. We ultimately revealed that at 12 h after reperfusion, 4 plasma EV genes (ITPKA, DDIT4L, CD19, and CYP4A11) correlated with both cardiomyocyte injury and primary graft dysfunction, suggesting that EVs are sensitive indicators of reperfusion injury reflecting lipid metabolism-induced stress and imbalance in calcium homeostasis. In conclusion, we show that profiling plasma EV gene expression may enlighten the mechanisms of heart transplant IRI.

Ultrasound-guided closure of the femoral artery during venoarterial decannulation using a large-bore closure device.

BACKGROUND: Peripheral femoro-femoral venoarterial extracorporeal membrane oxygenation (VA-ECMO) is viable for fast hemodynamic assistance during cardiogenic shock. Ultrasound-guided closure with a large-bore device (MANTA®) is a feasible option potentially replacing surgical arteriotomy closure in peripheral VA-ECMO decannulation. METHODS: This retrospective study included patients weaning from percutaneously inserted femoro-femoral VA-ECMO at the Helsinki University Hospital, Finland in 2012-2020. The primary endpoints were access-site complications, a composite of hematomas/seromas/surgical site infections (SSIs), and the safety endpoint of vascular complications (VCs). RESULTS: A total of 100 consecutive percutaneously implanted and weaned VA-ECMO patients were stratified into two groups by decannulation strategy: percutaneous ultrasound-guided MANTA device (n = 21, 21.0%) or surgical approach (n = 79, 79.0%). The mean age of the cohort was 51 ± 13 years and females represented 25.0%. The technical success rate of the percutaneous ultrasound-guided MANTA technique was 95.2%. In multivariate analysis, surgical closure was associated with a higher incidence of combined access site hematomas/seromas/SSIs compared to percutaneous ultrasound-guided deployment of MANTA device (44.3% versus 9.5%, odds ratio (OR): 7.162, 95% confidence interval (CI): 1.544-33.222; p = 0.012). Similarly, access-site complications necessitating interventions were more frequent in the surgical closure group compared to US-MANTA (ultrasound-guided MANTA) group (26.6% versus 0.0%, p = 0.005). VCs were infrequent in both groups without any significant intergroup difference (p > 0.99). CONCLUSIONS: Percutaneous ultrasound-guided MANTA closure of the femoral artery after VA-ECMO decannulation was associated with high technical success rate and low incidence of VCs. Compared to surgical closure, access-site complications were significantly less frequent, along with access-site complications necessitating interventions.

Severe primary graft dysfunction of the heart transplant is associated with increased plasma and intragraft proinflammatory cytokine expression.

INTRODUCTION: Heart transplant results have constantly improved but primary left ventricle graft dysfunction (LV-PGD) remains a devastating complication early after transplantation. Donor and recipient systemic inflammatory response may be involved in immune activation of the transplant, and LV-PGD development. Here, we investigated donor and recipient plasma and intragraft cytokine profiles preoperatively and during LV-PGD and searched for predictive markers for LV-PGD. METHODS: Donor and recipient plasma samples (n = 74) and myocardial biopsies of heart transplants (n = 64) were analyzed. Plasma and intragraft cytokine levels were determined by multiplexed and next-generation sequencing platforms, respectively. The development of LV-PGD during the first 24 hours, and graft function and mortality up to 1 year after transplantation, were examined. RESULTS: Severe LV-PGD, but not mild or moderate LV-PGD, was significantly associated with early mortality, plasma high-sensitivity troponin elevation, and an increase in intragraft and plasma proinflammatory cytokines during reperfusion. Preoperative donor and recipient plasma cytokine levels failed to predict LV-PGD. Cytokine network analysis identified interleukins -6, -8, -10, and -18 as key players during reperfusion. Prolonged cold and total ischemia time, and increased need for red blood cell transfusions during operation were identified as clinical risk factors for severe LV-PGD. CONCLUSIONS: Severe LV-PGD was associated with a poor clinical outcome. Donor and recipient plasma cytokine profile failed to predict LV-PGD, but severe LV-PGD was associated with an increase in post-reperfusion intragraft and recipient plasma proinflammatory cytokines. Identified key cytokines may be potential therapeutic targets to improve early and long-term outcomes after heart transplantation.

Plasma proteome of brain-dead organ donors predicts heart transplant outcome.

BACKGROUND: The pathophysiological changes related to brain death may affect the quality of the transplanted organs and expose the recipients to risks. We probed systemic changes reflected in donor plasma proteome and investigated their relationship to heart transplant outcomes. METHODS: Plasma samples from brain-dead multi-organ donors were analyzed by label-free protein quantification using high-definition mass spectrometry. Unsupervised and supervised statistical models were used to determine proteome differences between brain-dead donors and healthy controls. Proteome variation and the corresponding biological pathways were analyzed and correlated with transplant outcomes. RESULTS: Statistical models revealed that donors had a unique but heterogeneous plasma proteome with 237 of 463 proteins being changed compared to controls. Pathway analysis showed that coagulation, gluconeogenesis, and glycolysis pathways were upregulated in donors, while complement, LXR/RXR activation, and production of nitric oxide and reactive oxygen species in macrophages pathways were downregulated. In point-biserial correlation analysis, lysine-specific demethylase 3A was moderately correlated with any grade and severe PGD. In univariate and multivariate Cox regression analyses myosin Va and proteasome activator complex subunit 2 were significantly associated with the development of acute rejections with hemodynamic compromise within 30 days. Finally, we found that elevated levels of lysine-specific demethylase 3A and moesin were identified as predictors for graft-related 1-year mortality in univariate analysis. CONCLUSIONS: We show that brain death significantly changed plasma proteome signature Donor plasma protein changes related to endothelial cell and cardiomyocyte function, inflammation, and vascular growth and arteriogenesis could predict transplant outcome suggesting a role in donor evaluation.

Epitranscriptomics of Ischemic Heart Disease-The IHD-EPITRAN Study Design and Objectives.

Epitranscriptomic modifications in RNA can dramatically alter the way our genetic code is deciphered. Cells utilize these modifications not only to maintain physiological processes, but also to respond to extracellular cues and various stressors. Most often, adenosine residues in RNA are targeted, and result in modifications including methylation and deamination. Such modified residues as N-6-methyl-adenosine (m6A) and inosine, respectively, have been associated with cardiovascular diseases, and contribute to disease pathologies. The Ischemic Heart Disease Epitranscriptomics and Biomarkers (IHD-EPITRAN) study aims to provide a more comprehensive understanding to their nature and role in cardiovascular pathology. The study hypothesis is that pathological features of IHD are mirrored in the blood epitranscriptome. The IHD-EPITRAN study focuses on m6A and A-to-I modifications of RNA. Patients are recruited from four cohorts: (I) patients with IHD and myocardial infarction undergoing urgent revascularization; (II) patients with stable IHD undergoing coronary artery bypass grafting; (III) controls without coronary obstructions undergoing valve replacement due to aortic stenosis and (IV) controls with healthy coronaries verified by computed tomography. The abundance and distribution of m6A and A-to-I modifications in blood RNA are charted by quantitative and qualitative methods. Selected other modified nucleosides as well as IHD candidate protein and metabolic biomarkers are measured for reference. The results of the IHD-EPITRAN study can be expected to enable identification of epitranscriptomic IHD biomarker candidates and potential drug targets.

Challenges and satisfaction in Cardiothoracic Surgery Residency Programmes: insights from a Europe-wide survey.

OBJECTIVES: The increasing complexity of surgical patients and working time constraints represent challenges for training. In this study, the European Association for Cardio-Thoracic Surgery Residents' Committee aimed to evaluate satisfaction with current training programmes across Europe. METHODS: We conducted an online survey between October 2018 and April 2019, completed by a total of 219 participants from 24 countries. RESULTS: The average respondent was in the fourth or fifth year of training, mostly on a cardiac surgery pathway. Most trainees follow a 5-6-year programme, with a compulsory final certification exam, but no regular skills evaluation. Only a minority are expected to take the examination by the European Board of Cardiothoracic Surgery. Participants work on average 61.0 ± 13.1 h per week, including 27.1 ± 20.2 on-call. In total, only 19.7% confirmed the implementation of the European Working Time Directive, with 42.0% being unaware that European regulations existed. Having designated time for research was reported by 13.0%, despite 47.0% having a postgraduate degree. On average, respondents rated their satisfaction 7.9 out of 10, although 56.2% of participants were not satisfied with their training opportunities. We found an association between trainee satisfaction and regular skills evaluation, first operator experience and protected research time. CONCLUSIONS: On average, residents are satisfied with their training, despite significant disparities in the quality and structure of cardiothoracic surgery training across Europe. Areas for potential improvement include increasing structured feedback, research time integration and better working hours compliance. The development of European guidelines on training standards may support this.

Randomised sham-controlled double-blind trial evaluating remote ischaemic preconditioning in solid organ transplantation: a study protocol for the RIPTRANS trial.

INTRODUCTION: Remote ischaemic preconditioning (RIPC) using a non-invasive pneumatic tourniquet is a potential method for reducing ischaemia-reperfusion injury. RIPC has been extensively studied in animal models and cardiac surgery, but scarcely in solid organ transplantation. RIPC could be an inexpensive and simple method to improve function of transplanted organs. Accordingly, we aim to study whether RIPC performed in brain-dead organ donors improves function and longevity of transplanted organs. METHODS AND ANALYSES: RIPTRANS is a multicentre, sham-controlled, parallel group, randomised superiority trial comparing RIPC intervention versus sham-intervention in brain-dead organ donors scheduled to donate at least one kidney. Recipients of the organs (kidney, liver, pancreas, heart, lungs) from a randomised donor will be included provided that they give written informed consent. The RIPC intervention is performed by inflating a thigh tourniquet to 300 mm Hg 4 times for 5 min. The intervention is done two times: first right after the declaration of brain death and second immediately before transferring the donor to the operating theatre. The sham group receives the tourniquet, but it is not inflated. The primary endpoint is delayed graft function (DGF) in kidney allografts. Secondary endpoints include short-term functional outcomes of transplanted organs, rejections and graft survival in various time points up to 20 years. We aim to show that RIPC reduces the incidence of DGF from 25% to 15%. According to this, the sample size is set to 500 kidney transplant recipients. ETHICS AND DISSEMINATION: This study has been approved by Helsinki University Hospital Ethics Committee and Helsinki University Hospital's Institutional Review Board. The study protocol was be presented at the European Society of Organ Transplantation congress in Copenhagen 14-15 September 2019. The study results will be submitted to an international peer-reviewed scientific journal for publication. TRIAL REGISTRATION NUMBER: NCT03855722.

Donor Simvastatin Treatment in Heart Transplantation.

BACKGROUND: Ischemia-reperfusion injury may compromise the short-term and long-term prognosis after heart transplantation. Experimental studies show that simvastatin administered to the organ donor is vasculoprotective and inhibits cardiac allograft ischemia-reperfusion injury. METHODS: Eighty-four multiorgan donors were randomly assigned to receive 80 mg of simvastatin (42 donors) via nasogastric tube after declaration of brain death and upon acceptance as a cardiac donor, or to receive no simvastatin (42 donors). The primary efficacy end point was postoperative plasma troponin T and I levels during the first 24 hours after heart transplantation. Secondary end points included postoperative hemodynamics, inflammation, allograft function, rejections and rejection treatments, and mortality. Results: Organ donor simvastatin treatment significantly reduced the heart recipient plasma levels of troponin T by 34% (14 900 ± 12 100 ng/L to 9800 ± 7900 ng/L, P=0.047), and troponin I by 40% (171 000 ± 151 000 ng/L to 103 000 ± 109 000 ng/L, P=0.023) at 6 hours after reperfusion, the levels of NT-proBNP (N-terminal pro-B-type natriuretic peptide) by 36% (32 800 ± 24 300 ng/L to 20 900 ± 15 900 ng/L; P=0.011) at 1 week, and the number of rejection treatments with hemodynamic compromise by 53% within the first 30 days (P=0.046). Donor simvastatin treatment did not affect donor lipid levels but was associated with a specific transplant myocardial biopsy gene expression profile, and a decrease in recipient postoperative plasma levels of CXCL10 (C-X-C motif chemokine 10), interleukin-1α, placental growth factor, and platelet-derived growth factor-BB. Postoperative hemodynamics, biopsy-proven acute rejections, and mortality were similar. No adverse effects were seen in recipients receiving noncardiac solid organ transplants from simvastatin-treated donors. CONCLUSIONS: Donor simvastatin treatment reduces biomarkers of myocardial injury after heart transplantation, and-also considering its documented general safety profile-may be used as a novel, safe, and inexpensive adjunct therapy in multiorgan donation. CLINICAL TRIAL REGISTRATION: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01160978.

Hypoxia-inducible factor controls immunoregulatory properties of myeloid cells in mouse cardiac allografts - an experimental study.

Hypoxia-inducible factors (HIFs) play a critical role in inflammatory properties of myeloid-derived cells. The effect of HIFs on myeloid-derived cell functions in organ transplantation remains unknown, however. We transplanted hearts into transgenic mice with myeloid cell-targeted deletions of HIF-1α or its negative regulator von Hippel-Lindau (VHL) to investigate the effects of HIF-1α inactivation or HIF pathway activation, respectively, on ischemia-reperfusion injury (IRI) and acute rejection. Deletion of VHL in myeloid cells enhanced mRNA expression of anti-inflammatory genes IDO, Arg-1, and HO-1 in vitro. In vivo, VHL-/- myeloid-derived cells of allograft recipients alleviated IRI and acute rejection, evidenced by reduced cardiomyocyte damage, decreased proinflammatory cytokine mRNA levels, and absence of inflammatory infiltrate at 5 days after transplantation. Ultimately, allograft survival was significantly prolonged. In vitro, VHL-/- myeloid-derived cells dose-dependently inhibited T-cell proliferation. Myeloid cells with HIF-1α-deletion retained proinflammatory qualities in vitro and in vivo. Deletion of VHL in myeloid cells of nonimmunosuppressed cardiac allograft recipients reduced myocardial injury and acute rejection. We suggest that HIF transcription factors induce a regulatory phenotype in myeloid-derived cells, which may be harnessed as a novel therapeutic strategy to regulate immune responses after heart transplantation.

Dual antiplatelet and anticoagulant APAC prevents experimental ischemia-reperfusion-induced acute kidney injury.

BACKGROUND: Renal ischemia-reperfusion predisposes to acute kidney injury (AKI) and mortality. APAC, mast cell heparin proteoglycan mimetic is a potent dual antiplatelet and anticoagulant inhibiting thrombosis in several vascular models. METHODS: Clinically relevant (0.06 and 0.13 mg/kg) and high (0.32 and 7.3 mg/kg) heparin doses of APAC and unfractionated heparin (UFH) were administered i.v. in pharmacological studies. Antithrombotic action of APAC and UFH was assessed with platelet aggregation to collagen, activated partial thromboplastin (APTT) and prothrombin (PT) times. Pharmacodynamics of [64Cu]-APAC or -UFH were monitored by PET/CT. Next, APAC and UFH doses (0.06 and 0.13 mg/kg) were i.v. administered 10 min prior to renal ischemia-reperfusion injury (IRI) in rats. RESULTS: APAC in contrast to UFH inhibited platelet aggregation. During 0.06 and 0.13 mg/kg dose regimens APTT and PT remained at baseline, but at the high APTT prolonged fourfold to sixfold. Overall bio-distribution and clearance of APAC and UFH were similar. After bilateral 30-min renal artery clamping, creatinine, urea nitrogen and neutrophil gelatinase-associated lipocalin concentrations and histopathology indicated faster renal recovery by APAC (0.13 mg/kg). APAC, unlike UFH, prevented expression of innate immune ligand hyaluronan and tubulointerstitial injury marker Kim-1. Moreover, in severe bilateral 1-h renal artery clamping, APAC (0.13 mg/kg) prevented AKI, as demonstrated both by biomarkers and survival. Compatible with kidney protection APAC reduced the circulating levels of vascular destabilizing and pro-inflammatory angiopoietin-2 and syndecan-1. No tissue bleeding ensued. CONCLUSION: APAC and UFH were similarly eliminated via kidneys and liver. In contrast to UFH, APAC (0.13 mg/kg) was reno-protective in moderate and even severe IRI by attenuating vascular injury and innate immune activation.

Vascular Endothelial Growth Factor-B Overexpressing Hearts Are Not Protected From Transplant-Associated Ischemia-Reperfusion Injury.

OBJECTIVES: Cardiac vascular endothelial growth factor-B transgene limits myocardial damage in rat infarction models. We investigated whether heart transplant vascular endothelial growth factor-B overexpression protected against ischemia-reperfusion injury. MATERIALS AND METHODS: We transplanted hearts heterotopically from Dark Agouti to Wistar Furth rats. To characterize the role of vascular endothelial growth factor-B in ischemia-reperfusion injury, we transplanted either long-term human vascular endothelial growth factor-B transgene overexpressing hearts from Wistar Furth rats or short-term adeno-associated virus 9-human vascular endothelial growth factor-B-transduced hearts from Dark Agouti rats into Wistar Furth rats. Heart transplants were subjected to 2 hours of cold and 1 hour of warm ex vivo ischemia. Samples were collected 6 hours after reperfusion. RESULTS: Two hours of cold and 1 hour of warm ischemia increased vascular endothelial growth factor-B mRNA levels 2-fold before transplant and 6 hours after reperfusion. Transgenic vascular endothelial growth factor-B overexpression caused mild cardiac hypertrophy and elevated cardiac troponin T levels 6 hours after reperfusion. Laser Doppler measurements indicated impaired epicardial tissue perfusion in these transgenic transplants. Recombinant human vascular endothelial growth factor-B increased mRNA levels of cytochrome c oxidase and extracellular ATPase CD39, suggesting active oxidative phosphorylation and high ATP production. Adeno-associated virus 9-mediated vascular endothelial growth factor-B overexpression in transplanted hearts increased intragraft macrophages 1.5-fold and proinflammatory cytokine interleukin 12 p35 mRNA 1.6-fold, without affecting recipient serum cardiac troponin T concentration. CONCLUSIONS: Vascular endothelial growth factor-B expression in transplanted hearts is linked to ischemia and ischemia-reperfusion injury. Cardiac transgenic vascular endothelial growth factor-B overexpression failed to protect heart transplants from ischemia-reperfusion injury.

Systemic overexpression of matricellular protein CCN1 exacerbates obliterative bronchiolitis in mouse tracheal allografts.

Obliterative bronchiolitis (OB) involves airway epithelial detachment, fibroproliferation, and inflammation, resulting in chronic rejection and transplant failure. Cysteine-rich 61 (CCN1) is an integrin receptor antagonist with a context-dependent role in inflammatory and fibroproliferative processes. We used a mouse tracheal OB model to investigate the role of CCN1 in the development of lung allograft OB. C57Bl/6 mice received a systemic injection of CCN1-expressing adenoviral vectors 2 days prior to subcutaneous implantation of tracheal allografts from major MHC-mismatched BALB/c mice. We treated another group of tracheal allograft recipients with cyclic arginine-glycine-aspartic acid peptide to dissect the role of αvß3-integrin signaling in mediating CCN1 effects in tracheal allografts. Allografts were removed 4 weeks after transplantation and analyzed for luminal occlusion, inflammation, and vasculogenesis. CCN1 overexpression induced luminal occlusion (P < 0.05), fibroproliferation, and smooth muscle cell proliferation (P < 0.05). Selective activation of αvß3-integrin receptor failed to mimic the actions of CCN1, and blocking failed to inhibit the effects of CCN1 in tracheal allografts. In conclusion, CCN1 exacerbates tracheal OB by enhancing fibroproliferation via an αvß3-integrin-independent pathway. Further experiments are required to uncover its potentially harmful role in the development of OB after lung transplantation.

Ex vivo intracoronary gene transfer of adeno-associated virus 2 leads to superior transduction over serotypes 8 and 9 in rat heart transplants.

Heart transplant gene therapy requires vectors with long-lasting gene expression, high cardiotropism, and minimal pathological effects. Here, we examined transduction properties of ex vivo intracoronary delivery of adeno-associated virus (AAV) serotype 2, 8, and 9 in rat syngenic and allogenic heart transplants. Adult Dark Agouti (DA) rat hearts were intracoronarily perfused ex vivo with AAV2, AAV8, or AAV9 encoding firefly luciferase and transplanted heterotopically into the abdomen of syngenic DA or allogenic Wistar-Furth (WF) recipients. Serial in vivo bioluminescent imaging of syngraft and allograft recipients was performed for 6 months and 4 weeks, respectively. Grafts were removed for PCR-, RT-PCR, and luminometer analysis. In vivo bioluminescent imaging of recipients showed that AAV9 induced a prominent and stable luciferase activity in the abdomen, when compared with AAV2 and AAV8. However, ex vivo analyses revealed that intracoronary perfusion with AAV2 resulted in the highest heart transplant transduction levels in syngrafts and allografts. Ex vivo intracoronary delivery of AAV2 resulted in efficient transgene expression in heart transplants, whereas intracoronary AAV9 escapes into adjacent tissues. In terms of cardiac transduction, these results suggest AAV2 as a potential vector for gene therapy in preclinical heart transplants studies, and highlight the importance of delivery route in gene transfer studies.

Combined donor simvastatin and methylprednisolone treatment prevents ischemia-reperfusion injury in rat cardiac allografts through vasculoprotection and immunomodulation.

BACKGROUND: Ischemia-reperfusion injury (IRI) and allograft dysfunction remain as two of the major clinical challenges after heart transplantation. Here, we investigated the effect of donor treatment with simvastatin and methylprednisolone on microvascular dysfunction and immunomodulation during IRI in rat cardiac allografts subjected to prolonged ischemia time. METHODS: The DA rats received simvastatin, methylprednisolone, or both 2 hr before heart donation. The allografts were subjected to 4-hr hypothermic preservation and transplanted to the fully major histocompatibility complex-mismatched WF rat recipients. RESULTS: Six hours after reperfusion, donor treatment either with simvastatin alone or with high dose of methylprednisolone alone or in combination with simvastatin and methylprednisolone significantly reduced cardiac troponin T release and the number of allograft infiltrating ED1 macrophages MPO neutrophils. However, the combination donor treatment was superior in the prevention of IRI and significantly prolonged allograft survival. Donor simvastatin treatment inhibited allograft microvascular RhoA GTPase pathway activation, whereas methylprednisolone prevented activation of innate immune response and mRNA expression of hypoxia-inducible factor-1α and its multiple target genes. CONCLUSIONS: Our results show that donor treatment in combination with simvastatin and methylprednisolone prevents IRI and has beneficial effect on allograft survival in rat cardiac allografts. Minimizing microvascular injury and the activation of innate immunity may offer a novel therapeutic strategy to expand the donor pool and furthermore improve the function of the marginal donor organs.

Effect of simvastatin on development of obliterative airway disease: an experimental study.

BACKGROUND: Obliterative bronchiolitis after lung transplantation is characterized by airway inflammation leading to obliteration of small airways. Statins are known to have lipid-independent immunomodulatory properties. We investigated the effect of simvastatin treatment on innate and adaptive immune responses and the development of obliterative airway disease (OAD). METHODS: In fully MHC-mismatched rat tracheal allograft recipients, we used simvastatin at different doses (0.1 to 20 mg/kg/day orally) to assess its effect on OAD development. No immunosuppressive treatment was administered. Histologic, immunohistochemical and real-time RT-PCR analyses were performed 3, 10 and 30 days after transplantation. RESULTS: Simvastatin treatment with doses ranging from 0.5 to 20 mg/kg/day significantly enhanced early epithelial recovery and reduced the development of OAD. No dose response was observed. Simvastatin treatment markedly reduced IL-23 mRNA and lymphocyte chemokine CCL20 production, and the infiltration of CD4(+) and CD8(+) T cells into allografts already at 3 days. At 10 days, simvastatin significantly attenuated the production of pro-inflammatory cytokines, IL-1ß, TNF-α, MCP-1 and IP-10, and Th17-polarizing cytokines, IL-6 and IL-17e, and inhibited allograft infiltration by inflammatory cells. The protective effects of simvastatin on inflammation and OAD were partially mediated through nitric oxide synthase. CONCLUSIONS: Simvastatin treatment inhibited adaptive T-cell alloimmune activation as depicted by reduced expression of lymphocyte chemokine and pro-inflammatory cytokine mRNA and reduced allograft infiltration by inflammatory cells. Importantly, simvastatin inhibits the development of OAD and this effect is partially mediated by increased nitric oxide activity. These results suggest a role for simvastatin in the prevention of obliterative bronchiolitis.

Donor simvastatin treatment abolishes rat cardiac allograft ischemia/reperfusion injury and chronic rejection through microvascular protection.

BACKGROUND: Ischemia/reperfusion injury may have deleterious short- and long-term consequences for cardiac allografts. The underlying mechanisms involve microvascular dysfunction that may culminate in primary graft failure or untreatable chronic rejection. METHODS AND RESULTS: Here, we report that rat cardiac allograft ischemia/reperfusion injury resulted in profound microvascular dysfunction that was prevented by donor treatment with peroral single-dose simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A reductase and Rho GTPase inhibitor, 2 hours before graft procurement. During allograft preservation, donor simvastatin treatment inhibited microvascular endothelial cell and pericyte RhoA/Rho-associated protein kinase activation and endothelial cell-endothelial cell gap formation; decreased intragraft mRNA levels of hypoxia-inducible factor-1α, inducible nitric oxide synthase, and endothelin-1; and increased heme oxygenase-1. Donor, but not recipient, simvastatin treatment prevented ischemia/reperfusion injury-induced vascular leakage, leukocyte infiltration, the no-reflow phenomenon, and myocardial injury. The beneficial effects of simvastatin on vascular stability and the no-reflow phenomenon were abolished by concomitant nitric oxide synthase inhibition with N-nitro-l-arginine methyl ester and RhoA activation by geranylgeranyl pyrophosphate supplementation, respectively. In the chronic rejection model, donor simvastatin treatment inhibited cardiac allograft inflammation, transforming growth factor-ß1 signaling, and myocardial fibrosis. In vitro, simvastatin inhibited transforming growth factor-ß1-induced microvascular endothelial-to-mesenchymal transition. CONCLUSIONS: Our results demonstrate that donor simvastatin treatment prevents microvascular endothelial cell and pericyte dysfunction, ischemia/reperfusion injury, and chronic rejection and suggest a novel, clinically feasible strategy to protect cardiac allografts.

Innate and adaptive immune responses in obliterative airway disease in rat tracheal allografts.

BACKGROUND: We assessed cellular innate and adaptive immune responses in a rat heterotopic tracheal allograft model during the development of obliterative airway disease. METHODS: Syngeneic tracheal grafts were transplanted heterotopically from DA to DA rats and fully MHC-mismatched allografts from DA to WF rats. The recipients received either no immunosuppression or two different doses of cyclosporine and were euthanized at 3, 10 and 30 days. Non-transplanted DA tracheas served as controls. Histologic, immunohistochemical and real-time RT-PCR analyses were performed. RESULTS: The syngrafts had normal epithelium at 10 days and no tracheal occlusion was seen at 30 days. In non-immunosuppressed allografts, almost total loss of epithelium was observed at 10 days, culminating in tracheal occlusion at 30 days. The activation of innate immune response was observed during the ischemic period at 3 days in both groups. Influx of the infiltrating inflammatory cells was more prominent in the allografts. In syngrafts, mRNA expression of pro-inflammatory, but also tolerogenic, cytokines was significantly upregulated, whereas Th1 and Th17 priming factors were significantly downregulated. In allografts, prominent mRNA expression of pro-inflammatory cytokines was seen and adaptive Th1 and Th17 alloresponses were increased. Cyclosporine treatment reduced tracheal occlusion and inhibited both tolerogenic and pro-inflammatory T-cell responses in allografts. CONCLUSIONS: Ischemia induced a self-limiting, alloantigen-independent innate immune response in syngrafts. In allografts, the predominant pro-inflammatory milieu and alloantigen-dependent Th1 and Th17 responses were linked to the development of obliterative airway disease and were inhibited by cyclosporine treatment.

Increased Th17 rather than Th1 alloimmune response is associated with cardiac allograft vasculopathy after hypothermic preservation in the rat.

BACKGROUND: Preservation injury decreases patient survival and promotes the development of cardiac allograft vasculopathy. We investigated the sequential effects of hypothermic preservation on ischemia-reperfusion injury (IRI), subsequent innate immune activation, and adaptive immune response in rat cardiac allografts. METHODS: Allografts were transplanted from fully major histocompatibility complex-mismatched Dark Agouti to Wistar Furth rats without pre-operative hypothermia or after 4 hours of hypothermic preservation. Recipients received cyclosporine A immunosuppression. The allografts were recovered at 6 hours (n = 6, 7), 24 hours (n = 6), 10 days (n = 5), and 8 weeks (n = 5). Immunohistochemical, histologic, and reverse-transcription polymerase chain reaction analysis was performed. RESULTS: In IRI, significantly increased messenger RNA (mRNA) levels for Toll-like receptor 4, hyaluronan synthases (HAS)1-2 (p = 0.03), high-mobility group box 1 (p = 0.05), CD80/83 (p = 0.01, p = 0.048), and the cytokines tumor necrosis factor-alpha (p = 0.004), interferon-gamma (p = 0.012), and interleukin (IL)-6 (p = 0.019) were seen in allografts subjected to hypothermic preservation. During established alloimmune response, allografts subjected to hypothermic preservation expressed prominent infiltration of CD4+ T cells (p = 0.043) and dendritic cells (p = 0.029) and significantly up-regulated mRNA levels of CD80 (p = 0.036), chemokine (C-C motif) ligand 21 (p = 0.008), C-C chemokine receptor type 7 (p = 0.003), vascular endothelial growth factor-C (p = 0.016), and vascular endothelial growth factor receptor-3 (p = 0.02). These allografts also showed prominent mRNA upregulation of Foxp3 (p = 0.014), IL-17 (p = 0.038), and IL-23 (p = 0.043). Preservation significantly increased the incidence and intensity of allograft arteriosclerosis (p < 0.05) and cardiac fibrosis (p = 0.003) at 8 weeks. CONCLUSION: Our results demonstrate that preservation injury induced a cascade leading to an innate immune response that modulated the adaptive immune response towards Th17 rather than Th1 T-cell response in rat cardiac allografts and ultimately enhanced cardiac fibrosis and arterial occlusion. Our results also suggest that this immune response was not regulated by the calcineurin inhibitor cyclosporine A.