Human Orthotopic Liver Transplantation Research Articles

Transcriptional Profiling Underscores the Role of Preprocurement Allograft Metabolism and Innate Immune Status on Outcomes in Human Liver Transplantation.

The adverse effects of ischemia-reperfusion injury (IRI) remain a principal barrier to a successful outcome after lifesaving orthotopic liver transplantation (OLT). Gene expression during different phases of IRI is dynamic and modified by individual exposures, making it attractive for identifying potential therapeutic targets for improving the number of suitable organs for transplantation and patient outcomes. However, data remain limited on the functional landscape of gene expression during liver graft IRI, spanning procurement to reperfusion and recovery. Therefore, we sought to characterize transcriptomic profiles of IRI during multiple phases in human OLT. We conducted clinical data analyses, histologic evaluation, and RNA sequencing of 17 consecutive human primary OLT. We performed liver allograft biopsies at 4 time points: baseline (B, before donor cross-clamp), at the end of cold ischemia (CI), during early reperfusion (ER, after revascularization), and during late reperfusion (LR). Data were generated and then recipients grouped by post-OLT outcomes categories: immediate allograft function (IAF; n = 11) versus early allograft dysfunction (EAD; n = 6) groups. We observed that CI (vs B) modified a transcriptomic landscape enriched for a metabolic and immune process. Expression levels of hallmark inflammatory response genes were higher transitioning from CI to ER and decreased from ER to LR. IAF group predominantly showed higher bile and fatty acid metabolism activity during LR compared with EAD group, while EAD group maintained more immunomodulatory activities. Throughout all time points, EAD specimens exhibited decreased metabolic activity in both bile and fatty acid pathways. We report transcriptomic profiles of human liver allograft IRI from prepreservation in the donor to posttransplantation in the recipient. Immunomodulatory and metabolic landscapes across ER and LR phases were different between IAF and EAD allografts. Our study also highlights marker genes for these biological processes that we plan to explore as novel therapeutic targets or surrogate markers for severe allograft injury in clinical OLT.

Bile acids attenuate hepatic inflammation during ischemia/reperfusion injury

Background & AimsPersistent cholestasis has been associated with poor prognosis after orthotopic liver transplantation (OLT). In this study, we aimed to investigate how the accumulation of tauro-beta-muricholic acid (TβMCA), resulting from the reprogramming of bile acid (BA) metabolism during liver ischemia/reperfusion (IR) stress, attenuates liver inflammation. MethodsIngenuity Pathway Analysis (IPA) was performed using transcriptome data from a murine hepatic IR model. Three different models of hepatic IR (liver warm IR, bile duct separation-IR, common bile duct ligation-IR) were employed. We generated AAV-transfected mice and CD11b-DTR mice to assess the role of BAs in regulating the myeloid S1PR2-GSDMD axis. Hepatic BA levels were analyzed using targeted metabolomics. Finally, the correlation between the reprogramming of BA metabolism and hepatic S1PR2 levels was validated through RNA-seq of human OLT biopsies. ResultsWe found that BA metabolism underwent reprogramming in murine hepatocytes under IR stress, leading to increased synthesis of TβMCA, catalyzed by the enzyme CYP2C70. The levels of hepatic TβMCA were negatively correlated with the severity of hepatic inflammation, as indicated by the serum IL-1β levels. Inhibition of hepatic CYP2C70 resulted in reduced TβMCA production, which subsequently increased serum IL-1β levels and exacerbated IR injury. Moreover, our findings suggested that TβMCA could inhibit canonical inflammasome activation in macrophages and attenuate inflammatory responses in a myeloid-specific S1PR2-GSDMD-dependent manner. Additionally, Gly-βMCA, a derivative of TβMCA, could effectively attenuate inflammatory injury in vivo and inhibit human macrophage pyroptosis in vitro. ConclusionsIR stress orchestrates hepatic BA metabolism to generate TβMCA, which attenuates hepatic inflammatory injury by inhibiting the myeloid S1PR2-GSDMD axis. Bile acids have immunomodulatory functions in liver reperfusion injury that may guide therapeutic strategies. Impact and implicationsOur research reveals that liver IR stress triggers reprogramming of BA metabolism. This functions as an adaptive mechanism to mitigate inflammatory injury by regulating the S1PR2-GSDMD axis, thereby controlling the release of IL-1β from macrophages. Our results highlight the crucial role of bile acids in regulating hepatocyte-immune cell crosstalk, which demonstrates an immunomodulatory function in liver reperfusion injury that may guide therapeutic strategies targeting bile acids and their receptors.

Cold stress-induced ferroptosis in liver sinusoidal endothelial cells determines liver transplant injury and outcomes.

Although cold preservation remains the gold standard in organ transplantation, cold stress-induced cellular injury is a significant problem in clinical orthotopic liver transplantation (OLT). Because a recent study showed that cold stress activates ferroptosis, a form of regulated cell death, we investigated whether and how ferroptosis determines OLT outcomes in mice and humans. Treatment with ferroptosis inhibitor (ferrostatin-1) during cold preservation reduced lipid peroxidation (malondialdehyde; MDA), primarily in liver sinusoidal endothelial cells (LSECs), and alleviated ischemia/reperfusion injury in mouse OLT. Similarly, ferrostatin-1 reduced cell death in cold-stressed LSEC cultures. LSECs deficient in nuclear factor erythroid 2-related factor 2 (NRF2), a critical regulator of ferroptosis, were susceptible to cold stress-induced cell death, concomitant with enhanced endoplasmic reticulum (ER) stress and expression of mitochondrial Ca2+ uptake regulator (MICU1). Indeed, supplementing MICU1 inhibitor reduced ER stress, MDA expression, and cell death in NRF2-deficient but not WT LSECs, suggesting NRF2 is a critical regulator of MICU1-mediated ferroptosis. Consistent with murine data, enhanced liver NRF2 expression reduced MDA levels, hepatocellular damage, and incidence of early allograft dysfunction in human OLT recipients. This translational study provides a clinically applicable strategy in which inhibition of ferroptosis during liver cold preservation mitigates OLT injury by protecting LSECs from peritransplant stress via an NRF2-regulatory mechanism.

SIRT1 regulates hepatocyte programmed cell death via GSDME - IL18 axis in human and mouse liver transplantation

Sirtuin 1 (SIRT1) is a histone/protein deacetylase in the cellular response to inflammatory, metabolic, and oxidative stressors. We previously reported that myeloid SIRT1 regulates the inflamed liver’s canonical pyroptosis cell death pathway. However, whether/how hepatocyte SIRT1 is engaged in programmed cell death in the cold-stressed liver remains uncertain. Here, we undertook translational studies in human and mouse orthotopic liver transplantation (OLT) to interrogate the significance of hepatocyte-specific SIRT1 in cold-stored donor livers and liver grafts after reperfusion. In the clinical arm of sixty human OLT patients, hepatic SIRT1 levels in cold-preserved donor livers correlated with the anti-apoptotic Bcl-2 expression. After reperfusion, improved OLT function was accompanied by hepatic SIRT1 levels negatively associated with cleaved caspase-3 expression. In the experimental arm, we compared FLOX-control with hepatocyte-specific SIRT1-KO livers after orthotopic transplantation into WT mouse recipients, parallel with primary murine hepatocyte cultures subjected to cold activation with/without knockdown of SIRT1, GSDME, and IL18Rβ. Indeed, hepatocyte SIRT1 deficiency upregulated apoptosis and GSDME-mediated programmed cell death, deteriorating hepatocellular function and shortening OLT survival. Augmented GSDME processing, accompanied by increased secretion of IL18 by stressed hepatocytes, was prominent in SIRT1-deficient, cold-stored livers. Hepatocyte SIRT1 expression regulated anti-apoptotic Bcl-2/XIAP proteins, suppressed cold stress-triggered apoptosis, and mitigated GSDME licensing to release IL18. Notably, consistent with the ability of IL18 to depress hepatocyte SIRT1 and Bcl-2/XIAP in vitro, IL18 neutralization in vivo prevented hepatocellular damage and restored the anti-apoptotic phenotype in otherwise injury-prone SIRT1-deficient OLTs. In conclusion, this translational study identifies a novel hepatocyte SIRT1-IL18 molecular circuit as a therapeutic target in the mechanism underpinning hepatocyte death pathways in human and mouse liver transplantation.

T Cell CEACAM1–TIM-3 Crosstalk Alleviates Liver Transplant Injury in Mice and Humans

Carcinoembryonic antigen-related cell adhesion molecule 1 (CC1) acts through homophilic and heterophilic interactions with T cell immunoglobulin domain and mucin domain-containing protein 3 (TIM-3), which regulates innate immune activation in orthotopic liver transplantation (OLT). We investigated whether cluster of differentiation (CD) 4+ T cell-dependent CC1-TIM-3 crosstalk may affect OLT outcomes in mice and humans. Wild-type (WT) and CC1-deficient (CC1 knock-out [KO]) mouse livers were transplanted into WT, CC1KO, or T-cell TIM-3 transgenic (TIM-3Tg)/CC1KO double-mutant recipients. CD4+ T cells were adoptively transferred into T/B cell-deficient recombination activating gene 2 protein (Rag2) KO recipients, followed by OLT. The perioperative liver-associated CC1 increase was analyzed in 50 OLT patients. OLT injury in WT livers deteriorated in CC1KO compared with CC1-proficient (WT) recipients. The frequency of TIM-3+CD4+ T cells was higher in WT than CC1KO hosts. Reconstitution of Rag2KO mice with CC1KO-T cells increased nuclear factor (NF)-κB phosphorylation and OLT damage compared with recipients repopulated with WT T cells. T-cell TIM-3 enhancement in CC1KO recipients (WT→ TIM3Tg/CC1KO) suppressed NF-κB phosphorylation in Kupffer cells and mitigated OLT injury. However, TIM-3-mediated protection was lost by pharmacologic TIM-3 blockade or an absence of CC1 in the donor liver (CC1KO→ TIM-3Tg/CC1KO). The perioperative CC1 increase in human OLT reduced hepatocellular injury, early allograft dysfunction, and the cumulative rejection rate. This translational study identifies T cell-specific CC1 signaling as a therapeutic means to alleviate OLT injury by promoting T cell-intrinsic TIM-3, which in turn interacts with liver-associated CC1 to suppress NF-κB in Kupffer cells. By suppressing peritransplant liver damage, promoting T-cell homeostasis, and improving OLT outcomes, recipient CC1 signaling serves as a novel cytoprotective sentinel.

Recipient TIM4 signaling regulates ischemia reperfusion-induced ER stress and metabolic responses in liver transplantation: from mouse-to-human.

T-cell immunoglobulin and mucin (Tim)4 is expressed on APCs, including macrophages, as one of the main amplifiers in the mechanism of liver ischemia-reperfusion injury (IRI) following orthotopic liver transplantation (OLT). Though donor Tim4 selectively expressed on Kupffer cells serves as a checkpoint regulator of innate immune-driven IRI cascades, its role on cells outside the OLT remains unclear. To dissect the role of donor vs. recipient-specific Tim4 signaling in IR-induced stress and hepatocellular function, we employed a murine OLT model utilizing Tim4-knockout (KO) mice as either donor or recipient (WT → WT, WT → Tim4-KO, Tim4-KO → WT). In the experimental arm, disruption of donor Tim4 attenuated IRI-OLT damage, while recipient Tim4-null mutation aggravated hepatic IRI concomitant with disturbed lipid metabolism, enhanced endoplasmic reticulum stress, and activated pro-apoptotic signaling in the grafts. In the in vitro study, murine hepatocytes co-cultured with Tim4-null adipose tissue showed enhanced C/EBP homologous protein (CHOP) expression pattern and susceptibility to hepatocellular death accompanied by activated caspase cascade in response to TNF-α stimulation. In the clinical arm, liver grafts from forty-one transplant patients with enhanced TIM4 expression showed higher body mass index, augmented hepatic endoplasmic reticulum stress, enhanced pro-apoptotic markers, upregulated innate/adaptive immune responses, exacerbated hepatocellular damage, and inferior graft survival. In conclusion, although TIM4 is considered a principal villain in peri-transplant early tissue injury, recipient TIM4 signaling may serve as a savior of IR-triggered metabolic stress in mouse and human OLT recipients.

Neutrophil CEACAM1 determines susceptibility to NETosis by regulating the S1PR2/S1PR3 axis in liver transplantation.

Neutrophils, the largest innate immune cell population in humans, are the primary proinflammatory sentinel in the ischemia-reperfusion injury (IRI) mechanism in orthotopic liver transplantation (OLT). Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1, CC1, or CD66a) is essential in neutrophil activation and serves as a checkpoint regulator of innate immune-driven IRI cascade in OLT. Although CC1 alternative splicing generates two functionally distinct short and long cytoplasmic isoforms, their role in neutrophil activation remains unknown. Here, we undertook molecular and functional studies to interrogate the significance of neutrophil CC1 signaling in mouse and human OLT recipients. In the experimental arm, we employed a mouse OLT model to document that ablation of recipient-derived neutrophil CC1-long (CC1-L) isotype aggravated hepatic IRI by promoting neutrophil extracellular traps (NETs). Notably, by regulating the S1P-S1PR2/S1PR3 axis, neutrophil CC1-L determined susceptibility to NET formation via autophagy signaling. In the clinical arm, liver grafts from 55 transplant patients selectively enriched for neutrophil CC1-L showed relative resistance to ischemia-reperfusion (IR) stress/tissue damage, improved hepatocellular function, and clinical outcomes. In conclusion, despite neutrophils being considered a principal villain in peritransplant tissue injury, their CC1-L isoform may serve as a regulator of IR stress resistance/NETosis in human and mouse OLT recipients.

Research Highlights

Hepatic CEACAM1 Expression Indicates Donor Liver Quality and Prevents Early Transplantation Injury Nakamura K, Kageyama S, Kaldas FM, et al. J Clin Invest. 2020;130:2689–2704. Orthotopic liver transplantation (OLT) is accepted as the gold-standard treatment for patients with liver failure, yet it remains limited by significant donor shortage. A particular challenge is the quality of livers available for transplantation, with a large proportion of deceased donor livers not deemed to be appropriate for transplantation. It is recognized that ischemia-reperfusion injury (IRI) at the time of transplantation contributes to the development of early allograft dysfunction; however, the mechanisms underlying this process are incompletely understood.6 Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), also known as CD66a or biliary glycoprotein-1, is a transmembrane protein expressed in a number of immune and epithelial cells in which it acts as an adhesion molecule but also has a role in hepatic metabolic homeostasis. To investigate the role of CEACAM1 in IRI, Nakamura et al use a mouse model of OLT in which grafts from either wild-type or CC1−/− mice (which possess a global null mutation in CEACAM1) are subjected to extended cold storage (18 h) and subsequently transplanted into wild-type recipients.7 Mice in the CC1−/− group displayed increased evidence of IRI and higher levels of HMGB, a danger-associated molecular pattern, in addition to an enhanced infiltration with macrophages and neutrophils. The CC1−/− group also exhibited signs of injury resulting from cold storage, mediated by the ASK1/p-p38 pathway. Furthermore, preincubation of CC1−/− livers in cold storage with an ASK1 inhibitor improved outcomes. Having established a regulatory role for CEACAM1, the authors next validated the clinical relevance in human OLT. Liver graft biopsies were obtained from 60 patients at the time of liver transplantation. These were screened for CEACAM1 as well as ASK1 and p-p38, identifying a negative correlation between CEACAM1 levels and ASK1. To test the hypothesis that pretransplantation CEACAM1 levels impact clinical outcomes, samples were clustered into high or low CEACAM1 groups based on a median of CEACAM1 levels. Samples with low levels of CEACAM1 correlated with increased liver enzymes postoperatively as well as an augmented frequency of early allograft dysfunction and poorer long-term graft function, although the latter finding did not reach statistical significance. CEACAM1 may therefore represent a useful biomarker of liver quality, as well as a point for intervention, particularly through targeting the ASK1/p-p38 pathway. Transforming the Spleen Into a Liver-like Organ In Vivo Wang L, Wang C, Wang Z, et al. Sci Adv. 2020;6:eaaz9974. Regenerative medicine aims to solve several key challenges in organ transplantation—from the shortage of donor organs to the need for optimized immunosuppression. Significant progress has been made in recent years, but the field is still limited by the inability to generate the necessary vasculature to sustain the engineered tissues1. In the study from Wang et al, this limitation is addressed through a novel concept of transforming an existing organ, the spleen, to perform the function of the liver. The spleen is an ideal subject for this as it is large, has an excellent blood supply, and its absence can be tolerated well. The authors use a mouse model to transform the spleen by remodeling the tissue and subsequently transplanting liver cells into the organ. To facilitate the necessary transformation, the spleen was initially translocated surgically to a subcutaneous location. Thereafter, the spleen was injected with soluble tumor homogenate (STH, from a sarcoma cell line) on days 7, 10, and 14 to suppress immune function and enhance the extracellular matrix. Mice injected with this homogenate demonstrated an increase in spleen size, extracellular matrix composition, and angiogenesis (probably mediated through enhanced vascular endothelial growth factor expression). Interestingly, the transformed spleens provided an immunosuppressive microenvironment, with reduced T and B cells as well as an increase in regulatory T cells. The second step involved the transfer of liver cells into remodeled spleens (“hepatization”). Using luciferase-labeling, the authors were able to detect cells posttransplantation and demonstrated successful graft take of both allografted and xenografted human liver cells, although regular injection of STH was required to achieve this. Cessation of STH injection resulted in grafts being lost 2 wk after the last injection. Transformed spleens were able to produce albumin and metabolize drugs. Furthermore, in mice wherein 90% of the liver was removed, or wherein fulminant liver failure was induced, transformed spleens were able to take over functionality, and the mice survived. The strategy presented offers the exciting possibility of organ repurposing thus allowing organs to be engineered from a baseline prevascularized tissue in situ, providing an advantage over more traditional decellularization-recellularization techniques.

Hepatic CEACAM1 expression indicates donor liver quality and prevents early transplantation injury.

Although CEACAM1 (CC1) glycoprotein resides at the interface of immune liver injury and metabolic homeostasis, its role in orthotopic liver transplantation (OLT) remains elusive. We aimed to determine whether/how CEACAM1 signaling may affect hepatic ischemia-reperfusion injury (IRI) and OLT outcomes. In the mouse, donor liver CC1 null mutation augmented IRI-OLT (CC1-KO→WT) by enhancing ROS expression and HMGB1 translocation during cold storage, data supported by in vitro studies where hepatic flush from CC1-deficient livers enhanced macrophage activation in bone marrow-derived macrophage cultures. Although hepatic CC1 deficiency augmented cold stress-triggered ASK1/p-p38 upregulation, adjunctive ASK1 inhibition alleviated IRI and improved OLT survival by suppressing p-p38 upregulation, ROS induction, and HMGB1 translocation (CC1-KO→WT), whereas ASK1 silencing (siRNA) promoted cytoprotection in cold-stressed and damage-prone CC1-deficient hepatocyte cultures. Consistent with mouse data, CEACAM1 expression in 60 human donor liver biopsies correlated negatively with activation of the ASK1/p-p38 axis, whereas low CC1 levels associated with increased ROS and HMGB1 translocation, enhanced innate and adaptive immune responses, and inferior early OLT function. Notably, reduced donor liver CEACAM1 expression was identified as one of the independent predictors for early allograft dysfunction (EAD) in human OLT patients. Thus, as a checkpoint regulator of IR stress and sterile inflammation, CEACAM1 may be considered as a denominator of donor hepatic tissue quality, and a target for therapeutic modulation in OLT recipients.

Translationally controlled tumor protein exerts a proinflammatory role in acute rejection after liver transplantation

BackgroundTranslationally controlled tumor protein (TCTP), which has been verified to have a proinflammatory activity, plays an important role in allergy. However, it remains unclear whether TCTP has an impact on the acute rejection (AR) after liver transplantation. MethodsThree protocols were used to delineate the role of TCTP in AR after liver transplantation. First, in rat orthotopic liver transplantation (OLT), the expression of TCTP was measured by enzyme-linked immunosorbent assay (ELISA), real-time PCR, Western blot and immunofluorescence assays. Second, in mixed lymphocyte reaction (MLR), the role of TCTP in lymphocyte proliferation was measured by carboxyfluorescein succinimidyl ester (CFSE) labeling and the impact of TCTP on inflammatory factor release was detected by cytokine arrays. Third, in human OLT, the level of serum TCTP was detected by ELISA, and the relationship between TCTP and model for early allograft function (MEAF) score was assessed by Spearman's correlation. ResultsIn rat OLT, AR resulted in great harm to allografts, manifesting as deterioration of liver function, increasing inflammatory factors and infiltrating lymphocytes. Meanwhile, TCTP was overexpressed in serum and allografts. Higher level of TCTP was associated with higher rejection activity index (RAI). In an MLR protocol, TCTP knockdown inhibited the proliferation of mixed inflammatory cells and significantly suppressed the release of 15 cytokines and chemokines. In human OLT, the serum TCTP was up-regulated within a week after operation. Additionally, the increasing speed of serum TCTP positively correlated with MEAF scores (r = 0.449; P = 0.0088). ConclusionsUp-regulated TCTP positively affects AR after liver transplantation.

CD 44 as a Marker of Rejection in Patients with Post Liver Transplantation

Abstract Background The first human orthotopic liver transplantation (LT) in Europe was performed by Sir Roy Calne in Cambridge in 1968, only one year after the first successful human liver transplantation reported by Thomas Starzl in the United States. Since then LT has evolved rapidly, becoming the standard therapy for acute and chronic liver failure of all aetiologies, with more than 80,000 procedures performed to date. Survival rates have improved significantly in the last 25 years, achieving rates of 96% and 71% at 1 and 10 years after LT respectively. Objective To assessment of value of serum level of CD44 as a marker of rejection in patients with post liver transplantation. Patients and Methods The study was conducted on patient all were recruited from ASCOT from May 2017 till December 2018. It included 20 adult patients with Post LDLT with elevated liver function tests and they were reviewed to determine if the serum level of CD44 had a value in diagnosis of acute rejection depending on proving the rejection by liver biopsy. They were distributed as 14 males and 6 females with age ranging 16 - 59 years. After obtaining the results of liver biopsy we had 14 patients with rejection, 4 patients with cholangitis with biliary obstruction and 2 patient with rejection with vascular insult. Results The control group was with irrelevant past medical history and normal liver functions, normal liver biopsy and underwent measurement of CD44 serum level. They were distributed as 14 males and 6 females with age ranging 26 – 41 years. Conclusion There was correlation between different pathological findings in liver biopsy and Serum level of CD44 that the low levels being with rejection and higher levels being with control group, that the CD44 may be used as a marker for rejection.

Antibiotic pretreatment alleviates liver transplant damage in mice and humans.

Although modifications of gut microbiota with antibiotics (Abx) influence mouse skin and cardiac allografts, its role in orthotopic liver transplantation (OLT) remains unknown. We aimed to determine whether and how recipient Abx pretreatment may affect hepatic ischemia-reperfusion injury (IRI) and OLT outcomes. Mice (C57BL/6) with or without Abx treatment (10 days) were transplanted with allogeneic (BALB/c) cold-stored (18 hours) livers, followed by liver and blood sampling (6 hours). We divided 264 human OLT recipients on the basis of duration of pre-OLT Abx treatment into control (Abx-free/Abx <10 days; n = 108) and Abx treatment (Abx ≥10days; n = 156) groups; OLT biopsy (Bx) samples were collected 2 hours after OLT (n = 52). Abx in mice mitigated IRI-stressed OLT (IRI-OLT), decreased CCAAT/enhancer-binding protein homologous protein (CHOP) (endoplasmic reticulum [ER] stress), enhanced LC3B (autophagy), and inhibited inflammation, whereas it increased serum prostaglandin E2 (PGE2) and hepatic PGE2 receptor 4 (EP4) expression. PGE2 increased EP4, suppressed CHOP, and induced autophagosome formation in hepatocyte cultures in an EP4-dependent manner. An EP4 antagonist restored CHOP, suppressed LC3B, and recreated IRI-OLT. Remarkably, human recipients of Abx treatment plus OLT (Abx-OLT), despite severe pretransplantation clinical acuity, had higher EP4 and LC3B levels but lower CHOP levels, which coincided with improved hepatocellular function (serum aspartate aminotransferase/serum aspartate aminotransferase [sALT/sAST]) and a decreased incidence of early allograft dysfunction (EAD). Multivariate analysis identified "Abx-free/Abx <10 days" as a predictive factor of EAD. This study documents the benefits of Abx pretreatment in liver transplant recipients, identifies ER stress and autophagy regulation by the PGE2/EP4 axis as a homeostatic underpinning, and points to the microbiome as a therapeutic target in OLT.

Recipient HO-1 inducibility is essential for posttransplant hepatic HO-1 expression and graft protection: From bench-to-bedside.

By documenting potent antioxidative and anti-inflammatory functions, preclinical studies encourage heme oxygenase-1 (HO-1)-inducing regimens in clinical orthotopic liver transplantation (OLT). We aimed to determine the importance of recipient-derived HO-1 in murine and human OLTs. Hepatic biopsies from 51 OLT patients were screened for HO-1 expression (Western blots) prior to put-in (basal) and post reperfusion (stressed) and correlated with the hepatocellular function. In parallel, livers from HO-1 proficient mice (WT; C57/BL6), subjected to ex vivo cold storage (18hour), were transplanted to syngeneic myeloid HO-1 deficient (mHO-1 KO) or FLOX (control) hosts, and sampled postreperfusion (6hour). In human OLT, posttransplant but not pretransplant HO-1 expression correlated negatively with ALT levels (P=.0178). High posttransplant but not pretransplant HO-1 expression trended with improved OLT survival. Compared with controls, livers transplanted into mHO-1 KO recipient mice had decreased HO-1 levels, exacerbated hepatic damage/frequency of TUNEL+ cells, increased mRNA levels coding for TNFα/CXCL1/CXCL2/CXCL10, higher frequency of Ly6G+/4HN+ neutrophils; and enhanced MPO activity. Peritoneal neutrophils from mHO-1 KO mice exhibited higher CellRox+ ratio and increased TNFα/CXCL1/CXCL2/CXCL10 expression. By demonstrating the importance of posttransplant recipient HO-1 phenotype in hepatic macrophage/neutrophil regulation and function, this translational study identifies recipient HO-1 inducibility as a novel biomarker of ischemic stress resistance in OLT.

Heme oxygenase-1 regulates sirtuin-1-autophagy pathway in liver transplantation: From mouse to human.

Liver ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although graft autophagy is essential for resistance against hepatic IRI, its significance in clinical OLT remains unknown. Despite recent data identifying heme oxygenase-1 (HO-1) as a putative autophagy inducer, its role in OLT and interactions with sirtuin-1 (SIRT1), a key autophagy regulator, have not been studied. We aimed to examine HO-1-mediated autophagy induction in human OLT and in a murine OLT model with extended (20hours) cold storage, as well as to analyze the requirement for SIRT1 in autophagy regulation by HO-1. Fifty-one hepatic biopsy specimens from OLT patients were collected under an institutional review board protocol 2 hours after portal reperfusion, followed by Western blot analyses. High HO-1 levels correlated with well-preserved hepatocellular function and enhanced SIRT1/LC3B expression. In mice, HO-1 overexpression by genetically modified HO-1 macrophage therapy was accompanied by decreased OLT damage and increased SIRT1/LC3B expression, whereas adjunctive inhibition of SIRT1 signaling diminished HO-1-mediated hepatoprotection and autophagy induction. Our translational study confirms the clinical relevance of HO-1 cytoprotection and identifies SIRT1-mediated autophagy pathway as a new essential regulator of HO-1 function in IR-stressed OLT.

Liver transplantation in adults. An analysis of costs and benefits at the University of Pittsburgh.

Orthotopic liver transplantation in humans has come of age. After 20 yr of experimental effort in animals and human beings, this procedure has reached such a level of success that it has been reviewed by a National Institutes of Health consensus development conference (June 20-23, 1983) where the following conclusion was reached: “liver transplantation is a therapeutic modality for end stage liver disease that deserves broader application.” Moreover, the conference made the statement that the procedure should be applied both to children and adults who have “irreversible liver injury” and “who have exhausted alternative medical and surgical treatments and are approaching the terminal phase of their illness” (1). The specific indications for the procedure in adults have been identified previously (l-4). Although fulminant hepatic failure due to toxin or drug exposure may warrant liver transplantation, the rapid progression of the disease and the multisystem organ failure that is associated with the condition frequently precludes application of the procedure for this indication. Specifically, the logistic and technical problems of donor procurement and life support required in such cases, until transplantation is possible, make this condition an unlikely frequent cause for the procedure. The following is a cost-benefit analysis of liver transplantation as applied to a single institution, the

Alterations of Endothelial Glycocalyx During Orthotopic Liver Transplantation in Patients With End-Stage Liver Disease.

Endothelial glycocalyx participates in the maintenance of vascular integrity, and its perturbations cause capillary leakage, loss of vascular responsiveness, and enhanced adhesion of leukocytes and platelets. We hypothesized that marked shedding of the glycocalyx core protein, syndecan-1, occurs in end-stage liver disease (ESLD) and that it increases during orthotopic liver transplantation (OLT). We further evaluated the effects of general anesthesia on glycocalyx shedding and its association with acute kidney injury (AKI) after OLT. Thirty consecutive liver transplant recipients were enrolled in this prospective study. Ten healthy volunteers served as a control. Acute kidney injury was defined by Acute Kidney Injury Network criteria. Plasma syndecan-1 was significantly higher in ESLD patients than in healthy volunteers (74.3 ± 59.9 vs 10.7 ± 9.4 ng/mL), and it further increased significantly after reperfusion (74.3 ± 59.9 vs 312.6 ± 114.8 ng/mL). The type of general anesthesia had no significant effect on syndecan-1. Syndecan-1 was significantly higher during the entire study in patients with posttransplant AKI stage 2 or 3 compared to patients with AKI stage 0 or 1. The area under the curve of the receiver operating characteristics curve of syndecane-1 to predict AKI stage 2 or 3 within 48 hours after reperfusion was 0.76 (95% confidence interval, 0.57-0.89, P = 0.005). Patients with ESLD suffer from glycocalyx alterations, and ischemia-reperfusion injury during OLT further exacerbates its damage. Despite a higher incidence of AKI in patients with elevated syndecan-1, it is not helpful to predict de novo AKI. Volatile anesthetics did not attenuate glycocalyx shedding in human OLT.

Current developments in pediatric liver transplantation.

In 1953, the pioneer of human orthotopic liver transplantation (LT), Thomas E Starzl, was the first to attempt an orthotopic liver transplant into a 3 years old patient suffering from biliary atresia. Thus, the first LT in humans was attempted in a disease, which, up until today, remains the main indication for pediatric LT (pLT). During the last sixty years, refinements in diagnostics and surgical technique, the introduction of new immunosuppressive medications and improvements in perioperative pediatric care have established LT as routine procedure for childhood acute and chronic liver failure as well as inherited liver diseases. In contrast to adult recipients, pLT differs greatly in indications for LT, allocation practice, surgical technique, immunosuppression and post-operative life-long aftercare. Many aspects are focus of ongoing preclinical and clinical research. The present review gives an overview of current developments and the clinical outcome of pLT, with a focus on alternatives to full-size deceased-donor organ transplantation.

Biomarker differences between cadaveric grafts used in human orthotopic liver transplantation as identified by coulometric electrochemical array detection (CEAD) metabolomics.

Metabolomics in systems biology research unravels intracellular metabolic changes by high throughput methods, but such studies focusing on liver transplantation (LT) are limited. Microdialysate samples of liver grafts from donors after circulatory death (DCD; n=13) and brain death (DBD; n=27) during cold storage and post-reperfusion phase were analyzed through coulometric electrochemical array detection (CEAD) for identification of key metabolomics changes. Metabolite peak differences between the graft types at cold phase, post-reperfusion trends, and in failed allografts, were identified against reference chromatograms. In the cold phase, xanthine, uric acid, and kynurenine were overexpressed in DCD by 3-fold, and 3-nitrotyrosine (3-NT) and 4-hydroxy-3-methoxymandelic acid (HMMA) in DBD by 2-fold (p<0.05). In both grafts, homovanillic acid and methionine increased by 20%-30% with each 100 min increase in cold ischemia time (p<0.05). Uric acid expression was significantly different in DCD post-reperfusion. Failed allografts had overexpression of reduced glutathione and kynurenine (cold phase) and xanthine (post-reperfusion) (p<0.05). This differential expression of metabolites between graft types is a novel finding, meanwhile identification of overexpression of kynurenine in DCD grafts and in failed allografts is unique. Further studies should examine kynurenine as a potential biomarker predicting graft function, its causation, and actions on subsequent clinical outcomes.

Ulinastatin ameliorates acute kidney injury following liver transplantation in rats and humans.

Acute kidney injury (AKI) is a common complication following orthotopic liver transplantation (OLT) that evidently affects prognosis. However, no effective treatment exists for AKI. The aim of the present study was to elucidate whether ulinastatin application during OLT in humans can reduce kidney damage and improve renal function. In addition, the underlying mechanisms of ulinastatin were investigated on a rat autologous OLT (AOLT) model. In total, 60 patients undergoing an OLT were randomly selected to receive ulinastatin (U group; n=30) or saline (C group; n=30) during the OLT surgery. The patient demographics, AKI incidence rate, recovery indicators and renal injury indexes were measured during the perioperative period. In addition to the clinical trials, 40 rats were subjected to an AOLT and were divided into the control (C-R), sham-operation and ulinastatin treatment groups. Pathological renal damage, biomarkers of inflammation and oxidative stress were measured to investigate the effects and possible mechanisms of ulinastatin on AKI. In the clinical trials, ulinastatin application was shown to attenuate the incidence of AKI following OLT (P<0.05) and reduce the serum levels of cystatin C and urinary β2 microglobulin within 24 h of the OLT (P<0.05). Furthermore, ulinastatin was found to significantly improve the recovery of patients by reducing the time spent in the intensive care unit (P<0.01 vs. C group), the ventilation time and the hemodialysis rates (P<0.05 vs. C group). In the rat AOLT model, ulinastatin application was also shown to relieve renal pathological damage by reducing the serum cystatin C and creatinine levels. Notably, the levels of tumor necrosis factor-α, interleukin-6, hydrogen peroxide and reactive oxygen species were evidently reduced, while the level of superoxide dismutase was increased in the ulinastatin groups (P<0.05, vs. C-R group). In conclusion, ulinastatin application was demonstrated to protect against AKI following OLT by inhibiting inflammation and oxidation.

Evaluation of Syndecan-1 as a Marker of Ischemia and Reperfusion in Patients Undergoing Liver Transplantation.

Background. The endothelial glycocalyx is important for the maintenance of vascular integrity and changes in its structure result in disturbed capillary circulation and increased vascular permeability. Its shed compounds, such as Syndecan-1, can be quantified in the plasma in consequence of impairment of the endothelial cell barrier. This study aims to evaluate syndecan-1 to determine the effect of ischemia/reperfusion on the shedding of glycocalyx during orthotopic liver transplantation (OLT) and its impact on postoperative kidney function. Materials and Methods. In 22 patients undergoing OLT arterial blood samples were drawn at five different time points (TO: induction of anaesthesia, T1: clamping of inferior caval vein, T2: declamping/reperfusion, T3: end of surgery, T4: 24 hours after reperfusion) and analysed regarding syndecan-1 levels in the plasma by means of ELISA (Diaclone Research, Besancon, France). Acute kidney injury (AKI) following OLT was assessed according to the RIFLE criteria1. Data were compared using students's t-test, ROC-analysis was performed to determine the impact on AKI. Results. There was a significant increase of syndecan-1 level at T2 (273 ± 108 ng/ml) compared to T0 (77 ± 61 ng/ml) and T1 (92 ± 61 ng/ml), respectively (p < 0.0001). The sensitivity (67%) and specificity (70%) of increased syndecan-1 from T1 to T2 was very low to predict an AKI (stage: injury and failure) according to the RIFLE criteria (ROC 0.58; p = 0.5). Conclusion. This study provides evidence for shedding of the endothelial glycocalyx during ischemia/reperfusion in human OLT. The predictive value of syndecan-1 regarding the incidence of AKI is very low. 1. Ferreira AC, Nolasco F, Carvalho D, et al. Impact of RIFLE classification in liver transplantation. Clin Transplant 2010;24(3):394-400.