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BackgroundIschemia-reperfusion injury (IRI) is a significant clinical concern in liver transplantation, with a key influence on short-term and long-term allograft and patient survival. Myeloid cells trigger and sustain tissue inflammation and damage associated with IRI, but the mechanisms regulating these activities are unknown. To address this, we investigated the molecular characteristics of intragraft myeloid cells present in biopsy-proven IRI- and IRI+ liver transplants.MethodsRNA-sequencing was performed on 80 pre-reperfusion and post-reperfusion biopsies from 40 human recipients of liver transplantation (23 IRI+, 17 IRI-). We used transcriptional profiling and computational approaches to identify specific gene coexpression network modules correlated with functional subsets of MPO+, lysozyme+, and CD68+ myeloid cells quantified by immunohistochemistry on sequential sections from the same patient biopsies.ResultsA global molecular map showed gene signatures related to myeloid activation in all patients regardless of IRI status; however, myeloid cell subsets differed dramatically in their spatial morphology and associated gene signatures. IRI- recipients were found to have a natural corticosteroid production and response profile from pre-reperfusion to post-reperfusion, particularly among monocytes/macrophages. The pre-reperfusion signature of IRI+ recipients included acute inflammatory responses in neutrophils and increased translation of adaptive immune-related genes in monocytes/macrophages coupled with decreased glucocorticoid responses. Subsequent lymphocyte activation at post-reperfusion identified transcriptional programs associated with the transition to adaptive immunity found only among IRI+ recipients.ConclusionsMyeloid subset-specific genes and related signaling pathways provide targets for the development of therapeutic strategies aimed at limiting IRI in the clinical setting of liver transplantation.

Background and aimsThe sensitivity of current surveillance methods for detecting early-stage hepatocellular carcinoma (HCC) is suboptimal. Extracellular vesicles (EVs) are promising circulating biomarkers for early cancer detection. In this study, we aim to develop an HCC EV-based surface protein assay for early detection of HCC.Approach and resultsTissue microarray was used to evaluate four potential HCC-associated protein markers. An HCC EV surface protein assay, composed of covalent chemistry-mediated HCC EV purification and real-time immuno-polymerase chain reaction readouts, was developed and optimized for quantifying subpopulations of EVs. An HCC EV ECG score, calculated from the readouts of three HCC EV subpopulations ( E pCAM + CD63 + , C D147 + CD63 + , and G PC3 + CD63 + HCC EVs), was established for detecting early-stage HCC. A phase 2 biomarker study was conducted to evaluate the performance of ECG score in a training cohort ( n  = 106) and an independent validation cohort ( n  = 72).Overall, 99.7% of tissue microarray stained positive for at least one of the four HCC-associated protein markers (EpCAM, CD147, GPC3, and ASGPR1) that were subsequently validated in HCC EVs. In the training cohort, HCC EV ECG score demonstrated an area under the receiver operating curve (AUROC) of 0.95 (95% confidence interval [CI], 0.90-0.99) for distinguishing early-stage HCC from cirrhosis with a sensitivity of 91% and a specificity of 90%. The AUROCs of the HCC EV ECG score remained excellent in the validation cohort (0.93; 95% CI, 0.87-0.99) and in the subgroups by etiology (viral: 0.95; 95% CI, 0.90-1.00; nonviral: 0.94; 95% CI, 0.88-0.99).ConclusionHCC EV ECG score demonstrated great potential for detecting early-stage HCC. It could augment current surveillance methods and improve patients' outcomes.

Although glycogen synthase kinase β (Gsk3β) has been shown to regulate tissue inflammation, whether and how it regulates inflammation resolution versus inflammation activation is unclear. In a murine liver, partial warm ischemia/reperfusion injury (IRI) model, we found that Gsk3β inhibitory phosphorylation increased at both the early-activation and late-resolution stages of the disease. Myeloid Gsk3β deficiency not only alleviated liver injuries, it also facilitated the restoration of liver homeostasis. Depletion of Kupffer cells prior to the onset of liver ischemia diminished the differences between the WT and Gsk3β-KO mice in the activation of liver IRI. However, the resolution of liver IRI remained accelerated in Gsk3β-KO mice. In CD11b-DTR mice, Gsk3β-deficient BM-derived macrophages (BMMs) facilitated the resolution of liver IRI as compared with WT cells. Furthermore, Gsk3β deficiency promoted the reparative phenotype differentiation in vivo in liver-infiltrating macrophages and in vitro in BMMs. Gsk3 pharmacological inhibition promoted the resolution of liver IRI in WT, but not myeloid MerTK-deficient, mice. Thus, Gsk3β regulates liver IRI at both activation and resolution stages of the disease. Gsk3 inactivation enhances the proresolving function of liver-infiltrating macrophages in an MerTK-dependent manner.

This study investigated the effect of low-dose aspirin in primary adult liver transplantation (LT) on acute cellular rejection (ACR) as well as arterial patency rates. The use of low-dose aspirin after LT is practiced by many transplant centers to minimize the risk of hepatic artery thrombosis (HAT), although solid recommendations do not exist. However, aspirin also possesses potent anti-inflammatory properties and might mitigate inflammatory processes after LT, such as rejection. Therefore, we hypothesized that the use of aspirin after LT has a protective effect against ACR. This is an international, multicenter cohort study of primary adult deceased donor LT. The study included 17 high-volume LT centers and covered the 3-year period from 2013 to 2015 to allow a minimum 5-year follow-up. In this cohort of 2365 patients, prophylactic antiplatelet therapy with low-dose aspirin was administered in 1436 recipients (61%). The 1-year rejection-free survival rate was 89% in the aspirin group versus 82% in the no-aspirin group (hazard ratio [HR], 0.77; 95% confidence interval [CI], 0.63-0.94; p = 0.01). The 1-year primary arterial patency rates were 99% in the aspirin group and 96% in the no-aspirin group with an HR of 0.23 (95% CI, 0.13-0.40; p

Fractional flow reserve (FFR) determines the functional significance of epicardial stenoses assuming negligible venous pressure (Pv) and microvascular resistance. However, these assumptions may be invalid in end-stage liver disease (ESLD) because of fluctuating Pv and vasodilation. Accordingly, all patients with ESLD who underwent right-sided cardiac catheterization and coronary angiography with FFR as part of their orthotopic liver transplantation evaluation between 2013 and 2018 were included in the present study. Resting mean distal coronary pressure (Pd)/mean aortic pressure (Pa), FFR, and Pv were measured. FFR accounting for Pv (FFR - Pv) was defined as (Pd - Pv)/(Pa - Pv). The hyperemic effect of adenosine was defined as resting Pd/Pa - FFR. The primary outcome was all-cause mortality at 1 year. In 42 patients with ESLD, 49 stenoses were interrogated by FFR (90% were

Background and aimsEnvironmentally triggered chronic liver inflammation can cause collagen deposits, whereas early stages of fibrosis without any specific symptoms could hardly be detectable. We hypothesized that some of the human donor grafts in clinical liver transplantation (LT) might possess unrecognizable fibrosis, affecting their susceptibility to LT-induced stress and hepatocellular damage. This retrospective study aimed to assess the impact of occult hepatic fibrosis on clinical LT outcomes.Approach and resultsHuman (194) donor liver biopsies were stained for collagen with Sirius red, and positive areas (Sirius red-positive area; SRA) were measured. The body mass index, aspartate aminotransferase/alanine aminotransferase ratio, diabetes score was calculated using 962 cases of the donor data at the procurement. LT outcomes, including ischemia-reperfusion injury (IRI), early allograft dysfunction (EAD), and survival rates, were analyzed according to SRA and BARD scores. With the median SRA in 194 grafts of 9.4%, grafts were classified into low-SRA (

Background and aimsLiver ischemia reperfusion injury (IRI) remains an unresolved clinical problem. This study dissected roles of liver-resident macrophage Kupffer cells (KCs), with a functional focus on efferocytosis receptor T-cell immunoglobulin and mucin domain-containing protein-4 (TIM-4), in both the activation and resolution of IRI in a murine liver partial warm ischemia model.Approach and resultsFluorescence-activated cell sorting results showed that TIM-4 was expressed exclusively by KCs, but not infiltrating macrophages (iMФs), in IR livers. Anti-TIM-4 antibody depleted TIM-4+ macrophages in vivo, resulting in either alleviation or deterioration of liver IRI, which was determined by the repopulation kinetics of the KC niche with CD11b+ macrophages. To determine the KC-specific function of TIM-4, we reconstituted clodronate-liposome-treated mice with exogenous wild-type or TIM-4-deficient KCs at either 0 hour or 24 hours postreperfusion. TIM-4 deficiency in KCs resulted in not only increases in the severity of liver IRI (at 6 hours postreperfusion), but also impairment of the inflammation resolution (at 7 days postreperfusion). In vitro analysis revealed that TIM-4 promoted KC efferocytosis to regulate their Toll-like receptor response by up-regulating IL-10 and down-regulating TNF-α productions.ConclusionsTIM-4 is critical for KC homeostatic function in both the activation and resolution of liver IRI by efferocytosis.

BackgroundA major discrepancy between clinical and most experimental settings of liver ischemia-reperfusion injury (IRI) is the allogenicity.MethodsIn the current study, we first established a murine model of allogeneic orthotopic liver transplantation with extended cold ischemia time (18 h). Roles of CD4 T cells in the pathogenesis of IRI in liver allografts were determined using a depleting anti-CD4 antibody. The clinical relevance of CD4 as a marker of liver IRI was analyzed retrospectively in 55 liver transplant patients.ResultsCD4 depletion in both donors and recipients resulted in the most effective protection of liver allografts from IRI, as measured by serum transaminase levels and liver histology. CD4 depletion inhibited IR-induced intragraft neutrophil/macrophage infiltration and proinflammatory gene expressions. Quantitative reverse-transcriptase polymerase chain reaction analysis of human liver biopsies (2 h postreperfusion) revealed that posttransplant, rather than pretransplant, CD4 transcript levels correlated positively with proinflammatory gene expression profile. When we divided patients into subgroups according to intragraft CD4 levels, the high CD4 cohort developed a more severe hepatocellular damage than that with low CD4 levels.ConclusionsCD4 T cells play a key pathogenic role in IRI of allogeneic liver transplants, and intragraft CD4 levels in the early postreperfusion phase may serve as a potential biomarker and therapeutic target to ameliorate liver IRI and improve orthotopic liver transplantation outcomes.

IntroductionThe purpose of this study was to investigate the rates of complications and diagnostic yield of transjugular liver biopsy (TJLB) in deceased donor liver transplant (DDLT) recipients.MethodsFrom January 2009 to December 2019, 1,055 TJLBs were performed in 603 adult DDLT recipients with a mean age of 54 (±12 years). Data were retrospectively reviewed to determine the diagnostic efficacy and incidence of major and minor complications in the 3-day and 1-month period after TJLB. In addition, data were stratified according to platelet count and international normalized ratio to determine the safety of TJLB in patients with varying degrees of coagulopathy.ResultsTJLB yielded diagnostic rate of 98.1% (1,035/1,055), with an overall complication rate of 8.3% (88/1,055). Major complications accounted for 0.85% (9/1,055), and minor complications occurred in 7.48% (79/1,055). When patients were stratified by platelet count (0-50, 51-100, 101-200, 201-300, and >300 × 103 platelets/μL), no significant difference was noted in complication rates (9.5%, 8.6%, 7.6%, 8.5%, and 10.7%, respectively). When grouped by international normalized ratio (0-1, 1.1-2.0, 2.1-3.0, and >3.0), there was no statistical difference in complication rates (8.3%, 8.5%, 7.7%, and 0%, respectively).DiscussionTJLB is a safe, adequate, and effective method to investigate hepatic disorders in DDLT recipients with severe coagulopathy.

Transcriptomic profiling of tumor tissues introduces a large database, which has led to improvements in the ability of cancer diagnosis, treatment, and prevention. However, performing tumor transcriptomic profiling in the clinical setting is very challenging since the procurement of tumor tissues is inherently limited by invasive sampling procedures. Here, we demonstrated the feasibility of purifying hepatocellular carcinoma (HCC) circulating tumor cells (CTCs) from clinical patient samples with improved molecular integrity using Click Chips in conjunction with a multimarker antibody cocktail. The purified CTCs were then subjected to mRNA profiling by NanoString nCounter platform, targeting 64 HCC-specific genes, which were generated from an integrated data analysis framework with 8 tissue-based prognostic gene signatures from 7 publicly available HCC transcriptomic studies. After bioinformatics analysis and comparison, the HCC CTC-derived gene signatures showed high concordance with HCC tissue-derived gene signatures from TCGA database, suggesting that HCC CTCs purified by Click Chips could enable the translation of HCC tissue molecular profiling into a noninvasive setting.

The incidence of hepatocellular carcinoma (HCC) is growing in the United States, especially among the elderly. Older patients are increasingly receiving transplants as a result of HCC, but the impact of advancing age on long-term posttransplant outcomes is not clear. To study this, we used data from the US Multicenter HCC Transplant Consortium of 4980 patients. We divided the patients into 4 groups by age at transplantation: 18 to 64 years (n = 4001), 65 to 69 years (n = 683), 70 to 74 years (n = 252), and ≥75 years (n = 44). There were no differences in HCC tumor stage, type of bridging locoregional therapy, or explant residual tumor between the groups. Older age was confirmed to be an independent and significant predictor of overall survival even after adjusting for demographic, etiologic, and cancer-related factors on multivariable analysis. A dose-response effect of age on survival was observed, with every 5-year increase in age older than 50 years resulting in an absolute increase of 8.3% in the mortality rate. Competing risk analysis revealed that older patients experienced higher rates of non-HCC-related mortality (P = 0.004), and not HCC-related death (P = 0.24). To delineate the precise cause of death, we further analyzed a single-center cohort of patients who received a transplant as a result of HCC (n = 302). Patients older than 65 years had a higher incidence of de novo cancer (18.1% versus 7.6%; P = 0.006) after transplantation and higher overall cancer-related mortality (14.3% versus 6.6%; P = 0.03). Even carefully selected elderly patients with HCC have significantly worse posttransplant survival rates, which are mostly driven by non-HCC-related causes. Minimizing immunosuppression and closer surveillance for de novo cancers can potentially improve the outcomes in elderly patients who received a transplant as a result of HCC.

Foxo1 transcription factor is an evolutionarily conserved regulator of cell metabolism, oxidative stress, inflammation, and apoptosis. Activation of Hedgehog/Gli signaling is known to regulate cell growth, differentiation, and immune function. However, the molecular mechanisms by which interactive cell signaling networks restrain oxidative stress response and necroptosis are still poorly understood. Here, we report that myeloid-specific Foxo1 knockout (Foxo1M-KO) mice were resistant to oxidative stress-induced hepatocellular damage with reduced macrophage/neutrophil infiltration, and proinflammatory mediators in liver ischemia/reperfusion injury (IRI). Foxo1M-KO enhanced β-catenin-mediated Gli1/Snail activity, and reduced receptor-interacting protein kinase 3 (RIPK3) and NIMA-related kinase 7 (NEK7)/NLRP3 expression in IR-stressed livers. Disruption of Gli1 in Foxo1M-KO livers deteriorated liver function, diminished Snail, and augmented RIPK3 and NEK7/NLRP3. Mechanistically, macrophage Foxo1 and β-catenin colocalized in the nucleus, whereby the Foxo1 competed with T-cell factor (TCF) for interaction with β-catenin under inflammatory conditions. Disruption of the Foxo1-β-catenin axis by Foxo1 deletion enhanced β-catenin/TCF binding, activated Gli1/Snail signaling, leading to inhibited RIPK3 and NEK7/NLRP3. Furthermore, macrophage Gli1 or Snail knockout activated RIPK3 and increased hepatocyte necroptosis, while macrophage RIPK3 ablation diminished NEK7/NLRP3-driven inflammatory response. Our findings underscore a novel molecular mechanism of the myeloid Foxo1-β-catenin axis in regulating Hedgehog/Gli1 function that is key in oxidative stress-induced liver inflammation and necroptosis.

Background and aimsSterile inflammation is a major clinical concern during ischemia-reperfusion injury (IRI) triggered by traumatic events, including stroke, myocardial infarction, and solid organ transplantation. Despite high-mobility group box 1 (HMGB1) clearly being involved in sterile inflammation, its role is controversial because of a paucity of patient-focused research.Approach and resultsHere, we examined the role of HMGB1 oxidation states in human IRI following liver transplantation. Portal blood immediately following allograft reperfusion (liver flush; LF) had increased total HMGB1, but only LF from patients with histopathological IRI had increased disulfide-HMGB1 and induced Toll-like receptor 4-dependent tumor necrosis factor alpha production by macrophages. Disulfide HMGB1 levels increased concomitantly with IRI severity. IRI+ prereperfusion biopsies contained macrophages with hyperacetylated, lysosomal disulfide-HMGB1 that increased postreperfusion at sites of injury, paralleling increased histone acetyltransferase general transcription factor IIIC subunit 4 and decreased histone deacetylase 5 expression. Purified disulfide-HMGB1 or IRI+ blood stimulated further production of disulfide-HMGB1 and increased proinflammatory molecule and cytokine expression in macrophages through a positive feedback loop.ConclusionsThese data identify disulfide-HMGB1 as a mechanistic biomarker of, and therapeutic target for, minimizing sterile inflammation during human liver IRI.

Notch signaling plays important roles in the regulation of immune cell functioning during the inflammatory response. Activation of the innate immune signaling receptor NLRP3 promotes inflammation in injured tissue. However, it remains unknown whether Jagged1 (JAG1)-mediated myeloid Notch1 signaling regulates NLRP3 function in acute liver injury. Here, we report that myeloid Notch1 signaling regulates the NLRP3-driven inflammatory response in ischemia/reperfusion (IR)-induced liver injury. In a mouse model of liver IR injury, Notch1-proficient (Notch1FL/FL) mice receiving recombinant JAG1 showed a reduction in IR-induced liver injury and increased Notch intracellular domain (NICD) and heat shock transcription factor 1 (HSF1) expression, whereas myeloid-specific Notch1 knockout (Notch1M-KO) aggravated hepatocellular damage even with concomitant JAG1 treatment. Compared to JAG1-treated Notch1FL/FL controls, Notch1M-KO mice showed diminished HSF1 and Snail activity but augmented NLRP3/caspase-1 activity in ischemic liver. The disruption of HSF1 reduced Snail activation and enhanced NLRP3 activation, while the adoptive transfer of HSF1-expressing macrophages to Notch1M-KO mice augmented Snail activation and mitigated IR-triggered liver inflammation. Moreover, the knockdown of Snail in JAG1-treated Notch1FL/FL livers worsened hepatocellular functioning, reduced TRX1 expression and increased TXNIP/NLRP3 expression. Ablation of myeloid Notch1 or Snail increased ASK1 activation and hepatocellular apoptosis, whereas the activation of Snail increased TRX1 expression and reduced TXNIP, NLRP3/caspase-1, and ROS production. Our findings demonstrated that JAG1-mediated myeloid Notch1 signaling promotes HSF1 and Snail activation, which in turn inhibits NLRP3 function and hepatocellular apoptosis leading to the alleviation of IR-induced liver injury. Hence, the Notch1/HSF1/Snail signaling axis represents a novel regulator of and a potential therapeutic target for liver inflammatory injury.

Introduction: Randomized trials have demonstrated the safety and efficacy of one month of dual antiplatelet therapy (DAPT) after placement of drug-eluting stents in patients with high bleeding risk. Patients with end-stage liver disease (ESLD) are underrepresented in these trials. Patients who undergo percutaneous coronary intervention (PCI) in preparation for orthotopic liver transplantation (OLT) exhibit a high incidence of bleeding complications on DAPT. The rates of bleeding versus thrombotic complications in ESLD patients placed on DAPT following PCI are poorly described. Methods: We retrospectively collected data from 61 patients who were evaluated for OLT between 2016 and 2019 and underwent PCI prior to listing. Bleeding events were classified using the Bleeding Academic Research Consortium (BARC) definitions and included if the following criteria were met: events occurred in the setting of DAPT, were non-procedural in etiology, and occurred during the time following PCI and prior to OLT. Ischemic complications were evaluated by the incidence of myocardial infarction (MI), stent thrombosis, in-stent restenosis (>50%) and all-cause mortality at 1 year follow-up. Results: A total of 55/61 patients (90%) were placed on DAPT following PCI. Among them, 21/55 patients (38%) bled while taking DAPT, including 15 patients (27%) with BARC types 3-5 first-time bleeding events and 10 patients (18%) requiring early discontinuation of therapy. The median time to first bleeding event was 8 days (range 1 to 477 days, 85 th percentile 17 days). Among ischemic complications, MI occurred in 11/55 patients (20%) however only one patient had a type 1 MI with the remaining being type 2 in etiology. There were no episodes of stent thrombosis and 2 episodes of in-stent restenosis during the 1 year follow-up. A total of 12/55 patients (22%) went on to receive OLT and 18/55 (33%) passed away by 1 year post-PCI. Conclusions: Patients with ESLD exhibit a high rate of clinically significant bleeding on DAPT when compared to overall thrombotic events. The majority of bleeds occurred within the first month after PCI. These findings illustrate the need for larger studies to assess the safety of single instead of dual antiplatelet therapy in patients with ESLD who receive PCI.

Hepatocellular carcinoma (HCC) is a leading cause of mortality. Checkpoint inhibitors of programmed cell death protein-1 (PD-1) and programmed death-ligand 1 (PD-L1) have shown great efficacy, but lack biomarkers that predict response. Circulating tumor cells (CTCs) have promise as a liquid-biopsy biomarker; however, data on HCC CTCs expressing PD-L1 have not been reported. We sought to detect PD-L1-expressing HCC-CTCs and investigated their role as a prognostic and predictive biomarker. Using an antibody-based platform, CTCs were enumerated/phenotyped from a prospective cohort of 87 patients with HCC (49 early-stage, 22 locally advanced, and 16 metastatic), 7 patients with cirrhosis, and 8 healthy controls. Immunocytochemistry identified total HCC CTCs (4',6-diamidino-2-phenylindole-positive [DAPI+]/cytokeratin-positive [CK+]/clusters of differentiation 45-negative [CD45-]) and a subpopulation expressing PD-L1 (DAPI+/CK+/PD-L1+/CD45-). PD-L1+ CTCs were identified in 4 of 49 (8.2%) early-stage patients, but 12 of 22 (54.5%) locally advanced and 15 of 16 (93.8%) metastatic patients, accurately discriminating early from locally advanced/metastatic HCC (sensitivity = 71.1%, specificity = 91.8%, area under the receiver operating characteristic curve = 0.807; P

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%).

Background and aimsIschemia-reperfusion injury (IRI) represents a risk factor in liver transplantation (LT). We have shown that overexpression of heme oxygenase-1 (HO-1) mitigates hepatic IRI in LT recipients. Here, we hypothesized that human antigen R (HuR), the stabilizer of adenylate-uridylate (AU)-rich mRNAs, is required for hepatoprotection in LT.Approach and resultsIn an experimental arm, HuR/HO-1 protein expression was correlated with hepatic IRI phenotype. In an in vitro inflammation mimic model of hepatic warm IRI, induction of HuR/HO-1 and cytoplasmic localization following cytokine preconditioning were detected in primary hepatocyte cultures, whereas HuR silencing caused negative regulation of HO-1, followed by enhanced cytotoxicity. Using the HuR-inhibitor, we showed that HuR likely regulates HO-1 through its 3' untranslated region and causes neutrophil activation (CD69+/lymphocyte antigen 6 complex locus G [Ly6-G]). HuR silencing in bone marrow-derived macrophages decreased HO-1 expression, leading to the induction of proinflammatory cytokines/chemokines. RNA sequencing of HuR silenced transcripts under in vitro warm IRI revealed regulation of genes thymus cell antigen 1 (THY1), aconitate decarboxylase 1 (ACOD1), and Prostaglandin E Synthase (PTGES). HuR, but not hypoxia-inducible protein alpha, positively regulated HO-1 in warm, but not cold, hypoxia/reoxygenation conditions. HuR modulated HO-1 in primary hepatocytes, neutrophils, and macrophages under reperfusion. Adjunctive inhibition of HuR diminished microtubule-associated proteins 1A/1B light chain 3B (LC3B), a marker for autophagosome, under HO-1 regulation, suggesting a cytoprotective mechanism in hepatic IR. In a clinical arm, hepatic biopsies from 51 patients with LT were analyzed at 2 hours after reperfusion. Graft HuR expression was negatively correlated with macrophage (CD80/CD86) and neutrophil (Cathepsin G) markers. Hepatic IRI increased HuR/HO-1 expression and inflammatory genes. High HuR-expressing liver grafts showed lower serum alanine aminotransferase/serum aspartate aminotransferase levels and improved LT survival.ConclusionsThis translational study identifies HuR as a regulator of HO-1-mediated cytoprotection in sterile liver inflammation and a biomarker of ischemic stress resistance in LT.

Glycogen synthase kinase 3 (Gsk3) α and β are both constitutively active and inhibited upon stimulation by N-terminal serine phosphorylation. Although roles of active Gsk3 in liver ischemia reperfusion injury (IRI) have been well appreciated, whether Gsk3 N-terminal serine phosphorylation has any functional significance in the disease process remains unclear. In a murine liver partial warm ischemia model, we studied Gsk3 N-terminal serine mutant knock-in (KI) mice and showed that liver IRI was decreased in Gsk3αS21A but increased in Gsk3βS9A mutant KI mice. Bone marrow chimeric experiments revealed that the Gsk3α, but not β, mutation in liver parenchyma protected from IRI, and both mutations in bone marrow-derived cells exacerbated liver injuries. Mechanistically, mutant Gsk3α protected hepatocytes from inflammatory (TNF-α) cell death by the activation of HIV-1 TAT-interactive protein 60 (TIP60)-mediated autophagy pathway. The pharmacological inhibition of TIP60 or autophagy diminished the protection of the Gsk3α mutant hepatocytes from inflammatory cell death in vitro and the Gsk3α mutant KI mice from liver IRI in vivo. Thus, Gsk3 N-terminal serine phosphorylation inhibits liver innate immune activation but suppresses hepatocyte autophagy in response to inflammation. Gsk3 αS21, but not βS9, mutation is sufficient to sustain Gsk4 activities in hepatocytes and protect livers from IRI via TIP60 activation.

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.

Objective and backgroundPattern recognition receptors (PRRs) on immune and parenchymal cells can detect danger-associated molecular patterns (DAMPs) released from cells damaged during ischemia-reperfusion injury (IRI), in heart attack or stroke settings, but also as an unavoidable consequence of solid organ transplantation. Despite IRI being a significant clinical problem across all solid organ transplants, there are limited therapeutics and patient-specific diagnostics currently available.MethodsWe screened portal blood samples obtained from 67 human liver transplant recipients both pre- [portal vein (PV) sample] and post-(liver flush; LF) reperfusion for their ability to activate a panel of PRRs, and analyzed this reactivity in relation to biopsy-proven IRI.ResultsPV samples from IRI+ orthotopic liver transplantation (OLT) patients (n = 35) decreased activation of hTLR4- and hTLR9-transfected cells, whereas PV from IRI- patients (n = 32) primarily increased hTLR7 and hNOD2 activation. LF samples from OLT-IRI patients significantly increased activation of hTLR4 and hTLR9 over IRI- LF. In addition, the change from baseline reactivity to hTLR4/9/NOD2 was significantly higher in IRI+ than IRI- OLT patients.ConclusionsThese results demonstrate that TLR4/7/9 and NOD2 are involved in either promoting or attenuating hepatic IRI, and suggest a diagnostic screening of portal blood for reactivity to these PRRs might prove useful for prediction and/or therapeutic intervention in OLT patients before transplantation.

Farnesoid X receptor (FXR) is the nuclear receptor of bile acids and is involved in innate immune regulation. FXR agonists have been shown to protect multiple organs from inflammatory tissue injuries. Because liver expresses high levels of FXR, we explored the potential therapeutic benefits and underlying mechanisms of pharmacologic FXR activation in a murine model of partial liver warm ischemia. Pretreatment of mice with FXR agonist 3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064) attenuated liver ischemia/reperfusion injuries (IRIs) in wild-type but not FXR knockout mice. Posttreatment with GW4064 facilitated liver recovery from IRI. Mechanistically, Kupffer cells (KCs) expressed much higher levels of FXR than bone marrow-derived macrophages (BMMs). Pretreatment of KCs but not BMMs with GW4064 resulted in lower tumor necrosis factor α but higher interleukin-10 expressions following toll-like receptor stimulation. FXR-targeted gene small heterodimer partner (SHP) was critical for the regulation of KC response by GW4064. In vivo, the depletion of KCs but not cluster of differentiation (CD) 11b+ cells or knockdown of SHP diminished the immune regulatory effect of GW4064 in liver IRI. Thus, FXR activation protects liver from IRI by up-regulating SHP in KCs to inhibit the liver proinflammatory response.

BackgroundIntracranial hemorrhage (ICH) is a devastating complication. Although hypertension and thrombocytopenia are well-known risk factors for ICH in the general population, their roles in ICH after liver transplantation (LT) have not been well established.MethodsWe performed a retrospective study and hypothesized that intraoperative hypertension and thrombocytopenia were associated with posttransplant ICH. New onset of spontaneous hemorrhage in the central nervous system within 30 days after LT were identified by reviewing radiologic reports and medical records. Risk factors were identified by multivariate logistic regression. Receiver operating characteristic analysis and Youden index were used to find the cutoff value with optimal sensitivity and specificity.ResultsOf 1836 adult patients undergoing LT at University of California, Los Angeles, 36 (2.0%) developed ICH within 30 days after LT. Multivariate logistic regression demonstrated that intraoperative mean arterial pressure ≥105 mm Hg (≥10 min) (odds ratio, 6.5; 95% confidence interval, 2.7-7.7; P < 0.001) and platelet counts ≤30 × 10/L (odds ratio, 3.3; 95% confidence interval, 14-7.7; P = 0.006) were associated with increased risk of postoperative ICH. Preoperative total bilirubin ≥7 mg/dL was also a risk factor. Thirty-day mortality in ICH patients was 48.3%, significantly higher compared with the non-ICH group (3.0%; P < 0.001). Patients with all 3 risk factors had a 16% chance of developing ICH.ConclusionsIn the current study, postoperative ICH was uncommon but associated with high mortality. Prolonged intraoperative hypertension and severe thrombocytopenia were associated with postoperative ICH. More studies are warranted to confirm our findings and develop a strategy to prevent this devastating posttransplant complication.

Organ ischemia reperfusion injury (IRI), associated with acute hepatocyte death, remains an unresolved problem in clinical orthotopic liver transplantation (OLT). Autophagy, an intracellular self-digesting progress, is responsible for cell reprograming required to regain post-stress homeostasis. Methods: Here, we analyzed the cytoprotective mechanism of pituitary adenylate cyclase-activating polypeptide (PACAP)-promoted hepatocellular autophagy in a clinically relevant mouse model of extended hepatic cold storage (4 °C UW solution for 20 h) followed by syngeneic OLT. Results: In contrast to 41.7% of liver graft failure by day 7 post-transplant in control group, PACAP treatment significantly improved graft survival (91.7% by day 14), and promoted autophagy-associated regeneration programs in OLT. In parallel in vitro studies, PACAP-enhanced autophagy ameliorated cellular damage (LDH/ALT levels), and diminished necrosis in H2O2-stressed primary hepatocytes. Interestingly, PACAP not only induced nuclear cAMP response element-binding protein (CREB), but also triggered reprogramming factor Kruppel-like factor 4 (KLF4) expression in IR-stressed OLT. Indeed, CREB inhibition attenuated hepatic autophagy and recreated hepatocellular injury in otherwise PACAP-protected livers. Furthermore, CREB inhibition suppressed PACAP-induced KLF4 expression, whereas KLF4 blockade abolished PACAP-promoted autophagy and neutralized PACAP-mediated hepatoprotection both in vivo and in vitro. Conclusion: Current study documents the essential neural regulation of PACAP-promoted autophagy in hepatocellular homeostasis in OLT, which provides the emerging therapeutic principle to combat hepatic IRI in OLT.

Tissue inhibitor of metalloproteinase (TIMP) 3 is a naturally occurring inhibitor of a broad range of proteases, with key roles in extracellular matrix turnover and in the pathogenesis of various diseases. In this study, we investigated the response of mice lacking TIMP3 (TIMP3-/-) to hepatic ischemia/reperfusion injury (IRI). We report here that TIMP3-/- mice showed an enhanced inflammatory response, exacerbated organ damage, and further impaired liver function after IRI when compared with their wild-type littermates. Loss of TIMP3 led to the cleavage and shedding of E-cadherin during hepatic IRI; the full-length 120-kDa E-cadherin and the ratio of 38-kDa C-terminal fragment/120-kDa E-cadherin were decreased and increased, respectively, in TIMP3-/- livers after IRI. Moreover, GI254023X, a potent inhibitor of a disintegrin and metalloprotease (ADAM) 10, was capable of partially rescuing the expression of E-cadherin in the TIMP3-null hepatocytes. The proteolysis of E-cadherin in the TIMP3-/- livers was also linked to the loss of β-catenin from the hepatocyte membranes and to an increased susceptibility to apoptosis after liver IRI. In a similar fashion, depression of the E-cadherin/β-catenin complex mediated by TIMP3 deletion and knockdown of β-catenin by small interfering RNA were both capable of inducing caspase activation in isolated hepatocytes subjected to H2 O2 oxidative stress. Hence, these results support a protective role for TIMP3 expression in sheltering the hepatocyte E-cadherin/β-catenin complex from proteolytic processing and inhibiting apoptosis after hepatic IRI.

Somatic copy number alterations (SCNAs) are important genetic drivers of many cancers. We investigated the feasibility of obtaining SCNA profiles from circulating tumor cells (CTCs) as a molecular liquid biopsy for hepatocellular carcinoma (HCC). CTCs from ten HCC patients underwent SCNA profiling. The Cancer Genome Atlas (TCGA) SCNA data were used to develop a cancer origin classification model, which was then evaluated for classifying 44 CTCs from multiple cancer types. Sequencing of 18 CTC samples (median: 4 CTCs/sample) from 10 HCC patients using a low-resolution whole-genome sequencing strategy (median: 0.88 million reads/sample) revealed frequent SCNAs in previously reported HCC regions such as 8q amplifications and 17p deletions. SCNA profiling revealed that CTCs share a median of 80% concordance with the primary tumor. CTCs had SCNAs not seen in the primary tumor, some with prognostic implications. Using a SCNA profiling model, the tissue of origin was correctly identified for 32/44 (73%) CTCs from 12/16 (75%) patients with different cancer types.

Intestinal microbiota is thought to play an important role in hepatic ischemia/reperfusion injury (IRI) after liver transplantation (LT). Rifaximin, a nonabsorbable antibiotic used to treat encephalopathy, exhibits antibacterial activity within the gut. We report the first study examining the impact of pre-LT rifaximin use on reducing hepatic IRI and inflammatory cell infiltration after LT. This retrospective single-center study included adult LT recipients from January 2013 through June 2016. Patients were divided into 2 groups based on duration of rifaximin use before LT: rifaximin group (≥28 days) and control group (none or

The Hippo pathway, an evolutionarily conserved protein kinase cascade, tightly regulates cell growth and survival. Activation of yes-associated protein (YAP), a downstream effector of the Hippo pathway, has been shown to modulate tissue inflammation. However, it remains unknown as to whether and how the Hippo-YAP signaling may control NLR family pyrin domain containing 3 (NLRP3) activation in mesenchymal stem cell (MSC)-mediated immune regulation during liver inflammation. In a mouse model of ischemia/reperfusion (IR)-induced liver sterile inflammatory injury, we found that adoptive transfer of MSCs reduced hepatocellular damage, shifted macrophage polarization from M1 to M2 phenotype, and diminished inflammatory mediators. MSC treatment reduced mammalian Ste20-like kinase 1/2 and large tumor suppressor 1 phosphorylation but augmented YAP and β-catenin expression with increased prostaglandin E2 production in ischemic livers. However, disruption of myeloid YAP or β-catenin in MSC-transferred mice exacerbated IR-triggered liver inflammation, enhanced NLRP3/caspase-1 activity, and reduced M2 macrophage phenotype. Using MSC/macrophage coculture system, we found that MSCs increased macrophage YAP and β-catenin nuclear translocation. Importantly, YAP and β-catenin colocalize in the nucleus while YAP interacts with β-catenin and regulates its target gene X-box binding protein 1 (XBP1), leading to reduced NLRP3/caspase-1 activity after coculture. Moreover, macrophage YAP or β-catenin deficiency augmented XBP1/NLRP3 while XBP1 deletion diminished NLRP3/caspase-1 activity. Increasing NLRP3 expression reduced M2 macrophage arginase1 but augmented M1 macrophage inducible nitric oxide synthase expression accompanied by increased interleukin-1β release. Conclusion: MSCs promote macrophage Hippo pathway, which in turn controls NLRP3 activation through a direct interaction between YAP and β-catenin and regulates XBP1-mediated NLRP3 activation, leading to reprograming macrophage polarization toward an anti-inflammatory M2 phenotype. Moreover, YAP functions as a transcriptional coactivator of β-catenin in MSC-mediated immune regulation. Our findings suggest a therapeutic target in MSC-mediated immunotherapy of liver sterile inflammatory injury.

Background & aimsHepatic ischemia-reperfusion injury (IRI) is a major complication of hemorrhagic shock, liver resection and transplantation. YAP, a key downstream effector of the Hippo pathway, is essential for determining cell fate and maintaining homeostasis in the liver. We aimed to elucidate its role in IRI.MethodsThe role of YAP/Hippo signaling was systematically studied in biopsy specimens from 60 patients after orthotopic liver transplantation (OLT), and in a mouse model of liver warm IRI. Human biopsy specimens were collected after 2-10 h of cold storage and 3 h post-reperfusion, before being screened by western blot. In the mouse model, the role of YAP was probed by activating or inhibiting YAP prior to ischemia-reperfusion.ResultsIn human biopsies, high post-OLT YAP expression was correlated with well-preserved histology and improved hepatocellular function at postoperative day 1-7. In mice, the ischemia insult (90 min) triggered intrinsic hepatic YAP expression, which peaked at 1-6 h of reperfusion. Activation of YAP protected the liver against IR-stress, by promoting regenerative and anti-oxidative gene induction, while diminishing oxidative stress, necrosis/apoptosis and the innate inflammatory response. Inhibition of YAP aggravated hepatic IRI and suppressed repair/anti-oxidative genes. In mouse hepatocyte cultures, activating YAP prevented hypoxia-reoxygenation induced stress. Interestingly, YAP activation suppressed extracellular matrix synthesis and diminished hepatic stellate cell (HSC) activation, whereas YAP inhibition significantly delayed hepatic repair, potentiated HSC activation, and enhanced liver fibrosis at 7 days post-IRI. Notably, YAP activation failed to protect Nrf2-deficient livers against IR-mediated damage, leading to extensive fibrosis.ConclusionOur novel findings document the crucial role of YAP in IR-mediated hepatocellular damage and liver fibrogenesis, providing evidence of a potential therapeutic target for the management of sterile liver inflammation in transplant recipients.Lay summaryIn the clinical arm, graft YAP expression negatively correlated with liver function and tissue damage after human liver transplantation. YAP activation attenuated hepatocellular oxidative stress and diminished the innate immune response in mouse livers following ischemia-reperfusion injury. In the mouse model, YAP inhibited hepatic stellate cell activation, and abolished injury-mediated fibrogenesis up to 7 days after the ischemic insult.

Liver transplantation (LT) has become the standard of care for patients with end-stage liver disease and those with hepatic malignancies, while adaptive immune-dominated graft rejection remains a major challenge. Despite potent anti-inflammatory and cytoprotective functions of heme oxygenase-1 (HO-1) overexpression upon innate immune-driven hepatic ischemia reperfusion injury, its role in adaptive immune cell-driven responses remains to be elucidated. We analyzed human biopsies from LT recipients (n = 55) to determine putative association between HO-1 levels and adaptive/co-stimulatory gene expression programs in LT. HO-1 expression negatively correlated with innate (CD68, Cathepsin G, TLR4, CXCL10), adaptive (CD4, CD8, IL17) and co-stimulatory (CD28, CD80, CD86) molecules at the graft site. LT recipients with high HO-1 expression showed a trend towards improved overall survival. By demonstrating the association between graft HO-1 levels and adaptive/co-stimulatory gene programs, our study provides important insights to the role of HO-1 signaling in LT patients.

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

Guidelines to evaluate patients for coronary artery disease (CAD) during preoperative evaluation for orthotopic liver transplantation (OLT) are conflicting. Cardiac catheterization is not without risk in patients with end-stage liver disease. No study to date has looked at the utility of non-electrocardiogram-gated chest computed tomography (CT) in the preliver transplant population. Our hypothesis was that coronary artery calcium scores (CACSs) from chest CT scans ordered during the liver transplant workup can identify patients who would benefit from invasive angiography. Nine hundred and fifty-three patients who underwent coronary angiography as part of their OLT workup were considered. Charts were randomly selected and reviewed for the presence of a chest CT performed before coronary angiography during the OLT workup. Agatston and Weston scores were calculated. CACS results were compared with coronary angiography findings. Nine of 54 patients were found to have obstructive CAD by angiography. Receiver-operating characteristic analysis demonstrated that an Agatston score of 251 and a Weston score of 6 maximized sensitivity and specificity for detection of obstructive coronary disease. An Agatston score

OBJECTIVES:To investigate the safety profile and diagnostic efficacy of transjugular liver biopsy (TJLB), with a focus on patients with severe coagulopathies and with multiple biopsies. METHODS:Clinical, laboratory, and demographic information was collected on 1,321 TJLBs in 932 patients (mean age 43.5 ± 23.2 years) performed between January 2009 and May 2017 to determine the diagnostic success rate and incidence of both major and minor complications in the 3-day and 30-day period post-biopsies. These outcomes were also analyzed for severely coagulopathic patients and a subgroup of patients who underwent multiple biopsies. RESULTS:The overall success rate (diagnostic yield) of the TJLB procedure was 97.7% (1,291/1,321). Overall, the major and minor complication rates were 1.0% (13/1,321) and 9.5% (126/1,321), respectively. In patients with multiple biopsies, the overall complication rate was similar to the entire study cohort, which was 10.4% (57/550). Patients were also stratified according to the platelet counts of 0-50, 51-100, 101-200, 201-300 and >300 × 10 platelets/μL. The overall complication rates were 8.0% (10/124), 11.6% (36/310), 9.9% (54/547), 11.9% (28/235), and 14.3% (11/77), respectively, and these were not statistically significant from each other. Patients were also stratified by international normalized ratio into 0-1, 1.1-2, 2.1-3, and >3. The overall complication rates of these patients were 8.0% (19/237), 11.8% (113/954), 16.3% (7/43), and 0% (0/9), respectively, and were not statistically significant from each other. DISCUSSION:TJLB is a highly efficacious, well-tolerated and safe procedure. It can be safely performed multiple times in the same patient or in critically ill, severely coagulopathic patients with no significant increase in the rate of complication while maintaining an extremely favorable diagnostic yield.

BackgroundKidney delayed graft function (kDGF) remains a challenging problem following simultaneous liver and kidney transplantation (SLKT) with a reported incidence up to 40%. Given the scarcity of renal allografts, it is crucial to minimize the development of kDGF among SLKT recipients to improve patient and graft outcomes. We sought to assess the role of preoperative recipient and donor/graft factors on developing kDGF among recipients of SLKT.MethodsA retrospective review of 194 patients who received SLKT in the period from January 2004 to March 2017 in a single center was performed to assess the effect of preoperative factors on the development of kDGF.ResultsKidney delayed graft function was observed in 95 patients (49%). Multivariate analysis revealed that donor history of hypertension, cold static preservation of kidney grafts [versus using hypothermic pulsatile machine perfusion (HPMP)], donor final creatinine, physiologic MELD, and duration of delay of kidney transplantation after liver transplantation were significant independent predictors for kDGF. kDGF is associated with worse graft function and patient and graft survival.ConclusionsKidney delayed graft function has detrimental effects on graft function and graft survival. Understanding the risks and combining careful perioperative patient management, proper recipient selection and donor matching, and graft preservation using HPMP would decrease kDGF among SLKT recipients.

BACKGROUND:Hepatic ischemia-reperfusion injury (IRI) is a severe complication in liver transplantation, hepatectomy and hemorrhagic shock. As neuropeptides transmit the regulatory signal between nervous-immune systems communication, our previous study documented that pituitary adenylate cyclase-activating polypeptides (PACAP) depressed hepatic TLR4 immune response in liver IRI. METHODS:Here, we focused on how PACAP suppressed hepatocellular damage and enhanced hepatocyte regeneration in a murine model of partial liver warm IRI. RESULTS:Yes-associated protein (YAP), a cellular modulator of tissue regeneration, was readily induced in WT IR-livers. As its induction was failed in PACAP-deficient livers, PACAP supplement enhanced YAP expression in WT mouse, promoted its nuclear translocation and downstream anti-oxidative/regenerative genes expression both in vivo and in vitro. Further, verteporfin (VP), a YAP transcriptional inhibitor, abolished PACAP-mediated hepatoprotection significantly. Meanwhile, blockade of PKA-CREB signaling recreated liver damage in PACAP-protected liver, as well as impeded stimulation on YAP and its downstream gene expressions. Consistently, inhibition of PKA-CREB decreased PACAP promoted YAP expression in primary hepatocytes culture, and made them vulnerable to H2O2 stress in vitro. In addition, lysophosphatidic acid (LPA), another Hippo pathway inhibitor, failed to affect PACAP-mediated hepatoprotection or hepatocellular YAP induction. This implies that PACAP regulated YAP through PKA-CREB pathway at the transcriptional level rather than canonical hippo pathway. CONCLUSION:Our study discovered the neural modulation of PACAP-YAP axis in hepatic cytoprotection and homeostasis in liver IRI. These reveal a novel insight of neuropeptide PACAP in combating liver IRI in clinical patients.

BackgroundLiver ischemia and reperfusion injury (IRI) is a major complication of liver transplant, hepatectomy, and hemorrhagic shock. The cyclin-dependent kinase 2 (CDK2) acts as a pivotal regulator of cell cycle and proliferation.MethodsThis study evaluated the modulation and therapeutic potential of CDK2 inhibition in a mouse model of partial liver warm IRI.ResultsLiver IR-triggered intrinsic CDK2 expression, peaking by 0.5 hour of reperfusion and maintaining a high-level throughout 1 to 24 hours. Roscovitine, a specific CDK2 inhibitor, prevented liver IR-mediated damage with abolished serum alanine aminotransferase levels and reserved liver pathology. CDK2 inhibition-mediated liver protection was accompanied by decreased macrophage/neutrophil infiltration, diminished hepatocyte apoptosis, abolished toll like receptor 4 signaling and downstream gene inductions (C-X-C motif ligand-10, Tumor necrosis factor-α, interleukin-1β, and interleukin-6), yet augmented interleukin-10 expression. In vitro, CDK2 inhibition by Roscovitine suppressed macrophage TLR4 activation and further depressed downstream inflammatory signaling (myeloid differentiation factor 88, interferon regulatory transcription factor 3, p38, c-Jun N-terminal kinase, and extracellular-regulated kinase).ConclusionsOur novel findings revealed the critical role of CDK2 in hepatic cytoprotection and homeostasis against liver IRI. As CDK2 inhibition regulated local immune response and prevented hepatocyte death, this study provided the evidence for new treatment approaches to combat IRI in liver transplant.

Adverse cardiovascular events after liver transplantation (LT) are relatively common and are a significant source of early mortality. Although new-onset systolic dysfunction after LT is a reported phenomenon, there is little data regarding its incidence, risk factors, and outcomes.This single-center retrospective study included all adult patients from January 2002 to March 2015 with deceased-donor LT and available preoperative transthoracic echocardiograms (TTEs). In total, 1,760 patients were included in the study, 602 (34.2%) of whom had a postoperative TTE. The primary end point was development of new-onset cardiomyopathy, defined as a new left ventricular ejection fraction (LVEF) of

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 (18 hour), were transplanted to syngeneic myeloid HO-1 deficient (mHO-1 KO) or FLOX (control) hosts, and sampled postreperfusion (6 hour). 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.

The purpose of this study was to assess the significance of tenascin-C (Tnc) expression in steatotic liver ischemia/reperfusion injury (IRI). The critical shortage in donor organs has led to the use of steatotic livers in transplantation regardless of their elevated susceptibility to hepatic IRI. Tnc is an endogenous danger signal extracellular matrix molecule involved in various aspects of immunity and tissue injury. In the current study, mice were fed with a steatosis-inducing diet and developed approximately 50% hepatic steatosis, predominantly macrovesicular, before being subjected to hepatic IRI. We report here that lipid accumulation in hepatocytes inflated the production of Tnc in steatotic livers and in isolated hepatic stellate cells. Moreover, we show that the inability of Tnc-/- deficient steatotic mice to express Tnc significantly protected these mice from liver IRI. Compared with fatty controls, Tnc-/- steatotic mice showed significantly reduced serum transaminase levels and enhanced liver histological preservation at both 6 and 24 hours after hepatic IRI. The lack of Tnc expression resulted in impaired lymphocyte antigen 6 complex, locus (Ly6G) neutrophil and macrophage antigen-1 (Mac-1) leukocyte recruitment as well as in decreased expression of proinflammatory mediators (interleukin 1β, tumor necrosis factor α, and chemokine [C-X-C motif] ligand 2) after liver reperfusion. Myeloperoxidase (MPO) is the most abundant cytotoxic enzyme secreted by neutrophils and a key mediator of neutrophil-induced oxidative tissue injuries. Using an in vitro model of steatosis, we also show that Tnc markedly potentiated the effect of steatotic hepatocytes on neutrophil-derived MPO activity. In conclusion, our data support the view that inhibition of Tnc is a promising therapeutic approach to lessen inflammation in steatotic livers and to maximize their successful use in organ transplantation.

Nonselective Beta blockade (NSBB) is commonly prescribed for liver transplantation (LT) candidates, but its impact on intraoperative hemodynamics is not well understood. In this study, we investigated if preoperative NSBB was associated with severe bradycardia during LT and if severe intraoperative bradycardia was associated with 30-day mortality. Adult patients undergoing LT between 2005 and 2014 were included. Propensity matching was used to control selection bias. Intraoperative hemodynamics were compared between patients with and without preoperative NSBB. Univariate and multivariate methods were used in statistical analysis. Of 1452 patients, 370 who received preoperative NSBB were matched in a 1:1 ratio with those who did not. Propensity matching eliminated all significant differences between the two groups. Patients who received preoperative NSBB had a significantly higher incidence of severe intraoperative bradycardia compared with the non-BB group (9.6% vs 3.2%, P = 0.001, OR 2.95, 95% CI 1.42-6.12, P = 0.004). Intraoperative hypotension and postreperfusion syndrome were not significantly different between the two groups. Severe intraoperative bradycardia was associated with increased 30-day mortality. In conclusion, preoperative NSBB was associated with severe intraoperative bradycardia in LT. In patients who receive preoperative NSBB, severe intraoperative bradycardia should be closely monitored in LT. Further studies assessing safety of preoperative NSBB and intraoperative bradycardia in LT are warranted.

Liver ischemia-reperfusion injury (IRI) represents a risk factor for early graft dysfunction and an obstacle to expanding donor pool in orthotopic liver transplantation (OLT). We have reported on the crucial role of macrophage Notch1 signaling in mouse warm hepatic IRI model. However, its clinical relevance or therapeutic potential remain unknown. Here, we used Serelaxin (SER), to verify Notch1 induction and putative hepatoprotective function in ischemia-reperfusion-stressed OLT. C57BL/6 mouse livers subjected to extended (18-hour) cold storage were transplanted to syngeneic recipients. SER treatment at reperfusion ameliorated IRI, improved post-OLT survival, decreased neutrophil/macrophage infiltration, and suppressed proinflammatory cytokine programs, while simultaneously increasing Notch intracellular domain (NICD) and hairy and enhancer of split 1 (Hes1) target genes. In bone marrow-derived macrophage cultures, SER suppressed proinflammatory while enhancing antiinflammatory gene expression concomitantly with increased NICD and Hes1. Hepatic biopsies from 21 adult primary liver transplant patients (2 hours postreperfusion) were divided into low-NICD (n = 11) and high-NICD (n = 10) expression groups (western blots). Consistent with our murine findings, human livers characterized by high NICD were relatively IRI resistant, as shown by serum alanine aminotransferase (ALT) levels at day 1 post-OLT. Our study documents the efficacy of SER-Notch1 signaling in mouse OLT and highlights the protective function of Notch1 in liver transplant patients.

Background & aimsGlycogen synthase kinase 3β (Gsk3β [Gsk3b]) is a ubiquitously expressed kinase with distinctive functions in different types of cells. Although its roles in regulating innate immune activation and ischaemia and reperfusion injuries (IRIs) have been well documented, the underlying mechanisms remain ambiguous, in part because of the lack of cell-specific tools in vivo.MethodsWe created a myeloid-specific Gsk3b knockout (KO) strain to study the function of Gsk3β in macrophages in a murine liver partial warm ischaemia model.ResultsCompared with controls, myeloid Gsk3b KO mice were protected from IRI, with diminished proinflammatory but enhanced anti-inflammatory immune responses in livers. In bone marrow-derived macrophages, Gsk3β deficiency resulted in an early reduction of Tnf gene transcription but sustained increase of Il10 gene transcription on Toll-like receptor 4 stimulation in vitro. These effects were associated with enhanced AMP-activated protein kinase (AMPK) activation, which led to an accelerated and higher level of induction of the novel innate immune negative regulator small heterodimer partner (SHP [Nr0b2]). The regulatory function of Gsk3β on AMPK activation and SHP induction was confirmed in wild-type bone marrow-derived macrophages with a Gsk3 inhibitor. Furthermore, we found that this immune regulatory mechanism was independent of Gsk3β Ser9 phosphorylation and the phosphoinositide 3-kinase-Akt signalling pathway. In vivo, myeloid Gsk3β deficiency facilitated SHP upregulation by ischaemia-reperfusion in liver macrophages. Treatment of Gsk3b KO mice with either AMPK inhibitor or SHP small interfering RNA before the onset of liver ischaemia restored liver proinflammatory immune activation and IRI in these otherwise protected hosts. Additionally, pharmacological activation of AMPK protected wild-type mice from liver IRI, with reduced proinflammatory immune activation. Inhibition of the AMPK-SHP pathway by liver ischaemia was demonstrated in tumour resection patients.ConclusionsGsk3β promotes innate proinflammatory immune activation by restraining AMPK activation.Lay summaryGlycogen synthase kinase 3β promotes macrophage inflammatory activation by inhibiting the immune regulatory signalling of AMP-activated protein kinase and the induction of small heterodimer partner. Therefore, therapeutic targeting of glycogen synthase kinase 3β enhances innate immune regulation and protects liver from ischaemia and reperfusion injury.

Current clinicopathologic staging systems and serum biomarkers poorly discriminate tumor biology in hepatocellular carcinoma (HCC), with high recurrence rates following curative-intent surgical resection and liver transplantation (LT). Identification of accurate biomarkers for improved prognostication and treatment selection is a critical unmet need. We sought to develop a novel "liquid-biopsy" assay capable of detecting HCC circulating tumor cells (CTCs) and characterizing phenotypic subpopulations with prognostic significance. Using HCC cell lines, a tissue microarray, and human blood samples, an antibody cocktail targeting the cell-surface markers asialoglycoprotein receptor (ASGPR), glypican-3, and epithelial cell adhesion molecule was optimized for HCC CTC capture using the NanoVelcro CTC Assay. The ability of HCC CTCs and vimentin (VIM)-positive CTCs (a subpopulation expressing an epithelial-to-mesenchymal phenotype) to accurately discriminate tumor stage, recurrence, progression, and overall survival (OS) was evaluated in a prospective study of 80 patients. Multimarker capture detected greater numbers of CTCs than any individual antibody alone for both cell line and patient samples (P < 0.001). HCC CTCs were identified in 59/61 (97%) patients, and HCC (median, 6 CTCs) and non-HCC patients (median, 1 CTC; area under the receiver operating characteristic curve [AUROC] = 0.92; P < 0.001; sensitivity = 84.2%; specificity = 88.5%) were accurately discriminated. VIM-positive CTCs accurately discriminated early-stage, LT eligible patients (median, 0 CTCs) from locally advanced/metastatic, LT ineligible patients (median, 6 CTCs; AUROC = 0.89; P = 0.001; sensitivity = 87.1%; specificity = 90.0%), and predicted OS for all patients (hazard ratio [HR], 2.21; P = 0.001), and faster recurrence after curative-intent surgical or locoregional therapy in potentially curable early-stage HCC (HR, 3.14; P = 0.002). In conclusion, we developed a novel multimarker CTC enrichment assay that detects HCC CTCs with high efficiency and accuracy. A phenotypic subpopulation of VIM-positive CTCs appears to signify the presence of aggressive underlying disease and occult metastases and may have important implications for treatment selection. Liver Transplantation 24 946-960 2018 AASLD.

BackgroundWe examined neurologic consultations for altered mental status in perioperative liver transplant patients to determine the overall incidence, to assess the presumed etiology and the data reviewed to determine that etiology, and to assess outcomes.MethodsRetrospective chart review conducted for all 728 adult patients receiving orthotopic liver transplantation (OLT) between January 01, 2010, to June 30, 2014, with identification of 218 receiving neurology consults between 30 days pre-OLT and 90 days post-OLT, with review of all records necessary to determine initial findings and follow-up examination.ResultsSeventy-three consults for 69 patients were identified, with 27 felt to be altered since a procedure, 20 with sudden-onset altered mentation, and 26 with gradual or waxing-waning course. A single underlying etiology was identified in only 19 cases, with multiple factors involved in all others, with metabolic, toxic, infectious, and structural etiologies most often implicated. There was no statistically significant difference in outcome for those with altered mental status consults versus the total OLT population, though the sudden-onset presentation group did show significantly increased mortality rates.ConclusionsThis systematic study illustrates the variety of potential causes of altered mentation within the perioperative setting of liver transplantation. Workup including neuroimaging (preferably magnetic resonance imaging), infectious cultures, and expanded metabolic laboratory tests should be undertaken.

Hepatic ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and acute/chronic rejection as well as a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although glucocorticoid receptor (GR) signaling may enhance cytoprotective programs, clinical use of glucocorticoid is limited because of adverse effects, whereas clinical relevance of GR-facilitated cytoprotection in OLT remains unknown. We aimed to evaluate the significance of hepatic GR in clinical OLT and verify the impact of recombinant human relaxin (rhRLX), which may function as a GR agonist in a tissue/disease-specific manner. Fifty-one OLT patients were recruited under an institutional research board (IRB) protocol. Liver biopsies were collected after cold storage (presurgery) and 2 hours postreperfusion (before abdominal closure), followed by western blotting-assisted hepatic analyses. Forty-three percent of OLTs failed to increase GR perioperatively under surgical stress. Post-/pre-GR ratios at postoperative day 1 correlated negatively with serum aspartate aminotransferase (AST)/cleaved caspase-3 and positively with B-cell lymphoma-extra large (Bcl-xL)/B-cell lymphoma 2 (Bcl-2) levels. In a murine OLT model with extended (18-hour) cold storage, treatment with rhRLX ameliorated ischemia-reperfusion (IR) damage and improved survival while up-regulating hepatocyte GR and Bcl-xL/Bcl-2 expression in OLT. rhRLX-induced GR suppressed hepatocyte high-mobility group box 1 (HMGB1) translocation/release, accompanied by decreased Toll-like receptor 4 (TLR4)/receptor for advanced glycation end products (RAGE), suppressed interleukin 1 beta (IL1β), chemokine (C-C motif) ligand 2 (CCL2), C-X-C motif chemokine (CXCL)10, tumor necrosis factor alpha (TNFα), CXCL1, and CXCL2 levels, and attenuated neutrophil/macrophage accumulation in OLT. Inhibition of GR in hepatocyte culture and in OLT diminished rhRLX-mediated cytoprotection.ConclusionThis translational study underscores the role of rhRLX-GR signaling as a regulator of hepatocellular protection against IR stress in OLT. In the context of a recent phase III clinical trial demonstrating positive outcomes of rhRLX in patients with acute heart failure, studies on rhRLX for the management of IRI in OLT recipients are warranted. (Hepatology 2018;68:258-273).

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 (20 hours) 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.