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 IL18Rß. 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.

Alternative splicing of CEACAM1 by hypoxia-inducible factor-1α enhances tolerance to hepatic ischemia in mice and humans.

Although alternative splicing (AS) drives transcriptional responses and cellular adaptation to environmental stresses, its contributions in organ transplantation have not been appreciated. We have shown that carcinoembryonic antigen-related cell adhesion molecule (Ceacam1; CD66a), a transmembrane biliary glycoprotein expressed in epithelial, endothelial, and immune cells, determines donor liver transplant quality. Here, we studied how AS of Ceacam1 affects ischemia-reperfusion injury (IRI) in mouse and human livers. We found that the short cytoplasmic isoform Ceacam1-S increased during early acute and late resolution phases of warm IRI injury in mice. Transfection of Ceacam1-deficient mouse hepatocytes with adenoviral Ceacam1-S mitigated hypoxia-induced loss of cellular adhesion by repressing the Ask1/p-p38 cell death pathway. Nucleic acid-blocking morpholinos, designed to selectively induce Ceacam1-S, protected hepatocyte cultures against temperature-induced stress in vitro. Luciferase and chromatin immunoprecipitation assays identified direct binding of hypoxia-inducible factor-1α (Hif-1α) to the mouse polypyrimidine tract binding protein 1 (Ptbp1) promoter region. Dimethyloxalylglycine protected mouse livers from warm IR stress and hepatocellular damage by inhibiting prolyl hydroxylase domain-containing protein 1 and promoting AS of Ceacam1-S. Last, analysis of 46 human donor liver grafts revealed that CEACAM1-S positively correlated with pretransplant HIF1A expression. This also correlated with better transplant outcomes, including reduced TIMP1, total bilirubin, proinflammatory MCP1, CXCL10 cytokines, immune activation markers IL17A, and incidence of delayed complications from biliary anastomosis. This translational study identified mouse Hif-1α-controlled AS of Ceacam1, through transcriptional regulation of Ptbp1 promoter region, as a functional underpinning of hepatoprotection against IR stress and tissue damage in liver transplantation.

Disulfide-HMGB1 signals through TLR4 and TLR9 to induce inflammatory macrophages capable of innate-adaptive crosstalk in human liver transplantation.

Ischemia-reperfusion injury (IRI) during orthotopic liver transplantation (OLT) contributes to graft rejection and poor clinical outcomes. The disulfide form of high mobility group box 1 (diS-HMGB1), an intracellular protein released during OLT-IRI, induces pro-inflammatory macrophages. How diS-HMGB1 differentiates human monocytes into macrophages capable of activating adaptive immunity remains unknown. We investigated if diS-HMGB1 binds toll-like receptor (TLR) 4 and TLR9 to differentiate monocytes into pro-inflammatory macrophages that activate adaptive immunity and promote graft injury and dysfunction. Assessment of 106 clinical liver tissue and longitudinal blood samples revealed that OLT recipients were more likely to experience IRI and graft dysfunction with increased diS-HMGB1 released during reperfusion. Increased diS-HMGB1 concentration also correlated with TLR4/TLR9 activation, polarization of monocytes into pro-inflammatory macrophages, and production of anti-donor antibodies. In vitro, healthy volunteer monocytes stimulated with purified diS-HMGB1 had increased inflammatory cytokine secretion, antigen presentation machinery, and reactive oxygen species production. TLR4 inhibition primarily impeded cytokine/chemokine and costimulatory molecule programs, whereas TLR9 inhibition decreased HLA-DR and reactive oxygen species production. diS-HMGB1-polarized macrophages also showed increased capacity to present antigens and activate T memory cells. In murine OLT, diS-HMGB1 treatment potentiated ischemia-reperfusion-mediated hepatocellular injury, accompanied by increased serum alanine transaminase levels. This translational study identifies the diS-HMGB1/TLR4/TLR9 axis as potential therapeutic targets in OLT-IRI recipients.

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

BACKGROUND & AIMS: 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. METHODS: 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. RESULTS: 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 â†’ 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 â†’ TIM-3Tg/CC1KO). The perioperative CC1 increase in human OLT reduced hepatocellular injury, early allograft dysfunction, and the cumulative rejection rate. CONCLUSIONS: 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.

SIRT1 Regulates Hepatocyte Programmed Cell Death via GSDME - IL18 Axis in Human and Mouse Liver Transplantation.

Sirtuin 1 (SIRT1) is a histone/protein deacetylase involved in cellular senescence, inflammation, and stress resistance. We previously reported that myeloid SIRT1 signaling 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 signaling 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 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 IL18Rß signaling. Hepatocyte SIRT1 deficiency upregulated apoptosis and GSDME-mediated programmed cell death, which in turn deteriorated the hepatocellular function and shortened OLT survival. Augmented GSDME processing, accompanied by increased secretion of IL18 by stressed hepatocytes, was prominent in SIRT1-deficient, cold-stored livers. Hepatocyte SIRT1 signaling regulated anti-apoptotic Bcl-2/XIAP proteins, suppressed cold stress-triggered apoptosis, and mitigated GSDME licensing to release IL18. Notably, while crosslinking IL18R depressed SIRT1 and Bcl-2/XIAP signaling 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 signaling circuit as a therapeutic target in the mechanism underpinning hepatocyte death in human and mouse liver transplantation.

In Vivo Regeneration of Tubular Small Intestine With Motility: A Novel Approach by Orthotopic Transplantation of Decellularized Scaffold.

BACKGROUND: Whole-intestine engineering can provide a therapeutic alternative to bowel transplantation. Intestinal components including the mucosa, muscular layer, enteric nervous system, and vasculature must be reestablished as a tubular organ to generate an artificial small intestine. This study proposes a novel approach to produce a transplantable, well-organized tubular small intestine using a decellularized scaffold. METHODS: Male Lewis rat intestines were used to generate decellularized scaffolds. Patch or tubular grafts were prepared from the decellularized intestine and transplanted into the rat intestine orthotopically. Histological analysis of the decellularized intestine was performed up to 12 wk after transplantation. RESULTS: Histological examination revealed abundant vascularization into the decellularized patch graft 1 wk after transplantation. Muscular and nervous components, as well as cryptogenesis, were observed in the decellularized patch graft 2 wk after transplantation. Sixteen of the 18 rats survived with normal intake of food and water after the decellularized tubular graft transplantation. Compared with silicone tube grafts, the decellularized tubular grafts significantly promoted the infiltration and growth of intestinal components including the mucosa, muscular layer, and nerve plexus from the recipients. Circular and longitudinal muscle with a well-developed myenteric plexus was regenerated, and intestinal motility was confirmed in the decellularized tubular graft 12 wk after transplantation. CONCLUSIONS: Orthotopic transplantation of decellularized intestine enhanced the reconstruction of the well-organized tubular small intestine with an enteric nervous system in vivo. Our method using a decellularized scaffold represents a promising approach toward whole-intestine engineering and provides a therapeutic alternative for the irreversible intestinal failure.

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.

CD4+ T Cell NRF2 Signaling Improves Liver Transplantation Outcomes by Modulating T Cell Activation and Differentiation.

Aims: Innate and adaptive immune responses regulate hepatic ischemia-reperfusion injury (IRI) in orthotopic liver transplantation (OLT). While the mechanism of how nuclear factor erythroid 2-related factor 2 (NRF2) plays a role in liver IRI has been studied, the contribution of T cell-specific NRF2 in OLT remains unknown. In the current translational study, we investigated whether and how CD4+ T cell-specific NRF2 signaling affects liver transplant outcomes in mice and humans. Results: In the experimental arm, cold-stored (4°C/18 h) wild-type (WT) mouse livers transplanted to NRF2-deficient (NRF2-knockout [NRF2-KO]) recipients experienced greater hepatocellular damage than those in Nrf2-proficient (WT) counterparts, evidenced by Suzuki's histological scores, frequency of TdT-mediated dUTP nick end labeling (TUNEL)+ cells, and elevated serum aspartate aminotransferase/alanine aminotransferase (AST/ALT) levels. In vitro studies showed that NRF2 signaling suppressed CD4+ T cell differentiation to a proinflammatory phenotype (Th1, Th17) while promoting the regulatory (Foxp3+) T cell lineage. Furthermore, OLT injury deteriorated in immune-compromised RAG2-KO test recipients repopulated with CD4+ T cells from NRF2-KO compared with WT donor mice. In the clinical arm of 45 human liver transplant patients, the perioperative increase of NRF2 expression in donor livers negatively regulated innate and adaptive immune activation, resulting in reduced hepatocellular injury in NRF2-proficient OLT. Innovation and Conclusion: CD4+ T cell population expressing NRF2 attenuated ischemia and reperfusion (IR)-triggered hepatocellular damage in a clinically relevant mouse model of extended donor liver cold storage, followed by OLT, whereas the perioperative increase of NRF2 expression reduced hepatic injury in human liver transplant recipients. Thus, CD4+ T cell NRF2 may be a novel cytoprotective sentinel against IR stress in OLT recipients. Antioxid. Redox Signal. 38, 670-683.

Myeloid Ikaros-SIRT1 signaling axis regulates hepatic inflammation and pyroptosis in ischemia-stressed mouse and human liver.

BACKGROUND & AIMS: Although Ikaros (IKZF1) is a well-established transcriptional regulator in leukocyte lymphopoiesis and differentiation, its role in myeloid innate immune responses remains unclear. Sirtuin 1 (SIRT1) is a histone/protein deacetylase involved in cellular senescence, inflammation, and stress resistance. Whether SIRT1 signaling is essential in myeloid cell activation remains uncertain, while the molecular communication between Ikaros and SIRT1, two major transcriptional regulators, has not been studied. METHODS: We undertook molecular and functional studies to interrogate the significance of the myeloid Ikaros-SIRT1 axis in innate immune activation and whether it may serve as a homeostatic sentinel in human liver transplant recipients (hepatic biopsies) and murine models of sterile hepatic inflammation (liver warm ischemia-reperfusion injury in wild-type, myeloid-specific Sirt1-knockout, and CD11b-DTR mice) as well as primary bone marrow-derived macrophage (BMM) cultures (Ikaros silencing vs. overexpression). RESULTS: In our clinical study, we identified increased post-reperfusion hepatic Ikaros levels, accompanied by augmented inflammasome signaling yet depressed SIRT1, as a mechanism of hepatocellular damage in liver transplant recipients. In our experimental studies, we identified infiltrating macrophages as the major source of Ikaros in IR-stressed mouse livers. Then, we demonstrated that Ikaros-regulated pyroptosis - induced by canonical inflammasome signaling in BMM cultures - was SIRT1 dependent. Consistent with the latter, myeloid-specific Ikaros signaling augmented hepatic pyroptosis to aggravate pro-inflammatory responses in vivo by negatively regulating SIRT1 in an AMPK-dependent manner. Finally, myeloid-specific SIRT1 was required to suppress pyroptosis, pro-inflammatory phenotype, and ultimately mitigate hepatocellular injury in ischemia-stressed murine livers. CONCLUSION: These findings identify the Ikaros-SIRT1 axis as a novel mechanistic biomarker of pyroptosis and a putative checkpoint regulator of homeostasis in response to acute hepatic stress/injury in mouse and human livers. LAY SUMMARY: This report describes how crosstalk between Ikaros and SIRT1, two major transcriptional regulators, influence acute hepatic inflammation in murine models of liver ischemia-reperfusion injury and liver transplant recipients. We show that the myeloid Ikaros-SIRT1 axis regulates inflammasome-pyroptotic cell death and hepatocellular damage in stressed livers. Thus, the Ikaros-SIRT1 axis may serve as a novel checkpoint regulator that is required for homeostasis in response to acute liver injury in mice and humans.

Donor Hepatic Occult Collagen Deposition Predisposes to Peritransplant Stress and Impacts Human Liver Transplantation.

BACKGROUND AND AIMS: Environmentally 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 RESULTS: Human (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 (<15%; n = 140) and high-SRA (≥15%; n = 54) groups. Grafts with high SRA suffered from higher rates of IRI and EAD (P < 0.05) as compared to those with low SRA. Interestingly, high SRA was identified as an independent risk factor for EAD and positively correlated with the donor BARD score. When comparing low-BARD (n = 692) with high-BARD (n = 270) grafts in the same period, those with high BARD showed significantly higher post-LT transaminase levels and higher rates of IRI and EAD. CONCLUSIONS: These findings from the largest clinical study cohort to date document the essential role of occult collagen deposition in donor livers on LT outcomes. High-SRA and donor BARD scores correlated with an increased incidence of hepatic IRI and EAD in LT recipients. This study provides the rationale for in-depth and prospective assessment of occult fibrosis for refined personalized LT management.

Ischemia-reperfusion injury and its relationship with early allograft dysfunction in liver transplant patients.

Ischemia-reperfusion injury (IRI) is believed to contribute to graft dysfunction after liver transplantation (LT). However, studies on IRI and the impact of early allograft dysfunction (EAD) in IRI grafts are limited. Histological IRI was graded in 506 grafts from patients who had undergone LT and classified based on IRI severity (no, minimal, mild, moderate, and severe). Of the 506 grafts, 87.4% had IRI (no: 12.6%, minimal: 38.1%, mild: 35.4%, moderate: 13.0%, and severe: 0.8%). IRI severity correlated with the incidence of EAD and graft survival at 6 months. Longer cold/warm ischemia time, recipient/donor hypertension, and having a male donor were identified as independent risk factors for moderate to severe IRI. Among 70 grafts with moderate to severe IRI, 42.9% of grafts developed EAD, and grafts with EAD had significantly inferior survival compared to grafts without EAD. Longer cold ischemia time and large droplet macrovesicular steatosis (≥20%) were identified as independent risk factors for EAD. Our study demonstrated that increased IRI severity was correlated with inferior short-term graft outcomes. Careful consideration of IRI risk factors during donor-recipient matching may assist in optimizing graft utilization and LT outcomes. Furthermore, identification of risk factors of IRI-associated EAD may guide patient management and possible timely graft replacement.

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.

Therapeutic targets for liver regeneration after acute severe injury: a preclinical overview.

Introduction: Liver transplantation is the only viable treatment with a proven survival benefit for acute liver failure (ALF). Donor organ shortage is, however, a major hurdle; hence, alternative approaches that enable liver regeneration and target acute severe hepatocellular damage are necessary.Areas covered: This article sheds light on therapeutic targets for liver regeneration and considers their therapeutic potential. ALF following extensive hepatocyte damage and small-for-size syndrome (SFSS) are illuminated for the reader while the molecular mechanisms of liver regeneration are assessed in accordance with relevant therapeutic strategies. Furthermore, liver background parameters and predictive biomarkers that might associate with liver regeneration are reviewed.Expert opinion: There are established and novel experimental strategies for liver regeneration to prevent ALF resulting from SFSS. Granulocyte-colony stimulating factor (G-CSF) is a promising agent targeting liver regeneration after acute severe injury. Autophagy and hepatocyte senescence represent attractive new targets for liver regeneration in acute severe hepatic injury. Liver support strategies, including tissue engineering, constitute novel regenerative means; the success of this is dependent on stem cell research advances. However, there is no firm clinical evidence that these supportive strategies may alleviate hepatocellular damage until liver transplantation becomes available or successful self-liver regeneration occurs.

Human Antigen R (HuR): A Regulator of Heme Oxygenase-1 Cytoprotection in Mouse and Human Liver Transplant Injury.

BACKGROUND AND AIMS: Ischemia-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 RESULTS: In 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. CONCLUSIONS: This 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.

Novel hybrid three-dimensional artificial liver using human induced pluripotent stem cells and a rat decellularized liver scaffold.

INTRODUCTION: Liver transplantation is currently the only curative therapy for end-stage liver failure; however, establishment of alternative treatments is required owing to the serious donor organ shortage. Here, we propose a novel model of hybrid three-dimensional artificial livers using both human induced pluripotent stem cells (hiPSCs) and a rat decellularized liver serving as a scaffold. METHODS: Rat liver harvesting and decellularization were performed as reported in our previous studies. The decellularized liver scaffold was recellularized with hiPSC-derived hepatocyte-like cells (hiPSC-HLCs) through the biliary duct. The recellularized liver graft was continuously perfused with the culture medium using a pump at a flow rate of 0.5 mL/min in a standard CO2 (5%) cell incubator at 37 °C. RESULTS: After 48 h of continuous perfusion culture, the hiPSC-HLCs of the recellularized liver distributed into the parenchymal space. Furthermore, the recellularized liver expressed the albumin (ALB) and CYP3A4 genes, and secreted human ALB into the culture medium. CONCLUSION: Novel hybrid artificial livers using hiPSCs and rat decellularized liver scaffolds were successfully generated, which possessed human hepatic functions.

CD90 expression in human intrahepatic cholangiocarcinoma is associated with lymph node metastasis and poor prognosis.

BACKGROUND AND OBJECTIVES: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. However, its prognosis remains poor. Expression of cluster of differentiation 90 (CD90) has been identified as an indicator of poor prognosis in many cancers. Here, we examined the importance of CD90 expression in ICC. METHODS: We performed immunohistological assays for CD90 in human ICC surgical specimens and assessed its relationship with clinicopathological findings and prognosis. Moreover, we analyzed the characteristics of CD90+/- cells, mainly with respect to metastatic potential, using human ICC cell lines. RESULTS: CD90 expression was significantly associated with lymph node metastasis and was revealed to be an independent prognostic factor. The CD90+ cells present in ICC specimens did not appear to be cancer-associated fibroblasts, as they did not express α-smooth muscle actin. In vitro, CD90 + cells exhibited greater migratory ability and higher expression of epithelial-mesenchymal transition (EMT)-related genes, including CXCR4 and MMP7, than CD90- cells. Wnt/ß-catenin signaling pathway activation was also heightened in CD90+ cells. In such cells, EMT appeared to be induced by CXCR4 and MMP7 expression through activation of Wnt/ß-catenin signaling. CONCLUSION: CD90+ cells demonstrate high metastatic potential owing to Wnt/ß-catenin signaling activation and are associated with poor prognosis in ICC.

Establishment of practical recellularized liver graft for blood perfusion using primary rat hepatocytes and liver sinusoidal endothelial cells.

Tissue decellularization produces a three-dimensional scaffold that can be used to fabricate functional liver grafts following recellularization. Inappropriate cell distribution and clotting during blood perfusion hinder the practical use of recellularized livers. Here we aimed to establish a seeding method for the optimal distribution of parenchymal and endothelial cells, and to evaluate the effect of liver sinusoidal endothelial cells (LSECs) in the decellularized liver. Primary rat hepatocytes and LSECs were seeded into decellularized whole-liver scaffolds via the biliary duct and portal vein, respectively. Biliary duct seeding provided appropriate hepatocyte distribution into the parenchymal space, and portal vein-seeded LSECs simultaneously lined the portal lumen, thereby maintaining function and morphology. Hepatocytes co-seeded with LSECs retained their function compared with those seeded alone. Platelet deposition was significantly decreased and hepatocyte viability was maintained in the co-seeded group after extracorporeal blood perfusion. In conclusion, our seeding method provided optimal cell distribution into the parenchyma and vasculature according to the three-dimensional structure of the decellularized liver. LSECs maintained hepatic function, and supported hepatocyte viability under blood perfusion in the engineered liver graft owing to their antithrombogenicity. This recellularization procedure could help produce practical liver grafts with blood perfusion.

Identification of keratin 19-positive cancer stem cells associating human hepatocellular carcinoma using CYFRA 21-1.

The current lack of an easily measurable surrogate marker of cancer stem cells (CSCs) prevents the clinical application of CSCs for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC-CSC marker associated with transforming growth factor beta (TGFß)/Smad signaling, and that K19+ HCC-CSCs could be a new therapeutic target of TGFß receptor 1 inhibitor LY2157299. In this study, we examined whether K19+ HCC-CSCs can be tracked using cytokeratin 19 fragment CYFRA 21-1. In 147 HCC patients who underwent curative resection and evaluated K19 expression by immunohistochemistry, preoperative serum CYFRA 21-1 levels were significantly higher in K19+ patients than in K19- patients (P < 0.01). Receiver operating characteristic analyses revealed that serum CYFRA 21-1 was the statistically significant and the most sensitive predictor of tumor K19 expression among preoperative laboratory test values (P < 0.001). In HCC cells encoding with a K19 promoter-driven enhanced green fluorescent protein, fluorescence-activated cell sorting (FACS)-isolated K19+ cells displayed significantly higher levels of supernatant CYFRA 21-1 than K19- cells (P < 0.01). Gain/loss of K19 function experiments confirmed that CYFRA 21-1 levels were regulated by K19 function in HCC cells. Furthermore, CYFRA 21-1 levels reflected the treatment efficacy of LY2157299 in K19+ cells. In conclusion, CYFRA 21-1 can be used to predict K19 expression in HCC, and should thereby aid in the development of novel therapeutic strategies targeting K19+ HCC-CSCs.