Pirfenidone attenuates acetaminophen-induced liver injury via suppressing c-Jun N-terminal kinase phosphorylation.

Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in Western countries. Pirfenidone (PFD), an orally bioavailable pyridone derivative, is clinically used for idiopathic pulmonary fibrosis treatment and has antifibrotic, anti-inflammatory, and antioxidant effects. Here we examined the PFD effect on APAP-induced liver injury. In a murine model, APAP caused serum alanine aminotransferase elevation attenuated by PFD treatment. We performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and vital propidium iodide (PI) stainings simultaneously. APAP induced TUNEL-positive/PI-negative necrosis around the central vein and subsequent TUNEL-negative/PI-positive oncotic necrosis with hemorrhage and caused the upregulation of hypercoagulation- and hypoxia-associated gene expressions. PFD treatment suppressed these findings. Western blotting revealed PFD suppressed APAP-induced c-Jun N-terminal kinase (JNK) phosphorylation despite no effect on JNK phosphatase expressions. In conclusion, simultaneous TUNEL and vital PI staining is useful for discriminating APAP-induced necrosis from typical oncotic necrosis. Our results indicated that PFD attenuated APAP-induced liver injury by suppressing TUNEL-positive necrosis by directly blocking JNK phosphorylation. PFD is promising as a new option to prevent APAP-induced liver injury.

Corticosteroid suppresses urea-cycle-related gene expressions in ornithine transcarbamylase deficiency.

BACKGROUND: Ornithine transcarbamylase deficiency (OTCD) is most common among urea cycle disorders (UCDs), defined by defects in enzymes associated with ureagenesis. Corticosteroid administration to UCD patients, including OTCD patients, is suggested to be avoided, as it may induce life-threatening hyperammonemia. The mechanism has been considered nitrogen overload due to the catabolic effect of corticosteroids; however, the pathophysiological process is unclear. METHODS: To elucidate the mechanism of hyperammonemia induced by corticosteroid administration in OTCD patients, we analyzed a mouse model by administering corticosteroids to OTCspf-ash mice deficient in the OTC gene. Dexamethasone (DEX; 20 mg/kg) was administered to the OTCspf-ash and wild-type (WT) mice at 0 and 24 h, and the serum ammonia concentrations, the levels of the hepatic metabolites, and the gene expressions related with ammonia metabolism in the livers and muscles were analyzed. RESULTS: The ammonia levels in Otcspf-ash mice that were administered DEX tended to increase at 24 h and increased significantly at 48 h. The metabolomic analysis showed that the levels of citrulline, arginine, and ornithine did not differ significantly between Otcspf-ash mice that were administered DEX and normal saline; however, the level of aspartate was increased drastically in Otcspf-ash mice owing to DEX administration (P < 0.01). Among the enzymes associated with the urea cycle, mRNA expressions of carbamoyl-phosphate synthase 1, ornithine transcarbamylase, arginosuccinate synthase 1, and arginosuccinate lyase in the livers were significantly downregulated by DEX administration in both the Otcspf-ash and WT mice (P < 0.01). Among the enzymes associated with catabolism, mRNA expression of Muscle RING-finger protein-1 in the muscles was significantly upregulated in the muscles of WT mice by DEX administration (P < 0.05). CONCLUSIONS: We elucidated that corticosteroid administration induced hyperammonemia in Otcspf-ash mice by not only muscle catabolism but also suppressing urea-cycle-related gene expressions. Since the urea cycle intermediate amino acids, such as arginine, might not be effective because of the suppressed expression of urea-cycle-related genes by corticosteroid administration, we should consider an early intervention by renal replacement therapy in cases of UCD patients induced by corticosteroids to avoid brain injuries or fatal outcomes.

Discriminant equation using mucosally expressed cytokines and transcription factor for making definite diagnosis of inflammatory bowel disease unclassified.

BACKGROUND: The pathological conditions of UC and CD involved in inflammatory bowel disease-unclassified (IBD-U), UC with primary sclerosing cholangitis (PSC-UC), and UC with autoimmune pancreatitis type 2 (AIP-UC) remain unclear. Therefore, it is difficult to decide the appropriate treatments for these subtypes of UC. Our aim was to examine whether the discriminant equation using the mucosally expressed mediators designed as our previous study for IBD, could characterize IBD-U, PSC-UC, or AIP-UC. METHODS: A total of 56 patients including UC (n = 24), CD (n = 15), IBD-U (n = 10), PSC-UC (n = 4), and AIP-UC (n = 3), along with 9 control patients were enrolled in this study. Mucosally expressed inflammatory mediators related to Th1, Th2, Th17, and Treg were measured using quantitative PCR in endoscopic biopsies from the inflamed intestines of the patients. The IBD-U, PSC-UC or AIP-UC were characterized using discriminant analysis and principle component analysis. RESULTS: Through discriminant analyses, combinations of 3 to 7 inflammatory mediators were used to discriminate between UC and CD. Moreover, the identified 3 markers could diagnose patients with IBD-U as UC or CD with high accuracy. The distribution graph of inflammatory mediators using the principal component analysis revealed that PSC-UC and AIP-UC exhibited CD-like and UC-like features, respectively. CONCLUSIONS: The discriminant equation using mucosally expressed mediators of IL-13, IL-21 and T-bet can be used as a universal diagnostic tool not only for IBD-U but also to assess pathological conditions in PSC-UC and AIP-UC.

Portal Vein Thrombosis Repeatedly Observed in a Cirrhotic Patient with Antiphospholipid Antibody Syndrome.

Background: Although portal vein thrombosis in cirrhotic patients is frequently observed, the detailed process remains to be clarified, and the role of anticardiolipin antibody in the development of portal vein thrombosis has been controversial. Case Report: A 52-year-old man, who had been diagnosed with alcoholic cirrhosis of the liver, was admitted to our hospital suffering from dyspnea and ascites. Just after being diagnosed as having antiphospholipid antibody syndrome with lung thrombosis and delivering a positive result for the ß 2-glycoprotein I-dependent anticardiolipin antibody, he sustained rupture of the esophageal varices with rapid development of portal vein thrombosis, which resolved under anticoagulant therapy. Two years later, he was admitted again on suspicion of thrombosis because of an elevation in the serum D-dimer level, and computed tomography showed portal and upper mesenteric vein thrombosis. Although immediate anticoagulant therapy resulted in complete recanalization, he suffered the same episode 2 months later, which occurred with re-elevation of the serum D-dimer level. Conclusion: A positive finding of an anticardiolipin antibody in cirrhotic patients has been considered to be nonspecific and not related to the development of thrombus in the portal vein. This case, however, seems to indicate that cirrhotic patients with the ß2-glycoprotein I-dependent anticardiolipin antibody should be regarded as being at high risk for portal vein thrombosis. Monitoring with the serum D-dimer was useful in detecting portal vein thrombosis in its early stage.

Clinicopathologic Features of Adult-onset Still's Disease Complicated by Severe Liver Injury.

Adult-onset Still's disease (AOSD) is a systemic inflammatory disorder. Severe liver injury has rarely been reported, although liver enzyme elevation is a common complication of AOSD. We herein report four cases of relapsed AOSD with severe liver disorder by tapering or terminating corticosteroids. Liver specimens revealed robust infiltration of inflammatory cells throughout the lobule, especially cluster of differentiation (CD) 8-positive cells. Relapsed AOSD was refractory to corticosteroid reintroduction and required immunosuppressants. Severe liver injury with AOSD is pathologically characterized by extensive lobular infiltration of CD8-positive cells, and we should consider additive immunosuppressive agents on corticosteroids for treatment.

Microcirculatory disturbance in acute liver injury is triggered by IFNγ-CD40 axis.

BACKGROUND: Acute liver failure (ALF) is a life-threatening disorder that progresses from self-limiting acute liver injury (ALI). Microcirculatory disturbance characterized by sinusoidal hypercoagulation and subsequent massive hypoxic hepatocyte damage have been proposed to be the mechanism by which ALI deteriorates to ALF; however, the precise molecular pathway of the sinusoidal hypercoagulation remains unknown. Here, we analyzed ALI patients and mice models to uncover the pathogenesis of ALI with microcirculatory disturbance. METHODS: We conducted a single-center retrospective study for ALI and blood samples and liver tissues were analyzed to evaluate the microcirculatory disturbance in ALI patients (n = 120). Single-cell RNA sequencing analysis (scRNA-seq) was applied to the liver from the concanavalin A (Con A)­induced mouse model of ALI. Interferon-gamma (IFNγ) and tumor necrosis factor-alpha knockout mice, and primary human liver sinusoidal endothelial cells (LSECs) were used to assess the mechanism of microcirculatory disturbance. RESULTS: The serum IFNγ concentrations were significantly higher in ALI patients with microcirculatory disturbance than in patients without microcirculatory disturbance, and the IFNγ was upregulated in the Con A mouse model which presented microcirculatory disturbance. Hepatic IFNγ expression was increased as early as 1 hour after Con A treatment prior to sinusoidal hypercoagulation and hypoxic liver damage. scRNA-seq revealed that IFNγ was upregulated in innate lymphoid cells and stimulated hepatic vascular endothelial cells at the early stage of liver injury. In IFNγ knockout mice treated with Con A, the sinusoidal hypercoagulation and liver damage were remarkably attenuated, concomitant with the complete inhibition of CD40 and tissue factor (TF) upregulation in vascular endothelial cells. By ligand-receptor analysis, CD40-CD40 ligand interaction was identified in vascular endothelial cells. In human LSECs, IFNγ upregulated CD40 expression and TF was further induced by increased CD40-CD40 ligand interaction. Consistent with these findings, hepatic CD40 expression was significantly elevated in human ALI patients with microcirculatory disturbance. CONCLUSION: We identified the critical role of the IFNγ-CD40 axis as the molecular mechanism of microcirculatory disturbance in ALI. This finding may provide novel insights into the pathogenesis of ALI and potentially contribute to the emergence of new therapeutic strategies for ALI patients.

Acute kidney injury is an unfavorable prognostic factor in acute liver failure and is associated with tumor necrosis factor-alpha.

Acute kidney injury (AKI) is a common complication of acute liver failure (ALF); but its pathogenesis is unknown. ALF was divided into 2 subgroups; ALF with hepatic coma, which corresponds to ALF in the US and Europe, and ALF without hepatic coma. AKI has been shown to worsen the prognosis of ALF patients with hepatic coma; however, its prognostic significance in ALF without hepatic coma remains unknown. A single-center retrospective study of 174 patients with ALF was performed. AKI was defined according to KDIGO criteria. AKI developed in 29 (66.0%) of 44 ALF patients with hepatic coma and 27 (38.5%) of 130 ALF patients without hepatic coma. Systemic inflammatory response syndrome (SIRS) was found to be significantly associated with AKI incidence in ALF patients (P < .001). Tumor necrosis factor-alpha (TNF-α) was found to be significantly associated with the presence and severity of AKI (P = .0039 and P = .0140, respectively). On multivariate analysis, TNF-α was an independent risk factor linked with AKI (P = .0103). Even in the absence of hepatic coma, the transplant-free survival rate of ALF was significantly associated with the presence and severity of AKI. Even when hepatic coma is absent, AKI complicated in ALF is strongly associated with TNF-α and worsens the transplant-free survival rate. Before the onset of hepatic coma, plasma exchange, or extracorporeal blood purification to remove inflammatory cytokines should be considered in ALF patients.

Transcatheter arterial steroid injection therapy improves the prognosis of patients with acute liver failure.

Acute liver failure (ALF) is a disorder defined by coagulopathy and encephalopathy with a poor prognosis. No effective therapies have been established except for liver transplantation. We previously reported a subgroup of patients with acute liver injury who developed microcirculatory disturbance. We also established and reported transcatheter arterial steroid injection therapy (TASIT) as a new treatment of ALF. Here, we analyze the effectiveness of TASIT in a larger cohort and evaluate the impact on ALF patients with or without microcirculatory disturbance. We conducted a single-center retrospective study to evaluate the effectiveness of TASIT in patients with ALF admitted at Kyushu University Hospital between January 2005 and March 2018. TASIT is performed by injecting methylprednisolone via the proper hepatic artery for 3 days. One hundred ninety-4 patients with ALF were enrolled and analyzed in this study. Of the 87 patients given TASIT, 71 (81.6%) recovered without any complications and 16 (18.4%) died or underwent liver transplantation. Of the 107 patients not administered TASIT, 77 (72.0%) recovered and 30 (28.0%) progressed to irreversible liver failure. In the high-lactate dehydrogenase subgroup, 52 (86.7%) of the 60 patients with TASIT recovered, and the survival rate was significantly higher than that in patients who did not receive TASIT. Multivariate regression analysis revealed that the TASIT procedure was one of the significant prognostic factors in the high-lactate dehydrogenase subgroup and was significantly associated with prothrombin activity percentage improvement. TASIT is an effective treatment for patients with ALF, especially in those with microcirculatory disturbance.

FOXK1 promotes nonalcoholic fatty liver disease by mediating mTORC1-dependent inhibition of hepatic fatty acid oxidation.

Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disorder caused by overnutrition and can lead to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). The transcription factor Forkhead box K1 (FOXK1) is implicated in regulation of lipid metabolism downstream of mechanistic target of rapamycin complex 1 (mTORC1), but its role in NAFLD-NASH pathogenesis is understudied. Here, we show that FOXK1 mediates nutrient-dependent suppression of lipid catabolism in the liver. Hepatocyte-specific deletion of Foxk1 in mice fed a NASH-inducing diet ameliorates not only hepatic steatosis but also associated inflammation, fibrosis, and tumorigenesis, resulting in improved survival. Genome-wide transcriptomic and chromatin immunoprecipitation analyses identify several lipid metabolism-related genes, including Ppara, as direct targets of FOXK1 in the liver. Our results suggest that FOXK1 plays a key role in the regulation of hepatic lipid metabolism and that its inhibition is a promising therapeutic strategy for NAFLD-NASH, as well as for HCC.

Direct Conversion of Human Endothelial Cells Into Liver Cancer-Forming Cells Using Nonintegrative Episomal Vectors.

Liver cancer is an aggressive cancer associated with a poor prognosis. Development of therapeutic strategies for liver cancer requires fundamental research using suitable experimental models. Recent progress in direct reprogramming technology has enabled the generation of many types of cells that are difficult to obtain and provide a cellular resource in experimental models of human diseases. In this study, we aimed to establish a simple one-step method for inducing cells that can form malignant human liver tumors directly from healthy endothelial cells using nonintegrating episomal vectors. To screen for factors capable of inducing liver cancer-forming cells (LCCs), we selected nine genes and one short hairpin RNA that suppresses tumor protein p53 (TP53) expression and introduced them into human umbilical vein endothelial cells (HUVECs), using episomal vectors. To identify the essential factors, we examined the effect of changing the amounts and withdrawing individual factors. We then analyzed the proliferation, gene and protein expression, morphologic and chromosomal abnormality, transcriptome, and tumor formation ability of the induced cells. We found that a set of six factors, forkhead box A3 (FOXA3), hepatocyte nuclear factor homeobox 1A (HNF1A), HNF1B, lin-28 homolog B (LIN28B), MYCL proto-oncogene, bHLH transcription factor (L-MYC), and Kruppel-like factor 5 (KLF5), induced direct conversion of HUVECs into LCCs. The gene expression profile of these induced LCCs (iLCCs) was similar to that of human liver cancer cells, and these cells effectively formed tumors that resembled human combined hepatocellular-cholangiocarcinoma following transplantation into immunodeficient mice. Conclusion: We succeeded in the direct induction of iLCCs from HUVECs by using nonintegrating episomal vectors. iLCCs generated from patients with cancer and healthy volunteers will be useful for further advancements in cancer research and for developing methods for the diagnosis, treatment, and prognosis of liver cancer.

Upregulated expression of hypoxia reactive genes in peripheral blood mononuclear cells from chronic liver disease patients.

Liver fibrosis induces intrahepatic microcirculation disorder and hypoxic stress. Hypoxic stress has the potential for an increase in the possibility of more liver fibrosis and carcinogenesis. Liver biopsy is a standard method that evaluates of intrahepatic hypoxia, however, is invasive and has a risk of bleeding as a complication. Here, we investigated the hypoxia reactive gene expressions in peripheral blood mononuclear cells (PBMC) from chronic liver disease patients to evaluate intrahepatic hypoxia in a non-invasive manner. The subjects enrolled for this study were composed of 20 healthy volunteers (HV) and 48 patients with chronic liver disease (CLD). CLD patients contained 24 patients with chronic hepatitis（CH）and 24 patients with liver cirrhosis (LC). PBMC were isolated from heparinized peripheral blood samples. We measured the transcriptional expression of hypoxia reactive genes and inflammatory cytokines by quantitative RT-PCR. mRNA expression of adrenomedullin (AM), vascular endothelial growth factor A (VEGFA) superoxide dismutase (SOD), glutathione peroxidase (GPx) (p < 0.05), Interleukin-6 (IL-6), transforming growth factor-beta (TGF-ß) and heme oxygenase-1 (HO-1) in CLD group were significantly higher than HV. AM mRNA expression is correlated with serum lactate dehydrogenase (LDH), serum albumin (Alb), IL6, and SOD mRNA expression. The hypoxia reactive gene expression in PBMCs from CLD patients was more upregulated than HV. Especially, angiogenic genes were notably upregulated and correlated with liver fibrosis. Here, we suggest that mRNA expression of AM in PBMCs could be the biomarker of intrahepatic hypoxia.

Metabolic Alteration in Hepatocellular Carcinoma: Mechanism of Lipid Accumulation in Well-Differentiated Hepatocellular Carcinoma.

Objective: Metabolic alteration is widely considered as one of the hallmarks of cancer. Hepatocellular carcinoma (HCC) presents a unique pathological feature in which lipid accumulation is common in well-differentiated HCC and rare in poorly differentiated HCC; however, the underlying mechanism remains unclear. Methods: Tissue samples were obtained from 103 HCC patients who had undergone hepatic resection and 12 living donors of liver transplantation. We evaluated metabolic gene expressions in cancer tissues as well as background noncancer tissues and compared the expressions by the degree of cancer differentiation and by liver disease states. Besides, the metabolomics was evaluated and integrated to gene expressions in nonalcoholic steatohepatitis (NASH)-HCC model mice. Results: In cancer tissues, the expression levels of enzymes related to glycolysis, pentose phosphate pathway (PPP), and fatty acid (FA) synthesis were increased and that of tricarboxylic acid (TCA) cycle and ß-oxidation were suppressed. Same metabolic alterations were observed in noncancer tissue as the liver disease progresses from healthy liver to chronic hepatitis, cirrhosis, and HCC. Similar alterations of metabolic genes were detected in NASH-HCC mice, which were consistent with the results of metabolomics. As the degree of cancer differentiation decreased, glycolysis and PPP were accelerated; however, FA synthesis and uptake were diminished. Conclusions: The metabolic alterations including glycolysis, PPP, TCA cycle, and ß-oxidation became more prominent as liver disease progresses from normal, chronic hepatitis, cirrhosis, well-, moderately, and poorly differentiated HCC. FA synthesis and uptake were highest in well-differentiated HCC, which could explain the lipid accumulation.

Direct reprogramming of human umbilical vein- and peripheral blood-derived endothelial cells into hepatic progenitor cells.

Recent advances have enabled the direct induction of human tissue-specific stem and progenitor cells from differentiated somatic cells. However, it is not known whether human hepatic progenitor cells (hHepPCs) can be generated from other cell types by direct lineage reprogramming with defined transcription factors. Here, we show that a set of three transcription factors, FOXA3, HNF1A, and HNF6, can induce human umbilical vein endothelial cells to directly acquire the properties of hHepPCs. These induced hHepPCs (hiHepPCs) propagate in long-term monolayer culture and differentiate into functional hepatocytes and cholangiocytes by forming cell aggregates and cystic epithelial spheroids, respectively, under three-dimensional culture conditions. After transplantation, hiHepPC-derived hepatocytes and cholangiocytes reconstitute damaged liver tissues and support hepatic function. The defined transcription factors also induce hiHepPCs from endothelial cells circulating in adult human peripheral blood. These expandable and bipotential hiHepPCs may be useful in the study and treatment of human liver diseases.

Efficacy of tolvaptan for the patients with advanced hepatocellular carcinoma.

AIM: To investigate the factors influenced the efficacy of tolvaptan (TLV) in liver cirrhosis. METHODS: We retrospectively enrolled 61 consecutive patients with refractory hepatic ascites. All of them had been treated with furosemide and spironolactone before admission, and treated with TLV for 7 d in our hospital. The effect of TLV was defined by the rate of body weight loss, and the factors that influenced TLV efficacy were analyzed using multiple regression. RESULTS: Coexistent hepatocellular carcinoma (HCC) was the only significant predictive variable that attenuated the efficacy of TLV. In stratified analysis, high doses of furosemide decreased the efficacy of TLV in patients with HCC, and increased efficacy in those without HCC. In the latter, a high Child-Pugh-Turcotte score had a positive influence and a high concentration of lactate dehydrogenase had a negative influence on the effectiveness of TLV. CONCLUSION: Development of ascites may differ between patients with liver failure and those with HCC progression. A sufficient preceding dose of furosemide decreases diuretic effect of TLV.