Kojiro Taura

CCR2 promotes hepatic fibrosis in mice

Chemokines and chemokine receptors contribute to the migration of hepatic stellate cells (HSCs) and Kupffer cells, two key cell types in fibrogenesis. Here, we investigate the role of CCR2, the receptor for monocyte chemoattractant protein (MCP)‐1, MCP‐2, and MCP‐3, in hepatic fibrosis. Hepatic CCR2, MCP‐1, MCP‐2, and MCP‐3 messenger RNA expression was increased after bile duct ligation (BDL). Both Kupffer cells and HSCs, but not hepatocytes, expressed CCR2. BDL‐ and CCl4‐induced fibrosis was markedly reduced in CCR2−/− mice as assessed through collagen deposition, α‐smooth muscle actin expression, and hepatic hydroxyproline content. We generated CCR2 chimeric mice by the combination of clodronate, irradiation, and bone marrow (BM) transplantation allowing full reconstitution of Kupffer cells, but not HSCs, with BM cells. Chimeric mice containing wild‐type BM displayed increased macrophage recruitment, whereas chimeric mice containing CCR2−/− BM showed less macrophage recruitment at 5 days after BDL. Although CCR2 expressed in the BM enhanced macrophage recruitment in early phases of injury, CCR2 expression on resident liver cells including HSCs, but not on the BM, was required for fibrogenic responses in chronic fibrosis models. In vitro experiments demonstrated that HSCs deficient in CCR2−/− or its downstream mediator p47phox−/− did not display extracellular signal‐regulated kinase and AKT phosphorylation, chemotaxis, or reactive oxygen species production in response to MCP‐1, MCP‐2, and MCP‐3. Conclusion: Our results indicate that CCR2 promotes HSC chemotaxis and the development of hepatic fibrosis. (HEPATOLOGY 2009.)

Hepatocytes do not undergo epithelial‐mesenchymal transition in liver fibrosis in mice

The origin of fibrogenic cells in liver fibrosis remains controversial. We assessed the emerging concept that hepatocytes contribute to production of extracellular matrix (ECM) in liver fibrosis through epithelial‐mesenchymal transition (EMT). We bred triple transgenic mice expressing ROSA26 stop β‐galactosidase (β‐gal), albumin Cre, and collagen α1(I) green fluorescent protein (GFP), in which hepatocyte‐derived cells are permanently labeled by β‐gal and type I collagen‐expressing cells are labeled by GFP. We induced liver fibrosis by repetitive carbon tetrachloride (CCl4) injections. Liver sections and isolated cells were evaluated for GFP and β‐gal as well as expression of α‐smooth muscle actin (α‐SMA) and fibroblast‐specific protein 1 (FSP‐1). Upon stimulation with transforming growth factor β‐1, cultured hepatocytes isolated from untreated liver expressed both GFP and β‐gal with a fibroblast‐like morphological change but lacked expression of other mesenchymal markers. Cells from CCl4‐treated livers never showed double‐positivity for GFP and β‐gal. All β‐gal‐positive cells exhibited abundant cytoplasm, a typical morphology of hepatocytes, and expressed none of the mesenchymal markers including α‐SMA, FSP‐1, desmin, and vimentin. In liver sections of CCl4‐treated mice, GFP‐positive areas were coincident with fibrotic septa and never overlapped X‐gal‐positive areas. Conclusion: Type I collagen‐producing cells do not originate from hepatocytes. Hepatocytes in vivo neither acquire mesenchymal marker expression nor exhibit a morphological change clearly distinguishable from normal hepatocytes. Our results strongly challenge the concept that hepatocytes in vivo acquire a mesenchymal phenotype through EMT to produce the ECM in liver fibrosis. (HEPATOLOGY 2009.)

Hepatitis C virus–induced oxidative stress suppresses hepcidin expression through increased histone deacetylase activity

Chronic hepatitis C is characterized by iron accumulation in the liver, and excessive iron is hepatotoxic. However, the mechanism by which hepatitis C virus (HCV) regulates iron metabolism is poorly understood. Hepcidin plays a pivotal role as a negative regulator of iron absorption. The aim of the current study was to elucidate the mechanisms that govern hepcidin expression by HCV. Huh 7 cells, Huh7.5 cells, full‐length HCV replicon cells established from Huh7.5 cells, and adenoviruses expressing HCV‐core or HCV nonstructural proteins 3 through 5 (NS3‐5) were used. Hepcidin expression was significantly lower in HCV replicon cells and in HCV core–expressing Huh7 cells. The expression was inversely correlated with the amount of reactive oxygen species (ROS) production. Anti‐oxidants restored hepcidin expression in HCV replicon cells and Huh7 cells expressing HCV core. In HCV replicon cells, histone deacetylase (HDAC) activity was elevated at baseline and after exposure to hydrogen peroxide. Anti‐oxidants reduced HDAC activity in a dose‐dependent manner. HDAC inhibition increased hepcidin expression without affecting ROS production in HCV replicon cells. HCV‐induced ROS stabilized the expression of two negative hepcidin regulators, HIF1α and HIF2α, and its expression was decreased by a HDAC inhibitor or an anti‐oxidant. HCV‐induced ROS also caused hypoacetylation of histones and inhibited binding of two positive regulators, C/EBPα and STAT3, to the hepcidin promoter, whereas anti‐oxidant treatment of cells recovered C/EBPα and STAT3 binding to the hepcidin promoter. In addition, an HDAC inhibitor restored their binding to the hepcidin promoter via acetylation of histones. Conclusion: HCV‐induced oxidative stress suppresses hepcidin expression through increased HDAC activity. (HEPATOLOGY 2008.)

Hepatic Stellate Cells Secrete Angiopoietin 1 That Induces Angiogenesis in Liver Fibrosis

BACKGROUND & AIMS Although angiogenesis is closely associated with liver fibrosis, the angiogenic factors involved in liver fibrosis are not well characterized. Angiopoietin 1 is an angiogenic cytokine indispensable for vascular development and remodeling. It functions as an agonist for the receptor tyrosine kinase with immunoglobulin G-like and endothelial growth factor-like domains 2 (Tie2) and counteracts apoptosis, promotes vascular sprouting or branching, and stabilizes vessels. METHODS Liver samples from patients with liver fibrosis were evaluated for mRNA expression of angiogenic cytokines. Liver fibrosis was induced in BALB/c mice by either carbon tetrachloride (CCl(4)) or bile duct ligation (BDL). Hepatic stellate cells (HSCs) were isolated from BALB/c mice. We used an adenovirus expressing the extracellular domain of Tie2 (AdsTie2) to block angiopoietin signaling in mice and evaluated its effect on liver fibrosis. RESULTS mRNA expression level of angiopoietin 1 was increased in human fibrotic livers and correlated with the expression level of CD31, an endothelial cell marker. During experimental models of murine liver fibrosis, angiopoietin 1 was expressed by activated HSCs. In primary cultures, activated HSCs express and secrete angiopoietin 1 more abundantly than quiescent HSCs, and the inflammatory cytokine tumor necrosis factor-alpha stimulates its expression in an nuclear factor-kappaB-dependent manner. AdsTie2 inhibits angiogenesis and liver fibrosis induced by either CCl(4) or BDL. CONCLUSIONS These results reveal an angiogenic role of HSCs mediated by angiopoietin 1, which contributes to development of liver fibrosis. Thus, angiogenesis and hepatic fibrosis are mutually stimulatory, such that fibrosis requires angiogenesis and angiogenesis requires angiopoietin 1 from activated HSCs.

CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury

CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) is a key component in endoplasmic reticulum (ER) stress-mediated apoptosis. The goal of the study was to investigate the role of CHOP in cholestatic liver injury. Acute liver injury and liver fibrosis were assessed in wild-type (WT) and CHOP-deficient mice following bile duct ligation (BDL). In WT livers, BDL induced overexpression of CHOP and Bax, a downstream target in the CHOP-mediated ER stress pathway. Liver fibrosis was attenuated in CHOP-knockout mice. Expression levels of alpha-smooth muscle actin and transforming growth factor-beta1 were reduced, and apoptotic and necrotic hepatocyte death were both attenuated in CHOP-deficient mice. Hepatocytes were isolated from WT and CHOP-deficient mice and treated with 400 microM glycochenodeoxycholic acid (GCDCA) for 8 h to examine bile acid-induced apoptosis and necrosis. GCDCA induced overexpression of CHOP and Bax in isolated WT hepatocytes, whereas CHOP-deficient hepatocytes had reduced cleaved caspase-3 expression and a lower propidium iodide index after GCDCA treatment. In conclusion, cholestasis induces CHOP-mediated ER stress and triggers hepatocyte cell death, and CHOP deficiency attenuates this cell death and subsequent liver fibrosis. The results demonstrate an essential role of CHOP in development of liver fibrosis due to cholestatic liver damage.

c-Jun N-terminal kinase-1 from hematopoietic cells mediates progression from hepatic steatosis to steatohepatitis and fibrosis in mice.

BACKGROUND & AIMS c-Jun N-terminal kinase (JNK) plays a pivotal role in the development of the metabolic syndrome including nonalcoholic fatty liver disease. However, the mechanism underlying the contribution of JNK to the progression from simple steatosis to steatohepatitis and liver fibrosis is unresolved. METHODS Hepatic steatosis, inflammation, and fibrosis were examined in wild-type, jnk1(-/-), or jnk2(-/-) mice fed a choline-deficient L-amino acid-defined (CDAA) diet for 20 weeks. The functional contribution of JNK isoforms in Kupffer cells was assessed in vitro and in vivo using chimeric mice in which the hematopoietic compartment including Kupffer cells was replaced by wild-type, jnk1(-/-), or jnk2(-/-) cells. RESULTS CDAA diet induced significantly less hepatic inflammation and less liver fibrosis despite a similar level of hepatic steatosis in jnk1(-/-) mice as compared with wild-type or jnk2(-/-) mice. CDAA diet-induced hepatic inflammation was chronic and mediated by Kupffer cells. Pharmacologic inhibition of JNK or gene deletion of jnk1 but not jnk2 repressed the expression of inflammatory and fibrogenic mediators in primary Kupffer cells. In vivo, CDAA diet induced less hepatic inflammation and liver fibrosis despite an equivalent level of hepatic steatosis in chimeric mice with jnk1(-/-) hematopoietic cells as compared with chimeric mice with wild-type or jnk2(-/-) hematopoietic cells. CONCLUSIONS jnk1(-/-) mice are resistant to diet-induced steatohepatitis and liver fibrosis. JNK1 in hematopoietic cells, especially in Kupffer cells, contributes to the development of liver fibrosis by inducing chronic inflammation.

Implication of Frequent Local Ablation Therapy for Intrahepatic Recurrence in Prolonged Survival of Patients With Hepatocellular Carcinoma Undergoing Hepatic Resection: An Analysis of 610 Patients Over 16 Years Old

Objective:By comparing cohorts in 2 exclusive time frames, the factors that affected the surgical outcomes of patients with hepatocellular carcinoma (HCC) are presented. Summary Background Data:Reportedly, survival results of patients with HCC who underwent hepatectomy have improved in recent years. However, the major factors contributing to these favorable outcomes have not been fully explained. Methods:Between January 1985 and December 2000, 610 patients with HCC underwent liver resections as a primary and curative resection. They were categorized into 2 groups according to the year in which the surgeries were performed: before 1990 (n = 212; early group); and after 1991 (n = 398; late group). Clinicopathologic data, survival data, type of recurrence, and treatment of intrahepatic recurrence were compared between the 2 groups. Results:Clinicopathologic data were almost identical between the groups except for age, blood loss, and duration of surgery. The overall survival rate was significantly better in the late group compared with the early group (58.0% vs. 39.1% at 5 years, P < 0.0001). By contrast, disease-free survival remained unchanged (27.8% vs. 26.2% at 5 years, P = 0.2887). The most common type of recurrence was intrahepatic relapse, and there was no difference in the rate and the type of recurrence between the 2 groups. The 5-year survival rate after recurrence was increased in the late group (21.8% vs. 11.6%, P = 0.0002). Stratified analysis by the type of initial recurrence revealed that better survival in the late group was achieved only in solitary intrahepatic recurrences, not in multiple intrahepatic or extrahepatic recurrences. Changes in modality of treatment of recurrence were observed only in the management of solitary intrahepatic recurrences, where percutaneous ablation therapies were more frequently applied with new ablation techniques. Patients that had undergone ablation therapies in the late group had better postrecurrent survival than those in the early group. Multivariate analysis showed that presence of local ablation therapies was an independent favorable prognostic factor only in the late group. Conclusions:Significant improvements in outcomes were achieved in patients with HCC who underwent curative liver resections. Percutaneous ablation therapy for intrahepatic recurrence was considered to be a major contributory factor for improving survival after recurrence, as well as for overall survival.

Protection from liver fibrosis by a peroxisome proliferator-activated receptor δ agonist

Peroxisome proliferator-activated receptor delta (PPARδ), a member of the nuclear receptor family, is emerging as a key metabolic regulator with pleiotropic actions on various tissues including fat, skeletal muscle, and liver. Here we show that the PPARδ agonist KD3010, but not the well-validated GW501516, dramatically ameliorates liver injury induced by carbon tetrachloride (CCl4) injections. Deposition of extracellular matrix proteins was lower in the KD3010-treated group than in the vehicle- or GW501516-treated group. Interestingly, profibrogenic connective tissue growth factor was induced significantly by GW501516, but not by KD3010, following CCl4 treatment. The hepatoprotective and antifibrotic effect of KD3010 was confirmed in a model of cholestasis-induced liver injury and fibrosis using bile duct ligation for 3 wk. Primary hepatocytes treated with KD3010 but not GW501516 were protected from starvation or CCl4-induced cell death, in part because of reduced reactive oxygen species production. In conclusion, our data demonstrate that an orally active PPARδ agonist has hepatoprotective and antifibrotic effects in animal models of liver fibrosis, suggesting a possible mechanistic and therapeutic approach in treating patients with chronic liver diseases.

Origin of myofibroblasts in liver fibrosis

Most chronic liver diseases of all etiologies result in progressive liver fibrosis. Myofibroblasts produce the extracellular matrix, including type I collagen, which constitutes the fibrous scar in liver fibrosis. Normal liver has little type I collagen and no detectable myofibroblasts, but myofibroblasts appear early in experimental and clinical liver injury. The origin of the myofibroblast in liver fibrosis is still unresolved. The possibilities include activation of endogenous mesenchymal cells including fibroblasts and hepatic stellate cells, recruitment from the bone marrow, and transformation of epithelial or endothelial cells to myofibroblasts. In fact, the origin of myofibroblasts may be different for different types of chronic liver diseases, such as cholestatic liver disease or hepatotoxic liver disease. This review will examine our current understanding of the liver myofibroblast.

Antiapoptotic Effect of c-Jun N-terminal Kinase-1 through Mcl-1 Stabilization in TNF-Induced Hepatocyte Apoptosis

BACKGROUND & AIMS c-Jun N-terminal Kinase (JNK) is a key regulator in tumor necrosis factor (TNF)-mediated liver injury. However, distinct roles for JNK1 and JNK2 in hepatocyte apoptosis are still unresolved. Although myeloid cell leukemia-1 (Mcl-1) has been reported as a substrate of JNK, the role of Mcl-1 and its functional regulation by JNK in TNF-induced hepatocyte apoptosis and liver injury remain to be elucidated. METHODS TNF-induced hepatocyte apoptosis was investigated in wild-type, jnk1-/- and jnk2-/- mice in vitro and in the galactosamine/TNF (GalN/TNF) liver injury model. For further analysis, we used adenoviruses expressing wild-type Mcl-1 or its substitution mutant, and the Cre/loxP system (mcl-1f/f) to delete mcl-1. RESULTS jnk2-/- Hepatocytes showed increased Mcl-1 expression and were more resistant to TNF-induced apoptosis compared with wild-type or jnk1-/- hepatocytes. Increased Mcl-1 expression in jnk2-/- hepatocytes correlated with their JNK activity, which is mediated by residual JNK1 and higher than in wild-type or jnk1-/- hepatocytes. JNK activation led to phosphorylation of Mcl-1 in hepatocytes, and this increased the half-life of the Mcl-1 protein. Overexpression of Mcl-1 confirmed its antiapoptotic effect in TNF-induced hepatocyte apoptosis in vitro and in vivo. Deletion of mcl-1 in jnk2-/- hepatocytes increased TNF-induced hepatocyte apoptosis both in vitro and in GalN/TNF-induced liver injury model. CONCLUSIONS jnk2-/- Hepatocytes are resistant to TNF-induced apoptosis. Activated JNK1 contributes to this antiapoptotic phenotype of jnk2-/- hepatocytes through phosphorylation-mediated stabilization of Mcl-1.