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Background & Aims: Primary sclerosing cholangitis (PSC) has a variable, often progressive, course. Magnetic resonance cholangiography (MRC) is used in the diagnosis of PSC. Magnetic resonance risk scoring systems, called Anali without and with gadolinium, are used to predict disease progression, determined by radiologic factors. We aimed to assess the prognostic value of Anali scores in patients with PSC and validate our findings in a separate cohort., Methods: We performed a retrospective study of patients with large-duct PSC (internal cohort, 119 patients in France; external cohort, 119 patients in Canada, Italy, and the United Kingdom). All the first-available MRC results were reviewed by 2 radiologists and the Anali scores were calculated as follows: Anali without gadolinium = (1× dilatation of intrahepatic bile ducts) + (2× dysmorphy) + (1× portal hypertension); Anali with gadolinium = (1× dysmorphy) + (1× parenchymal enhancement heterogeneity). The primary end point was survival without liver transplantation or cirrhosis decompensation. The prognostic value of Anali scores was assessed by Cox regression modeling., Results: During a total of 549 patient-years for the internal cohort and 497 patient-years for the external cohort, we recorded 2 and 8 liver transplantations, 4 and 3 liver-related deaths, and 26 and 25 cirrhosis decompensations, respectively. In the univariate analysis, factors associated with survival without liver transplantation or cirrhosis decompensation in the internal cohort were as follows: serum levels of bilirubin, aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase, albumin, and Anali scores. Anali scores without and with gadolinium identified patients' survival without liver transplantation or cirrhosis decompensation with a c-statistic of 0.89 (95% CI, 0.84-0.95) and 0.75 (95% CI, 0.64-0.87), respectively. Independent prognostic factors identified by multivariate analysis were Anali scores and bilirubinemia. The prognostic value of Anali scores was confirmed in the external cohort., Conclusions: In internal and external cohorts, we found that Anali scores, determined from MRC, were associated with outcomes of patients with PSC. These scores might be used as prognostic factors., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Portal myofibroblasts (PMF) form a sub-population of highly proliferative and proangiogenic liver myofibroblasts that derive from portal mesenchymal progenitors. Endoplasmic reticulum (ER) stress was previously shown to modulate fibrogenesis, notably in the liver. Our aim was to determine if ER stress occurred in PMF and affected their functions. PMF were obtained after their expansion in vivo from bile duct-ligated (BDL) rats and referred to as BDL PMF. Compared to standard PMF obtained from normal rats, BDL PMF were more myofibroblastic, as assessed by higher alpha-smooth muscle actin expression and collagen 1 production. Their proangiogenic properties were also higher, whereas their proliferative and migratory capacities were lower. CHOP expression was detected in the liver of BDL rats, at the leading edge of portal fibrosis where PMF accumulate. BDL PMF displayed ER dilatation and an overexpression of the PERK pathway downstream targets, Chop, Gadd34 and Trb3, in comparison with standard PMF. In vitro, the induction of ER stress by tunicamycin in standard PMF, caused a decrease in their proliferative and migratory activity, and an increase in their proangiogenic activity, without affecting their myofibroblastic differentiation. Conversely, the treatment of BDL PMF with the PERK inhibitor GSK2656157 reduced ER stress, which caused a decrease in their angiogenic properties, and restored their proliferative and migratory capacity. In conclusion, PMF develop ER stress as they expand with the progression of fibrosis, which further increases their proangiogenic activity, but also inhibits their proliferation and migration. This phenotypic switch may restrict PMF expansion while they support angiogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Nuclear receptors (NR), the largest family of transcription factors, control many physiological and pathological processes. To gain insight into hepatic NR and their potential as therapeutic targets in cholestatis, we determined their expression in individual cell types of the mouse liver in normal and cholestatic conditions. Hepatocytes, cholangiocytes, hepatic stellate cells (HSC), sinusoidal endothelial cells (SEC) and Kupffer cells (KC) were isolated from the liver of mice with acute or chronic cholestasis (i.e. bile duct-ligated or Abcb4 -/- mice, respectively) and healthy controls. The expression of 43 out of the 49 NR was evidenced by RT-qPCR in one or several liver cell types. Expression of four NR was restricted to non-parenchymal liver cells. In normal conditions, NR were expressed at higher levels in individual cell types when compared to total liver. Half of the NR expressed in the liver had maximal expression in non-parenchymal cells. After bile duct ligation, NR mRNA changes occurred mostly in non-parenchymal cells and mainly consisted in down-regulations. In Abcb4 -/- mice, NR mRNA changes were equally frequent in hepatocytes and non-parenchymal cells. Essentially down-regulations were found in hepatocytes, HSC and cholangiocytes, as opposed to up-regulations in SEC and KC. While undetectable in total liver, Vdr expression was up-regulated in all non-parenchymal cells in Abcb4 -/- mice. In conclusion, non-parenchymal liver cells are a major site of NR expression. During cholestasis, NR expression is markedly altered mainly by down-regulations, suggesting major changes in metabolic activity. Thus, non-parenchymal cells are important new targets to consider in NR-directed therapies., (Copyright © 2017. Published by Elsevier B.V.)

We have developed a culture model to assess antifibrotic drugs using normal human liver myofibroblasts (HLMFs) obtained from 31 subjects. Activation was evaluated in terms of α-smooth muscle actin (α-SMA) and collagen 1 (Coll1) expression using RT-PCR, and proliferation as the uptake of 5-ethynil-2'-deoxyuridine. Under analysis of variance, between-subject differences accounted for 70% of all variability and inter-experiment differences for 30%. The sensitivity of the model was determined by quantifying the effects in terms of relative expression, which were 0.74±0.03 for cyclosporine A (CsA) and 2.4±0.10 for transforming growth factor-beta (TGF-β) (P<0.0001 vs no treatment) for α-SMA expression. Inter-subject variations in α-SMA and Coll1 expression enabled the classification of subjects as potentially low or high fibrosers. Finally, we observed that pirfenidone (which has beneficial effects in vivo) significantly reduced the expressions of α-SMA and Coll1, whereas the angiotensin-converting enzyme inhibitor losartan (which has no effect in vivo) had no significant effect. Our model may thus detect the antifibrotic properties of drugs. Antifibrotic drugs with promising clinical relevance could possibly be selected using a bank of HLMFs from high fibrosers.

Background: Chronic hepatitis C is a major cause of liver fibrosis and cirrhosis. It is generally accepted that inflammation that occurs in response to hepatocyte infection by the hepatitis C virus (HCV) is the main mechanism that triggers myofibroblast differentiation and stimulation in chronic hepatitis C. The aim of this study was to determine if HCV might infect human liver myofibroblasts (HLMF) and directly stimulate their fibrogenic activities., Methods: We evaluated the expression of the viral entry receptors, levels of HCV-RNA and HCV-protein and the expression of fibrosis markers in HLMF by using quantitative PCR, western blot and immunofluorescence analyses. Pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) were used to study the ability of HLMF to support viral entry, replication and fibrosis induction., Results: We showed that HLMF expressed all known molecules of the HCV receptor complex, i.e. CD81, LDL-R, scavenger receptor-BI, claudin-1 and occludin. These cells were also permissive to HCVpp entry. Inoculation with HCVcc caused short-term infection of these cells, as shown by their content in positive- and negative-strand HCV RNA, in core and NS3 viral proteins, and by their release of core protein levels in the culture supernatants. HCV infection stimulated myofibroblastic differentiation, proliferation and collagen production in these cells. In addition, evidence of in vivo infection was provided by the detection of positive- and negative-strand HCV RNA in preparations of HLMF obtained from HCV-infected patients., Conclusion: These findings indicate that HCV infection of HLMF can occur and trigger extracellular matrix overproduction, thereby contributing to the development of HCV-related liver fibrosis.

Unlabelled: Liver fibrosis expanding from portal tracts and vascular remodeling are determinant factors in the progression of liver diseases to cirrhosis. In the present study, we examined the potential contribution of portal myofibroblasts (PMFs) to the vascular changes leading to cirrhosis. The analyses of liver cells based on the transcriptome of rat PMFs, compared to hepatic stellate cell HSC-derived myofibroblasts in culture, identified collagen, type XV, alpha 1 (COL15A1) as a marker of PMFs. Normal liver contained rare COL15A1-immunoreactive cells adjacent to the bile ducts and canals of Hering in the portal area. A marked increase in COL15A1 expression occurred together with that of the endothelial marker, von Willebrand factor, in human and rat liver tissue, at advanced stages of fibrosis caused by either biliary or hepatocellular injury. In cirrhotic liver, COL15A1-expressing PMFs adopted a perivascular distribution outlining vascular capillaries proximal to reactive ductules, within large fibrotic septa. The effect of PMFs on endothelial cells (ECs) was evaluated by in vitro and in vivo angiogenesis assays. PMF-conditioned medium increased the migration and tubulogenesis of liver ECs as well as human umbilical vein ECs and triggered angiogenesis within Matrigel plugs in mice. In coculture, PMFs developed intercellular junctions with ECs and enhanced the formation of vascular structures. PMFs released vascular endothelial growth factor (VEGF)A-containing microparticles, which activated VEGF receptor 2 in ECs and largely mediated their proangiogenic effect. Cholangiocytes potentiated the angiogenic properties of PMFs by increasing VEGFA expression and microparticle shedding in these cells., Conclusion: PMFs are key cells in hepatic vascular remodeling. They signal to ECs through VEGFA-laden microparticles and act as mural cells for newly formed vessels, driving scar progression from portal tracts into the parenchyma., (© 2014 by the American Association for the Study of Liver Diseases.)

Hepatic fibrosis results from the accumulation of extracellular matrix-producing myofibroblasts in the liver. The mechanisms leading to the activation of hepatic stellate cells (HSCs) into myofibroblasts have been well described. By contrast, few molecular pathways leading to myofibroblast deactivation have been documented. Recently, the vitamin D-VDR axis has been shown to modulate HSC activity through a complex mechanism involving epigenetic modifications induced by the SMAD pathway., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Unlabelled: Alterations in apical junctional complexes (AJCs) have been reported in genetic or acquired biliary diseases. The vitamin D nuclear receptor (VDR), predominantly expressed in biliary epithelial cells in the liver, has been shown to regulate AJCs. The aim of our study was thus to investigate the role of VDR in the maintenance of bile duct integrity in mice challenged with biliary-type liver injury. Vdr(-/-) mice subjected to bile duct ligation (BDL) displayed increased liver damage compared to wildtype BDL mice. Adaptation to cholestasis, ascertained by expression of genes involved in bile acid metabolism and tissue repair, was limited in Vdr(-/-) BDL mice. Furthermore, evaluation of Vdr(-/-) BDL mouse liver tissue sections indicated altered E-cadherin staining associated with increased bile duct rupture. Total liver protein analysis revealed that a truncated form of E-cadherin was present in higher amounts in Vdr(-/-) mice subjected to BDL compared to wildtype BDL mice. Truncated E-cadherin was also associated with loss of cell adhesion in biliary epithelial cells silenced for VDR. In these cells, E-cadherin cleavage occurred together with calpain 1 activation and was prevented by the silencing of calpain 1. Furthermore, VDR deficiency led to the activation of the epidermal growth factor receptor (EGFR) pathway, while EGFR activation by EGF induced both calpain 1 activation and E-cadherin cleavage in these cells. Finally, truncation of E-cadherin was blunted when EGFR signaling was inhibited in VDR-silenced cells., Conclusion: Biliary-type liver injury is exacerbated in Vdr(-/-) mice by limited adaptive response and increased bile duct rupture. These results indicate that loss of VDR restricts the adaptation to cholestasis and diminishes bile duct integrity in the setting of biliary-type liver injury., (Copyright © 2013 The Authors. HEPATOLOGY published by Wiley on behalf of the American Association for the Study of Liver Diseases.)

Aim: Ezrin and radixin are actin-binding proteins that contribute to the integrity of epithelia. Abnormalities of bile secretion occur primarily in cholestatic liver diseases and are associated with changes in cell cytoskeleton. Expression of these proteins during liver development and in cholestatic liver diseases remains poorly investigated., Methods: Ezrin and radixin expression was analyzed in fetal, adult and pediatric cholestatic human liver (i.e. biliary atresia, sclerosing cholangitis) by immunohistochemistry., Results: In adult and fetal livers, ezrin was expressed exclusively in the cells of the biliary lineage (i.e. biliary epithelial cells and ductal cells) whereas radixin was located not only in hepatocytes but also in cells of the biliary lineage. In the lobule of mature livers, radixin displayed a zonal distribution with predominant expression in the periportal region. In cholestatic diseases, both proteins were expressed in cells of the ductular reaction. An aberrant expression of ezrin was detected in hepatocytes of cirrhotic nodules with a CK7-positive pattern and in malignant hepatocytes in a course of cholestatic disease toward cancer., Conclusions: Among the components of the liver epithelial cells, ezrin was exclusively expressed in biliary phenotype cells, while radixin was found in biliary and hepatocytic lineages, with a periportal zonal expression. In cholestatic diseases, ezrin was expressed in hepatocytes supporting the appearance of a biliary phenotype., (Copyright © 2013. Published by Elsevier Masson SAS.)

Nonalcoholic fatty liver disease (NAFLD) is associated with all features of the metabolic syndrome. Although deposition of excess triglycerides within liver cells, a hallmark of NAFLD, is associated with a loss of insulin sensitivity, it is not clear which cellular abnormality arises first. We have explored this in mice overexpressing carbohydrate responsive element-binding protein (ChREBP). On a standard diet, mice overexpressing ChREBP remained insulin sensitive, despite increased expression of genes involved in lipogenesis/fatty acid esterification and resultant hepatic steatosis (simple fatty liver). Lipidomic analysis revealed that the steatosis was associated with increased accumulation of monounsaturated fatty acids (MUFAs). In primary cultures of mouse hepatocytes, ChREBP overexpression induced expression of stearoyl-CoA desaturase 1 (Scd1), the enzyme responsible for the conversion of saturated fatty acids (SFAs) into MUFAs. SFA impairment of insulin-responsive Akt phosphorylation was therefore rescued by the elevation of Scd1 levels upon ChREBP overexpression, whereas pharmacological or shRNA-mediated reduction of Scd1 activity decreased the beneficial effect of ChREBP on Akt phosphorylation. Importantly, ChREBP-overexpressing mice fed a high-fat diet showed normal insulin levels and improved insulin signaling and glucose tolerance compared with controls, despite having greater hepatic steatosis. Finally, ChREBP expression in liver biopsies from patients with nonalcoholic steatohepatitis was increased when steatosis was greater than 50% and decreased in the presence of severe insulin resistance. Together, these results demonstrate that increased ChREBP can dissociate hepatic steatosis from insulin resistance, with beneficial effects on both glucose and lipid metabolism.

NME/NDPK family proteins are involved in the control of intracellular nucleotide homeostasis as well as in both physiological and pathological cellular processes, such as proliferation, differentiation, development, apoptosis, and metastasis dissemination, through mechanisms still largely unknown. One family member, NME1/NDPK-A, is a metastasis suppressor, yet the primary physiological functions of this protein are still missing. The purpose of this study was to identify new NME1/NDPK-A-dependent biological functions and pathways regulated by this gene in the liver. We analyzed the proteomes of wild-type and transgenic NME1-null mouse livers by combining two-dimensional gel electrophoresis and mass spectrometry (matrix-assisted laser desorption/ionization time of flight and liquid chromatography-tandem mass spectrometry). We found that the levels of three proteins, namely, phenylalanine hydroxylase, annexin IV, and elongation factor 1 Bα (EF-1Bα), were strongly reduced in the cytosolic fraction of NME1(-/-) mouse livers when compared to the wild type. This was confirmed by immunoblotting analysis. No concomitant reduction in the corresponding messenger RNAs or of total protein level was observed, however, suggesting that NME1 controls annexin IV and EF-1Bα amounts by post-translational mechanisms. NME1 deletion induced a change in the subcellular location of annexin IV in hepatocytes resulting in enrichment of this protein at the plasma membrane. We also observed a redistribution of EF-1Bα in NME1(-/-) hepatocytes to an intracytoplasmic compartment that colocalized with a marker of the reticulum endoplasmic. Finally, we found reduced expression of annexin IV coincident with decreased NME1 expression in a panel of different carcinoma cell lines. Taken together, our data suggest for the first time that NME1 might regulate the subcellular trafficking of annexin IV and EF-1Bα. The potential role of these proteins in metastatic dissemination is discussed.

Vitamin D through the vitamin D nuclear receptor (VDR) plays a key role in mineral ion homeostasis. The liver is central in vitamin D synthesis, however the direct involvement of the vitamin D-VDR axis on the liver remains to be evaluated. In this review, we will describe vitamin D metabolism and the mechanisms of homeostatic control. We will also address the associations between the vitamin D-VDR axis and pathological liver entities, such as non-alcoholic fatty liver disease, autoimmune liver disease, viral hepatitis and liver cancer. The link between liver diseases and the vitamin D-VDR axis will be discussed in light of evidences arising from in vitro and in vivo studies. Finally, we will consider the therapeutic potential of the vitamin D-VDR axis in liver diseases., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Background: Hepatic fibrosis, an outcome of chronic liver diseases, is characterized by an accumulation of collagen, which is produced by activated human intrahepatic fibroblasts (HIF). Transforming growth factor (TGF) β is an important inducer of fibrogenesis, in collaboration with other cytokines, such as interleukin (IL) 4. IL-4 is overexpressed in severe recurrent hepatitis C after liver transplantation, exerting profibrotic effects. In contrast, cyclosporine (CsA) had been shown to decrease fibroblast activation and collagen production. We therefore investigated the effects of CsA on TGF-β and IL-4 profibrotic activities on HIF in vitro., Methods: Isolated HIF were cultured without or with human TGF-β, human IL-4, CsA, or combined TGF- β+CsA or IL-4+CsA. We performed real-time polymerase chain reaction for collagen types I, III, and IV and alpha-SMA, a marker of fibroblast activation we also measured total collagen in supernates. TGF-β and IL-4 increased the expressions of alpha smooth muscle action (SMA) collagen I, III, and IV mRNAs (P < .05 vs untreated cells) as well as the overall collagen level in the supernates (P < .01). CsA decreased the expression of mRNAs encoding alpha-SMA and collagens (P < .01). Expressions of alpha-SMA and collagens I, III, and IV mRNAs were significantly lower under combined treatments (TGF-β vs TGF-β+CsA [P < .01] and IL-4 vs IL-4+CsA [P < .01]). Collagen level was decreased by combined treatments (TGF-β vs TGF-β+CsA [P < .05] and IL-4 vs IL-4+CsA [P = .05])., Conclusion: CsA inhibited the profibrotic effects of TGF-β and IL-4 by decreasing the activation and production of collagen by HIF. CsA may decrease fibroblast activation and collagen accumulation, exerting beneficial effects on fibrosis progression, particularly among patients with recurrent hepatitis C., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Liver injury causes vascular disorganization and local tissue hypoxia starting early in disease course. In this context, hypoxia acts not only as an aggravating factor of cell damage and inflammation, but also as an inhibitor of liver regeneration, a major stimulus of angiogenesis and fibrogenesis, and a promoter of liver carcinogenesis. Many of the effects of hypoxia are mediated by hypoxia-inducible factor-1alpha (HIF-1alpha), an oxygen-sensitive transcription factor. Compared to cells in the periportal area, intralobular hepatic stellate cells (HSCs) are more responsive to hypoxia and like other pericytes play a key role in angiogenesis through interactions with endothelial cells VIA platelet-derived growth factor (PGDF) and vascular endothelial growth factor (VEGF) signaling, at the leading edge of fibrotic septa. Although required for successful liver repair, angiogenesis in cirrhosis may be inefficient because of the immaturity and permeability of VEGF-induced neo-vessels, and thereby may fail to correct liver hypoxia. The multiple receptor tyrosine kinase inhibitors, acting on VEGF and PDGF receptors, initially designed for cancer treatment, show in addition to therapeutic efficacy in patients with hepatocellular carcinoma, beneficial effects on many aspects of the progression of liver diseases, including, fibrosis, inflammation and portal hypertension., (Copyright Thieme Medical Publishers.)

Aims: The aim of this study was to determine whether serum levels of adipokines, including the ratio of serum adiponectin to leptin (A/L) levels could predict the severity of liver injury in patients with non-alcoholic fatty liver disease (NAFLD)., Patients and Methods: Fifty-seven patients with biopsy-proven non-alcoholic steatohepatitis (NASH) (mean age 51+/-12, sex ratio 1), 17 with simple steatosis (mean age 47+/-12, sex ratio 1.4) and 10 controls without steatosis (mean age 51+/-11, sex ratio 4) were investigated. In all subjects, serum concentrations of triglycerides, ultrasensitive C reactive protein, leptin, adiponectin, soluble tumour necrosis factor receptor 1, interleukin (IL)-6 and Homeostasis Model Assessment Method (HOMA) were measured. Hepatic expression of adiponectin and its two receptors was assessed by quantitative reverse transcriptase polymerase chain reaction., Results: Body mass index (BMI) and HOMA were correlated positively with leptin levels (r=0.44 and 0.28 respectively) and negatively with the A/L ratio (r=0.51 and 0.41 respectively). Independent parameters associated with NASH vs steatosis were HOMA>3 [odds ratio (OR)=6.9] and A/L ratio <1.4 10(3) (OR=5.2). The combination of HOMA with A/L ratio showed an area under the receiver operating characteristic curve of 0.82 for distinguishing between NASH and steatosis. Extensive portal fibrosis was present in 17 (23%) patients with NAFLD. Three independent parameters were associated with fibrosis: age (OR=1.1), BMI (OR=1.3) and high IL-6 levels (OR=1.6). The hepatic expression of adiponectin receptor 2 was significantly higher in patients with NASH compared with controls and was related with necroinflammatory injury., Conclusions: This study shows that in patients with NAFLD, the combination of HOMA with A/L ratio may be a useful non-invasive approach to appreciate the severity of liver damage.

Recent evidence indicates that growth hormone (GH) is involved in liver regeneration. To test whether insulin-like growth factor I (IGF-I) mediates this effect, we studied liver regeneration induced by partial hepatectomy in liver-specific IGF type 1 receptor knockout (LIGFREKO) mice. The absence of IGF-1R caused a significant decrease in hepatocyte proliferation in males (-52%), but not in females, as assessed by Ki67 immunohistochemistry. Cyclin D1 and cyclin A protein levels in the livers of LIGFREKO males were only half those in controls, indicating that cyclin induction during liver regeneration is dependent on IGF-1R signaling. Analyzing the signaling cascade initiated by IGF-1R, we observed a lack of IRS-1 induction in LIGFREKO livers. In contrast, the induction of IRS-2 synthesis was similar in LIGFREKO and control groups, suggesting the existence of differential regulation of IRS synthesis during liver regeneration. Regenerating livers from LIGFREKO animals also showed significantly less activated ERKs than controls. Our findings demonstrate that IGF-1R makes a significant contribution to liver regeneration. Using the LIGFREKO model, we provide new evidence that IGF-1R/IRS-1/ERK signaling may be the intracellular pathway controlling the cell cycle via cyclin D1 and cyclin A in the regenerating liver.

During cholestasis, bile acids accumulate in the liver, and induce cellular alterations. Cholestasis is a major cause of liver fibrosis. We have used precision-cut liver slices (PCLS) in culture to investigate the effects of bile acids on hepatic cells. Rat PCLS were placed on an insert in a vial containing culture medium, and gently agitated on a roller platform. PCLS were treated with 100 microM taurolithocholate (TLC), taurodeoxycholate (TDC) or taurocholate (TC) for 24 or 48 h. PCLS viability was measured, and immunohistochemistry was performed with antibodies against active caspase 3, platelet-derived growth factor (PDGF) receptor-beta and ED-A fibronectin. TDC and TLC, two hydrophobic bile acids, induced hepatocyte necrosis and apoptosis, whereas TC, an hydrophilic bile acid, improved slice viability as compared with controls. Both TDC and TC induced biliary epithelial cell proliferation, together with portal fibroblast proliferation and activation, as shown by PDGF receptor-beta and ED-A fibronectin expression. TLC induced biliary epithelial cell apoptosis. Our results indicate that individual bile acids induce cell type-specific effects in a complex liver microenvironment. The fact that PCLS support biliary epithelial cell and portal fibroblast proliferation will make this model very useful for the study of the mechanisms involved in portal fibrosis.

Purpose: To examine the potential of cultured human liver slices to predict cytochrome P450 (CYP) inducibility, regarding global and zonal CYP expression, together with drug-induced histologic changes., Methods: We first assessed whether CYP2B6, 3A4, and 2C9 expression was maintained in cultured liver slices. Cultured hepatocytes were used as the reference culture system. Then we tested the effects of phenobarbital and cyclophosphamide on CYP expression in both models., Results: Morphologic features are preserved in slices. Basal CYP expression declines with time in culture in both models. Slices display the same region specificity of CYP2B6, 2C9, and 3A4 expression as intact liver. CYP2B6 and 3A4 mRNA, apoprotein, and enzyme-related activities were induced by phenobarbital and cyclophosphamide, whereas CYP2C9 apoprotein was not. Their immunoreactivities were also increased, while their zonal distribution was preserved on slice tissue sections. Microsomal enzyme induction was confirmed by histology., Conclusions: Cultured human liver slices are an attractive alternative to hepatocyte culture for the prediction of human CYP isoenzyme induction by xenobiotics.

Aims: Interactions between polymorphonuclear leukocytes (PMN) and sinusoidal endothelial cells (SEC) may contribute to ischemia-reperfusion injury. The aim of the study was to determine the influence of PMN hypoxia-reoxygenation and degranulation, on SEC toxic response., Methods: PMNs collected from rat pleural cavity underwent hypoxia- reoxygenation or N-formyl-methionyl-leucyl-phenylalanine (fMLP) degranulation treatment, and were then separated from their conditioned medium. Rat SECs were incubated either with PMNs in coculture or with their conditioned medium, for 210 min. Oxidative metabolism in PMNs was measured by chemiluminescence. LDH release and elastase activity were measured in SEC supernatants., Results: PMN-conditioned medium induced an increase in LDH release in SECs. Hypoxia-reoxygenation of PMNs induced an increase in their chemiluminescent response without increasing the cytotoxic effect of their conditioned medium. By contrast, the cytotoxic effect of conditioned medium was increased following PMN treatment with fMLP. In the latter case, cytotoxicity was combined with a rise in the elastase activity released in the supernatants, but was not reduced by inhibitors of elastase or of other proteases., Conclusions: The results indicate that toxic products are released, at least in part through degranulation, by PMNs, and induce cytotoxicity in SECs. This mechanism may contribute to SEC injury during hypoxia-reoxygenation.

We tested the effects of low (20% O2) and high (70% O2) oxygen tension on the morphological and biochemical integrity of human liver slices incubated for up to 72 h in supplemented Williams' E medium in a dynamic rotating culture system. High oxygen tension was more effective than low oxygen tension for preserving morphological integrity in long-term culture (48-72 h). After 72 h of culture with 70% O2, the lobular pattern was well preserved, and the survival of hepatocytes (approximately 80%) and other cell types was good. Immunohistochemical studies showed good preservation of the region-specific expression of CYP2EI and CYP3A4 isoenzymes for up to 72 h of incubation in 70% O2. As compared to 20% O2, the oxidized glutathione content and reactive oxygen species production were slightly increased in 70% O2, suggesting that minimal oxidative stress occurred with the high oxygen tension. In conclusion, despite slight oxidative stress associated with high oxygen tension, 70% O2 appeared more appropriate than 20% O2 for preserving the morphological and biochemical integrity of human liver slices cultured in a dynamic organ culture system for up to 72 h.

Summary: After liver injury, hepatic stellate cells (HSC) undergo a pleiotropic response termed "activation" that also occurs in culture models and ultimately leads to the conversion of HSC into myofibroblasts expressing smooth muscle alpha-actin (alpha-SMA). The onset of HSC proliferation in primary culture coincides with the induction of platelet-derived growth factor receptor-beta (PDGFR-beta) expression, while platelet-derived growth factor (PDGF) is the most potent mitogen for culture-activated HSC. Yet, the mechanisms and the stage of activation required for HSC proliferation in the intact liver are still uncertain. In the present study, we analyzed the proliferative response of HSC to rat cholestatic liver injury and the role of PDGF in this response. After in vivo incorporation of bromodeoxyuridine (BrdU), pure vitamin A-containing HSC were isolated at different time points after bile duct ligation (BDL) or sham operation and were analyzed by means of flow cytometry. The induction of HSC proliferation, as ascertained by BrdU incorporation, occurred between 24 and 48 hours and reached a plateau as soon as 48 hours after BDL. Flow cytometry and immunoblot analyses of HSC indicated that the induction of proliferation in HSC coincided with the up-regulation of PDGFR-beta protein on their surface but preceded that of alpha-SMA. A dose-dependent inhibition of PDGF-BB-induced HSC proliferation by STI571, a PDGF receptor tyrosine kinase inhibitor, was documented in vitro. Daily intraperitoneal injections of STI571 (20 mg/kg) caused a 60% reduction in BrdU positive isolated HSC and in the amount of desmin-immunoreactive sinusoidal cells on liver tissue sections in 48-hour bile duct-ligated rats. These results indicate that cholestatic liver injury elicits an early proliferative response in HSC that is mainly mediated by PDGF, and which precedes HSC phenotypic conversion into myofibroblasts.

Flucloxacillin, an isoxazolyl-penicillin, causes cholestasis and biliary epithelium injury. The aim of the study was to determine whether flucloxacillin, either directly or through metabolite formation, may induce cytotoxicity in hepatic or biliary cells. Cytotoxicity was assessed by lactate dehydrogenase release in primary cultures of human hepatocytes and of gallbladder-derived biliary epithelial cells (BEC). Metabolite production in microsome and cell preparations was analyzed by chromatography, nuclear magnetic resonance spectroscopy, and mass spectrometry. While flucloxacillin induced no direct cytotoxicity in any of the hepatocyte (n = 12) and BEC (n = 19) preparations, the conditioned media from cultured hepatocytes preincubated with flucloxacillin (50-500 mg/L) triggered a significant increase in lactate dehydrogenase release over controls in approximately 50% of BEC preparations (7/12), and this effect depended upon flucloxacillin concentration. Remaining BEC preparations exhibited no toxic response. Cytotoxicity in BEC preparations (9/13) was also induced by the supernatants of human liver microsomes and of recombinant human cytochrome P450 (CYP)3A4 preincubated with flucloxacillin (500 mg/L). Supernatants from both liver microsome and CYP3A4 preparations contained one major metabolite which was identified as 5'-hydroxymethylflucloxacillin. The production of this metabolite was inhibited following CYP3A4 inhibition by troleandomycin in human liver microsomes, and markedly enhanced following CYP3A induction by dexamethasone in rat liver microsomes. As opposed to BEC, cultured hepatocytes displayed significant CYP3A activity and produced low amounts of this metabolite. The purified metabolite (0.01-5 mg/L) exerted toxic effects in BEC but not in hepatocytes. In conclusion, hepatocytes mainly via CYP3A4 activity, generate flucloxacillin metabolite(s) including 5'-hydroxymethylflucloxacillin that may induce cytotoxicity in susceptible BEC. These metabolic events may contribute to the pathogenesis of drug-induced cholangiopathies.

Two types of epithelial cells derived from a unique embryonic stem cell coexist in the liver: hepatocytes, which form the liver parenchyma, and cholangiocytes which line intrahepatic bile ducts. Both cell types represent more than 70% and around 3% of hepatic cells, respectively. Initially secreted in the canalicular space formed by the apical domain of hepatocytes, bile is subsequently collected and modified in bile ducts. Both types of epithelia can regenerate as a result of differentiated cell proliferation. However, massive or chronic injury may induce the emergence and proliferation of oval cells, a heterogeneous population of small cells expressing embryonic markers that may subsequently differentiate into hepatocytes or cholangiocytes. Oval cells may also give rise to hepatocellular or cholangiocellular carcinomas. They may derive not only from hepatic cells (including hepatocytes and cholangiocytes themselves), but also from extrahepatic haematopoietic stem cells.

The authors examined the expression of cystic fibrosis transmembrane conductance regulator (CFTR) and its relationship to histopathological changes in cystic fibrosis (CF) liver tissue. Immunohistochemistry was used to examine expression of CFTR, intercellular adhesion molecule-1 (ICAM-1) and liver cell-type markers in liver cryosections in 11 patients with CF-associated liver disease, and non-CF controls with (n = 17) and without (n = 3) liver disease. In CF patients prominent inflammatory infiltrates were not found, yet hepatic stellate cells were identified within fibrotic areas around bile ducts. Proliferating bile ducts displayed ICAM-1 immunoreactivity in 3 cases, but bile ducts were otherwise negative. In 2 patients homozygous for R764X and for 1112delT no CFTR immunoreactivity was detected. Bile-duct epithelial cells in patients carrying the DeltaF508 mutation displayed aberrant cytoplasmic immunolocalization of CFTR, as determined with confocal laser scanning microscopy, in contrast to the distinct CFTR expression at the luminal surface seen in controls. No clear relationship between CFTR expression and fibrosis or inflammation was evidenced in CF patients. In conclusion, these findings are consistent with an impairment of DeltaF508 CFTR processing in intrahepatic biliary epithelium. ICAM-1 expression on bile-duct epithelial cells and inflammatory infiltrates were rare findings in CF liver tissue, indicating that immunological mechanisms are unlikely to be involved in initiation of CF-associated liver disease.

The accumulation of myofibroblasts and fibrosis around proliferating bile ducts in cholestatic liver disease has been attributed to the proliferation and phenotypic modulation of portal fibroblasts, whereas the contribution of hepatic stellate cells remains uncertain. There is increasing evidence to indicate that bile ducts may stimulate chemoattraction of hepatic stellate cells (HSC). In the present study, we undertook dynamic tests to examine such a possibility and to investigate the role of two potential mediators: platelet-derived growth factor-BB (PDGF-BB) and endothelin-1. Cholestasis was induced by bile duct ligation in rats. HSC were isolated from normal rats and culture activated into myofibroblasts expressing PDGF-beta receptors. Migration of myofibroblastic HSC was investigated in a Transwell chemotaxis filter assay. As compared with basal conditions, PDGF-BB (100 microg/l) and endothelin-1 (10(-8) M) induced a 3-fold and 1.7-fold increase in HSC migration, respectively. Bile duct segments isolated from cholestatic rats triggered a 3-fold increase in migration. This stimulation was significantly more potent than that observed in the presence of normal bile ducts. It was inhibited by neutralizing anti-PDGF antibodies and by STI571 PDGF receptor tyrosine kinase inhibitor, by 60% and 85%, respectively, whereas Bosentan, an endothelin receptor antagonist, had no significant inhibiting effect. In bile duct segments from cholestatic rats PDGF-B chain mRNA was detected at higher levels than in controls, whereas PDGF-BB was immunolocalized in bile duct epithelial cells. The results indicate that chemotaxis of HSC towards bile duct structures may contribute to the development of periductular fibrosis in cholestatic disorders, and that PDGF-BB is the major mediator in this process. In addition, anti-liver fibrogenic properties of STI571 are suggested by potent inhibition of myofibroblastic HSC function.

Objectives: To determine the viability and differentiation of human hepatocytes immunoprotected by encapsulation and transplanted in rats without immunosuppression., Methods: Freshly isolated human hepatocytes were encapsulated in hollow fibers and transplanted in the peritoneal cavity of immunocompetent rats. The fibers were explanted for analysis at D3, D7 and D14 following transplantation. Morphological features under light and electron microscopy and gene expression were compared to those of non-transplanted encapsulated hepatocytes (D0). Human cytochrome P450 3A and albumin mRNAs were quantified by Northern blot. Cytochrome P450 3A proteins were detected by Western blot and cytochrome P450 3A enzyme activity was assessed by measuring the formation of 6beta-hydroxytestosterone by high performance liquid chromatography., Results: Transplanted hepatocytes were more than 60 % viable and exhibited morphological criteria of hepatocytic differentiation up to D7. Albumin and cytochrome P450 3A transcripts were also detected up to D14. At D3 and D7, albumin mRNA levels were of 30 %, compared to control D0 hepatocytes, while cytochrome P450 3A5 and cytochrome P450 3A4 mRNA levels were 65 % and 0 %, respectively. Cytochrome P450 3A immunoreactivity was detected by Western blot up to D14 and 6beta-hydroxylase activity was 17 % at D3 compared to D0, supporting with disappearance of cytochrome P450 3A4 mRNA., Conclusions: Human hepatocytes remain viable for a short period, following encapsulation and intraperitoneal transplantation in rat. Other experimental conditions need to be tested to prevent or delay a decrease in hepatocyte specific gene expression.

Aims/background: Sinusoidal endothelial cells are the primary target of ischemia-reperfusion injury following liver preservation. The present study was undertaken to examine the susceptibility of sinusoidal endothelial cells to hypoxia-reoxygenation and the potential role of oxygen free radicals in the induction of cell injury., Methods: Sinusoidal endothelial cells were isolated from rat liver. After 2 3 days of primary culture, the cells were exposed to hypoxia (N2/CO2 95/5) for 120 min and reoxygenation (O2/CO2 95/5) for 90 min. Control cells were exposed to hypoxia alone, to 95% O2 alone or were maintained under normoxic conditions. Human umbilical vein endothelial cells were used as a model of vascular endothelial cells and submitted to the same protocol. Cell viability and lipid peroxidation were assessed by LDH leakage and malondialdehyde production, respectively. In order to test the potential role of xanthine oxidase and mitochondrial dysfunction in cell injury, the cells were treated with allopurinol and potassium cyanide (KCN) respectively., Results: The different gaseous treatments did not affect LDH leakage in human umbilical vein endothelial cells. In sinusoidal endothelial cells, the sequential hypoxia-reoxygenation caused a significant increase in LDH release, malondialdehyde production and xanthine oxidase activity while hypoxia alone had no effect except on xanthine oxidase activity. Allopurinol inhibited xanthine oxidase without preventing cell injury or lipid peroxidation in this latter cell type., Conclusions: The results suggest that sinusoidal endothelial cells, as opposed to vascular endothelial cells, are susceptible to a direct cytotoxic effect of hypoxia-reoxygenation. This effect occurs in combination with an increase in xanthine oxidase activity and lipid peroxidation, although cell injury is mediated at least in part by mechanisms independent of xanthine oxidase such as mitochondrial dysfunction.

Lipocytes are the predominant target for ET-1 within the normal and fibrotic liver. ET-1-induced contraction of lipocytes becomes prominent in liver injury. In this setting, ET-1 may have an autocrine role in regulating sinusoidal flow, in association with eicosanoids and other endothelium derived factors such as nitric oxide (NO). In more advanced fibrosis, ET-1 may participate in contraction of collagen bands causing distortion of the lobular structure. A direct role of ET-1 in the activation of lipocytes is still hypothetical but could represent additional effects of ET-1 in various stages of liver fibrogenesis via paracrine and/or autocrine routes.

The endothelins (ETs) form a group of three vasoactive peptides (ET-1, ET-2, and ET-3) for which two types of cellular receptors have been identified, types A and B ET receptors (ETA and ETB receptors, respectively). To address possible targets for ETs within the liver, we isolated the four principal liver cell populations and placed them in short-term primary culture. By ligand-binding assay and mRNA levels, expression of ET receptors was greatest on hepatic lipocytes (Ito cells or fat-storing cells), which are perisinusoidal cells exhibiting features of smooth muscle cells. Moreover, lipocytes expressed both ETA and ETB receptors. The mRNA for ETB receptor, but not for ETA receptor, was detectable in sinusoidal endothelial cells and Kupffer cells; neither mRNA was detectable in hepatocytes. Both ET-1 and ET-3 elicited contraction of activated lipocytes cultured on collagen lattices; the EC50 value for ET-1 was 3 +/- 1 nM and for ET-3 was 17 +/- 12 nM. In cell isolates from injured liver (after administration of carbon tetrachloride), expression of ET receptors was unchanged. However, mRNA for ET-1 was significantly increased in activated lipocytes, suggesting an autocrine loop for the initiation of lipocyte contraction. The findings imply that ET-1 may play a role in regulating sinusoidal perfusion through its effect on lipocytes, particularly in injury states.

Hepatic lipocytes are perisinusoidal cells that have been thought to be analogous to tissue pericytes, a cell type with purported vasoregulatory properties. However, we and others have recently demonstrated that lipocytes acquire markers of smooth muscle cells or myofibroblasts only after liver injury, via a process termed "activation." In this study, we document lipocyte contractility on collagen lattices and examine the importance of activation in this process. In culture, lipocytes became contractile only after spreading and activating, coincident with expression of smooth muscle alpha actin, a marker of activation (1990. Virchows Arch. B Cell Pathol. 59:349). After 5 d in culture, lipocytes induced rapid and sustained contraction of collagen lattices (to 43.7 +/- 2.3% of their original size 24 h after detachment). There was no contraction of lattices containing hepatocytes. Scanning electron microscopy demonstrated intimate associations of lipocyte cell membranes and collagen fibrils. Reduction in cell volume during contraction was also prominent. Lattice contraction by lipocytes was proportional to cell number. Serum was a potent stimulator of lipocyte contraction, as were endothelin types 1, 2, and 3; the effect of serum and endothelin 1 were additive. Neither thrombin, angiotensin-II, serotonin, nor the cytokines PDGF and TGF beta induced contraction. Cytochalasin B treatment resulted in concentration-dependent inhibition of contraction. As a test of the in vivo relevance of the culture findings, lipocytes were isolated from fibrotic animals and examined immediately after adherence. Whereas lipocytes from normal liver were initially compact, smooth muscle alpha actin negative and noncontractile, cells from animals with hepatic injury due to CCl4 displayed an activated appearance, expressed smooth muscle alpha actin, and were contractile immediately after adherence. Additionally, IFN-gamma, an agent which blocks lipocyte activation (1992. Hepatology. 16:776), inhibited lipocyte contraction. The data document that normal (i.e., quiescent) lipocytes are not contractile, but that activation is associated with the development of contractility. These findings suggest that a role for lipocytes in organ contraction or vasoregulation may be confined to injured, not normal liver.

The infection of liver cells by HIV-1 was investigated in vivo. Liver biopsies from 13 anti-HIV-1 antibody-positive patients were studied and HIV-1 DNA was revealed by polymerase chain reaction (PCR) in eight. In situ hybridization demonstrated the presence of HIV-1 RNA in all eight PCR-positive liver specimens. Mononuclear inflammatory cells in the portal tracts and Kupffer cells were labeled by a HIV-1 35S-RNA probe in all cases and by an anti-p24 monoclonal antibody, in seven cases. In addition, hepatocytes also clearly scored positive for HIV-1 RNA in three cases. These results demonstrate the infection of both parenchymal and non-parenchymal liver cells by HIV-1 in vivo and therefore show that HIV-1 can infect an epithelial CD4-negative cell type.

In situ hybridization was used to identify the cell types infected by hepatitis C virus (HCV) in the liver. Using an antisense HCV-RNA probe from the 5' non-coding region, HCV-RNAs molecules were detected in liver sections of 4/11 patients with chronic hepatitis C. These 4 positive subjects were also infected by HIV. HCV-RNA-positive strands were detected in scattered hepatocytes as well as in cells identified as mononuclear cells within the inflammatory infiltrates. HCV-RNA negative strands, likely replicative intermediates, were also detected in these cells. This study therefore indicates that replication of HCV may occur in both hepatocytes and mononuclear liver cells.

Abstract: Cholestasis is frequently encountered in the ICU and is associated with a poor outcome. Ischaemia should be considered among the numerous aetiologic factors that may trigger cholestasis in the ICU. Blood supply to biliary tract is mainly provided by the hepatic artery, throughout a peribiliary vascular plexus. Interruption of the hepatic artery blood supply leads to cholestasis with a concomitant proliferative biliary reaction. Bile duct proliferation persists, while bile flow restores and biologic cholestasis syndrome spontaneously resolves in several weeks. Liver fibrosis related to the activation of periportal mesenchymental cells is observed in the close vicinity of proliferative bile ducts. Ischaemic cholestasis can be ascribed, at least partly, to hypoxia-induced disorders in the expression of hepatocytes biliary salts membrane transporters. [Copyright &y& Elsevier]