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Competing Interests: Mario Strazzabosco consults for Engitix. The remaining authors have no conflicts to report.

Competing Interests: Mario Strazzabosco consults for Engitix. The remaining authors have no conflicts to report.

Background and Aims: Cholangiopathies are an important cause of morbidity and mortality. Their pathogenesis and treatment remain unclear in part because of the lack of disease models relevant to humans. Three-dimensional biliary organoids hold great promise; however, the inaccessibility of their apical pole and the presence of extracellular matrix (ECM) limits their application. We hypothesized that signals coming from the extracellular matrix regulate organoids' 3-dimensional architecture and could be manipulated to generate novel organotypic culture systems., Approach and Results: Biliary organoids were generated from human livers and grown embedded into Culturex Basement Membrane Extract as spheroids around an internal lumen (EMB). When removed from the EMC, biliary organoids revert their polarity and expose the apical membrane on the outside (AOOs). Functional, immunohistochemical, and transmission electron microscope studies, along with bulk and single-cell transcriptomic, demonstrate that AOOs are less heterogeneous and show increased biliary differentiation and decreased expression of stem cell features. AOOs transport bile acids and have competent tight junctions. When cocultured with liver pathogenic bacteria (Enterococcus spp.), AOOs secrete a range of proinflammatory chemokines (ie, MCP1, IL8, CCL20, and IP-10). Transcriptomic analysis and treatment with a beta-1-integrin blocking antibody identified beta-1-integrin signaling as a sensor of the cell-extracellular matrix interaction and a determinant of organoid polarity., Conclusions: This novel organoid model can be used to study bile transport, interactions with pathobionts, epithelial permeability, cross talk with other liver and immune cell types, and the effect of matrix changes on the biliary epithelium and obtain key insights into the pathobiology of cholangiopathies., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Association for the Study of Liver Diseases.)

Type 2 diabetes mellitus is a recognized risk factor for HCC in patients with liver disease, independent from the etiology of their liver disease. Hence, prevention and treatment of type 2 diabetes mellitus and its underlying cause, insulin resistance, should be considered a treatment target for patients with liver disease. The drug armamentarium for diabetes is wide and consists of agents with insulin-sensitizing activity, agents that stimulate insulin secretion, insulin itself, and agents that reduce gastrointestinal and urinary glucose absorption. From an endocrinology perspective, the main goal of treatment is the achievement of euglycemia; however, in patients at risk of, or with known underlying liver disease, the choice of diabetic medication as it relates to potential hepatic carcinogenesis remains complex and should be carefully considered. In the last decade, increasing evidence has suggested that metformin may reduce the risk of HCC, whereas evidence for other classes of diabetic medications, particularly some of the newer agents including the sodium glucose cotransporter-2 inhibitors and glucagon-like peptide-1 receptor agonists, is fewer and often inconsistent. In this review, we aim to summarize the current evidence on the potential effects of the most widely used diabetic agents on liver cancer tumorigenesis., (© 2022 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Fluid and bicarbonate secretion is a principal function of cholangiocytes, and impaired secretion results in cholestasis. Cholangiocyte secretion depends on peri-apical expression of the type 3 inositol trisphosphate receptor (ITPR3), and loss of this intracellular Ca 2+ release channel is a final common event in most cholangiopathies. Here we investigated the mechanism by which ITPR3 localizes to the apical region to regulate secretion. Isolated bile duct units, primary mouse cholangiocytes, and polarized Madin-Darby canine kidney (MDCK) cells were examined using a combination of biochemical and fluorescence microscopy techniques to investigate the mechanism of ITPR3 targeting to the apical region. Apical localization of ITPR3 depended on the presence of intact lipid rafts as well as interactions with both caveolin 1 (CAV1) and myosin heavy chain 9 (MYH9). Chemical disruption of lipid rafts or knockdown of CAV1 or MYH9 redistributed ITPR3 away from the apical region. MYH9 interacted with the five c-terminal amino acids of the ITPR3 peptide. Disruption of lipid rafts impaired Ca 2+ signaling, and absence of CAV1 impaired both Ca 2+ signaling and fluid secretion. Conclusion: A cooperative mechanism involving MYH9, CAV1, and apical lipid rafts localize ITPR3 to the apical region to regulate Ca 2+ signaling and secretion in cholangiocytes., (© 2022 The Authors. Hepatology Communications published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Intrahepatic cholangiocarcinoma (iCCA) has over the last 10-20 years become the focus of increasing concern, largely due to its rising incidence and high mortality rates worldwide. The significant increase in mortality rates from this primary hepatobiliary cancer, particularly over the past decade, has coincided with a rapidly growing interest among clinicians, investigators, and patient advocates to seek greater mechanistic insights and more effective biomarker-driven targeted approaches for managing and/or preventing this challenging liver cancer. In addition to discussing challenges posed by this aggressive cancer, this review will emphasize recent epidemiological, basic, and translational research findings for iCCA. In particular, we will highlight emerging demographic changes and evolving risk factors, the critical role of the tumor microenvironment, extracellular vesicle biomarkers and therapeutics, intertumoral and intratumoral heterogeneity, and current and emerging targeted therapies regarding iCCA. Specifically, recent evidence linking non-bile duct medical conditions, such as nonalcoholic fatty liver disease and nonspecific cirrhosis, to intrahepatic cholangiocarcinogenesis together with geographic and ethnic variation will be assessed. Recent developments concerning the roles played by transforming growth factor-β and platelet-derived growth factor D in driving the recruitment and expansion of cancer-associated myofibroblasts within cholangiocarcinoma (CCA) stroma as well as their therapeutic implications will also be discussed. In addition, the potential significance of extracellular vesicles as bile and serum biomarkers and therapeutic delivery systems for iCCA will be described. An integrated systems approach to classifying heterogeneous iCCA subtypes will be further highlighted, and recent clinical trials and emerging targeted therapies will be reviewed, along with recommendations for future translational research opportunities. Established international CCA networks are now facilitating collaborations aimed at advancing iCCA translational and clinical research., (© 2018 by the American Association for the Study of Liver Diseases.)

Congenital hepatic fibrosis (CHF), a genetic disease caused by mutations in the polycystic kidney and hepatic disease 1 (PKHD1) gene, encoding for the protein fibrocystin/polyductin complex, is characterized by biliary dysgenesis, progressive portal fibrosis, and a protein kinase A-mediated activating phosphorylation of β-catenin at Ser675. Biliary structures of Pkhd1 del4/del4 mice, a mouse model of CHF, secrete chemokine (C-X-C motif) ligand 10 (CXCL10), a chemokine able to recruit macrophages. The aim of this study was to clarify whether CXCL10 plays a pathogenetic role in disease progression in CHF/Caroli disease and to understand the mechanisms leading to increased CXCL10 secretion. We demonstrate that treatment of Pkhd1 del4/del4 mice for 3 months with AMG-487, an inhibitor of CXC chemokine receptor family 3, the cognate receptor of CXCL10, reduces the peribiliary recruitment of alternative activated macrophages (cluster of differentiation 45 + F4/80 + cells), spleen size, liver fibrosis (sirius red), and cyst growth (cytokeratin 19-positive area), consistent with a pathogenetic role of CXCL10. Furthermore, we show that in fibrocystin/polyductin complex-defective cholangiocytes, isolated from Pkhd1 del4/del4 mice, CXCL10 production is mediated by Janus kinase/signal transducer and activator of transcription 3 in response to interleukin 1beta (IL-1β) and β-catenin. Specifically, IL-1β promotes signal transducer and activator of transcription 3 phosphorylation, whereas β-catenin promotes its nuclear translocation. Increased pro-IL-1β was regulated by nuclear factor kappa-light-chain-enhancer of activated B cells, and increased secretion of active IL-1β was mediated by the activation of Nod-like receptors, pyrin domain containing 3 inflammasome (increased expression of caspase 1 and Nod-like receptors, pyrin domain containing 3)., Conclusion: In fibrocystin/polyductin complex-defective cholangiocytes, β-catenin and IL-1β are responsible for signal transducer and activator of transcription 3-dependent secretion of CXCL10; in vivo experiments show that the CXCL10/CXC chemokine receptor family 3 axis prevents the recruitment of macrophages, reduces inflammation, and halts the progression of the disease; the increased production of IL-1β highlights the autoinflammatory nature of CHF and may open novel therapeutic avenues. (Hepatology 2018;67:1903-1919)., (© 2017 by the American Association for the Study of Liver Diseases.)

Cystic fibrosis transmembrane conductance regulator (CFTR), the channel mutated in cystic fibrosis (CF), is expressed by the biliary epithelium (i.e., cholangiocytes) of the liver. Progressive clinical liver disease (CF-associated liver disease; CFLD) occurs in around 10% of CF patients and represents the third leading cause of death. Impaired secretion and inflammation contribute to CFLD; however, the lack of human-derived experimental models has hampered the understanding of CFLD pathophysiology and the search for a cure. We have investigated the cellular mechanisms altered in human CF cholangiocytes using induced pluripotent stem cells (iPSCs) derived from healthy controls and a ΔF508 CFTR patient. We have devised a novel protocol for the differentiation of human iPSC into polarized monolayers of cholangiocytes. Our results show that iPSC-cholangiocytes reproduced the polarity and the secretory function of the biliary epithelium. Protein kinase A/cAMP-mediated fluid secretion was impaired in ΔF508 cholangiocytes and negligibly improved by VX-770 and VX-809, two small molecule drugs used to correct and potentiate ΔF508 CFTR. Moreover, ΔF508 cholangiocytes showed increased phosphorylation of Src kinase and Toll-like receptor 4 and proinflammatory changes, including increased nuclear factor kappa-light-chain-enhancer of activated B cells activation, secretion of proinflammatory chemokines (i.e., monocyte chemotactic protein 1 and interleukin-8), as well as alterations of the F-actin cytoskeleton. Treatment with Src inhibitor (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine) decreased the inflammatory changes and improved cytoskeletal defects. Inhibition of Src, along with administration of VX-770 and VX-809, successfully restored fluid secretion to normal levels., Conclusion: Our findings have strong translational potential and indicate that targeting Src kinase and decreasing inflammation may increase the efficacy of pharmacological therapies aimed at correcting the basic ΔF508 defect in CF liver patients. These studies also demonstrate the promise of applying iPSC technology in modeling human cholangiopathies. (Hepatology 2018;67:972-988)., (© 2017 by the American Association for the Study of Liver Diseases.)

The migration from legacy fee-for-service reimbursement to payments linked to high-value health care is accelerating in the United States because of new legislation and redesign of payments from the Centers for Medicare and Medicaid Services. Because patients with chronic diseases account for substantial use of health care resources, payers and health systems are focusing on maximizing the value of care for these patients. Because chronic liver diseases impose a major health burden worldwide affecting the health and lives of many individuals and families as well as substantial costs for individuals and payers, hepatologists must understand how they can improve their practices. Hepatologists practice a high-intensity cognitive subspecialty, using complex and costly procedures and medications. High-value patient care requires multidisciplinary coordination, labor-intensive support for critically ill patients, and effective chronic disease management. Under current fee-for-service reimbursement, patient values, medical success, and financial success can all be misaligned. Many current attempts to link health outcomes to reimbursement are based on compliance with process measures, with less emphasis on outcomes that matter most to patients, thus slowing transformation to higher-value team-based care. Outcome measures that reflect the entire cycle of care are needed to assist both clinicians and administrators in improving the quality and value of care. A comprehensive set of outcome measures for liver diseases is not currently available. Numerous researchers now are attempting to fill this gap by devising and testing outcome indicators and patient-reported outcomes for the major liver conditions. These indicators will provide tools to implement a value-based approach for patients with chronic liver diseases to compare results and value of care between referral centers, to perform health technology assessment, and to guide decision-making processes for health authorities. This review sets the groundwork for implementing a value-based, patient-centered approach to chronic liver diseases within a health system. (Hepatology 2017;65:1749-1755)., (© 2017 by the American Association for the Study of Liver Diseases.)

In the liver, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) regulates bile secretion and other functions at the apical membrane of biliary epithelial cells (i.e., cholangiocytes). CF-related liver disease is a major cause of death in patients with CF. CFTR dysfunction affects innate immune pathways, generating a para-inflammatory status in the liver and other epithelia. This study investigates the mechanisms linking CFTR to toll-like receptor 4 activity. We found that CFTR is associated with a multiprotein complex at the apical membrane of normal mouse cholangiocytes, with proteins that negatively control Rous sarcoma oncogene cellular homolog (Src) activity. In CFTR-defective cholangiocytes, Src tyrosine kinase self-activates and phosphorylates toll-like receptor 4, resulting in activation of nuclear factor kappa-light-chain-enhancer of activated B cells and increased proinflammatory cytokine production in response to endotoxins. This Src/nuclear factor kappa-light-chain-enhancer of activated B cells-dependent inflammatory process attracts inflammatory cells but also generates changes in the apical junctional complex and loss of epithelial barrier function. Inhibition of Src decreased the inflammatory response of CF cholangiocytes to lipopolysaccharide, rescued the junctional defect in vitro, and significantly attenuated endotoxin-induced biliary damage and inflammation in vivo (Cftr knockout mice)., Conclusion: These findings reveal a novel function of CFTR as a regulator of toll-like receptor 4 responses and cell polarity in biliary epithelial cells; this mechanism is pathogenetic, as shown by the protective effects of Src inhibition in vivo, and may be a novel therapeutic target in CF-related liver disease and other inflammatory cholangiopathies. (Hepatology 2016;64:2118-2134)., (© 2016 by the American Association for the Study of Liver Diseases.)

Unlabelled: In the current context of rising health care costs and decreasing sustainability, it is becoming increasingly common to resort to decision analytical modeling and health economics evaluations. Decision analytic models are analytical tools that help decision makers to select the best choice between alternative health care interventions, taking into consideration the complexity of the disease, the socioeconomic context, and the relevant differences in outcomes. We present a brief overview of the use of decision analytical models in health economic evaluations and their applications in the area of liver diseases. The aim is to provide the reader with the basic elements to evaluate health economic analysis reports and to discuss some limitations of the current approaches, as highlighted by the case of the therapy of chronic hepatitis C. To serve its purpose, health economics evaluations must be able to do justice to medical innovation and the market while protecting patients and society and promoting fair access to treatment and its economic sustainability., Conclusion: New approaches and methods able to include variables such as prevalence of the disease, budget impact, and sustainability into the cost-effectiveness analysis are needed to reach this goal. (Hepatology 2016;64:1331-1342)., (© 2016 by the American Association for the Study of Liver Diseases.)

Unlabelled: Congenital hepatic fibrosis (CHF) is a disease of the biliary epithelium characterized by bile duct changes resembling ductal plate malformations and by progressive peribiliary fibrosis, in the absence of overt necroinflammation. Progressive liver fibrosis leads to portal hypertension and liver failure; however, the mechanisms leading to fibrosis in CHF remain elusive. CHF is caused by mutations in PKHD1, a gene encoding for fibrocystin, a ciliary protein expressed in cholangiocytes. Using a fibrocystin-defective (Pkhd1(del4/del4)) mouse, which is orthologous of CHF, we show that Pkhd1(del4/del4) cholangiocytes are characterized by a β-catenin-dependent secretion of a range of chemokines, including chemokine (C-X-C motif) ligands 1, 10, and 12, which stimulate bone marrow-derived macrophage recruitment. We also show that Pkhd1(del4/del4) cholangiocytes, in turn, respond to proinflammatory cytokines released by macrophages by up-regulating αvβ6 integrin, an activator of latent local transforming growth factor-β1. While the macrophage infiltrate is initially dominated by the M1 phenotype, the profibrogenic M2 phenotype increases with disease progression, along with the number of portal myofibroblasts. Consistent with these findings, clodronate-induced macrophage depletion results in a significant reduction of portal fibrosis and portal hypertension as well as of liver cysts., Conclusion: Fibrosis can be initiated by an epithelial cell dysfunction, leading to low-grade inflammation, macrophage recruitment, and collagen deposition; these findings establish a new paradigm for biliary fibrosis and represent a model to understand the relationship between cell dysfunction, parainflammation, liver fibrosis, and macrophage polarization over time., (© 2015 by the American Association for the Study of Liver Diseases.)

Unlabelled: Polycystin-2 (PC2 or TRPPC2), a member of the transient receptor potential channel family, is a nonselective calcium channel. Mutations in PC2 are associated with polycystic liver diseases. PC2-defective cholangiocytes show increased production of cyclic adenosine monophosphate, protein kinase A-dependent activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, hypoxia-inducible factor 1α (HIF-1α)-mediated vascular endothelial growth factor (VEGF) production, and stimulation of cyst growth and progression. Activation of the ERK/HIF-1α/VEGF pathway in cholangiocytes plays a key role during repair from biliary damage. We hypothesized that PC2 levels are modulated during biliary damage/repair, resulting in activation of the ERK/HIF-1α/VEGF pathway. PC2 protein expression, but not its gene expression, was significantly reduced in mouse livers with biliary damage (Mdr2(-/-) knockout, bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine treatment). Treatment of cholangiocytes with proinflammatory cytokines, nitric oxide donors, and endoplasmic reticulum stressors increased ERK1/2 phosphorylation, HIF-1α transcriptional activity, secretion of VEGF, and VEGF receptor type 2 phosphorylation and down-regulated PC2 protein expression without affecting PC2 gene expression. Expression of homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1 protein and NEK, ubiquitin-like proteins that promote proteosomal PC2 degradation, was increased. Pretreatment with the proteasome inhibitor MG-132 restored the expression of PC2 in cells treated with cytokines but not in cells treated with nitric oxide donors or with endoplasmic reticulum stressors. In these conditions, PC2 degradation was instead inhibited by interfering with the autophagy pathway. Treatment of 3,5-diethoxycarbonyl-1,4-dihydrocollidine mice and of Mdr2(-/-) mice with the proteasome inhibitor bortezomib restored PC2 expression and significantly reduced the ductular reaction, fibrosis, and phosphorylated ERK1/2., Conclusion: In response to biliary damage, PC2 expression is modulated posttranslationally by the proteasome or the autophagy pathway, and PC2 down-regulation is associated with activation of ERK1/2 and an increase of HIF-1α-mediated VEGF secretion; treatments able to restore PC2 expression and to reduce ductular reaction and fibrosis may represent a new therapeutic approach in biliary diseases., (© 2015 by the American Association for the Study of Liver Diseases.)

Unlabelled: Cystic fibrosis-associated liver disease is a chronic cholangiopathy that negatively affects the quality of life of cystic fibrosis patients. In addition to reducing biliary chloride and bicarbonate secretion, up-regulation of toll-like receptor 4/nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB)-dependent immune mechanisms plays a major role in the pathogenesis of cystic fibrosis-associated liver disease and may represent a therapeutic target. Nuclear receptors are transcription factors that regulate several intracellular functions. Some nuclear receptors, including peroxisome proliferator-activated receptor-γ (PPAR-γ), may counterregulate inflammation in a tissue-specific manner. In this study, we explored the anti-inflammatory effect of PPAR-γ stimulation in vivo in cystic fibrosis transmembrane conductance regulator (Cftr) knockout mice exposed to dextran sodium sulfate and in vitro in primary cholangiocytes isolated from wild-type and from Cftr-knockout mice exposed to lipopolysaccharide. We found that in CFTR-defective biliary epithelium expression of PPAR-γ is increased but that this does not result in increased receptor activity because the availability of bioactive ligands is reduced. Exogenous administration of synthetic agonists of PPAR-γ (pioglitazone and rosiglitazone) up-regulates PPAR-γ-dependent genes, while inhibiting the activation of NF-κB and the secretion of proinflammatory cytokines (lipopolysaccharide-induced CXC chemokine, monocyte chemotactic protein-1, macrophage inflammatory protein-2, granulocyte colony-stimulating factor, keratinocyte chemoattractant) in response to lipopolysaccharide. PPAR-γ agonists modulate NF-κB-dependent inflammation by up-regulating nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha, a negative regulator of NF-κB. Stimulation of PPAR-γ in vivo (rosiglitazone) significantly attenuates biliary damage and inflammation in Cftr-knockout mice exposed to a dextran sodium sulfate-induced portal endotoxemia., Conclusion: These studies unravel a novel function of PPAR-γ in controlling biliary epithelium inflammation and suggest that impaired activation of PPAR-γ contributes to the chronic inflammatory state of CFTR-defective cholangiocytes., (© 2015 by the American Association for the Study of Liver Diseases.)

This review critically discusses the most recent advances in the role of Notch signaling in liver development, homeostasis, and disease. It is now clear that the significance of Notch in determining mammalian cell fates and functions extends beyond development, and Notch is a major regular of organ homeostasis. Moreover, Notch signaling is reactivated upon injury and regulates the complex interactions between the distinct liver cell types involved in the repair process. Notch is also involved in the regulation of liver metabolism, inflammation, and cancer. The net effects of Notch signaling are highly variable and finely regulated at multiple levels, but also depend on the specific cellular context in which Notch is activated. Persistent activation of Notch signaling is associated with liver malignancies, such as hepatocellular carcinoma with stem cell features and intrahepatic cholangiocarcinoma. The complexity of the pathway provides several possible targets for agents able to inhibit Notch. However, further cell- and context-specific in-depth understanding of Notch signaling in liver homeostasis and disease will be essential to translate these concepts into clinical practice and be able to predict benefits and risks of evolving therapies., (© 2014 by the American Association for the Study of Liver Diseases.)

Unlabelled: Genetically determined loss of fibrocystin function causes congenital hepatic fibrosis (CHF), Caroli disease (CD), and autosomal recessive polycystic kidney disease (ARPKD). Cystic dysplasia of the intrahepatic bile ducts and progressive portal fibrosis characterize liver pathology in CHF/CD. At a cellular level, several functional morphological and signaling changes have been reported including increased levels of 3'-5'-cyclic adenosine monophosphate (cAMP). In this study we addressed the relationships between increased cAMP and β-catenin. In cholangiocytes isolated and cultured from Pkhd1(del4/del4) mice, stimulation of cAMP/PKA signaling (forskolin 10 μM) stimulated Ser(675) -phosphorylation of β-catenin, its nuclear localization, and its transcriptional activity (western blot and TOP flash assay, respectively) along with a down-regulation of E-cadherin expression (immunocytochemistry and western blot); these changes were inhibited by the PKA blocker, PKI (1 μM). The Rho-GTPase, Rac-1, was also significantly activated by cAMP in Pkhd1(del4/del4) cholangiocytes. Rac-1 inhibition blocked cAMP-dependent nuclear translocation and transcriptional activity of pSer(675) -β-catenin. Cell migration (Boyden chambers) was significantly higher in cholangiocytes obtained from Pkhd1(del4/del4) and was inhibited by: (1) PKI, (2) silencing β-catenin (siRNA), and (3) the Rac-1 inhibitor NSC 23766., Conclusion: These data show that in fibrocystin-defective cholangiocytes, cAMP/PKA signaling stimulates pSer(675) -phosphorylation of β-catenin and Rac-1 activity. In the presence of activated Rac-1, pSer(675) -β-catenin is translocated to the nucleus, becomes transcriptionally active, and is responsible for increased motility of Pkhd1(del4/del4) cholangiocytes. β-Catenin-dependent changes in cell motility may be central to the pathogenesis of the disease and represent a potential therapeutic target., (© 2013 by the American Association for the Study of Liver Diseases.)

Unlabelled: Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)β. PDGF-D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and c-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration., Conclusion: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRβ and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach., (Copyright © 2013 by the American Association for the Study of Liver Diseases.)

Unlabelled: Mutations in polycystins are a cause of polycystic liver disease. In polycystin-2 (PC2)-defective mice, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA)-dependent activation of the Rat Sarcoma (Ras)/rapidly accelerated fibrosarcoma (Raf)/mitogen signal-regulated kinase-extracellular signal-regulated kinase (ERK) 1/2 pathway stimulates the growth of liver cysts. To test the hypothesis that sorafenib, a Raf inhibitor used for the treatment of liver and kidney cancers, inhibits liver cyst growth in PC2-defective mice, we treated PC2 (i.e., Pkd2(flox/-) :pCxCreER(TM) [Pkd2cKO]) mice with sorafenib-tosylate for 8 weeks (20-60 mg/kg/day). Sorafenib caused an unexpected increase in liver cyst area, cell proliferation (Ki67), and expression of phosphorylated ERK (pERK) compared with Pkd2cKO mice treated with vehicle. When given to epithelial cells isolated from liver cysts of Pkd2cKO mice (Pkd2cKO-cells), sorafenib progressively stimulated pERK1/2 and cell proliferation [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assay (MTS)] at doses between 0.001 and 1 μM; however, both pERK1/2 and cell proliferation significantly decreased at the dose of 10 μM. Raf kinase activity assay showed that whereas B-Raf is inhibited by sorafenib in both wild-type (WT) and Pkd2cKO cells, Raf-1 is inhibited in WT cells but is significantly stimulated in Pkd2cKO cells. In Pkd2cKO cells pretreated with the PKA inhibitor 14-22 amide, myristolated (1 μM) and in mice treated with octreotide in combination with sorafenib, the paradoxical activation of Raf/ERK1/2 was abolished, and cyst growth was inhibited., Conclusion: In PC2-defective cells, sorafenib inhibits B-Raf but paradoxically activates Raf-1, resulting in increased ERK1/2 phosphorylation, cell proliferation, and cyst growth in vivo. These effects are consistent with the ability of Raf inhibitors to transactivate Raf-1 when a PKA-activated Ras promotes Raf-1/B-Raf heterodimerization, and are inhibited by interfering with cAMP/PKA signaling both in vitro and in vivo, as shown by the reduction of liver cysts in mice treated with combined octreotide and sorafenib., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Unlabelled: Mutations in polycystins (PC1 or PC2/TRPP2) cause progressive polycystic liver disease (PLD). In PC2-defective mice, cyclic 3',5'-adenosine monophosphate/ protein kinase A (cAMP/PKA)-dependent activation of extracellular signal-regulated kinase/ mammalian target of rapamycin (ERK-mTOR) signaling stimulates cyst growth. We investigated the mechanisms connecting PC2 dysfunction to altered Ca(2+) and cAMP production and inappropriate ERK signaling in PC2-defective cholangiocytes. Cystic cholangiocytes were isolated from PC2 conditional-KO (knockout) mice (Pkd2(flox/-) :pCxCreER™; hence, called Pkd2KO) and compared to cholangiocytes from wild-type mice (WT). Our results showed that, compared to WT cells, in PC2-defective cholangiocytes (Pkd2KO), cytoplasmic and ER-Ca(2+) (measured with Fura-2 and Mag-Fluo4) levels are decreased and store-operated Ca(2+) entry (SOCE) is inhibited, whereas the expression of Ca(2+) -sensor stromal interaction molecule 1 (STIM1) and store-operated Ca(2+) channels (e.g., the Orai1 channel) are unchanged. In Pkd2KO cells, ER-Ca(2+) depletion increases cAMP and PKA-dependent ERK1/2 activation and both are inhibited by STIM1 inhibitors or by silencing of adenylyl cyclase type 6 (AC6)., Conclusion: These data suggest that PC2 plays a key role in SOCE activation and inhibits the STIM-dependent activation of AC6 by ER Ca(2+) depletion. In PC2-defective cells, the interaction of STIM-1 with Orai channels is uncoupled, whereas coupling to AC6 is maximized. The resulting overproduction of cAMP, in turn, potently activates the PKA/ERK pathway. PLD, because of PC2 deficiency, represents the first example of human disease linked to the inappropriate activation of store-operated cAMP production., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Unlabelled: Chronic cholangiopathies have limited therapeutic options and represent an important indication for liver transplantation. The nuclear farnesoid X receptor (FXR) and the membrane G protein-coupled receptor, TGR5, regulate bile acid (BA) homeostasis and inflammation. Therefore, we hypothesized that activation of FXR and/or TGR5 could ameliorate liver injury in Mdr2(-/-) (Abcb4(-/-)) mice, a model of chronic cholangiopathy. Hepatic inflammation, fibrosis, as well as bile secretion and key genes of BA homeostasis were addressed in Mdr2(-/-) mice fed either a chow diet or a diet supplemented with the FXR agonist, INT-747, the TGR5 agonist, INT-777, or the dual FXR/TGR5 agonist, INT-767 (0.03% w/w). Only the dual FXR/TGR5 agonist, INT-767, significantly improved serum liver enzymes, hepatic inflammation, and biliary fibrosis in Mdr2(-/-) mice, whereas INT-747 and INT-777 had no hepatoprotective effects. In line with this, INT-767 significantly induced bile flow and biliary HCO 3- output, as well as gene expression of carbonic anhydrase 14, an important enzyme able to enhance HCO 3- transport, in an Fxr-dependent manner. In addition, INT-767 dramatically reduced bile acid synthesis via the induction of ileal Fgf15 and hepatic Shp gene expression, thus resulting in significantly reduced biliary bile acid output in Mdr2(-/-) mice., Conclusion: This study shows that FXR activation improves liver injury in a mouse model of chronic cholangiopathy by reduction of biliary BA output and promotion of HCO 3--rich bile secretion., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Unlabelled: Cholangiocarcinoma (CCA) carries a severe prognosis because of its strong invasiveness and early metastasization. In several patients, otherwise eligible for surgical resection, micrometastasis are already present at the time of surgery. The mechanisms responsible for CCA invasiveness are unclear. S100A4, a member of the S100 family of small Ca(2+)-binding proteins, is expressed in mesenchymal cells, regulates cell motility in several cell types, and is expressed in some epithelial cancers. Thus, we aimed to study the role of S100A4 in CCA invasiveness and metastasization. The expression of S100A4 was studied by immunohistochemistry in 93 human liver samples of CCA patients undergoing surgical resection and correlated with metastases development (67 cases) and patient survival following surgery using log rank tests and multivariate analysis. S100A4 expression was studied in EGI-1 and TFK-1, human CCA cell lines with and without nuclear S100A4 expression, respectively. Metastatic properties of CCA cells were assessed by xenotransplantation in severe combined immunodeficiency (SCID) mice after transduction with lentiviral vectors encoding firefly luciferase gene. Proliferation, motility (wound healing), invasiveness (Boyden chamber), and metalloproteinases (MMPs) secretion were studied in CCA cells, with or without lentiviral silencing of S100A4. Nuclear expression of S100A4 by neoplastic ducts was a strong predictor of metastasization and reduced survival after resection (P < 0.01). EGI-1 CCA cells showed stronger metastatic properties than TFK-1 when xenotransplanted in SCID mice. S100A4-silenced EGI-1 cells showed significantly reduced motility, invasiveness, and MMP-9 secretion in vitro, without changes in cell proliferation., Conclusion: Nuclear S100A4 identifies a subset of CCA patients with a poor prognosis after surgical resection. Nuclear expression of S100A4 increases CCA cells invasiveness and metastasization, indicating that S100A4 may also represent a potential therapeutic target., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Unlabelled: Polycystic liver disease may complicate autosomal dominant polycystic kidney disease (ADPKD), a disease caused by mutations in polycystins, which are proteins that regulate signaling, morphogenesis, and differentiation in epithelial cells. The cystic biliary epithelium [liver cystic epithelium (LCE)] secretes vascular endothelial growth factor (VEGF), which promotes liver cyst growth via autocrine and paracrine mechanisms. The expression of insulin-like growth factor 1 (IGF1), insulin-like growth factor 1 receptor (IGF1R), and phosphorylated mammalian target of rapamycin (p-mTOR) and the protein kinase A (PKA)-dependent phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) are also up-regulated in LCE. We have hypothesized that mammalian target of rapamycin (mTOR) represents a common pathway for the regulation of hypoxia-inducible factor 1 alpha (HIF1alpha)-dependent VEGF secretion by IGF1 and ERK1/2. Conditional polycystin-2-knockout (Pkd2KO) mice were used for in vivo studies and to isolate cystic cholangiocytes [liver cystic epithelial cells (LCECs)]. The expression of p-mTOR, VEGF, cleaved caspase 3 (CC3), proliferating cell nuclear antigen (PCNA), IGF1, IGF1R, phosphorylated extracellular signal-regulated kinase, p-P70S6K, HIF1alpha, and VEGF in LCE, LCECs, and wild-type cholangiocytes was studied with immunohistochemistry, western blotting, or enzyme-linked immunosorbent assays. The cystic area was measured by computer-assisted morphometry of pancytokeratin-stained sections. Cell proliferation in vitro was studied with 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and bromodeoxyuridine assays. The treatment of Pkd2KO mice with the mTOR inhibitor rapamycin significantly reduced the liver cyst area, liver/body weight ratio, pericystic microvascular density, and PCNA expression while increasing expression of CC3. Rapamycin inhibited IGF1-stimulated HIF1alpha accumulation and VEGF secretion in LCECs. IGF1-stimulated LCEC proliferation was inhibited by rapamycin and SU5416 (a vascular endothelial growth factor receptor 2 inhibitor). Phosphorylation of the mTOR-dependent kinase P70S6K was significantly reduced by PKA inhibitor 14-22 amide and by the mitogen signal-regulated kinase inhibitor U1026., Conclusion: These data demonstrate that PKA-dependent up-regulation of mTOR has a central role in the proliferative, antiapoptotic, and pro-angiogenic effects of IGF1 and VEGF in polycystin-2-defective mice. This study also highlights a mechanistic link between PKA, ERK, mTOR, and HIF1alpha-mediated VEGF secretion and provides a proof of concept for the potential use of mTOR inhibitors in ADPKD and conditions with aberrant cholangiocyte proliferation.

Transplantation is the treatment of choice for hepatocellular carcinoma (HCC) meeting the Milan criteria. HCC and chronic liver diseases have distinct natural histories for which an equitable transplant policy must account. We enrolled and prospectively followed at a single center 206 consecutive HCC patients that presented within the Milan criteria. Patients were treated per the Barcelona Clinic Liver Cancer (BCLC) algorithm; 95% received resection, ablation, or transarterial chemoembolization. The median follow-up was 16 months. Progression occurred in 84 patients, and 8 patients died. Risk factors for the time to disease progression (death or progression beyond T2) were analyzed in 170 patients with a complete data set. Risk factors with the strongest relationship to progression included tumor diameter and tumor persistence/recurrence after local therapy (hazard ratios of 1.51 and 2.75, respectively, when transplanted patients were censored at the time of transplantation and hazard ratios of 1.53 and 3.66, respectively, when transplantation was counted as an event; P < or = 0.0001). To evaluate the current Model for End-Stage Liver Disease (MELD) exception, we compared the expected progression rate (PR) with our observed PR in 133 stage T2 patients. The current policy resulted in a large overestimation of the PR for T2 HCC and an unsatisfactory performance [Harrell's concordance index (C index) = 0.60, transplant censored; C index = 0.55, transplant as progression]. Risk factors for progression that were identified by univariate analysis were considered for multivariate analysis. With these risk factors and the patients' natural MELD scores, an adjusted model applicable to organ allocation was generated, and this decreased the discrepancy between the expected and observed PRs (C index = 0.66, transplant censored; C index = 0.69, transplant as progression). In conclusion, the current MELD exception largely overestimates progression in T2 patients treated according to the BCLC guidelines. The tumor response to resective or ablative treatment can predict tumor progression beyond the Milan criteria, and it should be taken into account in models designed to prioritize organ allocation., ((c) 2010 AASLD.)

Unlabelled: Cyclic adenosine monophosphate (cAMP) is generated by adenylyl cyclases (ACs), a group of enzymes with different tissue specificity and regulation. We hypothesized that AC isoforms are heterogeneously expressed along the biliary tree, are associated with specific secretory stimuli, and are differentially modulated in cholestasis. Small duct and large duct cholangiocytes were isolated from controls and from lipopolysaccharide-treated or alpha-naphthylisothiocyanate-treated rats. AC isoform expression was assessed via real-time polymerase chain reaction. Secretion and cAMP levels were measured in intrahepatic bile duct units after stimulation with secretin, forskolin, HCO(3)(-)/CO(2), cholinergic agonists, and beta-adrenergic agonists, with or without selected inhibitors or after silencing of AC8 or soluble adenylyl cyclase (sAC) with small interfering RNA. Gene expression of the Ca(2+)-insensitive isoforms (AC4, AC7) was higher in small duct cholangiocytes, whereas that of the Ca(2+)-inhibitable (AC5, AC6, AC9), the Ca(2+)/calmodulin-stimulated AC8, and the soluble sAC was higher in large duct cholangiocytes. Ca(2+)/calmodulin inhibitors and AC8 gene silencing inhibited choleresis and cAMP production stimulated by secretin and acetylcholine, but not by forskolin. Secretion stimulated by isoproterenol and calcineurin inibitors was cAMP-dependent and gamma-aminobutyric acid-inhibitable, consistent with activation of AC9. Cholangiocyte secretion stimulated by isohydric changes in [HCO(3)(-)](i) was cAMP-dependent and inhibited by sAC inhibitor and sAC gene silencing. Treatment with lipopolysaccharide or alpha-naphthylisothiocyanate increased expression of AC7 and sAC but decreased expression of the other ACs., Conclusion: These studies demonstrate a previously unrecognized role of ACs in biliary pathophysiology. In fact: (1) AC isoforms are differentially expressed in cholangiocyte subpopulations; (2) AC8, AC9, and sAC mediate cholangiocyte secretion in response to secretin, beta-adrenergic agonists, or changes in [HCO(3)(-)](i), respectively; and (3) AC gene expression is modulated in experimental cholestasis.

Unlabelled: 24-norursodeoxycholic acid (norUDCA), a side chain-modified ursodeoxycholic acid derivative, has dramatic therapeutic effects in experimental cholestasis and may be a promising agent for the treatment of cholestatic liver diseases. We aimed to better understand the physiologic and therapeutic properties of norUDCA and to test if they are related to its side chain length and/or relative resistance to amidation. For this purpose, Mdr2(-/-) mice, a model for sclerosing cholangitis, received either a standard diet or a norUDCA-, tauro norursodeoxycholic acid (tauro- norUDCA)-, or di norursodeoxycholic acid (di norUDCA)-enriched diet. Bile composition, serum biochemistry, liver histology, fibrosis, and expression of key detoxification and transport systems were investigated. Direct choleretic effects were addressed in isolated bile duct units. The role of Cftr for norUDCA-induced choleresis was explored in Cftr(-/-) mice. norUDCA had pharmacologic features that were not shared by its derivatives, including the increase in hepatic and serum bile acid levels and a strong stimulation of biliary HCO(3)(-)-output. norUDCA directly stimulated fluid secretion in isolated bile duct units in a HCO(3)(-)-dependent fashion to a higher extent than the other bile acids. Notably, the norUDCA significantly stimulated HCO(3)(-)-output also in Cftr(-/-) mice. In Mdr2(-/-) mice, cholangitis and fibrosis strongly improved with norUDCA, remained unchanged with tauro- norUDCA, and worsened with di norUDCA. Expression of Mrp4, Cyp2b10, and Sult2a1 was increased by norUDCA and di norUDCA, but was unaffected by tauro- norUDCA., Conclusion: The relative resistance of norUDCA to amidation may explain its unique physiologic and pharmacologic properties. These include the ability to undergo cholehepatic shunting and to directly stimulate cholangiocyte secretion, both resulting in a HCO(3)(-)-rich hypercholeresis that protects the liver from cholestatic injury.

Type I autoimmune hepatitis usually has an indolent presentation and course, and is classically thought of as a disease of young women, but can in fact occur across all age ranges. Although its etiology remains unclear, it is hypothesized that an environmental antigen may trigger the disease in a genetically susceptible individual. Here, we report the unusual case of a woman in her seventh decade who presented with acute liver failure as her initial manifestation of autoimmune hepatitis, and who had been a long-time ingestor of hand-picked, wild mushrooms.

Unlabelled: Intrahepatic bile ducts maintain a close anatomical relationship with hepatic arteries. During liver ontogenesis, the development of the hepatic artery appears to be modulated by unknown signals originating from the bile duct. Given the capability of cholangiocytes to produce angiogenic growth factors and influence peribiliary vascularization, we studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1, angiopoietin-2, and their cognate receptors (VEGFR-1, VEGFR-2, Tie-2) in fetal human livers at different gestational ages and in mice characterized by defective biliary morphogenesis (Hnf6(-/-)). The results showed that throughout the different developmental stages, VEGF was expressed by developing bile ducts and angiopoietin-1 by hepatoblasts, whereas their cognate receptors were variably expressed by vascular cells according to the different maturational stages. Precursors of endothelial and mural cells expressed VEGFR-2 and Tie-2, respectively. In immature hepatic arteries, endothelial cells expressed VEGFR-1, whereas mural cells expressed both Tie-2 and Angiopoietin-2. In mature hepatic arteries, endothelial cells expressed Tie-2 along with VEGFR-1. In early postnatal Hnf6(-/-) mice, VEGF-expressing ductal plates failed to incorporate into the portal mesenchyma, resulting in severely altered arterial vasculogenesis., Conclusion: The reciprocal expression of angiogenic growth factors and receptors during development supports their involvement in the cross talk between liver epithelial cells and the portal vasculature. Cholangiocytes generate a VEGF gradient that is crucial during the migratory stage, when it determines arterial vasculogenesis in their vicinity, whereas angiopoietin-1 signaling from hepatoblasts contributes to the remodeling of the hepatic artery necessary to meet the demands of the developing epithelium.

End-stage liver disease after hepatitis C virus (HCV) infection is the most common indication for liver transplantation, accounting for over 40% of liver transplants performed. Combined liver/kidney transplantation is being performed more frequently, in part because HCV infection may coexist with conditions that damage the kidney, such as diabetes and cryoglobulinemia. Unfortunately, HCV hepatitis and cryoglobulinemia may recur after liver transplantation and adversely affect graft and patient survival. In immunocompetent patients, interferon (IFN) and ribavirin (RBV) combination therapy is often able to control cryoglobulinemic syndrome. Very little data are available on liver transplant recipients, whereas IFN usually is not indicated in kidney transplant recipients because of early reports of steroid-induced rejection after its administration. Successful treatment of cryoglobulinemia with IFN/RBV in recipients of combined liver/kidney transplant has not been previously reported. We treated 1 recipient of a combined liver and kidney transplant with pegylated-IFN/RBV combination therapy. The patient developed HCV recurrence associated with cryoglobulinemia and severe cutaneous peripheral and neurologic manifestations. Treatment with pegylated-IFN-alpha2b and RBV for 12 months cured the cryoglobulinemic vasculitis and allowed the sustained eradication of HCV with no significant changes in kidney function.

Liver involvement in autosomal dominant polycystic kidney disease (ADPKD) is characterized by altered remodeling of the embryonic ductal plate (DP) with presence of biliary cysts and aberrant portal vasculature. The genetic defect causing ADPKD has been identified, but mechanisms of liver cyst growth remain uncertain. To investigate the possible role of angiogenic mechanisms, we have studied the immunohistochemical expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2) and their receptors (VEGFR-1, VEGFR-2, Tie-2) in ADPKD, Caroli's disease, normal and fetal livers. In ADPKD and control livers Ang-1 and Ang-2 gene expression was studied by real-time-PCR. Effects of VEGF on cholangiocyte proliferation were studied by PCNA Western Blot in isolated rat cholangiocytes and by MTS assay in cultured cholangiocytes isolated from ADPKD patients and from an ADPKD mouse model (Pkd2(WS25/-)). Cholangiocytes were strongly positive for VEGF, VEGFR-1, VEGFR-2 and Ang-2 in ADPKD and Caroli, and also for Ang-1 and Tie-2 in ADPKD, similar to fetal ductal plate cells. VEGF stimulated proliferation in both normal and ADPKD cholangiocytes, but the effect was particularly evident in the latter. Ang-1 alone had no effect, but was synergic to VEGF. VEGF expression on cholangiocytes positively correlated with microvascular density. In conclusion, consistent with the immature phenotype of the cystic epithelium, expression of VEGF, VEGFRs, Ang-1 and Tie-2 is strongly upregulated in cholangiocytes from polycystic liver diseases. VEGF and Ang-1 have autocrine proliferative effect on cholangiocyte growth and paracrine effect on portal vasculature, thus promoting the growth of the cysts and their vascular supply. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).

The diseases of the intrahepatic biliary tree are a large group of potentially evolutive congenital and acquired liver disorders affecting both the adult and pediatric populations. They represent a relevant cause of liver-related morbidity and mortality and an important indication for liver transplantation, particularly in children. While the practical approach to patients affected by biliary tree diseases has not significantly changed yet, the conceptual approach to the pathophysiology of cholangiopathies has witnessed important advances that will be discussed. The primary cell target of the pathogenetic sequence of these disorders is the biliary epithelium. Cholangiocytes have multifaceted functions, not limited to bile production. Their capability to secrete a range of different pro-inflammatory mediators, cytokines, and chemokines indicates a major role of cholangiocytes in the inflammatory reaction. Furthermore, paracrine secretion of growth factors and peptides mediates an extensive cross-talk with other liver cell types, including hepatocytes, stellate, and endothelial and inflammatory cells. Cholangiopathies share a number of pathogenetic mechanisms, including inflammation, cholestasis, fibrosis, apoptosis, altered development, and neoplastic transformation. These basic disease mechanisms will be discussed in detail, along with the distinct features of a number of cholangiopathies. Furthermore, an increase in the biliary cell compartment is a common response to many forms of liver injury, from cholangiopathies to viral and fulminant hepatitis. Elucidation of these pathophysiologic mechanisms will likely provide clues for future therapeutic strategies. Furthermore, understanding the role of cholangiocytes in liver regeneration/repair and the mechanisms of cholangiocyte activation and their relationship with liver progenitor cell will be of further interest.

Primary biliary cirrhosis (PBC) is a disorder of unknown origin with autoimmune features. Recently, impaired biliary secretion of bicarbonate has been shown in patients with PBC. Here we have investigated whether bile duct epithelial cells isolated from PBC patients exhibit defects in transepithelial bicarbonate transport by analyzing the activities of 2 ion exchangers, Cl(-)/HCO3(-) anion exchanger 2 (AE2) and Na(+)/H(+) exchanger (NHE) in isolated cholangiocytes. AE2 and NHE activities were studied in basal conditions and after stimulation with cyclic adenosine monophosphate (cAMP) and extracellular adenosine triphosphate (ATP), respectively. Cholangiocytes were grown from needle liver biopsies from 12 PBC patients, 8 normal controls, and 9 patients with other liver diseases. Also, intrahepatic cholangiocytes were cultured after immunomagnetic isolation from normal liver tissue (n = 6), and from recipients undergoing liver transplantation for end-stage PBC (n = 9) and other forms of liver disease (n = 8). In needle-biopsy cholangiocytes, basal AE2 activity was significantly decreased in PBC as compared with normal livers and disease controls. In addition, we observed that though cAMP increased AE2 activity in cholangiocytes from both normal and non-PBC livers, this effect was absent in PBC cholangiocytes. Similarly, though in cholangiocytes from normal and disease control livers extracellular ATP induced a marked enhancement of NHE activity, cholangiocytes from PBC patients failed to respond to purinergic stimulation. In conclusion, our findings provide functional evidence that PBC cholangiocytes exhibit a widespread failure in the regulation of carriers involved in transepithelial H(+)/HCO3(-) transport, thus, providing a molecular basis for the impaired bicarbonate secretion in this cholestatic syndrome.

Intravenous methylprednisolone is used in most liver transplant centers as first-line therapy of acute hepatic cellular rejection in patients who undergo liver transplant. However, no controlled study has been performed to date to define the optimal dose and duration of the steroid regimen. The schedules that actually are used in most transplant centers are drawn from those that were developed empirically for the treatment of acute renal graft rejection. Thus, the aim of the study was to compare two schedules of steroid treatment of acute hepatic cellular rejection among those most widely used. Thirty-eight eligible patients with grade II or III acute hepatic cellular rejection were randomized to receive two different high-dose methylprednisolone schedules. Eighteen patients were randomized in group A (intravenous dose of 1,000 mg of methylprednisolone followed by a 6-day taper from 200 to 20 mg/d). Twenty patients were randomized in group B (intravenous dose of 1,000 mg of methylprednisolone for three consecutive days). The response to treatment was evaluated by means of a second liver biopsy. The treatment of group A proved to be more effective than treatment of group B. The resolution of acute hepatic cellular rejection was observed in 83.3% of cases in group A and 50.0% of cases in group B (P <.05). The treatment of group A proved to be safer also than treatment of group B. Patients randomized in group B showed a higher prevalence of infections (90.0% of cases versus 55.5% of cases; P <.01) mainly because of bacterial (80.0% versus 50.0%; P <.05) and viral (50.0% versus 16.6%; P <.05) agents. In conclusion, the study shows that intravenous administration of 1,000 mg of methylprednisolone followed by a 6-day taper from 200 to 20 mg/d is more effective and safer than intravenous dose of 1,000 mg of methylprednisolone for three consecutive days in the treatment of acute cellular rejection in patients with liver transplantation.

In view of the occurrence of hepatobiliary disorders in cystic fibrosis (CF) this study addresses the role of the cystic fibrosis transmembrane conductance regulator (CFTR) and of Ca(2+)-activated Cl(-) channels in promoting HCO3- secretion in bile ductular cells. Human cholangiocytes were isolated from control livers and from 1 patient with CF (DeltaF508/G542X mutations). Single channel and whole cell currents were analyzed by patch clamp techniques, and HCO3- secretion was determined by fluorometric analysis of the rate of recovery of intracellular pH following alkaline loading. In control cholangiocytes, both cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) catalytic subunit, activated CFTR Cl(-) channels that exhibited a nonrectifying conductance of 8 pS and appeared in clusters. Activation of Cl(-) current by cAMP was associated with an increase in the rate of HCO3- secretion. The basal rate of HCO3- secretion was lower in CF than in control cholangiocytes. In both control and CF cholangiocytes, raising intracellular Ca(2+) concentrations with ionomycin led to a parallel activation of Cl(-) current and HCO3- secretion. Consistent with reports that premature stop codon mutations (class I; e.g., G542X) can be read over by treatment with aminoglycoside antibiotics, exposure of CF cholangiocytes to gentamicin restored activation by cAMP of Cl(-) current and HCO3- secretion. The observation that activation of Ca(2+)-dependent Cl(-) channels can substitute for cystic fibrosis transmembrane conductance regulator (CFTR) in supporting HCO3- secretion and the efficacy of gentamicin in restoring CFTR function and HCO3- secretion in class I mutations are of potential clinical interest.

Pathogenic mechanisms and dynamics of hepatitis C virus (HCV) reinfection in orthotopic liver transplantation (OLT) are poorly defined. This study focuses on these aspects by studying 55 frozen biopsy specimens from transplant recipients with various histological diagnoses obtained from 4 days to 4 years post-OLT and 10 patients with HCV-related chronic hepatitis. The percentage of HCV-infected hepatocytes, number and distribution of CD8 and natural killer cells, and rates of hepatocellular apoptosis and proliferation were quantified by immunohistochemistry. HCV antigens were detected in 37% of biopsy specimens obtained within 20 days and 90% of biopsy specimens obtained from 21 days to 6 months after OLT. The number of HCV-infected hepatocytes was never less than 40% in acute hepatitis specimens and never greater than 30% in the other cases. Hepatocellular apoptosis was high in biopsy specimens of acute hepatitis and moderate in those from transplant recipients with normal histological characteristics, but still greater than in specimens of chronic active hepatitis. Proliferation correlated significantly with apoptosis. Lymphocyte infiltration was high and similar among cases of acute hepatitis, chronic hepatitis, and rejection. These data: (1) show that the detection of liver HCV antigens is sensitive enough to be used in clinical practice as a diagnostic tool to detect infection of the transplanted liver and might be useful, combined with conventional histological evaluation to detect hepatitic damage, for therapeutic decision making; (2) suggest direct cytotoxicity of HCV, as well as immunologic mechanisms possibly prevalent in chronic hepatitis and rejection, at least in the phase of acute massive liver infection; and (3) show that hepatocellular apoptosis and regeneration might be active enough to lead to replacement of the entire transplanted liver in 2 weeks.

Biliary epithelial cells (cholangiocytes) are responsible for rapid regulation of bile volume and alkalinity. Secretin and other hormones raising intracellular cyclic adenosine monophosphate (cAMP) concentrations promote biliary HCO3 secretion by stimulating apical Cl- channels and Cl-/HCO3- exchange (AE2). Cholangiocyte ion transport may also be stimulated by locally acting mediators; for example, adenosine 5'-triphosphate (ATP), a secretagogue that can be released into the bile by hepatocytes and cholangiocytes, activates Cl- conductances and Na+/H+ exchange (NHE) in cholangiocyte cell lines. To further explore the role of extracellular ATP in the paracrine regulation of carrier mechanisms regulating cholangiocyte H+/HCO3- secretion, we investigated the effects of nucleotides on intracellular pH regulation (measured by microfluorimetry with 2'7'-bis(2-carboxyethyl)-5,6,carboxyfluorescein [BCECF]) in human (MZ-ChA-1) and rat (NRC-1) cholangiocyte cell lines. In MZ-ChA-1 cells, 10 mol/L ATP, uridine 5'-triphosphate (UTP), and ATPgammas significantly increased NHE activity. The pharmacological profile of agonists was consistent with that anticipated for receptors of the P2Y2 class. ATP did not increase AE2 activity, but, when given to cells pretreated with agents raising intracellular cAMP, had a synergistic stimulatory effect that was inhibited by amiloride. To assess the polarity of purinergic receptors, monolayers of NRC-1 cells were exposed to apical or basolateral nucleotides. Apical administration of purinergic agonists, but not adenosine, increased basolateral NHE activity (ATPgammaS > UTP > ATP). Basolateral administration of purinergic agonists induced a weaker activation of NHE, which was instead strongly stimulated by adenosine and by adenosine receptor agonists (NECA = R-PIA = S-PIA). In conclusion, this study demonstrates that, consistent with the proposed role for biliary ATP in paracrine and autocrine control of cholangiocyte ion secretion, extracellular ATP stimulates cholangiocyte basolateral NHE activity through P2Y2 receptors that are predominantly expressed at the apical cell membrane.

Biliary epithelial cells (cholangiocytes) modulate bile fluidity and alkalinity absorbing and/or secreting fluid and electrolytes, particularly HCO3- and Cl-. Mechanisms responsible for transepithelial H+/HCO3- secretion in human cholangiocytes are largely unknown. Human cholangiocytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatric liver transplantation were cultured in the presence of human hepatocyte growth factor. Maintenance of cholangiocyte phenotypic features was assessed using markers such as cytokeratin 19, gamma-glutamyltranspeptidase, vimentin, factor VIII-related antigen, desmin, epithelial membrane antigen (EMA), and human epithelial antigen (HEA) 125. Intracellular pH (pHi) transients were measured microfluorimetrically 2'7'-Bis(2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCO3-, pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na(+)-dependent and amiloride-inhibitable. No Na(+)-independent recovery was recorded even after stimulation with agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCO3-, recovery from an intracellular acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4-diisothiocyan atostilben-2,2-disulfonic acid (DIDS) and by intracellular Cl- depletion. Acute removal of extracellular Cl induced a pHi alkalinization that was inhibited by DIDS. pHi recovery from an intracellular alkaline load (isohydric CO2 changes) was Cl(-)-dependent and DIDS-inhibitable. Administration of agents raising intracellular cAMP concentrations increased both Na(+)-dependent and Na(+)-independent Cl-/HCO-3 exchange activity. Stimulation of Cl-/HCO3- exchange activity was not prevented by the Cl- channel inhibitor 5'-nitro-2(2)-phenylpropyl-amino-benzoate(NPPB). In conclusion, human cholangiocytes possess two acid extruders (Na+/H+exchanger and Na(+)-dependent Cl-/HCO3- exchange) and an acid loader (Cl-/HCO3- exchange), whereas no evidence was found for cAMP activated H(+)-ATPase. Bicarbonate influx is thus mainly mediated by Na-dependent Cl-/HCO3- exchange, whereas Na+:HCO-3 cotransport is not active in the physiological range of pHi. Stimulation of Na(+)-independent Cl-/HCO3- exchanger by cAMP does not require activation of Cl- conductances. These mechanisms may underlay hormone-regulated biliary HCO3- secretion in the human biliary tree.

Intracellular pH (pHi) plays an important role in the metabolic activation of quiescent cells after a proliferative stimulus, and Na+/H+ exchange activity is required for growth in some extrahepatic tumors. To investigate intracellular acid/base homeostasis in hepatoma cells and the effects of putative liver growth factors on Na+/h+ exchange activity, we have studied intracellular pH (pHi) regulation in Hep G2 cells, a well-differentiated hepatoma cell line, both in resting conditions and after administration of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulinlike growth factor-II (IGF-II). The effects of fetal calf serum, TGF alpha, and amiloride on 3H-Thymidine incorporation were also studied. Amiloride (1 mmol/L) and external Na+ removal decreased baseline pHi in both HEPES and KRB. In HEPES, cells recovered from an acid load (20 mmol/L NH4Cl) by an amiloride inhibitable Na+/H+ exchange. In KRB, an additional, DIDS-inhibitable, Na(+)- and HCO3- dependent, but Cl(-)-independent acid extruder (Na:HCO3 cotransport) was activated. No evidence was found for a Cl/HCO3 exchange acting as acid loader. Administration of EGF and TGF alpha, but not of IGF-II, induced a dose-dependent, amiloride-inhibitable increase in baseline pHi, together with an increase in Na+/H+ exchange activity, shifting to the right the JH/pHi curve. Finally, 3H-thymidine incorporation in Hep G2 cells, in the presence of FCS or TGF alpha, was strongly inhibited by amiloride. In conclusion, in Hep G2 cells, pHi is mainly regulated by Na+/H+ exchange, which activity can be stimulated by EGF and TGF alpha, but not by IGF-II. Administration of TGF alpha stimulates DNA synthesis, an effect that is blocked by amiloride, an inhibitor of Na+/H+ exchanger. These data suggest that Na+/H+ exchange activation may play a critical role in the growth of some hepatic tumors.

Recent studies in perfused livers and isolated hepatocytes indicate that ursodeoxycholic acid-induced HCO3-rich hypercholeresis originates at the ductule/duct level. The bile duct epithelium may be involved in bile alkalinization by passively reabsorbing the protonated unconjugated ursodeoxycholic acid, by directly secreting in response to an ursodeoxycholic acid-induced increase in acid/base transporter activity or by taking up UDCA- in exchange for a base equivalent. To investigate these processes in more detail, we studied the effects of ursodeoxycholic acid on intracellular pH in SK-ChA-1, a well-differentiated human cholangiocarcinoma cell line similar to bile duct epithelium in terms of intracellular pH regulatory mechanisms and morphological markers. Intracellular pH changes were monitored with a microfluorimetric setup using the fluorescent indicator 2'-7'-bis(2-carboxyethyl)-5,6,carboxy fluorescein. Administration of 50 to 1,000 mumol/L UDCA in the absence of HCO3 caused dose-dependent intracellular acidification (intracellular pH = -0.13 +/- 0.03 pH/U after 500 mumol/L ursodeoxycholic acid). Acidification was not prevented by preincubation of cells with 0.5 mmol/L 4,4-diisothiocyanatostilbene-2,2,-disulfonic acid (DIDS) for 30 min or by furosemide administration (1 mmol/L), thus ruling out the stimulation of Cl/HCO3 exchange or the presence of an ursodeoxycholic acid/base exchange. Ursodeoxycholic acid also acidified human fibroblasts, a cell type with no transport capability for ursodeoxycholic acid. In addition, direct measurement of the activities of the three major acid/base transporters in Sk-ChA-1 cells (Na+/H+ exchange, sodium-dependent and sodium-independent Cl/HCO3 exchange) failed to show significative differences between cells treated with 500 mumol/L UDCA and controls.(ABSTRACT TRUNCATED AT 250 WORDS)