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The most studied physiological function of biliary epithelial cells (cholangiocytes) is to regulate bile flow and composition, in particular the hydration and alkalinity of the primary bile secreted by hepatocytes. After almost three decades of studies it is now become clear that cholangiocytes are also involved in epithelial innate immunity, in inflammation, and in the reparative processes in response to liver damage. An increasing number of evidence highlights the ability of cholangiocyte to undergo changes in phenotype and function in response to liver damage. By participating actively to the immune and inflammatory responses, cholangiocytes represent a first defense line against liver injury from different causes. Indeed, cholangiocytes express a number of receptors able to recognize pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), such as Toll-like receptors (TLR), which modulate their pro-inflammatory behavior. Cholangiocytes can be both the targets and the initiators of the inflammatory process. Derangements of the signals controlling these mechanisms are at the basis of the pathogenesis of different cholangiopathies, both hereditary and acquired, such as cystic fibrosis-related liver disease and sclerosing cholangitis. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Prognosis of cholangiocarcinoma, a devastating liver epithelial malignancy characterized by early invasiveness, remains very dismal, though its incidence has been steadily increasing. Evidence is mounting that in cholangiocarcinoma, tumor epithelial cells establish an intricate web of mutual interactions with multiple stromal components, largely determining the pervasive behavior of the tumor. The main cellular components of the tumor microenvironment (i.e. myofibroblasts, macrophages, lymphatic endothelial cells), which has been recently termed as ‘tumor reactive stroma’, are recruited and activated by neoplastic cells, and in turn, deleteriously mold tumor behavior by releasing a huge variety of paracrine signals, including cyto/chemokines, growth factors, morphogens and proteinases. An abnormally remodeled and stiff extracellular matrix favors and supports these cell interactions. Although the mechanisms responsible for the generation of tumor reactive stroma are largely uncertain, hypoxia presumably plays a central role. In this review, we will dissect the intimate relationship among the different cell elements cooperating within this complex ‘ecosystem’, with the ultimate goal to pave the way for a deeper understanding of the mechanisms underlying cholangiocarcinoma aggressiveness, and possibly, to foster the development of innovative, combinatorial therapies aimed at halting tumor progression. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by Jesus Banales, Marco Marzioni, Nicholas LaRusso and Peter Jansen.

Liver disease is a severe complication in patients with Cystic Fibrosis (CF), a genetic disease caused by mutations in the gene encoding for cystic fibrosis transmembrane conductance regulator (CFTR) channel. The sequence of events leading to CFLD is still unclear and has limited the development of more specific treatments other than the bile acid UDCA. However, in the last twenty years, several gaps have been filled, which have mainly been possible due to the availability of different animal models that mimic CF. CF mice, although they lack a spontaneous liver manifestation, have been essential to better understand the multiple functions of CFTR expression on the apical membrane of cholangiocytes, from chloride channel to regulator of epithelial innate immunity. Additionally, we have learned that the gut microbiota might be a pathogenetic factor for the development of liver disease. The recent creation of novel CF animal models (i.e. pig and ferret) that better reproduce the human disease, will allow for comparative studies with species that spontaneously develop the liver disease and will hopefully lead to novel therapeutic treatments. In this review, we have compared and summarized the main features of the current available CF animal models and their applicability for the study of the liver phenotype.

Cholestasis is a frequent clinical condition initiating or complicating chronic liver diseases, particularly cholangiopathies, where the biliary epithelium is the primary target of the pathogenetic sequence. Until a few decades ago, understanding of cholestasis relied mostly on the experimental model of bile duct ligation in rodents. However, a simple model of biliary obstruction cannot reproduce the complex mechanisms and networks leading to cholestasis in cholangiopathies. These networks are underpinned by an intricate dysregulation of pro-inflammatory and pro-fibrotic signals involving besides cholangiocytes, multiple cell elements of both innate and adaptive immunity. Therefore, in the last years, a wide range of animal models of biliary injury have been developed, mostly in mice, following three main approaches, chemical induction, immunization and genetic manipulation. In this review, we will give an update of the animal models of the two main cholangiopathies, primary sclerosing cholangitis and primary biliary cholangitis, which have provided us with the most relevant insights into the pathogenesis of these still controversial diseases.

Bile duct epithelial cells, also known as cholangiocytes, regulate the composition ofbileand its flow. Acquired, congenital and genetic dysfunctions in these cells give rise to asetof diverse and complex diseases, often of unknown aetiology, called cholangiopathies. New knowledge has been steadily acquired about genetic and congenital cholangiopathies, and this has led to a better understanding of the mechanisms of acquired cholangiopathies. This Review focuses on findings from studies on Alagille syndrome, polycystic liver diseases, fibropolycystic liver diseases (Caroli disease and congenital hepatic fibrosis) and cystic fibrosis-related liver disease. In particular, knowledge on the role of Notch signalling in biliary repair and tubulogenesis has been advanced by work on Alagille syndrome, and investigations in polycystic liver diseases have highlighted the role of primary cilia in biliary pathophysiology and the concept of biliary angiogenic signalling and its role in cyst growth and biliary repair. In fibropolycystic liver disease, research has shown that loss of fibrocystin generates a signalling cascade that increases β-catenin signalling, activates the NOD-, LRR- and pyrin domain-containing 3 inflammasome, and promotes production of IL-1β and other chemokines that attract macrophages and orchestrate the process of pericystic and portal fibrosis, which are the main mechanisms of progression in cholangiopathies. In cystic fibrosis-related liver disease, lack of cystic fibrosis transmembrane conductance regulator increases the sensitivity of epithelial Toll-like receptor 4 that sustains the secretion of nuclear factor-κB-dependent cytokines and peribiliary inflammation in response to gut-derived products, providing a model for primary sclerosing cholangitis. These signalling mechanisms may be targeted therapeutically and they offer a possibility for the development of novel treatments for acquired cholangiopathies.

The cholangipathies are a class of liver diseases that specifically affects the biliary tree. These pathologies may have different etiologies (genetic, autoimmune, viral, or toxic) but all of them are characterized by a stark inflammatory infiltrate, increasing overtime, accompanied by an excess of periportal fibrosis. The cellular types that mount the regenerative/reparative hepatic response to the damage belong to different lineages, including cholagiocytes, mesenchymal and inflammatory cells, which dynamically interact with each other, exchanging different signals acting in autocrine and paracrine fashion. Those messengers may be proinflammatory cytokines and profibrotic chemokines (IL-1, and 6; CXCL1, 10 and 12, or MCP-1), morphogens (Notch, Hedgehog, and WNT/β-catenin signal pathways) and finally growth factors (VEGF, PDGF, and TGFβ, among others). In this review we will focus on the main molecular mechanisms mediating the establishment of a fibroinflammatory liver response that, if perpetuated, can lead not only to organ dysfunction but also to neoplastic transformation. Primary Sclerosing Cholangitis and Congenital Hepatic Fibrosis/Caroli’s disease, two chronic cholangiopathies, known to be prodrome of cholangiocarcinoma, for which several murine models are also available, were also used to further dissect the mechanisms of fibroinflammation leading to tumor development.

The acquisition of invasive functions by tumor cells is a first and crucial step toward the development of metastasis, which nowadays represents the main cause of cancer-related death. Cholangiocarcinoma (CCA), a primary liver cancer originating from the biliary epithelium, typically develops intrahepatic or lymph node metastases at early stages, thus preventing the majority of patients from undergoing curative treatments, consistent with their very poor prognosis. As in most carcinomas, CCA cells gradually adopt a motile, mesenchymal-like phenotype, enabling them to cross the basement membrane, detach from the primary tumor, and invade the surrounding stroma. Unfortunately, little is known about the molecular mechanisms that synergistically orchestrate this proinvasive phenotypic switch. Autocrine and paracrine signals (cyto/chemokines, growth factors, and morphogens) permeating the tumor microenvironment undoubtedly play a prominent role in this context. Moreover, a number of recently identified signaling systems are currently drawing attention as putative mechanistic determinants of CCA cell invasion. They encompass transcription factors, protein kinases and phosphatases, ubiquitin ligases, adaptor proteins, and miRNAs, whose aberrant expression may result from either stochastic mutations or the abnormal activation of upstream pro-oncogenic pathways. Herein we sought to summarize the most relevant molecules in this field and to discuss their mechanism of action and potential prognostic relevance in CCA. Hopefully, a deeper knowledge of the molecular determinants of CCA invasiveness will help to identify clinically useful biomarkers and novel druggable targets, with the ultimate goal to develop innovative approaches to the management of this devastating malignancy.

Despite recent advances, pathogenesis of cholangiocarcinoma, a highly lethal cancer, remains enigmatic. Furthermore, treatment options are still limited and often disappointing. For this reason, in the last few years there has been a mounting interest towards the generation of experimental models able to reproduce the main features associated with this aggressive behavior. Toxic and infestation-induced, genetically engineered and cell implantation rodent models have been generated, contributing to a deeper understanding of the complex cell biology of the tumor, sustained by multiple cell interactions and driven by a huge variety of molecular perturbations. Herein, we will overview the most relevant animal models of biliary carcinogenesis, highlighting the methodological strategy, the molecular, histological and clinical phenotypes consistent with the human condition, their particular strengths and weaknesses and the novel therapeutic approaches that have been developed.

Background & Aims In cholangiocarcinoma, early metastatic spread via lymphatic vessels often precludes curative therapies. Cholangiocarcinoma invasiveness is fostered by an extensive stromal reaction, enriched in cancer-associated fibroblasts (CAFs) and lymphatic endothelial cells (LECs). Cholangiocarcinoma cells recruit and activate CAFs by secreting PDGF-D. Herein, we investigated the role of PDGF-D and liver myofibroblasts in promoting lymphangiogenesis in cholangiocarcinoma. Methods Human cholangiocarcinoma specimens were immunostained for podoplanin (LEC marker), α-SMA (CAF marker), VEGF-A, VEGF-C, and their cognate receptors (VEGFR2, VEGFR3). VEGF-A and VEGF-C secretion was evaluated in human fibroblasts obtained from primary sclerosing cholangitis explants. Using human LECs incubated with conditioned medium from PDGF-D-stimulated fibroblasts we assessed migration, 3D vascular assembly, transendothelial electric resistance and transendothelial migration of cholangiocarcinoma cells (EGI-1). We then studied the effects of selective CAF depletion induced by the BH3 mimetic navitoclax on LEC density and lymph node metastases in vivo. Results In cholangiocarcinoma specimens, CAFs and LECs were closely adjacent. CAFs expressed VEGF-A and VEGF-C, while LECs expressed VEGFR2 and VEGFR3. Upon PDGF-D stimulation, fibroblasts secreted increased levels of VEGF-C and VEGF-A. Fibroblasts, stimulated by PDGF-D induced LEC recruitment and 3D assembly, increased LEC monolayer permeability, and promoted transendothelial EGI-1 migration. These effects were all suppressed by the PDGFRβ inhibitor, imatinib. In the rat model of cholangiocarcinoma, navitoclax-induced CAF depletion, markedly reduced lymphatic vascularization and reduced lymph node metastases. Conclusion PDGF-D stimulates VEGF-C and VEGF-A production by fibroblasts, resulting in expansion of the lymphatic vasculature and tumor cell intravasation. This critical process in the early metastasis of cholangiocarcinoma may be blocked by inducing CAF apoptosis or by inhibiting the PDGF-D-induced axis. Lay summary Cholangiocarcinoma is a highly malignant cancer affecting the biliary tree, which is characterized by a rich stromal reaction involving a dense population of cancer-associated fibroblasts that promote early metastatic spread. Herein, we show that cholangiocarcinoma-derived PDGF-D stimulates fibroblasts to secrete vascular growth factors. Thus, targeting fibroblasts or PDGF-D-induced signals may represent an effective tool to block tumor-associated lymphangiogenesis and reduce the invasiveness of cholangiocarcinoma.

Glioblastoma multiforme (GBM) is a grade IV astrocytoma and the most common form of malignant brain tumor in adults. GBM remains one of the most fatal and least successfully treated solid tumors: current therapies provide a median survival of 12-15 months after diagnosis, due to the high recurrence rate. Glioma Stem Cells (GSCs) are believed to be the real driving force of tumor initiation, progression and relapse. Therefore, better therapeutic strategies GSCs-targeted are needed. Resveratrol is a polyphenolic phytoalexin found in fruits and vegetables displaying pleiotropic health benefits. Many studies have highlighted its chemo-preventive and chemotherapeutic activities in a wide range of solid tumors. In this work, we analyzed the effects of Resveratrol exposure on cell viability, proliferation and motility in seven GSC lines isolated from GBM patients. For the first time in our knowledge, we investigated Resveratrol impact on Wnt signaling pathway in GSCs, evaluating the expression of seven Wnt signaling pathway-related genes and the protein levels of c-Myc and β-catenin. Finally, we analyzed Twist1 and Snail1 protein levels, two pivotal activators of epithelial-mesenchymal transition (EMT) program. Results showed that although response to Resveratrol exposure was highly heterogeneous among GSC lines, generally it was able to inhibit cell proliferation, increase cell mortality, and strongly decrease cell motility, modulating the Wnt signaling pathway and the EMT activators. Treatment with Resveratrol may represent a new interesting therapeutic approach, in order to affect GSCs proliferation and motility, even if further investigations are needed to deeply understand the GSCs heterogeneous response.

Chronic diseases of the biliary tree (cholangiopathies) represent one of the major unmet needs in clinical hepatology and a significant knowledge gap in liver pathophysiology. The common theme in cholangiopathies is that the target of the disease is the biliary tree. After damage to the biliary epithelium, inflammatory changes stimulate a reparative response with proliferation of cholangiocytes and restoration of the biliary architecture, owing to the reactivation of a variety of morphogenetic signals. Chronic damage and inflammation will ultimately result in pathological repair with generation of biliary fibrosis and clinical progression of the disease. The hallmark of pathological biliary repair is the appearance of reactive ductular cells, a population of cholangiocyte-like epithelial cells of unclear and likely mixed origin that are able to orchestrate a complex process that involves a number of different cell types, under joint control of inflammatory and morphogenetic signals. Several questions remain open concerning the histogenesis of reactive ductular cells, their role in liver repair, their mechanism of activation, and the signals exchanged with the other cellular elements cooperating in the reparative process. This review contributes to the current debate by highlighting a number of new concepts derived from the study of the pathophysiology of chronic cholangiopathies, such as congenital hepatic fibrosis, biliary atresia, and Alagille syndrome.

Resistance to conventional chemotherapeutic agents, a typical feature of cholangiocarcinoma, prevents the efficacy of the therapeutic arsenal usually used to combat malignancy in humans. Mechanisms of chemoresistance by neoplastic cholangiocytes include evasion of drug-induced apoptosis mediated by autocrine and paracrine cues released in the tumor microenvironment. Here, recent evidence regarding molecular mechanisms of chemoresistance is reviewed, as well as associations between well-developed chemoresistance and activation of the cancer stem cell compartment. It is concluded that improved understanding of the complex interplay between apoptosis signaling and the promotion of cell survival represent potentially productive areas for active investigation, with the ultimate aim of encouraging future studies to unveil new, effective strategies able to overcome current limitations on treatment.

Nuclear expression of the calcium-binding protein S100A4 is a biomarker of increased invasiveness in cholangiocarcinoma, a primary liver cancer with scarce treatment opportunities and dismal prognosis. In this study, we provide evidence that targeting S100A4 nuclear import by low-dose paclitaxel, a microtubule-stabilizing agent, inhibits cholangiocarcinoma invasiveness and metastatic spread. Administration of low-dose paclitaxel to established (EGI-1) and primary (CCA-TV3) cholangiocarcinoma cell lines expressing nuclear S100A4 triggered a marked reduction in nuclear expression of S100A4 without modifying its cytoplasmic levels, an effect associated with a significant decrease in cell migration and invasiveness. While low-dose paclitaxel did not affect cellular proliferation, apoptosis, or cytoskeletal integrity, it significantly reduced SUMOylation of S100A4, a critical posttranslational modification that directs its trafficking to the nucleus. This effect of low-dose paclitaxel was reproduced by ginkolic acid, a specific SUMOylation inhibitor. Downregulation of nuclear S100A4 by low-dose paclitaxel was associated with a strong reduction in RhoA and Cdc42 GTPase activity, MT1-MMP expression, and MMP-9 secretion. In an SCID mouse xenograft model, low-dose metronomic paclitaxel treatment decreased lung dissemination of EGI-1 cells without significantly affecting their local tumor growth. In the tumor mass, nuclear S100A4 expression by cholangiocarcinoma cells was significantly reduced, whereas rates of proliferation and apoptosis were unchanged. Overall, our findings highlight nuclear S100A4 as a candidate therapeutic target in cholangiocarcinoma and establish a mechanistic rationale for the use of low-dose paclitaxel in blocking metastatic progression of cholangiocarcinoma. Cancer Res; 76(16); 4775–84. ©2016 AACR.

Cholangiocarcinoma is an aggressive, strongly chemoresistant liver malignancy. Leukemia inhibitory factor (LIF), an IL-6 family cytokine, promotes progression of various carcinomas. To investigate the role of LIF in cholangiocarcinoma, we evaluated the expression of LIF and its receptor (LIFR) in human samples. LIF secretion and LIFR expression were assessed in established and primary human cholangiocarcinoma cell lines. In cholangiocarcinoma cells, we tested LIF effects on proliferation, invasion, stem cell-like phenotype, chemotherapy-induced apoptosis (gemcitabine+cisplatin), expression levels of pro-apoptotic (Bax) and anti-apoptotic (Mcl-1) proteins, with/without PI3K inhibition, and of pSTAT3, pERK1/2, pAKT. LIF effect on chemotherapy-induced apoptosis was evaluated after LIFR silencing and Mcl-1 inactivation. Results show that LIF and LIFR expression were higher in neoplastic than in control cholangiocytes; LIF was also expressed by tumor stromal cells. LIF had no effects on cholangiocarcinoma cell proliferation, invasion, and stemness signatures, whilst it counteracted drug-induced apoptosis. Upon LIF stimulation, decreased apoptosis was associated with Mcl-1 and pAKT up-regulation and abolished by PI3K inhibition. LIFR silencing and Mcl-1 blockade restored drug-induced apoptosis. In conclusion, autocrine and paracrine LIF signaling promote chemoresistance in cholangiocarcinoma by up-regulating Mcl-1 via a novel STAT3- and MAPK-independent, PI3K/AKT-dependent pathway. Targeting LIF signaling may increase CCA responsiveness to chemotherapy.

Background/Aims In routine examination of liver biopsies isolated ductular hyperplasia (IDH) may be the only histopathological change. Here we describe the clinical and immunophenotypic features of a number of cases retrospectively identified reviewing consecutive liver biopsies from five Italian centers over 4 years. Methods We reviewed 1235 cases biopsied for chronic liver disease (1078 for viral hepatitis). Records of cases fulfilling the inclusion criteria for IDH were reviewed to identify possible aetiologies. Biopsies showing IDH and control biopsies were studied by immunohistochemistry for cytokeratin-7, epithelial-membrane-antigen (EMA), neural-cell-adhesion-molecule (NCAM), Ki-67. Results Out of 70 biopsies fulfilling IDH criteria, 16 (22.8%) were of unknown aetiology. Patients with idiopathic IDH (age 38.2±11 years) were asymptomatic with mild, long-lasting ALT and/or γGT increases. A significant increase of well-differentiated (EMA-positive; NCAM-negative) bile ductules localized at the portal interface and inside the lobule was found in idiopathic IDH. Conclusions Idiopathic IDH was present in 10% of adults biopsied for persistent mild liver function test abnormalities unrelated to viral hepatitis. In contrast with the ductular reaction seen in many forms of liver disease, it is characterized by well-differentiated hyperplastic ductules in absence of significant inflammation, and may represent a non-specific pattern of reaction to mild liver damages.

Cholangiocarcinoma (CCA) is a devastating malignancy arising from the bile ducts. Cancer-associated fibroblasts (CAFs) are key players in CCA invasiveness and in the generation of a desmoplastic reaction. The aim of the present study was to develop a novel model by which to study tumor-stroma interactions using primary cultures of human biliary epithelial cells (hBECs) and stromal cells (SCs) in CCA. hBECs and SCs, isolated from surgical resections (n=10), were semi-purified by centrifugation on a Percoll gradient; hBECs were further immunopurified. hBECs and SCs were characterized using epithelial [cytokeratin 7 (CK7) and CK19] and mesenchymal [vimentin (VMN), α-smooth muscle actin (α-SMA), CD68] cell markers. The purity of cultured cells was assessed by fluorescent immunocytochemistry. hBECs were HEA125/CK7/CK19-positive and VMN/α-SMA-negative. SCs were VMN/α-SMA-positive and CK7/CK19-negative. CCA 2-D culture models have been described but they use long-standing CCA cell lines of various biliary tumor cell origins with stromal cells derived from non-cholangiocarcinoma tissues. Recently, a novel 3-D organotypic co-culture model of rat cholangiocarcinoma was described. In the present study, we obtained pure and stable primary cultures of hBECs and SCs from CCA surgical specimens. These cell cultures may provide a useful tool by which to study CCA tumor-stroma interactions.

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. (Hepatology 2013;53:1042–1053)

Background and Aims: Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC). Methods: After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ-/- mice. Results: The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features. Conclusions: Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution. © 2011 Colombo et al.

OBJECTIVE: To study the mechanism(s) linking insulin resistance (IR) to hepatic fibrosis and the role of the epithelial component in tissue repair and fibrosis in chronic hepatitis C (CHC). DESIGN: Prospective observational study. SETTING: Tertiary care academic centre. PATIENTS: 78 consecutive patients with CHC. MAIN OUTCOME MEASURES: IR, calculated by the oral glucose insulin sensitivity during oral glucose tolerance test; necroinflammatory activity and fibrosis, defined according to Ishak's score; steatosis, graded as 0 (66%). To evaluate the role of the epithelial component in tissue repair and fibrosis, the expansion of the ductular reaction (DR) was calculated by keratin-7 (CK7) morphometry. Nuclear expression of Snail, downregulation of E-cadherin and expression of fibroblast specific protein-1 (FSP1) and vimentin by CK7-positive cells were used as markers of epithelial-mesenchymal transition in DR elements. RESULTS: IR, the degree of necroinflammation and expansion of the DR (stratified as reactive ductular cells (RDCs), hepatic progenitor cells and intermediate hepatobiliary cells according to morphological criteria) were all associated with the stage of fibrosis. Nuclear Snail expression, E-cadherin downregulation and vimentin upregulation were observed in RDCs. By dual immunofluorescence for CK7 and FSP1, the number of RDCs undergoing epithelial-mesenchymal transition progressively increased together with the necroinflammatory score. By multivariate analysis, total inflammation and insulin resistance were the only factors significantly predicting the presence of advanced fibrosis (Ishak score ≥3) and the expansion of RDCs. CONCLUSION: This study indicates that IR is associated with the degree of necroinflammatory injury in CHC and contributes to hepatic fibrosis by stimulating the expansion of RDCs that express epithelial-mesenchymal transition markers.

Cholangiocarcinoma (CCA) is the second most common primary malignancy of the liver; the incidence of intrahepatic CCA in Western countries has been steadily growing in the last two decades.1 In spite of the rising incidence, treatment options for CCA remain unsatisfactory,1,2 particularly because of the strong and early invasiveness of the tumor. In many patients, lymphnodal or distant metastasis or micrometastasis are present already at the time of the diagnosis, limiting and worsening the prognosis in patients otherwise eligible for surgical resection. However, a subset of patients with less aggressive CCA may even undergo liver transplantation after neoadjuvant radiochemotherapy and have excellent survival. Biomarkers able to predict tumor invasiveness and prognosis would be an important decision-making tool. Unfortunately, mechanisms that determine CCA invasiveness are largely unknown. Cancer invasiveness and metastasization requires tightly adherent epithelial cells to convert to a more motile phenotype expressing several mesenchymal features. 3 During this process, some molecular programs typical of the mesenchymal phenotype are activated, as shown by the expression of specific cell surface proteins, cytoskeletal proteins, extracellular matrix-degrading enzymes, and transcription factors.4 One such proteins is S100A4, a member of the S100 family of small calcium-binding proteins, expressed by mesenchymal cells, macrophages,5 and by epithelial cells in mesenchymal transition (EMT). Expression of S100A4 was shown to be a predictor of metastasization in colon cancer.6,7 The mechanisms of action of A100A4 depends on the cellular localization of the protein. In the cytoplasm S100A4 interacts with a number of partner proteins in cytoskeleton and in the plasma membrane (such as myosin IIa or liprin-β1). When localized in the nucleus, S100A4 may exert transcriptional functions that affect several genes, including matrix metalloproteinase (MMP)-98 and E-cadherin.9 However, the mechanism of action of S100A4 remains largely unknown, as it remains unclear whether S100A4 is just a biomarker of cancer cell aggressiveness or actually represents a functional target amenable of therapeutic intervention. While examining the expression of EMT markers in CCA specimens, we noticed that a subgroup of CCAs expressed S100A4 in the nucleus. In this study we addressed: (1) the prognostic significance of S100A4 nuclear expression in a large series of patients undergoing surgical resection, and (2) the functional relevance of S100A4 expression on the metastatic potential, motility, and invasiveness of CCA cell lines in vivo and in vitro. Our results show that nuclear expression of S100A4 by neoplastic bile ducts significantly correlated with increased metastasization and reduced survival after surgery, that human CCA cells with nuclear expression of S100A4 have a much stronger metastatic ability when xenotransplanted into severe combined immunodeficiency (SCID) mice, and that silencing S100A4 in CCA cells that originally overexpressed S100A4 significantly reduced motility and invasive capabilities in vitro.

Hyperbilirubinemia is a common laboratory finding in clinical practice, being found in several haematological and liver diseases as well as in familial conditions (5-10% in Western countries). Although most of the familial forms of hyperbilirubinemia are classically viewed as benign conditions, they have gained an increased interest in the last few years since recent data have indicated that subjects with an impaired bilirubin metabolism may have an increased susceptibility to drug toxicity. The authors briefly review the main steps of bilirubin metabolism, with a special emphasis on the emerging concepts on the molecular mechanisms of regulation by nuclear receptors (NRs) and genetic factors. Then the different forms of isolated hyperbilirubinemia occurring in both adults and paediatrics are systematically analysed, and a new categorisation is also proposed in light of the recent advances in bilirubin research. Finally, a diagnostic algorithm is discussed, along with a correct approach to its management, in order to avoid unnecessary medical investigations. © 2009.

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.

Patients with Alagille syndrome (AGS), a genetic disorder of Notch signaling, suffer from severe ductopenia and cholestasis, but progression to biliary cirrhosis is rare. Instead, in biliary atresia (BA) severe cholestasis is associated with a pronounced "ductular reaction" and rapid progression to biliary cirrhosis. Given the role of Notch in biliary development, we hypothesized that defective Notch signaling would influence the reparative mechanisms in cholestatic cholangiopathies. Thus we compared phenotype and relative abundance of the epithelial components of the hepatic reparative complex in AGS (n = 10) and BA (n = 30) using immunohistochemistry and computer-assisted morphometry. BA was characterized by an increase in reactive ductular and hepatic progenitor cells, whereas in AGS, a striking increase in intermediate hepatobiliary cells contrasted with the near absence of reactive ductular cells and hepatic progenitor cells. Hepatocellular mitoinhibition index (p21(waf1)/Ki67) was similar in AGS and BA. Fibrosis was more severe in BA, where portal septa thickness positively correlated with reactive ductular cells and hepatic progenitor cells. AGS hepatobiliary cells failed to express hepatic nuclear factor (HNF) 1beta, a biliary-specific transcription factor. These data indicate that Notch signaling plays a role in liver repair mechanisms in postnatal life: its defect results in absent reactive ductular cells and accumulation of hepatobiliary cells lacking HNF1beta, thus being unable to switch to a biliary phenotype.

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).