Small Cholangiocytes Research Articles

3D organoid cultivation improves the maturation and functional differentiation of cholangiocytes from human pluripotent stem cells.

Idiopathic cholangiopathies are diseases that affect cholangiocytes, and they have unknown etiologies. Currently, orthotopic liver transplantation is the only treatment available for end-stage liver disease. Limited access to the bile duct makes it difficult to model cholangiocyte diseases. In this study, by mimicking the embryonic development of cholangiocytes and using a robust, feeder- and serum-free protocol, we first demonstrate the generation of unique functional 3D organoids consisting of small and large cholangiocytes derived from human pluripotent stem cells (PSCs), as opposed to traditional 2D culture systems. At day 28 of differentiation, the human PSC-derived cholangiocytes expressed markers of mature cholangiocytes, such as CK7, CK19, and cystic fibrosis transmembrane conductance regulator (CFTR). Compared with the 2D culture system-generated cholangiocytes, the 3D cholangiocyte organoids (COs) showed higher expression of the region-specific markers of intrahepatic cholangiocytes YAP1 and JAG1 and extrahepatic cholangiocytes AQP1 and MUC1. Furthermore, the COs had small-large tube-like structures and functional assays revealed that they exhibited characteristics of mature cholangiocytes, such as multidrug resistance protein 1 transporter function and CFTR channel activity. In addition to the extracellular matrix supports, the epidermal growth factor receptor (EGFR)-mediated signaling regulation might be involved in this cholangiocyte maturation and differentiation. These results indicated the successful generation of intrahepatic and extrahepatic cholangiocytes by using our 3D organoid protocol. The results highlight the advantages of our 3D culture system over the 2D culture system in promoting the functional differentiation and maturation of cholangiocytes. In summary, in advance of the previous works, our study provides a possible concept of small-large cholangiocyte transdifferentiation of human PSCs under cost-effective 3D culture conditions. The study findings have implications for the development of effective cell-based therapy using COs for patients with cholangiopathies.

Deconvolution analysis identified altered hepatic cell landscape in primary sclerosing cholangitis and primary biliary cholangitis.

Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are characterized by ductular reaction, hepatic inflammation, and liver fibrosis. Hepatic cells are heterogeneous, and functional roles of different hepatic cell phenotypes are still not defined in the pathophysiology of cholangiopathies. Cell deconvolution analysis estimates cell fractions of different cell phenotypes in bulk transcriptome data, and CIBERSORTx is a powerful deconvolution method to estimate cell composition in microarray data. CIBERSORTx performs estimation based on the reference file, which is referred to as signature matrix, and allows users to create custom signature matrix to identify specific phenotypes. In the current study, we created two custom signature matrices using two single cell RNA sequencing data of hepatic cells and performed deconvolution for bulk microarray data of liver tissues including PSC and PBC patients. Custom signature matrix files were created using single-cell RNA sequencing data downloaded from GSE185477 and GSE115469. Custom signature matrices were validated for their deconvolution performance using validation data sets. Cell composition of each hepatic cell phenotype in the liver, which was identified in custom signature matrices, was calculated by CIBERSORTx and bulk RNA sequencing data of GSE159676. Deconvolution results were validated by analyzing marker expression for the cell phenotype in GSE159676 data. CIBERSORTx and custom signature matrices showed comprehensive performance in estimation of population of various hepatic cell phenotypes. We identified increased population of large cholangiocytes in PSC and PBC livers, which is in agreement with previous studies referred to as ductular reaction, supporting the effectiveness and reliability of deconvolution analysis in this study. Interestingly, we identified decreased population of small cholangiocytes, periportal hepatocytes, and interzonal hepatocytes in PSC and PBC liver tissues compared to healthy livers. Although further studies are required to elucidate the roles of these hepatic cell phenotypes in cholestatic liver injury, our approach provides important implications that cell functions may differ depending on phenotypes, even in the same cell type during liver injury. Deconvolution analysis using CIBERSORTx could provide a novel approach for studies of specific hepatic cell phenotypes in liver diseases.

Unique DUOX2+ACE2+ small cholangiocytes are pathogenic targets for primary biliary cholangitis

Cholangiocytes play a crucial role in bile formation. Cholangiocyte injury causes cholestasis, including primary biliary cholangitis (PBC). However, the etiology of PBC remains unclear despite being characterized as an autoimmune disease. Using single-cell RNA sequencing (scRNA-seq), fluorescence-activated-cell-sorting, multiplex immunofluorescence (IF) and RNAscope analyses, we identified unique DUOX2+ACE2+ small cholangiocytes in human and mouse livers. Their selective decrease in PBC patients was associated with the severity of disease. Moreover, proteomics, scRNA-seq, and qPCR analyses indicated that polymeric immunoglobulin receptor (pIgR) was highly expressed in DUOX2+ACE2+ cholangiocytes. Serum anti-pIgR autoantibody levels were significantly increased in PBC patients, regardless of positive and negative AMA-M2. Spatial transcriptomics and multiplex IF revealed that CD27+ memory B and plasma cells accumulated in the hepatic portal tracts of PBC patients. Collectively, DUOX2+ACE2+ small cholangiocytes are pathogenic targets in PBC, and preservation of DUOX2+ACE2+ cholangiocytes and targeting anti-pIgR autoantibodies may be valuable strategies for therapeutic interventions in PBC.

Mast cells selectively target large cholangiocytes during biliary injury via H2HR-mediated cAMP/pERK1/2 signaling.

Bile ducts are heterogenous in structure and function, and primary sclerosing cholangitis (PSC) damages specific bile ducts leading to ductular reaction (DR), mast cell (MC) infiltration, increased histamine release, inflammation, and fibrosis. Bile duct ligation (BDL) induces large duct damage via cyclic adenosine monophosphate (cAMP)/extracellular signal‐related protein kinase (ERK) signaling, and large cholangiocytes express H2 histamine receptor (H2HR). We evaluated how MCs interact with large cholangiocytes during cholestasis. Male wild‐type (WT) and MC‐deficient (Kit W‐sh ) mice 10–12 weeks of age were subjected to BDL for 7 days. Select Kit W‐sh mice were injected with MCs pretreated with control or H2HR antagonist (ranitidine, 25 μm, 48 h) via tail vein injection. In vitro, MC migration toward small mouse cholangiocytes (SMCCs) and large mouse cholangiocytes (LMCCs) treated with lipopolysaccharide or histamine (±ranitidine) was measured. LMCCs were stimulated with MC supernatants pretreated with control, α‐methyl‐dl‐histidine (to block histamine release), or ranitidine. Liver damage, large duct DR/senescence, inflammation, fibrosis, and cAMP/ERK immunoreactivity increased in BDL WT and Kit W‐sh +MC mice but decreased in BDL Kit W‐sh and Kit W‐sh +MC‐H2HR mice. In vitro, MCs migrate toward damaged LMCCs (but not SMCCs) blocked by inhibition of H2HR. Loss of MC histamine or MC‐H2HR decreases LMCC proliferation, senescence, H2HR, and cAMP/ERK levels. Human PSC livers have increased MC number found near DR, senescent ducts, and H2HR‐positive ducts. Conclusion: Infiltrating MCs preferentially interact with large ducts via H2HR signaling promoting biliary and liver damage. Mediation of MCs may be a therapeutic strategy for PSC.

Identification of the chloride channel, leucine-rich repeat-containing protein 8, subfamily a (LRRC8A), in mouse cholangiocytes.

Chloride (Cl- ) channels in the apical membrane of biliary epithelial cells (BECs), also known as cholangiocytes, provide the driving force for biliary secretion. Although two Cl- channels have been identified on a molecular basis, the Cystic Fibrosis Transmembrane Conductance Regulator and Transmembrane Member 16A, a third Cl- channel with unique biophysical properties has been described. Leucine-Rich Repeat-Containing Protein 8, subfamily A (LRRC8A) is a newly identified protein capable of transporting Cl- in other epithelium in response to cell swelling. The aim of the present study was to determine if LRRC8A represents the volume-regulated anion channel in mouse BECs. Studies were performed in mouse small (MSC) and large (MLC) cholangiocytes. Membrane Cl- currents were measured by whole-cell patch-clamp techniques and cell volume measurements were performed by calcein-AM fluorescence. Exposure of either MSC or MLC to hypotonicity (190 mOsm) rapidly increased cell volume and activated Cl- currents. Currents exhibited outward rectification, time-dependent inactivation at positive membrane potentials, and reversal potential at 0 mV (ECl ). Removal of extracellular Cl- or specific pharmacological inhibition of LRRC8A abolished currents. LRRC8A was detected in both MSC and MLC by reverse transcription polymerase chain reaction and confirmed by western blot. Transfection with LRRC8A small interfering RNA decreased protein levels by >70% and abolished volume-stimulated Cl- currents. These results demonstrate that LRRC8A is functionally present in mouse BECs, contributes to volume-activated Cl- secretion, and, therefore, may be a target to modulate bile formation in the treatment of cholestatic liver disorders.

Localization of Orphan GPCRs within the Liver

G protein‐coupled receptors (GPCRs) are the largest gene family in the genome and are involved in almost every aspect of physiology ranging from hormone receptors to eyesight. They also happen to represent the largest class of “druggable” molecules; in fact, 20‐30% of all FDA‐approved drugs on the market target GPCRs. Despite this, the vast majority of GPCRs remain underappreciated with incomplete expression patterns and characterization. These receptors, termed GPRs, have been linked to metabolic pathways and are known to respond to naturally produced metabolites. We reasoned that the liver, the largest metabolic organ in the body, is primed to take advantage of some of these GPRs in order to regulate its internal environment. To identify these receptors, we performed a large‐scale screen for over 300 GPCRs on whole murine liver using TaqMan qPCR array cards. This screen identified a total of 57 GPRs – with 7 of the them deemed to be “high expressing” with ΔCt values comparable to the more well‐characterized hepatic GPCRs (i.e. glucagon receptor). We confirmed expression of each of these GPRs (GPRc5c, GPR125, GPR146, GPR137, GPR116, GPR108, GPR17) via gene‐specific PCR from multiple murine liver samples.In order to characterize these understudied receptors within the liver, we must first understand where they are localized and what activates them. Given the dearth of reliable antibodies, we began by examining expression using RNAscope, a powerful in situ technique that specifically localizes mRNA sequences. GPR108, which has previously been linked to regulation of Toll‐Like Receptor signaling, was found within hepatocytes. GPR146, a known C‐peptide receptor which has been shown to impact blood cholesterol levels, was highly expressed in hepatocytes, and weakly localized to cholangioctyes and vessels. GPR109a, a highly studied receptor known to respond to short chain fatty acids and a potential therapeutic target for liver disease, was found to be primarily expressed in cholangiocytes, which notably play a key role in modification of bile. Finally, GPR125, an atypical adhesion GPCR, exhibited weak expression in hepatocytes but rather strong staining in select cholangiocytes and the liver vasculature. This was confirmed using both immunohistochemistry and immunofluorescence microscopy where we noted basolateral staining along the small cholangiocytes and hepatocytes. Given its localization and function as an adhesion receptor, GPR125 is well‐positioned within the liver to respond to, and to regulate, extracellular matrix proteins and their composition in both health and disease. Efforts are currently underway to deorphanize these receptors using the PRESTO‐Tango assay and to better understand their roles in the pathogenesis of metabolic diseases in the liver.

Secretin Treatment Promotes Hepatic Progenitor Cell Activation, Ductal‐Canalicular Junction Formation and Amelioration of Liver Damage in a Model of Late‐Stage Primary Biliary Cholangitis

Introduction PBC is a cholestatic disease characterized by bile duct loss, ductular reaction and liver fibrosis. The canals of Hering (CoH) are the site of ductulo-canalicular junction (DCJ) that maintain proper bile drainage from the liver, and contain the hepatic progenitor cell (HPC) population. CoH loss is noted in PBC and correlates with scoring. Secretin binds to its receptor (SR, expressed by large cholangiocytes) to promote biliary proliferation during cholestasis; however, during large duct damage, small cholangiocytes acquire large cholangiocyte phenotypes including SR expression. Secretin/SR expression is downregulated in late-stage PBC; therefore, we aimed to understand the role of the secretin/SR axis during PBC. We hypothesized that secretin treatment restores DCJ through HPC activation and ameliorates liver phenotypes associated with late-stage PBC. Methods Female 34 wk old wild-type (WT) and dominant-negative transforming growth factor-β receptor II (dnTGFβRII, model of late-stage PBC) mice were treated with control or secretin (2.5 nmol/kg BW) for 8 wk by IP implanted minipumps. Cholangitis activity, hepatitis activity and bile duct loss were determined by the Nakanuma scoring system. Ductular reaction was measured by semi-quantitative CK-19 immunostaining. Liver fibrosis was evaluated by Sirius Red staining, collagen-1a immunostaining and qPCR for α-SMA. Hepatic stellate cell (HSC) presence was determined by desmin immunostaining. Cholestasis was indicated by hepatic total bile acid (TBA) levels using EIA. DCJ presence was evaluated by co-immunostaining for ABC transporter bile salt export pump (BSEP)/CK-19 and semi-quantification. HPC activation was assessed by Sox9 staining and semi-quantification. Results dnTGFβRII mice had an increase in cholangitis and hepatitis activity; however, 8 wks of secretin treatment significantly reduces cholangitis, but not hepatitis, activity. dnTGFβRII mice present with ductular reaction and significant bile duct loss, which was reversed with 8 wks secretin treatment. Hepatic TBA levels, liver fibrosis and HSC presence are increased in dnTGFβRII mice, but significantly reduced following 8 wks of secretin treatment. In line with human studies, dnTGFβRII mice have significant loss of DCJ and reduced HPC number; however, 8 wks of secretin treatment restores these parameters. Conclusion Long-term secretin treatment (i) reduces ductular reaction, (ii) reverses bile duct loss, (iii) decreases liver fibrosis, (iv) promotes HPC activity and (v) restores DCJ presence in a model of late-stage PBC. Secretin maintenance of the DCJ may be due to (i) the restoration of bile flow and release of toxic bile acids or (ii) secretin binding to SR acquired on small cholangiocytes/HPCs. Treatment with secretin may be a therapeutic option for late-stage PBC patients.

GASTROINTESTINAL COMPLICATIONS OF CORONAVIRUS DISEASE (COVID-19).

ABSTRACT Background: It is currently understood that severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) directly enters target cells by binding to the angiotensin-converting enzyme 2 (ACE2) receptor. Accordingly, tissues with high expression levels of ACE2 are more susceptible to infection, including pulmonary alveolar epithelial cells, small intestine enterocytes, cholangiocytes, and vascular endothelial cells. Considering the atypical manifestations of COVID-19 and the challenges of early diagnosis, this review addresses the possible gastrointestinal complications of the disease. Method: The phrase “Gastrointestinal complication of COVID” was searched in the PubMed, Medline, and SciELO databases. Due to the heterogeneity of the studies included in the present review, a narrative synthesis of the available qualitative data was performed. Result: The literature search retrieved 28 articles, primarily case reports and case series, for the qualitative analysis of gastrointestinal complications of COVID-19, in addition to two retrospective cohort and one case-control. The studies focused on hemorrhagic, thrombotic, ischemic, and perforation complications, in addition to acute pancreatitis and pneumatosis intestinalis. Conclusion: There is a straight relationship between high expression levels of ACE2 in the gastrointestinal tract and its greater susceptibility to direct infection by SARS-CoV-2. So, it is important to consider the gastrointestinal infection manifestations for early diagnosis and treatment trying to avoid more serious complications and death.

Different iron-handling in inflamed small and large cholangiocytes and in small and large-duct type intrahepatic cholangiocarcinoma.

Cholangiocarcinoma (CCA) represents the second most common primary hepatic malignancy and originates from the neoplastic transformation of the biliary cells. The intrahepatic subtype includes two morpho-molecular forms: large-duct type intrahepatic CCA (iCCA) and small-duct type iCCA. Iron is fundamental for the cellular processes, contributing in tumor development and progression. The aim of this study was to evaluate iron uptake, storage, and efflux proteins in both lipopolysaccharide-inflamed small and large cholangiocytes as well as in different iCCA subtypes. Our results show that, despite an increase in interleukin-6 production by both small and large cholangiocytes, ferroportin (Fpn) was decreased only in small cholangiocytes, whereas transferrin receptor-1 (TfR1) and ferritin (Ftn) did not show any change. Differently from in vitro models, Fpn expression was increased in malignant cholangiocytes of small-duct type iCCA in comparison to large-duct type iCCA and peritumoral tissues. TfR1, Ftn and hepcidin were enhanced, even if at different extent, in both malignant cholangiocytes in comparison to the surrounding samples. Lactoferrin was higher in large-duct type iCCA in respect to small-duct type iCCA and peritumoral tissues. These findings show a different iron handling by inflamed small and large cholangiocytes, and small and large-duct type iCCA. The difference in iron homeostasis by the iCCA subtypes may have implications for the tumor management.

Mechanosensor transient receptor potential vanilloid member 4 (TRPV4) regulates mouse cholangiocyte secretion and bile formation.

Mechanosensitive signaling has emerged as a mechanism for the regulation of cholangiocyte transport and bile formation. The mechanical effect of fluid-flow, or shear, at the apical membrane of cholangiocytes regulates secretion through a process involving increases in [Ca2+]i and activation of Ca2+-activated Cl- channels. However, the initiating steps translating shear force to increases in intracellular calcium concentration ([Ca2+]i) are unknown. Transient receptor potential vanilloid member 4 (TRPV4), a nonselective cation channel present in the apical membrane of cholangiocytes, has been proposed as a potential mechanosensor. The aim of the present studies was to determine the potential role of TRPV4 in initiating mechanosensitive signaling in response to fluid-flow in cholangiocytes. TRPV4 expression was confirmed in both small and large mouse cholangiocytes. Exposure of cells to either fluid flow or specific TRPV4 pharmacological agonists rapidly increased both [Ca2+]i and membrane cation currents. Both flow- and agonist-stimulated currents displayed identical biophysical properties and were inhibited in the presence of TRPV4 antagonists or in cells after transfection with TRPV4 small interfering RNA. Transfection of mouse cholangiocytes with a TRPV4-enhanced green fluorescent protein construct increased the expression of TRPV4 and the magnitude of flow-stimulated currents. A specific TRPV4 agonist significantly increased the biliary concentration of ATP and bile flow in live mice when administered intravenously and increased ATP release from cholangiocyte monolayers when applied exogenously. The findings are consistent with a model in which activation of cholangiocyte TRPV4 translates shear force into an acute rise in membrane cation permeability, [Ca2+]i, ATP release, and bile flow. Understanding the role of mechanosensitive transport pathways may provide novel insights to modulate bile flow for the treatment of cholestatic liver disorders.NEW & NOTEWORTHY These studies functionally characterize TRPV4 as a mechanosensitive channel in mouse cholangiocytes. By mediating a rapid rise in intracellular Ca2+, necessary for Ca2+-dependent secretion, TRPV4 represents a mechanosensor responsible for translating fluid flow into intracellular signaling and biliary secretion. Furthermore, intravenous infusion of a specific TRPV4 agonist increases bile flow in live mice. Understanding the role of TRPV4 in mechanosensitive transport pathways may provide novel insights to modulate bile flow during cholestasis.

From the Editor’s desk…: June 2019

From the Editor’s desk…: June 2019

Reactive cholangiocytes differentiate into proliferative hepatocytes with efficient DNA repair in mice with chronic liver injury

Chronic liver diseases are characterized by expansion of the small immature cholangiocytes - a mechanism named ductular reaction (DR) - which have the capacity to differentiate into hepatocytes. We investigated the kinetics of this differentiation, as well as analyzing several important features of the newly formed hepatocytes, such as functional maturity, clonal expansion and resistance to stress in mice with long-term liver damage. We tracked cholangiocytes using osteopontin-iCreERT2 and hepatocytes with AAV8-TBG-Cre. Mice received carbon tetrachloride (CCl4) for >24 weeks to induce chronic liver injury. Livers were collected for the analysis of reporter proteins, cell proliferation and death, DNA damage, and nuclear ploidy; hepatocytes were also isolated for RNA sequencing. During liver injury we observed a transient DR and the differentiation of DR cells into hepatocytes as clones that expanded to occupy 12% of the liver parenchyma by week 8. By lineage tracing, we confirmed that these new hepatocytes derived from cholangiocytes but not from native hepatocytes. They had all the features of mature functional hepatocytes. In contrast to the exhausted native hepatocytes, these newly formed hepatocytes had higher proliferative capability, less apoptosis, a lower proportion of highly polyploid nuclei and were better at eliminating DNA damage. In chronic liver injury, DR cells differentiate into stress-resistant hepatocytes that repopulate the liver. The process might account for the observed parenchymal reconstitution in livers of patients with advanced-stage hepatitis and could be a target for regenerative purposes. During chronic liver disease, while native hepatocytes are exhausted and genetically unstable, a subset of cholangiocytes clonally expand to differentiate into young, functional and robust hepatocytes. This cholangiocyte cell population is a promising target for regenerative therapies in patients with chronic liver insufficiency.

The role of the secretin/secretin receptor axis in inflammatory cholangiocyte communication via extracellular vesicles

Small and large intrahepatic bile ducts consist of small and large cholangiocytes, respectively, and these cholangiocytes have different morphology and functions. The gastrointestinal peptide hormone, secretin (SCT) that binds to secretin receptor (SR), is a key mediator in cholangiocyte pathophysiology. Extracellular vesicles (EVs) are membrane-bound vesicles and cell-cell EV communication is recognized as an important factor in liver pathology, although EV communication between cholangiocytes is not identified to date. Cholangiocytes secrete proinflammatory cytokines during bacterial infection leading to biliary inflammation and hyperplasia. We demonstrate that cholangiocytes stimulated with lipopolysaccharide (LPS), which is a membrane component of gram-negative bacteria, secrete more EVs than cholangiocytes incubated with vehicle. These LPS-derived EVs induce inflammatory responses in other cholangiocytes including elevated cytokine production and cell proliferation. Large but not small cholangiocytes show inflammatory responses against large but not small cholangiocyte-derived EVs. Large cholangiocytes with knocked down either SCT or SR by short hairpin RNAs show reduced EV secretion during LPS stimulation, and EVs isolated from SCT or SR knocked down cholangiocytes fail to induce inflammatory reactions in control large cholangiocytes. This study identifies cholangiocyte EV communication during LPS stimulation, and demonstrates that the SCT/SR axis may be important for this event.

H19 promotes cholestatic liver fibrosis by preventing ZEB1-mediated inhibition of epithelial cell adhesion molecule.

The activation of hepatic H19RNA promoted cholestatic liver fibrosis in mice through the ZEB1/EpCAM signaling pathway. (Hepatology 2017;66:1183-1196).

Vasopressin regulates the growth of the biliary epithelium in polycystic liver disease

The neurohypophysial hormone arginine vasopressin (AVP) acts by three distinct receptor subtypes: V1a, V1b, and V2. In the liver, AVP is involved in ureogenesis, glycogenolysis, neoglucogenesis and regeneration. No data exist about the presence of AVP in the biliary epithelium. Cholangiocytes are the target cells in a number of animal models of cholestasis, including bile duct ligation (BDL), and in several human pathologies, such as polycystic liver disease characterized by the presence of cysts that bud from the biliary epithelium. In vivo, liver fragments from normal and BDL mice and rats as well as liver samples from normal and ADPKD patients were collected to evaluate: (i) intrahepatic bile duct mass by immunohistochemistry for cytokeratin-19; and (ii) expression of V1a, V1b and V2 by immunohistochemistry, immunofluorescence and real-time PCR. In vitro, small and large mouse cholangiocytes, H69 (non-malignant human cholangiocytes) and LCDE (human cholangiocytes from the cystic epithelium) were stimulated with vasopressin in the absence/presence of AVP antagonists such as OPC-31260 and Tolvaptan, before assessing cellular growth by MTT assay and cAMP levels. Cholangiocytes express V2 receptor that was upregulated following BDL and in ADPKD liver samples. Administration of AVP increased proliferation and cAMP levels of small cholangiocytes and LCDE cells. We found no effect in the proliferation of large mouse cholangiocytes and H69 cells. Increases were blocked by preincubation with the AVP antagonists. These results showed that AVP and its receptors may be important in the modulation of the proliferation rate of the biliary epithelium.

Tu1618 Treatment of Biliary Injury With Small Cholangiocyte Therapy Decreases Stellate Cell Activation via Mediation of Cellular Senescence

Tu1618 Treatment of Biliary Injury With Small Cholangiocyte Therapy Decreases Stellate Cell Activation via Mediation of Cellular Senescence

Vasopressin induces cholangiocyte proliferation in experimental cholestasis and in Polycystic Liver Disease

The hormone vasopressin (hereafter AVP) is a neuropeptide mainly synthesized in the brain’s hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei, works by three distinct receptor subtypes: V1a, V1b, and V2 [1]. In liver, AVP is involved in glycogenolysis and neoglucogenesis and regenerative processes [2]. Cholangiocytes are the cells that line the biliary ducts and they are the target in a number of animal models of cholestasis including bile duct ligation (BDL) and in several human pathologies such as polycystic liver disease (PLD) characterized by the presence of numerous cysts within the liver that arise from biliary epithelium [3]. Since no data exist about the presence and the role of AVP and receptors in biliary epithelium, we aimed to evaluate the effects of AVP in experimental model of cholestasis and in course of PLD. In vivo, normal and BDL liver fragments from rats, normal and PLD from human patients were collected to evaluate: (i) intrahepatic bile duct mass (IBDM) by immunohistochemistry for citokeratin-19 (CK-19); and (ii) expression of V1a, V1b and V2 by immunohistochemistry, immunofluorescence and real time PCR. In vitro, small and large mouse cholangiocytes, H69 (non-malignant human cholangiocytes) and LCDE (human cholangiocytes from cystic epithelium) were stimulated with AVP in the absence/presence of antagonists such as OPC-31260 and Tolvaptan, before assessing cellular growth by MTT proliferation assay, cAMP levels by a RIA kit and the expression of some angiogenic factors, such as platelet-derived growth factor (PDGF) and Angiopoietins (Ang-1 and Ang-2). Cholangiocytes express V2 receptor that was upregulated following BDL and in course of polycystic disease. Treatment with AVP of cholangiocyte cultures increased proliferation, cAMP levels and expression of PDGF, Ang-1, Ang-2 in small cholangiocytes and LCDE cells. These increments were blocked by pre-incubation with the AVP antagonists. Our results showed that AVP play an important role in growth of the biliary epithelium during cholestasis and in cystic epithelium in course of PLD acting on the cAMP signalling pathway and increasing angiogenic factors. Additional studies are necessary, but these first results may be considered important in the regulation of the biliary growth/loss in course of cholangiopathies.

Ischemia reperfusion of the hepatic artery induces the functional damage of large bile ducts by changes in the expression of angiogenic factors.

Liver transplantation and cholangiocarcinoma induce biliary dysfunction following ischemia reperfusion (IR). The function of the intrahepatic biliary tree is regulated by both autocrine and paracrine factors. The aim of the study was to demonstrate that IR-induced damage of cholangiocytes is associated with altered expression of biliary angiogenic factors. Normal and bile duct ligation rats underwent 24-h sham or hepatic reperfusion after 30 min of transient occlusion of the hepatic artery (HAIR) or portal vein (PVIR) before collecting liver blocks and cholangiocyte RNA or protein. We evaluated liver histology, biliary apoptosis, proliferation and expression of VEGF-A/C, VEGFR-2/3, Ang-1/2, and Tie-1/2 in liver sections and isolated small and large cholangiocytes. Normal rat intrahepatic cholangiocyte cultures (NRICC) were maintained under standard conditions in normoxic or under a hypoxic atmosphere for 4 h and then transferred to normal conditions for selected times. Subsequently, we measured changes in biliary proliferation and apoptosis and the expression of VEGF-A/C and VEGFR-2/3. In vivo, HAIR (but not PVIR) induced damage of large bile ducts and decreased proliferation and secretin-stimulated cAMP levels. HAIR-induced damage of large bile ducts was associated with increased expression of VEGF-A/C, VEGFR-2/3, Ang-1/2, and Tie-1/2. In vitro, under hypoxic conditions, there was increased apoptosis and reduced proliferation of NRICC concomitant with enhanced expression of VEGF-A/C and VEGFR-2/3. The functional damage of large bile ducts by HAIR and hypoxia is associated with increased expression of angiogenic factors in small cholangiocytes, presumably due to a compensatory mechanism in response to biliary damage.

Functional and structural features of cholangiocytes in health and disease.

Cholangiocytes are the epithelial cells that line the bile ducts. Along the biliary tree, two different kinds of cholangiocytes exist: small and large cholangiocytes. Each type has important differences in their biological role in physiologic and pathologic conditions. In response to injury, cholangiocytes become reactive and acquire a neuroendocrine-like phenotype with the secretion of a number of peptides. These molecules act in an autocrine/paracrine fashion to modulate cholangiocyte biology and determine the evolution of biliary damage. The failure of such mechanisms is believed to influence the progression of cholangiopathies, a group of diseases that selectively target biliary cells. Therefore, the understanding of mechanisms regulating cholangiocyte response to injury is expected to foster the development of new therapeutic options to treat biliary diseases. In this review, we discuss the most recent findings in the mechanisms driving cholangiocyte adaptation to damage, with particular emphasis on molecular pathways that are susceptible of therapeutic intervention. Morphogenic pathways (Hippo, Notch, Hedgehog), which have been recently shown to regulate biliary ontogenesis and response to injury, are also reviewed as well as the results of ongoing clinical trials evaluating new drugs for the treatment of cholangiopathies.

Wnt/Beta‐Catenin Cooperates With Yap Signaling To Promote Hepatobiliary Repair In A Model Of Chronic Liver Injury

Proliferation of atypical ductules and compensatory hepatocyte transdifferentiation to cholangiocytes is a hallmark of 3,5‐diethoxycarbonyl‐ 1, 4‐dihydro‐collidine (DDC) diet‐induced injury in mice. Because liver‐specific beta‐catenin conditional KO mice exhibit fewer proliferating ductules after DDC, we hypothesized that Wnt signaling plays a role in this process. To test this, WT animals on control and DDC diet were analyzed for Wnt expression. We found upregulation of 3 Wnts – 7a, 7b, and 10a – in the portal triad of DDC‐fed mice, as determined by tissue microdissection. Cell sorting showed expression of these three Wnts exclusively in the EpCAM+ cell compartment. Addition of Wnt7b and 10a to small cholangiocyte cell line induced proliferation in an autocrine manner, consistent with an increase in CK19+/Ki67+ cells after DDC. In contrast, direct co‐culture of Hek293 cells expressing recombinant Wnt7a significantly increased Sox‐9 gene expression in AML12 cells and primary hepatocytes. To elucidate the role of beta‐catenin in transdifferentiation, we analyzed liver‐specific transgenic (TG) ser45‐mutant b‐catenin‐expressing mice after DDC diet. Compared to WT, TG had significantly upregulated expression of EpCAM and CK19, and more Sox‐9+ hepatocytes. Further, Yap, which regulates liver cell fate, is increased in TG hepatocyte nuclei, concomitant with increased expression of Yap target genes. Thus, Wnt/beta‐catenin signaling cooperates with Yap pathway to regulate hepatobiliary repair after DDC injury.