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Digestive Disease Research Center, Scott & White, TX, United StatesDepartment of Medicine, Division of Gastroenterology, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, TX, United States
Department of Medicine, Division of Gastroenterology, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, TX, United States
Department of Medicine, Division of Gastroenterology, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, TX, United StatesShengjing Hospital, China Medical University, Shenyang City, Liaoning Province, China
Digestive Disease Research Center, Scott & White, TX, United StatesDepartment of Medicine, Division of Gastroenterology, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, TX, United StatesCentral Texas Veterans Health Care System, Temple, TX, United States
Department of Medicine, Division of Gastroenterology, Scott & White and Texas A&M Health Science Center, College of Medicine, Temple, TX, United States
Corresponding author at: Department of Human Anatomy, University of Rome “La Sapienza”, Via Alfonso Borelli 50 00161 Rome, Rome 00161, Italy. Tel.: +39 06 4991 8060; fax: +39 06 4991 8062.
Bile duct epithelial cells (i.e., cholangiocytes), which line the intrahepatic biliary epithelium, are the target cells in a number of human cholestatic liver diseases (termed cholangiopathies). Cholangiocyte proliferation and death is present in virtually all human cholangiopathies. A number of recent studies have provided insights into the key mechanisms that regulate the proliferation and function of cholangiocytes during the pathogenesis of cholestatic liver diseases. In our review, we have summarised the most important of these recent studies over the past 3 years with a focus on those performed in the animal model of extrahepatic bile duct ligation. In the first part of the review, we provide relevant background on the biliary ductal system. We then proceed with a general discussion of the factors regulating biliary proliferation performed in the cholestatic animal model of bile duct ligation. Further characterisation of the factors that regulate cholangiocyte proliferation and function will help in elucidating the mechanisms regulating the pathogenesis of biliary tract diseases in humans and in devising new treatment approaches for these devastating diseases.
], these cells play an important pathophysiological role in the modification of the composition of bile during the transit in the bile ducts, which involves the secretion and absorption of water, electrolytes and other organic solutes from hepatocellular bile [
Cholangiocytes are the target cells of a number of diseases termed cholangiopathies. This disease class is made up of inherited disorders [Alagille syndrome and cystic fibrosis (CF)], autoimmune disorders [primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), autoimmune cholangitis (AIC), allograft rejection, graft-versus-host disease (GVHD)], infections (cholangitis due to bacteria, fungi, parasites or viruses), drug-induced injury, ischaemic injury and diseases of unknown aetiology (biliary atresia and idiopathic vanishing bile duct syndromes) [
]. Cholangiopathies are predominantly characterised by a bile duct-directed inflammatory response that leads to bile duct injury associated with biliary proliferation in the early stage of the disease course [
]. If the biliary injury is chronic there will be increased bile duct loss, biliary fibrosis and the increased incidence of bile duct cancer (i.e., cholangiocarcinoma) [
]. The intrahepatic bile ductal system consists of the portion of (i) bile canaliculi, small spaces localised between two adjacent hepatocytes (0.5–2 mm) forming a three-dimensional network that continues in (ii) bile ductules (canals of Hering), localised at the periphery of the hepatic lobule and characterised by 3–4 cholangiocytes, that form the junction between hepatocytes and cholangiocytes (ductular–canalicular junction) allowing the confluence of the bile in (iii) bile ducts (interlobular bile ducts) localised in the portal space. Interlobular bile ducts progressively continue in larger ducts until the right and left hepatic ducts, that, at the level of the hylus, determine the origin of extrahepatic biliary tree [
]. According to Ludwig, the human intrahepatic biliary epithelium is divided into small bile ductules (<15 μm), interlobular ducts (15–10 μm), septal ducts (100–300 μm), segmental ducts (400–800 μm) and hepatic ducts (>800 μm) [
]. The rodent intrahepatic bile duct system has been recently classified into small ducts (<15 μm in external diameter) lined by small cholangiocytes (approximately 8 μm in diameter characterised by high nucleus/cytoplasm ratio) and large bile ducts (>15 μm in diameter characterised by low nucleus/cytoplasm ratio) lined by large cholangiocytes (approximately 15 μm in diameter) [
]. These studies have also shown that a significant relationship exists between cholangiocyte area and external bile duct diameter, with small bile ducts lined by small cholangiocytes and large ducts lined by large cholangiocytes [
]. The latter finding is particularly relevant since it allows for the direct mapping of studies obtained in isolated small and large cholangiocytes to different portions (i.e., small and large) of the intrahepatic biliary epithelium ex situ[
]. In support of the morphological heterogeneity of the biliary epithelium, Masyuk et al. have reconstructed the intrahepatic biliary system that resembles a tree, with the common and hepatic ducts corresponding to the trunk, the intrahepatic bile ducts corresponding to the large branches and the small ductules corresponding to the smallest tree limbs of the tree [
]. “Typical” cholangiocyte proliferation is a hyperplastic reaction, which induces an increase in the number of intrahepatic bile ducts (with a well-defined lumen) confined to portal areas [
]. “Atypical” cholangiocyte proliferation is commonly seen in patients with prolonged cholestatic liver diseases such as PBC or PSC and is characterised by irregular proliferation of intrahepatic bile ducts sprouting into periportal and parenchymal regions and occasionally forming anastomosing cords with adjacent hepatocytes [
]. Oval cell proliferation takes place in the early stages of chemically induced hepatocarcinogenesis and is characterised by a disorganised proliferation of biliary structures with a poorly defined lumen [
]. In these hyperplastic models, cholangiocyte proliferation is closely associated with increased SR gene expression and secretin-stimulated cAMP levels [
Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-b/gastrin receptors via d-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms.
]. A recent study by Strazzabosco et al. demonstrates that differential expression of AC isoforms mediate the secretory functions of small and large cholangiocytes [
]. A number of animal models that mimic cholestatic liver diseases and liver injury have been utilised to expand our knowledge related to the mechanisms of cholangiocyte proliferation [
]. Of these models of bile duct injury, the BDL model has been the most commonly used. A number of coordinate factors (stimulatory or inhibitory) have been shown to regulate cholangiocyte growth in the cholestatic BDL model. It has been shown that increased biliary pressure is a trigger for the stimulation or inhibition of these putative growth factors [
] that increased biliary and portal hypertension (induced by the first), represent key proliferative triggers for the growth of bile ducts and hepatocytes. Similar to findings in human cholangiopathies (e.g., PBC and PSC), recent studies in rats have demonstrated that “typical” cholangiocyte proliferation occurs within a limited range of duct sizes [
]. A recent study in mice with BDL demonstrated that similar to rats, the mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous [
]. These findings are of importance due to increased availability and usage of transgenic mouse models for studying cholestatic liver disease pathogenesis [
]. The study indicates that the mouse is a suitable model for defining the heterogeneous responses of cholangiocytes during cholestasis and biliary damage [
], changes in the functional expression of this receptor have been suggested as a pathophysiological tool for evaluating changes in the degree of cholangiocyte growth/loss [
Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-b/gastrin receptors via d-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms.
]. Proliferating cholangiocytes acquire a neuroendocrine phenotype and secrete and respond to a number of hormones, neuropeptides and neurotransmitters [
]. The formation of a neuroendocrine compartment predominated by cholangiocytes represents a unique opportunity for cholangiocytes to regulate their own proliferation via autocrine pathways and for cholangiocytes to influence other nearby cell types, such as vascular endothelial cells, portal fibroblasts and hepatic stellate cells [
]. A number of recent studies have highlighted and expanded our knowledge of the concept that proliferating cholangiocytes display neuroendocrine features [
Fig. 1Immunolocalisation of cytokeratin-19 (CK-19) in proliferating cholangiocytes in the bile duct ligation (BDL) animal model. Original magnification 40×.
4. Update on neuroendocrine regulation of biliary proliferation during BDL
Over the past several years, a number of studies have explored the neuroendocrine regulation of cholangiocyte proliferation during extrahepatic cholestasis. The neuroendocrine factors contributing to cholangiocyte proliferation have been previously reviewed [
Table 2Other factors regulating cholangiocyte proliferation during cholestasis.
Regulatory factors
Effect on cholangiocyte growth
Second messenger/transduction pathways
References
Endocannabinoid system
Chronic treatment of rats with BDL with anandamide decreased cholangiocyte proliferation
Accumulation of reactive oxygen species; upregulation of the expression of TRX1, Ref1, c-Fos, and c-Jun expression; increase in the nuclear localisation of TRX1; increase in AP-1 transcriptional activity
CGRP (calcitonin gene-related peptide) is a potent vasodilator peptide that participates in the regulation of vascular tone and regional organ blood flow [
]. We have recently demonstrated that hepatic sensory innervation and cholangiocyte expression of α-CGRP (Fig. 2) play a key role in the regulation of cholangiocyte proliferation during cholestasis induced by BDL [
]. Both α- and β-CGRP stimulated proliferation of isolated BDL cholangiocytes by activation of protein kinase A (PKA) and cAMP response element binding (CREB) [
]. These studies indicate that sensory innervation plays a role in the regulation of biliary proliferation and other sensory neuropeptides may play a role in chronic inflammation during cholangiopathies
Fig. 2Localisation of α-CGRP (red) by immunofluorescence in normal and 3-day BDL WT mice liver sections. Bile ducts were stained with CK-7 (green). α-CGRP-positive staining and CK-7 colocalise in the bile ducts of 3-day BDL WT mice. The scale bar represents 20 μM. Arrows indicate bile ducts.
The aminergic peptide and neurotransmitter histamine regulates many functions in the body, such as neurogenic functions, inflammatory responses, allergic responses, and gastric secretion [
]. Following BDL, the expression of HR3R is significantly increased in proliferating cholangiocytes. Activation of HR3R by the chronic administration of the agonist (R)-(α)-(−)-methylhistamine dihydrobromide (RAMH) to rats for 7 days after BDL resulted in a decrease in the growth of the biliary tree with no difference in the rate of apoptosis [
]. In addition, administration of histamine to this animal model of cholestasis also resulted in a decrease in cholangiocyte proliferation, and blocking histamine actions by using the selective HR3R antagonist thioperamide maleate resulted in a partial reversal of these effects [
]. Both in vivo and in vitro, RAMH inhibition of cholangiocyte growth was associated with downregulation of cAMP-dependent PKA–ERK1/2-Elk-1 signalling pathway [
GLP-1 is secreted by a number of neuroendocrine cell types and plays a role in sustaining beta-cell survival in experimental models of diabetes and induces the transdifferentiation of pancreatic ductal cells [
]. GLP-1 and the GLP-1 receptor specific agonist exendin-4 had similar effects on cholangiocytes by stimulating proliferation in vivo in normal rats and in isolated cholangiocytes from normal and BDL rats [
]. Cholangiocytes from BDL but not normal rats express the message for the precursor for GLP-1, preproglucagon, which is a finding that suggests that GLP-1 is an important player in biliary growth during cholestasis [
]. The pro-proliferative effect of GLP-1 was mediated through phosphoinositide 3-kinase (PI3K), cAMP/PKA and Ca2+-CaMKII α (calmodulin-dependent protein kinase α) signalling mechanisms [
]. The mechanisms by which GLP-1 regulates cholangiocyte growth are depicted in Fig. 3. More recently, exendin-4 has been shown to protect cholangiocytes from apoptosis in in vivo and in vitro models of cholangiocyte apoptosis [
]. In vitro, exendin-4 prevented glycochenodeoxycholic acid-induced Bax mitochondrial translocation, cytochrome c release and caspase 3 activities, which was blocked by inhibition of PI3K [
]. In vivo, exendin-4 administration prevented the increase in TUNEL, positive cholangiocytes and the loss of bile ducts that is observed in BDL rats treated with CCl4[
]. Further studies are required to determine if exendin-4 will be effective for preventing the progression of cholangiopathies towards ductopenia.
Fig. 3Proposed sequence of intracellular events associated to GLP-1R activation in cholangiocytes. GLP-1R activation sustains cell growth enhancing the activation state of the PI3K and cAMP/PKA cascades. Cell proliferation is also elicited by the extracellular Ca2+-dependent activation of CaMKIIα that can modulate cholangiocyte proliferation both directly and by cross-talking with the cAMP/PKA cascade. In contrast, GLP-1R activation does not result in any change of ERK1/2 activation state and does not increase the IP3-PKCα signalling.
]. Interestingly, normal and BDL cholangiocytes expressed the biosynthetic pathway (i.e., steroidogenic acute regulatory protein or STAR, 3β-hydroxysteroid dehydrogenase or 3β-HSD, and cytochrome P450 side-chain cleavage or p450scc) for and secrete progesterone [
] (Fig. 4). In vitro, supernatants collected from normal and BDL cholangiocytes increased cholangiocyte proliferation, which was partially inhibited by preincubation with anti-progesterone and inhibition of progesterone steroidogenesis with aminoglutethimide prevented cholangiocyte proliferation [
]. These findings provide further support for the concept that neuroendocrine autocrine/paracrine mechanisms play a key role in the modulation of cholangiocyte proliferative responses to cholestasis
Fig. 4Immunofluorescence for key proteins in the progesterone steroidogenesis pathway [steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (p450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD)] in liver sections from normal and BDL female and male rats demonstrates that bile ducts express these steroidogenesis pathway proteins (red staining). Colocalisation with CK-19 (green staining, a cholangiocyte-specific marker) of the bile ducts expressing StAR, p450scc, and 3β-HSD is also visible.
]. Chronic administration of FSH to normal rats increased, whereas administration of antide (a gonadotropin-releasing hormone antagonist that blocks FSH secretion) and anti-FSH to normal rats decreased cholangiocyte proliferation and secretory responses [
]. In vitro, FSH increased cholangiocyte proliferation, cAMP levels, and ERK1/2 and Elk-1 phosphorylation, which were prevented by preincubation with anti-FSH [
]. Silencing of FSH expression also decreases basal cholangiocyte proliferation suggesting that FSH is a key autocrine factor regulating biliary mass [
]. These findings have important pathological implications since modulation of the expression and secretion of FSH may be used to modulate cholangiocyte proliferation during cholestatic liver diseases
4.5 Angiogenic factors
Vascular endothelial growth factor (VEGF) is a mitogen for vascular endothelial cells for vascular cells and regulates vascular pathophysiology [
]. We have previously shown that VEGF-A and VEGF-C are secreted by cholangiocytes and play an important role in the regulation of cholangiocyte proliferation and apoptosis during cholestasis and biliary injury induced by hepatic artery ligation [
]. We have also shown that the bile acid TC prevents cholangiocyte death by apoptosis, and the loss of proliferative and functional responses of cholangiocytes in response to CCl4 and cholinergic or adrenergic denervation [
A recent study has provided additional evidence that VEGF plays a role in both the protection of cholangiocytes from damage in an experimental model of cholestasis [
]. In an animal model of cholestasis and biliary damage induced by caffeic acid, the feeding of the protective bile acid, taurocholate prevented bile duct damage, which was associated with increased cholangiocyte VEGF-A, VEGF-C, VEGFR-2 and VEGFR-3 expression [
]. Recent evidence also indicates that the endocannabinoid system plays a role in regulating cholangiocyte proliferation during cholestasis induced by BDL [
]. Chronic treatment of rats with BDL with anandamide decreased cholangiocyte proliferation and induced the accumulation of reactive oxygen species, upregulated the expression of TRX1, Ref1, c-Fos and c-Jun expression, increased the nuclear localisation of TRX1 and increased AP-1 transcriptional activity [
]. This work demonstrated that modulation of the endocannabinoid system and/or the ROS/TRX1/Ref1/AP-1 pathway may have therapeutic implications for the treatment of early stage cholestatic liver diseases characterised by cholangiocyte proliferation [
Molecular organization, structural features, and ligand binding characteristics of CD44, a highly variable cell surface glycoprotein with multiple functions.
Significant correlations of e-cadherin, catenin, and CD44 variant form expression with carcinoma cell differentiation and prognosis of extrahepatic bile duct carcinomas.
High frequency of autoantibodies in patients with primary sclerosing cholangitis that bind biliary epithelial cells and induce expression of CD44 and production of interleukin 6.
]. He et al. provide evidence that suggests that the proliferative cholangiocytes lining the intrahepatic ducts are an important source of hepatic CD44 [
]. They demonstrated in vitro that cholangiocyte proliferation was stimulated by hyaluronan treatment, and blocked by siRNA for CD44 or anti-CD44 antibody [
] have shown that IGF1 plays a key role in mediating cholangiocyte proliferation after BDL and in protecting cholangiocytes from the cytotoxic effect of hydrophobic bile salts. More recently, we evaluated the expression of IGF1 isoforms in rat cholangiocytes, and evaluated their involvement in cell proliferation or damage induced by BDL or hydrophobic bile salts [
Alterations of growth plate and abnormal insulin-like growth factor I metabolism in growth-retarded hypokalemic rats: effect of growth hormone treatment.
]. After BDL for 3 h, the ‘locally acting’ IGF1 isoform decreases threefold in hepatocytes but remains stable in cholangiocytes with respect to sham-controls [
]. After 1 week of BDL, hepatocytes displays a further fivefold decrease of ‘locally acting’ IGF1 mRNA. In contrast, cholangiocytes show an eightfold increase of the ‘locally acting’ IGF1 mRNA. The effect of BDL for 3 h on IGF1 isoforms was reproduced in vitro by incubation with glycochenodeoxycholate (GCDC) [
]. The cytotoxic effects (inhibition of proliferation and induction of apoptosis) of GCDC on isolated cholangiocytes were more pronounced after silencing (siRNA) of ‘locally acting’ than ‘circulating’ IGF1 isoform [
]. Therefore, these findings demonstrate that rat cholangiocytes express the ‘locally acting’ IGF1 isoform, which decreases during cell damage and increases during cell mitosis [
]. These findings indicate that, besides muscle and neural tissues, also in liver cells the ‘locally acting’ IGF1 isoform is important in modulating response of cholangiocytes to damage.
4.6.4 Ezrin–radixin–moesin-binding phosphoprotein
Ezrin–radixin–moesin-binding phosphoprotein 50 (EBP50) is inducible by oestrogen and alters cell proliferation. This study demonstrated the expression and role of EBP50 in ductular reaction in normal human liver, human cholangiopathies (i.e., CF, PBC and PSC) and BDL rats [
]. The study demonstrated that in normal human liver, EBP50 is expressed in the canalicular membranes of hepatocytes and, together with ezrin and CFTR, in the apical domains of cholangiocytes [
]. These studies demonstrated that the expression and distribution of EBP50 (regulated by oestrogens) contribute to the proliferative responses of cholangiocytes [
Takeda et al. have demonstrated that the death signalling pathway mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) contributes to the pathogenesis of biliary cirrhosis [
]. Administration of an agonistic anti-DR5 monoclonal antibody triggered cholangiocyte apoptosis, cholangitis and cholestatic liver injury reminiscent of PSC [
]. DR5 and TRAIL expression were also found to be elevated in cholangiocytes of human PSC and PBC patient samples suggesting that modulation of DR5 death signalling might be a therapeutic option for chronic cholestatic liver diseases [
Foxl1 has been previously shown to be dramatically induced in cholangiocytes by both BDL and in response to 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet [
]. In Foxl1 knockout mice with BDL, they observed an increase in parenchymal necrosis and significantly impaired cholangiocyte and hepatocyte proliferation [
]. The regulation of cholangiocyte proliferation may provide a means to prevent biliary fibrosis during chronic cholestatic liver diseases.
5. Summary and future directions
We have reviewed recent studies that address factors that regulate cholangiocyte proliferation during extrahepatic cholestasis induced by BDL. Cholangiocyte proliferation is closely associated with transdifferentiation to a neuroendocrine phenotype. Future studies will be necessary to determine the role that proliferating cholangiocytes play in the pathogenesis of biliary fibrosis during cholestasis and how cholangiocytes interact with other cell types of the liver such as hepatic stellate cells during cholestatic liver diseases. Our developing knowledge of the fundamental factors that control cholangiocyte proliferation during cholestasis will aid in the development of therapies for the treatment of chronic cholestatic liver diseases.
Conflict of interest statement
None declared.
Acknowledgements
Portions of the studies discussed here were supported partly by a grant award from Scott & White and NIH RO1 DK081442 to Shannon Glaser, by the Dr. Nicholas C., Hightower Centennial Chair of Gastroenterology from Scott & White, the VA Research Career Scientist Award, a VA Merit Award and the NIH grants DK76898, DK58411 and DK62975 to Gianfranco Alpini, by University and Federate Athenaeum funds from University of Rome “La Sapienza” to Eugenio Gaudio and MIUR grants: PRIN #2007, prot. 2007HPT7BA_003 to Domenico Alvaro.
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Gastrin inhibits cholangiocyte growth in bile duct-ligated rats by interaction with cholecystokinin-b/gastrin receptors via d-myo-inositol 1,4,5-triphosphate-, Ca(2+)-, and protein kinase C alpha-dependent mechanisms.
Molecular organization, structural features, and ligand binding characteristics of CD44, a highly variable cell surface glycoprotein with multiple functions.
Significant correlations of e-cadherin, catenin, and CD44 variant form expression with carcinoma cell differentiation and prognosis of extrahepatic bile duct carcinomas.
High frequency of autoantibodies in patients with primary sclerosing cholangitis that bind biliary epithelial cells and induce expression of CD44 and production of interleukin 6.
Alterations of growth plate and abnormal insulin-like growth factor I metabolism in growth-retarded hypokalemic rats: effect of growth hormone treatment.
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