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Microbial products are thought to influence the progression of cholangiopathies, in particular primary sclerosing cholangitis (PSC). Inflammasomes are molecular platforms that respond to microbial products through the synthesis of proinflammatory cytokines. We investigated the role of inflammasome activation in cholangiocyte response to injury. Nucleotide-binding oligomerization domain (NOD)-like receptor family, pyrin domain-containing protein 3 (Nlrp3) expression was tested in cholangiocytes of normal and cholestatic livers. Effects of Nlrp3 activation induced by incubation with lipopolysaccharide and ATP was studied in vitro in normal and siRNA-Nlrp3 knocked-down cholangiocytes. Wild-type and Nlrp3 knockout (Nlrp3 -/- ) mice were fed 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; a model of sclerosing cholangitis) for 4 weeks. Nlrp3 and its components were overexpressed in cholangiocytes of mice subjected to DDC and in patients affected by PSC. In vitro, Nlrp3 activation stimulated expression of Il-18 but not of Il-1β and Il-6. Nlrp3 activation had no effect on cholangiocyte proliferation but significantly decreased the expression of Zonulin-1 and E-cadherin, whereas Nlrp3 knockdown increased the permeability of cholangiocyte monolayers. In vivo, the DDC-stimulated number of cytokeratin-19-positive cells in the liver of wild-type animals was slightly reduced in Nlrp3 -/- mice, and expression of E-cadherin was reestablished. In conclusion, Nlrp3 is expressed in reactive cholangiocytes, in both murine models and patients with PSC. Activation of Nlrp3 leads to synthesis of proinflammatory cytokines and influences epithelial integrity of cholangiocytes., (Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Endoscopic ultrasound-guided fine needle aspiration is routinely used in the diagnostic work up of pancreatic cancer but has a low sensitivity. Studies showed that Pancreatic Duodenal Homeobox-1 (PDX-1) is expressed in pancreatic cancer, which is associated with a worse prognosis. We aimed to verify whether the assessment of PDX-1 in endoscopic ultrasound-guided fine needle aspiration samples may be helpful for the diagnosis of pancreatic cancer., Methods: mRNA of 54 pancreatic cancer and 25 cystic lesions was extracted. PDX-1 expression was assessed by Real-Time PCR., Results: In all but two patients with pancreatic cancer, PDX-1 was expressed and was found positive in 7 patients with pancreatic cancer in which cytology was negative. The positivity was associated with a probability of 0.98 (95% CI 0.90-1.00) of having cancer and the negativity with one of 0.08 (95% CI 0.01-0.27). The probability of cancer rose to 1.00 (95% CI 0.97-1.00) for patients positive to both PDX-1 and cytology and fell to 0.0 (95% CI 0.00-0.15) in patients negative for both., Conclusions: PDX-1mRNA is detectable in samples of pancreatic cancer. Its quantification may be helpful to improve the diagnosis of pancreatic cancer., (Copyright © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Semaphorin7A (SEMA7A) is a membrane-anchored protein involved in immune and inflammatory responses, exerting an effect on pulmonary fibrosis. Thus, we aimed to investigate the role of SEMA7A in hepatic fibrosis. Liver injury was induced in vivo by carbon tetrachloride i.p. injection or bile duct ligation in wild-type and SEMA7A knockout (KO) mice. Human and mouse liver samples and primary mouse hepatic cell populations were used for Western blot analysis, quantitative real-time RT-PCR, and immunohistochemistry. SEMA7A is highly expressed in hepatic stellate cells (HSCs). The expression of SEMA7A and its receptor β1-integrin subunit increase during liver injury and in activated HSCs. Transforming growth factor β-stimulated HSCs showed increased expression of SEMA7A in a SMAD2/3-independent manner, leading to increased expression of fibrogenic and inflammation markers. This pattern was significantly blunted in SEMA7A KO HSCs. Overexpression of SEMA7A in HSCs showed increased fibrogenic and inflammation markers expression. In vivo, SEMA7A KO mice treated with carbon tetrachloride and bile duct ligation developed reduced fibrosis versus wild-type mice. Moreover, SEMA7A expression increased in liver samples of patients with fibrosis versus healthy controls. SEMA7A was expressed in the liver and was increased in the course of liver fibrosis, both in mice and in humans. SEMA7A was mainly expressed in HSCs with respect to other cell types in the liver and plays a critical role in regulating fibrosis., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Cholangiocyte proliferation plays a role in the progression of cholangiopathies, in particular in primary sclerosing cholangitis. The mechanisms regulating cholangiocyte proliferation are still undefined. Pancreatic Duodenal Homeobox protein 1 (PDX-1) is expressed by reactive cholangiocytes. In the adult pancreas, PDX-1 regulates the proliferative response to injury of ductal cells. Its effects can be counteracted by Hairy and enhancer of split 1 (Hes-1). We aimed at studying whether PDX-1/Hes-1 interactions regulate cholangiocyte proliferation in response to injury., Methods: The effect of the loss of PDX-1 on cholangiocyte proliferation was studied in vitro. In vivo PDX-1-heterozygous (+/-) mice were subjected to either DDC feeding (a model of sclerosing cholangitis) or to bile duct ligation (BDL). PDX-1/Hes-1 interactions on cell proliferation were determined by exposure to All-trans Retinoic Acid (At-RA), an inductor of Hes-1., Results: In vitro, cholangiocyte proliferation was undetectable in cells pre-treated with PDX-1 siRNA. In vivo, increases in bile duct mass and collagen deposition observed after DDC feeding or BDL were significantly reduced in PDX-1(+/-) mice. Hes-1 expression is reduced in proliferating cholangiocytes; At-RA induced a dose-dependent increase in Hes-1 and a decrease in PDX-1 expression. At-RA neutralized the increases in PDX-1 expression and cell proliferation, both in vitro and in vivo in DDC mice. PDX-1 is overexpressed and Hes-1 downregulated in cholangiocytes isolated from PSC livers., Conclusions: Hes-1 downregulation allows PDX-1 to act as a major determinant of cholangiocyte proliferation in response to cholestatic injury. These findings provide novel mechanistic insights into the pathophysiology of cholangiopathies., (Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Background: Cholangiocarcinoma cells over-express oestrogen receptor-β, which displays anti-proliferative and pro-apoptotic effects., Aim: To evaluate the effects of a newly developed and highly selective oestrogen receptor-β agonist (KB9520) on experimental intrahepatic cholangiocarcinoma., Methods: In vitro, the effects of KB9520 on apoptosis and proliferation of HuH-28 cells, HuH-28 cells with selective oestrogen receptor-β silencing (by small interfering RNA), HepG2 cells (oestrogen receptor-α and oestrogen receptor-β negative) and HepER3 cells (HepG2 cells transformed to stably express oestrogen receptor-α) were evaluated. In vivo, the effects of KB9520 on experimental intrahepatic cholangiocarcinoma, induced by thioacetamide administration were tested., Results: In vitro, KB9520 induced apoptosis and inhibited proliferation of HuH-28 cells. KB9520 effects were absent in cells lacking oestrogen receptor-α and β (HepG2) and in cells expressing only oestrogen receptor-α (HepER3); its pro-apoptotic effect was impaired in cells where oestrogen receptor-β expression was decreased by specific small interfering RNA. In vivo, KB9520 inhibited experimental intrahepatic cholangiocarcinoma development in thioacetamide-treated rats and promoted tumour regression in rats where tumour was already established. In treated animals, tumour areas showed reduced proliferation but increased apoptosis., Conclusions: KB9520 induced apoptosis in cholangiocarcinoma by selectively acting on oestrogen receptor-β, suggesting that oestrogen receptor-β selective agonists may be a novel and effective therapeutic option for the medical treatment of intrahepatic cholangiocarcinoma., (Copyright © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background & Aims: Reactive cholangiocytes acquire a neuroendocrine-like phenotype, with synthesis and local release of neuropeptides and hormones. The mechanism that drives such phenotypical changes is still undefined. Pancreatic Duodenal Homeobox-1 (PDX-1) is a transcription factor required for pancreatic development, that sustains pancreatic beta-cell response to injury and insulin synthesis. PDX-1 induces neuroendocrine-like transition of pancreatic ductal cells. Cholangiocyte response to injury is modulated by Glucagon-Like Peptide-1 Receptor (GLP-1R), which, in the pancreas, activates PDX-1. We wanted to verify whether PDX-1 plays any role in cholangiocyte neuroendocrine-like transdifferentiation in response to injury., Methods: PDX-1 expression was assessed in cholangiocytes from normal and one week bile duct ligated (BDL) rats. Changes in PDX-1 expression and activation upon GLP-1R activation were then assayed. The effects of the lack of PDX-1 in cholangiocytes were studied in vitro by siRNA and in vivo by the employment of PDX-1-deficient (+/-) mice., Results: BDL but not normal cholangiocytes express PDX-1. GLP-1R activation elicits, in a PI3K-dependent fashion, PDX-1 expression, together with its nuclear translocation. In vitro, GLP-1R-induced increases in VEGF and IGF-1 mRNA expression were blunted in cells with PDX-1 siRNA. In vivo, the VEGF and IGF-1 mRNA expression in the liver after one week BDL was markedly reduced in PDX-1-deficient mice, together with reduced bile duct mass., Conclusions: In response to injury, reactive cholangiocytes de novo express PDX-1, the activation of which allows cholangiocytes to synthesize IGF-1 and VEGF. These findings suggest that PDX-1 drives the acquisition of the neuroendocrine-like phenotype by cholangiocytes in response to cholestatic injury., (Copyright © 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Cholangiocytes are the epithelial cells that line the biliary tree; they are the target of chronic diseases termed cholangiopathies, which represent a daily challenge for clinicians, since definitive medical treatments are not available yet. It is generally accepted that the progression of injury in the course of cholangiopathies, and promotion and progression of cholangiocarcinoma are at least in part due to the failure of the cholangiocytes' mechanisms of adaptation to injury. Recently, several studies on the pathophysiology of the biliary epithelium have shed some light on the mechanisms that govern cholangiocyte response to injury. These studies provide novel information to help interpret some of the clinical aspects of cholangiopathies and cholangiocarcinoma; the purpose of this review is thus to describe some of these novel findings, focusing on their significance from a clinical perspective., (Copyright (c) 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Background/aims: Incidence of cholangiocarcinoma is increasing worldwide, yet remaining highly aggressive and with poor prognosis. The mechanisms that drive cholangiocyte transition towards malignant phenotype are obscure. Cholangiocyte benign proliferation is subjected to a self-limiting mechanism based on the autocrine release of endogenous opioid peptides. Despite the presence of both, ligands interact with delta opioid receptor (OR), but not with microOR, with the consequent inhibition of cell growth. We aimed to verify whether cholangiocarcinoma growth is associated with failure of opioidergic regulation of growth control., Methods: We evaluated the effects of OR selective agonists on cholangiocarcinoma cell proliferation, migration and apoptosis. Intracellular signals were also characterised., Results: Activation of microOR, but not deltaOR, increases cholangiocarcinoma cell growth. Such an effect is mediated by ERK1/2, PI3K and Ca(2+)-CamKIIalpha cascades, but not by cAMP/PKA and PKCalpha. microOR activation also enhances cholangiocarcinoma cell migration and reduces death by apoptosis. The anti-apoptotic effect of microOR was PI3K dependent., Conclusions: Our data indicate that cholangiocarcinoma growth is associated with altered opioidergic regulation of cholangiocyte biology, thus opening new scenarios for future surveillance or early diagnostic strategies for cholangiocarcinoma.

Background: Cells within the acidic extracellular environment of solid tumours maintain their intracellular pH through the activity of the Na(+)/H(+) exchanger and the Na(+) dependent Cl(-)/HCO(3)(-) exchanger. The inhibition of these mechanisms could therefore inhibit cancer cell growth., Aim: We evaluated the effect of two selective inhibitors of these transporters (cariporide and S3705) on proliferation and apoptosis of human cholangiocarcinoma cells (HUH-28 and Mz-ChA-1 cells) as a function of external pH (7.4 and 6.8)., Methods/results: HUH-28 cells incubated for 24h at external pH 7.4 or 6.8 without inhibitors maintained intracellular pH at physiological level, whereas incubation with cariporide and/or S3705 caused the intracellular pH of cells to drop. Incubation of HUH-28 cells with cariporide and/or S3705 was able to reduce proliferation, evaluated by a colorimetric ELISA method, and to induce apoptosis, evaluated by measuring caspase-3 activity and Annexin-V staining, and these effects were more evident at external pH 6.8. S3705 but not cariporide was able to inhibit serum-induced phosphorylation of ERK1/2, AKT and BAD, intracellular molecules involved in cancer cell proliferation and survival. Similar results were obtained in Mz-ChA-1 cells., Conclusions: (1) Inhibition of intracellular pH regulatory mechanisms by cariporide and S3705 reduces proliferation and induces apoptosis in cholangiocarcinoma cells; and (2) these drugs might have potential therapeutic value against cholangiocarcinoma.

Insulin resistance induces nonalcoholic fatty liver disease and nonalcoholic steatohepatitis (NASH). We used a high-fat, high-calorie solid diet (HFD) to create a model of insulin resistance and NASH in nongenetically modified rats and to study the relationship between visceral adipose tissue and liver. Obesity and insulin resistance occurred in HFD rats, accompanied by a progressive increase in visceral adipose tissue tumor necrosis factor (TNF)-alpha mRNA and in circulating free fatty acids. HFD also decreased adiponectin mRNA and peroxisome proliferator-activated receptor (PPAR)-alpha expression in the visceral adipose tissue and the liver, respectively, and induced hepatic insulin resistance through TNF-alpha-mediated c-Jun N-terminal kinase (JNK)-dependent insulin receptor substrate-1Ser307 phosphorylation. These modifications lead to hepatic steatosis accompanied by oxidative stress phenomena, necroinflammation, and hepatocyte apoptosis at 4 weeks and by pericentral fibrosis at 6 months. Supplementation of n-3 polyunsaturated fatty acid, a PPARalpha ligand, to HFD-treated animals restored hepatic adiponectin and PPARalpha expression, reduced TNF-alpha hepatic levels, and ameliorated fatty liver and the degree of liver injury. Thus, our model mimics the most common features of NASH in humans and provides an ideal tool to study the role of individual pathogenetic events (as for PPARalpha down-regulation) and to define any future experimental therapy, such as n-3 polyunsaturated fatty acid, which ameliorated the degree of liver injury.

Background/aims: Disapperacence of bile ducts (ductopenia) represents the terminal, common stage of human cholangiopathies, and estrogens exert a major role in stimulating cholangiocyte proliferation. We thus evaluated whether estrogen administration protect from the bile duct loss induced by the biliary-digestive diversion in bile duct ligated (BDL) rats., Methods: After 3 weeks of BDL, rats were subjected to biliary-digestive diversion and treated with daily injections of 17beta-estradiol or a control solution., Results: Both after 7 and 14 days from the biliary-digestive diversion a marked increase of the number of apoptotic cholangiocytes was observed. In contrast, 17beta-estradiol significantly reduced cholangiocyte apoptosis. 17beta-estradiol also prevented the biliary-digestive diversion-induced loss of PCNA-positive cholangiocytes and of the bile duct mass. Biliary-digestive diversion determined a marked reduction of ERK1/2 phopsphorylation in cholangiocytes that was reversed by the administration of 17beta-estradiol., Conclusions: This study indicates that estrogens prevent the increase of cholangiocyte apoptosis and loss of cholangiocyte proliferation induced by the biliary-digestive diversion in the BDL rat. In parallel, 17beta-estradiol also enhanced ERK1/2 phosphorylation, which is instead strongly reduced by the biliary-digestive diversion. These novel findings suggest that estrogens could prevent the evolution of cholangiopathies toward ductopenia.

Background/aims: The regulation of three major intracellular signalling protein kinases was investigated in two models of liver injury leading to hepatic fibrosis, dimethylnitrosamine administration (DMN) and bile duct ligation (BDL)., Methods: Extracellular signal-regulated kinases (ERK)1/2, c-Jun terminal kinase (JNK) and p70S6-kinase (p70(S6K)) were studied in vivo in the whole liver, in liver sections and in isolated hepatocytes, cholangiocytes and hepatic stellate cells (HSC)., Results: In the whole liver, activation of these kinases occurred with a different kinetic pattern in both models of liver injury. By immunohistochemistry and Western blot in isolated cells, phosphorylated kinases were detected in proliferating cells (i.e. hepatocytes and cholangiocytes after DMN and BDL, respectively), in addition to stellate-like elements. ERK1/2, JNK and p70(S6K) activation was associated with hepatocytes proliferation after DMN, while JNK activation was not associated with cholangiocytes proliferation after BDL. In HSC isolated from injured livers, protein kinases were differentially activated after BDL and DMN. Kinases activation in HSC in vivo preceded cell proliferation and alpha-smooth muscle actin appearance, a marker of HSC transformation in myofibroblast-like cells, and collagen deposition., Conclusions: Our findings indicate that these kinases are coordinately regulated during liver regeneration and suggest that their modulation could be considered as a future therapeutic approach in the management of liver damage.

Background/aims: Pirfenidone has been recently shown to reduce dimethynitrosamine-induced liver fibrosis in the rat, but no information are available on the effect of this drug on cultured hepatic stellate cells (HSC)., Methods: HSC proliferation was evaluated by measuring bromodeoxyuridine incorporation; PDGF-receptor autophosphorylation, extracellular signal-regulated kinase (ERK1/2) and pp70(S6K) activation were evaluated by western blot; protein kinase C activation was evaluated by western blot and by ELISA; type I collagen accumulation and alpha1(I) procollagen mRNA expression were evaluated by ELISA and northern blot, respectively., Results: Pirfenidone significantly inhibited PDGF-induced HSC proliferation, starting at a concentration of 1 microM, with a maximal effect at 1000 microM, without affecting HSC viability and without inducing apoptosis. The inhibition of PDGF-induced HSC proliferation was associated neither with variations in PDGF-receptor autophosphorylation, or with ERK1/2 and pp70(S6K) activation. On the other hand, pirfenidone was able to inhibit PDGF-induced activation of the Na(+)/H(+) exchanger, which is involved in PDGF-induced HSC proliferation in HSC, with a maximal effect at 1000 microM and inhibited PDGF-induced protein kinase C activation. Pirfenidone 100 and 1000 microM inhibited type I collagen accumulation in the culture medium induced by transforming growth factor(beta1) by 54% and 92%, respectively, as well as TGF(beta1)-induced alpha1(I) procollagen mRNA expression., Results: Pirfenidone could be a new candidate for antifibrotic therapy in chronic liver diseases., (Copyright 2002 European Association for the Study of the Liver)

Background/aims: Cytoskeletal reorganization plays an important role in the regulation of different cell functions, such as proliferation and migration. Since platelet-derived growth factor (PDGF) stimulates both proliferation and chemotaxis of hepatic stellate cells (HSC), we investigated the effects of this cytokine on cytoskeletal components of cultured rat HSC., Methods/results: Exposure of HSC to PDGF induced the formation of stress fibres and of a ruffled configuration of the plasma membrane, evaluated by both fluorescence and electron microscopy. These modifications were also induced by exposure to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA) and abolished by pretreatment with the PKC inhibitor calphostin C, with the Rho inhibitor C3 exoenzyme and with the intracellular calcium chelator MAPTAM, but not with the PI-3 kinase inhibitor wortmannin or with the mitogen-activated protein kinase kinase inhibitor PD 98059. PDGF induced a translocation of Rho from the cytosol to the membrane which was inhibited by C3 exoenzyme and by calpostin C, and which was also induced by PMA. Moreover, PDGF induced a rearrangement of vinculin which was prevented by C3 exoenzyme and calphostin C., Conclusions: PDGF-induced cytoskeletal reorganization in HSC is dependent on PKC and Rho, thus suggesting that these two pathways may play an important role in the response of liver to injury.

Background/aims: The Na+/H+ exchanger is involved in rat hepatic stellate cell (HSC) proliferation induced by platelet-derived growth factor (PDGF). We therefore evaluated in human HSC: (1) the mechanisms of intracellular pH regulation; (2) the relationship between Na+/H+ exchange activation and cell proliferation induced by PDGF, insulin-like growth factor 1 (IGF-1) and insulin., Methods/results: pH(i) regulation was mainly dependent on the activity of the Na+/H+ exchanger, which was evaluated by measuring pH(i) recovery from an acute acid load. PDGF (25 ng/ml) gradually increased the activity of the Na+/H+ exchanger which peaked at 18 h and remained stable until the 24th h. IGF-1 (10 nmol/l), but not insulin (100 nmol/l), slightly but significantly increased the activity of the Na+/H+ exchanger. Amiloride (100 micromol/l) and 20 micromol/l 5-N-ethyl-N-isopropyl-amiloride completely inhibited HSC proliferation (evaluated by measurement of bromodeoxyuridine incorporation) induced by PDGF and IGF-1, but did not affect proliferation of HSC induced by insulin. Finally, IGF-1 did not modify the activity of the Na+/Ca2+ exchanger., Conclusions: The Na+/H+ exchanger is involved in HSC proliferation induced by PDGF and IGF-1, whereas the proliferative effect of insulin is mediated by intracellular pathways which are Na+/H+ exchange-independent.