Biliary Proliferation Research Articles

Molecular mechanism of cholangiocarcinoma carcinogenesis.

Cholangiocarcinoma (CCA) is a highly malignant cancer of the biliary tract with a poor prognosis, which often arises from conditions causing long-term inflammation, injury, and reparative biliary epithelial cell proliferation. Several conditions are known to be major risk factors for cancer in the biliary tract or gallbladder, including primary sclerosing cholangitis, liver fluke infection, pancreaticobiliary maljunction, and chemical exposure in proof-printing workers. Abnormalities in various signaling cascades, molecules, and genetic mutations are involved in the pathogenesis of CCA. CCA is characterized by a series of highly recurrent mutations in genes, including KRAS, BRF, TP53, Smad, and p16(INK4a) . Cytokines that are affected by inflammatory environmental conditions, such as interleukin-6 (IL-6), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), and platelet-derived growth factor (PDGF), play an important role in cancer pathogenesis. Prominent signaling pathways important in carcinogenesis include TGF-β/Smad, IL-6/STAT-3, PI3K/AKT, Wnt, RAF/MEK/MAPK, and Notch. Additionally, some microRNAs regulate targets in critical pathways of CCA development and progression. This review article provides the understanding of the genetic and epigenetic mechanism(s) of carcinogenesis in CCA, which leads to the development of new therapeutic targets for the prevention and treatment of this devastating cancer.

Prolonged administration of secretin to normal rats increases biliary proliferation and secretin-induced ductal secretory activity.

Cholangiocyte proliferation is coordinately regulated by a number of gastrointestinal hormones/peptides, some of which display stimulatory effects and some have inhibitory actions on cholangiocyte proliferation. Enhanced biliary proliferation [for example after bile duct ligation (BDL) and partial hepatectomy] is associated with increased expression of secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR) and Cl(-)/HCO3 (-) anion exchanger 2 and secretin-stimulated ductal secretion, whereas loss/damage of bile ducts [for example after acute carbon tetrachloride (CCl4) administration] is associated with reduced secretin-stimulated ductal secretory activity. There is growing information regarding the role of gastrointestinal hormones the regulation of biliary growth. For example, while gastrin, somatostatin and serotonin inhibit bile duct hyperplasia of cholestatic rats by downregulation of cAMP signaling, secretin has been shown to stimulate the proliferation of normal mice by activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signaling. However, no information exists regarding the stimulatory effects of secretin on biliary proliferation of normal rats. Thus, we evaluated the in vivo and in vitro effect of secretin on biliary proliferation, the expression of markers key of ductal secretion and secretin-stimulated ductal secretion. Normal male rats were treated with saline or secretin (2.5 nmoles/kg BW/day by osmotic minipumps for one week). We evaluated: (I) intrahepatic bile duct mass (IBDM) in liver sections and PCNA expression in purified cholangiocytes; (II) SR and CFTR mRNA expression and secretin-stimulated cAMP levels in purified cholangiocytes; and (III) secretin-stimulated bile and bicarbonate secretion in bile fistula rats. In vitro, normal rat intrahepatic cholangiocyte lines (NRIC) were treated with BSA (basal) or secretin (100 nM) for 24 to 72 hours in the absence/presence of a PKA or a MEK inhibitor before evaluating proliferation by MTS assays. Prolonged administration of secretin to normal rats increased IBDM and PCNA expression in purified cholangiocytes compared to saline-treated normal rats. Also, secretin increased the expression of proteins (SR and CFTR) that are key in the regulating ductal secretion and enhanced secretin-stimulated cAMP levels and bile and bicarbonate secretion. In vitro, secretin increased the proliferation of NRIC, increase that was prevented by PKA and MAPK inhibitors. We have demonstrated that secretin stimulates both in vivo and in vitro biliary proliferation and secretin-stimulated ductal secretory activity in normal rats. We suggest that the stimulatory effect of secretin on biliary proliferation and secretion may be important for preventing biliary dysfunction during ductopenic disorders.

Seneciosis in cattle associated with photosensitization

Senecio spp. poisoning is the main cause of cattle mortality in the central region of Rio Grande do Sul. This paper reports an outbreak of seneciosis in cattle with high prevalence of photosensitization, where 83 out of 162 cows (51.3%) presented this clinical sign. The outbreak occurred in September 2013, affecting adult cows that were held in a 205 hectare-pasture from April to October 2013 with abundant Senecio brasiliensis infestation. Main clinical signs were weight loss, excessive lacrimation or mucopurulent ocular discharge, nasal serous discharge, ventral diphteric glossitis, crusts in the nose, teats, dorsum of ears, and vulva. Liver biopsy was performed in all the cows under risk; the histopathological findings in the liver biopsies consisted of fibrosis, megalocytosis, and biliary ductal proliferation and were present in 73.4% of the biopsied animals. Six cows had increased serum activity of gamma glutamyl transferase. Three affected cows were necropsied. The main necropsy findings were a hard liver, distended gall bladder, edema of the mesentery and abomasum. Liver histological changes in the necropsied cows were similar to those of the biopsied livers. Spongiosis was detected in the brain of necropsied cows and is characteristic of hepatic encephalopathy.

Sa1699 Local Inhibition of Hepatic GnRH by Vivo-Morpholino Reduces Biliary Proliferation and Ameliorates the Expression of Fibrotic Markers in Cholestatic Rats

A S L D A b st ra ct s increase in circulating Galanin in the serum. Galanin immunoreactivity was observed in both cholangiocytes and hepatocytes, whereas GalR1 was found predominantly in cholangiocytes. Systemic treatment of rats with M617 increased both CK-19 expression and IBDM in both sham and BDL-treated animals. In vitro, treatment of the mouse cholangiocyte cell line with M617 increased ERK1/2 and RSK-1 activity. There was a concomitant increase in cholangiocyte proliferation after M617 treatment that could be blocked by pretreatment with inhibitors for ERK1/2 and RSK-1. Conclusions: Data presented here demonstrate a direct stimulatory role for Galanin on cholangiocyte proliferation under physiological (sham) and pathological (BDL) conditions via a mechanism involving the activation of ERK1/2 and subsequent phosphorylation of RSK-1. Targeting Galanin or GalR1 may prove a useful strategy to regulate biliary mass during cholestatic liver injury.

Secretin Stimulates Biliary Cell Proliferation by Regulating Expression of MicroRNA 125b and MicroRNA let7a in Mice

Proliferating cholangiocytes secrete and respond to neuroendocrine hormones, including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice. Cholestasis was induced in secretin knockout (Sct(-/-)) and wild-type (control) mice by bile duct ligation (BDL). At days 3 and 7 after BDL, control and Sct(-/-) mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected. Immunohistochemical, immunoblot, luciferase reporter, and real-time polymerase chain reaction assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells and in serum and bile. Secretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile after BDL in control mice. BDL Sct(-/-) mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor (VEGF) A and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA, and those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue. After liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in up-regulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases.

Inhibition of the liver expression of arylalkylamine N-acetyltransferase increases the expression of angiogenic factors in cholangiocytes.

Reduction of biliary serotonin N-acetyltransferase (AANAT) expression and melatonin administration/secretion in cholangiocytes increases biliary proliferation and the expression of SR, CFTR and Cl(-)/HCO3 (-) AE2. The balance between biliary proliferation/damage is regulated by several autocrine neuroendocrine factors including vascular endothelial growth factor-A/C (VEGF-A/C). VEGFs are secreted by several epithelia, where they modulate cell growth by autocrine and paracrine mechanisms. No data exists regarding the effect of AANAT modulation on the expressions of VEGFs by cholangiocytes. In this study, we evaluated the effect of local modulation of biliary AANAT expression on the cholangiocytes synthesis of VEGF-A/C. The decrease in AANAT expression and subsequent lower melatonin secretion by cholangiocytes was associated with increased expression of VEGF-A/C. Overexpression of AANAT in cholangiocyte lines decreased the expression of VEGF-A/C. Modulation of melatonin synthesis may affect the expression of VEGF-A/C by cholangiocytes and may modulate the hepatic microvascularization through the regulation of VEGF-A/C expression regulating biliary functions.

HISTOPATHOLOGY ASPECTS AND FIBROSIS EVALUATION OF BOVINE NATURALLY INFECTED LIVERS BY Fasciola hepatica

Fasciolose é uma zoonose que infecta especialmente os ruminantes e os danos ao fígado causado pela infecção crônica pelo parasito são graves. Objetiva-se com este estudo avaliar os achados histopatológicos no fígado de bovinos naturalmente infectados com Fasciola hepatica, bem como propor a classificação de lesão hepática causada por essa doença baseado na inflamação crônica e fibrose. Foram utilizados fragmentos do lobo direito e esquerdo de 100 fígados bovinos com fasciolose. Os fragmentos foram submetidos a processamento histológico e coloração com HE e Tricrômico de Masson. A classificação das lesões foi realizada por meio da atribuição de uma classificação gradual de I a III, de acordo com a intensidade das alterações inflamatórias e fibróticas. As lesões mais prevalentes foram a proliferação do ducto biliar, infiltração de células mononucleares, arteriosclerose e fibrose periportal e periductal. Lesões fibróticas foram mais intensas do que a inflamação e 45% das amostras pertenciam à categoria II, seguido de 39% na categoria I e 16% na categoria III. A proposta de categorização histopatológica da lesão hepática em fasciolose crônica em bovinos naturalmente infectados provou ser um método valioso de avaliação microscópica, uma vez que permite determinar a evolução da doença.

Protective Effects of Guava Pulp on Cholestatic Liver Injury.

Background. Cholestatic liver injury is a leading cause of chronic liver diseases involved with oxidative stress changes and inflammation; thus, antioxidant and anti-inflammation compound-rich guava may play a pivotal role in protecting against the cholestatic liver damages. Our aims for this study are to determine whether guava pulp (GP) has protective effects on cholestatic liver injury-induced mouse model and on interleukin-6 (IL-6) mediated proliferation of QBC939 cholangiocarcinoma cell line. Methods. Mice were induced to cholestatic liver damage by left and median bile duct ligation (LMBDL) surgery and then treated with GP. Plasma and liver samples were collected for biochemical and pathological assays. 5-Bromo-2′-deoxyuridine (BrdU) assay and Western blots were used to detect proliferation and gene expression in QBC939 cells, respectively. Results. Compared with LMBDL only group, in GP-treated mice, the levels of alanine aminotransferase (ALT) and bilirubin decreased, biliary epithelial cell proliferation and liver fibrogenesis were suppressed, Src/MEK/ERK1/2/c-Myc pathway and expressions of transforming growth factor β1(TGF-β1), tissue inhibitor of metalloproteinases TIMP), and procollagen 1α1(COL1α1) were downregulated significantly. Moreover, the GP extract reduced IL-6-enhanced QBC939 cell proliferation, p-ERK, and c-Myc expression as well. Conclusions. GP may provide a new perspective for the treatment of cholestatic liver injury.

Nonselective inhibition of prostaglandin-endoperoxide synthases by naproxen ameliorates acute or chronic liver injury in animals

The rising prevalence of hepatic injury due to toxins, metabolites, viruses, etc., necessitates development of further mechanisms for protecting the liver and for treating acute or chronic liver diseases. To examine whether inhibition of inflammation is directed by cyclo-oxygenase pathways, we performed animal studies with naproxen, which inhibits prostaglandin-endoperoxide synthases 1 and 2 and is in extensive clinical use. We administered carbon tetrachloride to induce acute liver injury and ligated the common bile duct to induce chronic liver injury in adult rats. These experimental manipulations produced abnormalities in liver tests, tissue necrosis, compensatory hepatocyte or biliary proliferation, and onset of fibrosis, particularly after bile duct ligation. After carbon tetrachloride-induced acute injury, naproxen decreased liver test abnormalities, tissue necrosis and compensatory hepatocellular proliferation. After bile duct ligation-induced chronic injury, naproxen decreased liver test abnormalities, tissue injury and compensatory biliary hyperplasia. Moreover, after bile duct ligation, naproxen-treated rats showed more periductular oval liver cells, which have been classified as hepatic progenitor cells. In naproxen-treated rats, we found greater expression in hepatic stellate cells and mononuclear cells of cytoprotective factors, such as vascular endothelial growth factor. The ability of naproxen to induce expression of vascular endothelial growth factor was verified in cell culture studies with CFSC-8B clone of rat hepatic stellate cells. Whereas assays for carbon tetrachloride toxicity using cultured primary hepatocytes established that naproxen was not directly cytoprotective, we found conditioned medium containing vascular endothelial growth factor from naproxen-treated CFSC-8B cells protected hepatocytes from carbon tetrachloride toxicity. Therefore, naproxen was capable of ameliorating toxic liver injury, which involved naproxen-induced release of physiological cytoprotective factors in nonparenchymal liver cells. Such drug-induced release of endogenous cytoprotectants will advance therapeutic development for hepatic injury.

Liver Failure and the Need for Transplantation in 6 Patients With Hepatoportal Sclerosis

Hepatoportal sclerosis (HPS), first reported by Mikkelsen et al in 1965, is a pathologic condition that does not cause cirrhotic portal hypertension. The primary hepatic lesion in HPS is found in portal vein branches with preserved synthetic function. Rarely do patients with HPS need liver transplantation. The aim of this study was to describe the clinical and pathologic features of 6 HPS cases who underwent liver transplantation (OLT). From 2000 to 2008, 6 OLT candidates were diagnosed with HPS: 3 displayed bleeding varices and 4 ascites. Child-Pugh evaluation was class B (n = 4) or C (n = 2). The Model for End-stage Liver Disease scores were 18 (n = 2), 20 (n = 3), and 22 (n = 1). Cirrhosis resulted from presumed diagnoses of alcohol n = (1), autoimmune n = (2) or cryptogenic cirrhosis n = (3). On histologic examination, there was marked phlebosclerosis in all cases, including nonocclusive portal vein thrombosis (n = 3), intense portal fibrosis (n = 1), moderate portal fibrosis (n = 5), and uniform moderate sinusoidal dilatation without megasinusoid formation, but with ductal biliary proliferation and ductal biliary fibrosis in all cases. Cholestasis was observed in 1 and incomplete septal cirrhosis in 4 cases. None of the subjects showed histological features of the presumed underlying liver disease. The overall survival of this group was no different from that of other OLT patients. HPS causing hepatic failure may require liver transplantation. Fhlebosclerosis andportal fibrosis may contribute to the loss of hepatic synthesis leading to the need for hepatic transplant. Significant portal fibrosis and phlebosclerosis can contribute to hepatic parenchymal and posterior synthetic loss.

Chronic nicotine exposure stimulates biliary growth and fibrosis in normal rats.

Epidemiological studies have indicated smoking to be a risk factor for the progression of liver diseases. Nicotine is the chief addictive substance in cigarette smoke and has powerful biological properties throughout the body. Nicotine has been implicated in a number of disease processes, including increased cell proliferation and fibrosis in several organ systems. The aim of this study was to evaluate the effects of chronic administration of nicotine on biliary proliferation and fibrosis in normal rats. In vivo, rats were treated with nicotine by osmotic minipumps for two weeks. Proliferation, α7-nicotinic receptor and profibrotic expression were evaluated in liver tissue, cholangiocytes and a polarized cholangiocyte cell line (normal rat intrahepatic cholangiocyte). Nicotine-dependent activation of the Ca(2+)/IP3/ERK 1/2 intracellular signalling pathway was also evaluated in normal rat intrahepatic cholangiocyte. Cholangiocytes express α7-nicotinic receptor. Chronic administration of nicotine to normal rats stimulated biliary proliferation and profibrotic gene and protein expression such as alpha-smooth muscle actin and fibronectin 1. Activation of α7-nicotinic receptor stimulated Ca(2+)/ERK1/2-dependent cholangiocyte proliferation. Chronic exposure to nicotine contributes to biliary fibrosis by activation of cholangiocyte proliferation and expression of profibrotic genes. Modulation of α7-nicotinic receptor signalling axis may be useful for the management of biliary proliferation and fibrosis during cholangiopathies.

Omeprazole inhibits biliary proliferation during extrahepatic cholestasis

Omeprazole (OME) has been shown to reduce liver regeneration and enhance gastrin serum levels. Since gastrin inhibits biliary growth, we tested the hypothesis that omeprazole inhibits cholangiocyte proliferation in BDL rats by increasing gastrin serum levels.MethodsIn vivo, male Fischer 344 rats (immediately BDL) were treated with vehicle or OME (40 mg/Kg BW daily) for 1 wk. We measured changes in: liver histology, and intrahepatic bile duct mass in liver sections; H3 histone and PCNA and secretin receptor (SR) gene expression; basal and secretin‐stimulated cAMP levels in cholangiocytes bile and bicarbonate secretion in bile fistula rats. Changes in SR and secretin‐stimulated secretion are key functional tools for evaluating the degree of biliary hyperplasia.ResultsChronic in vivo administration of OME to BDL rats increased gastrin serum levels and reduced biliary proliferation and hyperplasia and decreased SR expression and significantly inhibited the stimulatory effects of secretin on cAMP levels and ductal secretion compared to vehicle treated controls.ConclusionThe usage of OME may be important for the management of chronic cholestatic liver diseases.

Histidine decarboxylase regulates biliary homeostasis via autocrine histamine signaling

Cholangiocyte homeostasis is regulated by both autocrine/paracrine signaling. In bile duct ligated (BDL) rats large cholangiocyte proliferate, whereas CCl4 induces large cholangiocyte apoptosis. Histidine decarboxylase (HDC) regulates histamine synthesis. We have shown that: (i) cholangiocytes express HDC and secrete histamine; (ii) histamine stimulates biliary growth; and (ii) HDC inhibition decreases biliary growth. No information exists that HDC/histamine regulates cholangiocyte homeostasis. In vivo, normal and 1 wk BDL rats were treated with saline or histamine (0.5 mg/kg) and given CCl4 by gavage (0.5 ml/100 gm). Liver blocks and large cholangiocytes were obtained. HDC, PCNA, TUNEL and CK‐19 biliary expression was measured by immunohistochemistry in liver sections and by qPCR and immunoblots in large cholangiocytes. In vitro, control (MSE‐neg) or stable transfected HDC knockdown large cholangiocytes (MSE‐HDC) were treated with CCl4 (5 mM) in the absence/presence of histamine (10 μM) before evaluating apoptosis and proliferation. After BDL, there was enhanced cholangiocyte proliferation, ductal mass and biliary HDC expression. CCl4 treatment (i) enhanced biliary apoptosis; and (ii) decreased HDC and serum/biliary histamine levels and biliary proliferation and ductal mass, changes that were prevented by histamine. In MSE‐HDC cells there was reduced biliary growth and enhanced apoptosis compared to Mz‐neg. In MSE‐HDC cells treated with CCl4, apoptosis increased compared to MSE‐neg cells treated with CCl4. In Mz‐HDC, CCl4‐induced apoptosis and loss of proliferation was prevented by histamine. HDC is a key regulator of cholangiocyte homeostasis and histamine may be a protective factor after toxic injury. Treatment of cholangiopathies characterized by proliferation or loss may be regulated by the modulation of histamine levels.

Modulation of the biliary expression of arylalkylamine N-acetyltransferase alters the autocrine proliferative responses of cholangiocytes in rats

Secretin stimulates ductal secretion by interacting with secretin receptor (SR) activating cyclic adenosine 3',5'-monophosphate/cystic fibrosis transmembrane conductance regulator/chloride bicarbonate anion exchanger 2 (cAMP⇒CFTR⇒Cl(-) /HCO 3- AE2) signaling that is elevated by biliary hyperplasia. Cholangiocytes secrete several neuroendocrine factors regulating biliary functions by autocrine mechanisms. Melatonin inhibits biliary growth and secretin-stimulated choleresis in cholestatic bile-duct-ligated (BDL) rats by interaction with melatonin type 1 (MT1) receptor through down-regulation of cAMP-dependent signaling. No data exist regarding the role of melatonin synthesized locally by cholangiocytes in the autocrine regulation of biliary growth and function. In this study, we evaluated the (1) expression of arylalkylamine N-acetyltransferase (AANAT; the rate-limiting enzyme for melatonin synthesis from serotonin) in cholangiocytes and (2) effect of local modulation of biliary AANAT expression on the autocrine proliferative/secretory responses of cholangiocytes. In the liver, cholangiocytes (and, to a lesser extent, BDL hepatocytes) expressed AANAT. AANAT expression and melatonin secretion (1) increased in BDL, compared to normal rats and BDL rats treated with melatonin, and (2) decreased in normal and BDL rats treated with AANAT Vivo-Morpholino, compared to controls. The decrease in AANAT expression, and subsequent lower melatonin secretion by cholangiocytes, was associated with increased biliary proliferation and increased SR, CFTR, and Cl(-) /HCO 3- AE2 expression. Overexpression of AANAT in cholangiocyte cell lines decreased the basal proliferative rate and expression of SR, CFTR, and Cl(-) /HCO 3- AE2 and ablated secretin-stimulated biliary secretion in these cells. Local modulation of melatonin synthesis may be important for management of the balance between biliary proliferation/damage that is typical of cholangiopathies. (HEPATOLOGY 2013).

Involvement of ERK1/2 signaling in proliferation of eight liver cell types during hepatic regeneration in rats

It has been well established that ERK1/2 signaling, often subdivided into nine types of pathways, can regulate the hepatocyte proliferative response during liver regeneration. However, the effect of ERK1/2 signaling on the proliferation of other hepatic cell types remains unclear. We isolated and purified 8 liver cell types at 10 time points after 2/3 hepatectomy in adult rats. For each cell type, mRNA expression changes for ERK1/2 signaling-involved genes were monitored up to 168 h, using microarrays. Real-time PCR assays were performed for array data verification. The expression levels of these genes varied considerably between different cell types. Integrating microarray results with gene synergical analysis, at the priming phase, activation of integrin/Grb2/Ras pathway in hepatocytes apparently contributed to G0/G1 transition. Two other pathways, G-protein/EPAC/Rap1 and G-protein/PKA/Rap1, were stimulated in hepatic stellate cells, while RTK/PKC/Ras and RTK/Grb2/Ras were stimulated in Kupffer cells. At the progressive phase, the ERK1/2 pathway is involved in hepatocyte replication; three pathways, namely Ca(2+)/PKC/Ras, RTK/Grb2/Ras and G-protein/EPAC/Rap1, were found to play roles in biliary epithelial cell proliferation, while RTK/PKC/Ras and RTK/Grb2/Ras were involved in Kupffer cell proliferation, and G-protein/PKC/Ras in pit cell proliferation. At the terminal phase, the promotive effect of the ERK1/2 pathway on replication of hepatocytes, biliary epithelial cells, oval cells, hepatic stellate cells, Kupffer cells, and dendritic cells was considerably reduced, possibly due to their differentiation at the end of regeneration. G-protein/PKC/Ras, integrin/Grb2/Ras and G-protein/ PKA/Rap1 pathways were active in sinusoidal endothelial cells, perhaps to aid in their proliferation. We conclude that ERK1/2 has a signaling role in the regulation of proliferation of 8 cell types during liver regeneration process.

Notch‐dependent expression of epithelial–mesenchymal transition markers in cholangiocytes after liver transplantation

Epithelial-mesenchymal transition (EMT) has been identified in chronic cholestatic liver diseases, which are characterized by biliary proliferation and fibrosis. Activation of Notch signaling mediates EMT in a variety of epithelial cell types. In the present study, we investigated the role of Notch signaling in the regulation of EMT marker expression in cholangiocytes after liver transplantation. Orthotopic liver transplantation was performed in Sprague-Dawley rats. Liver tissues and isolated cholangiocytes were collected 1 week after transplantation. The expression of mesenchymal and biliary epithelial markers was evaluated by immunohistochemistry, quantitative polymerase chain reaction (PCR) and western blotting in liver sections and isolated cholangiocytes. Quantitative real-time PCR and western blotting for Jagged1 and HES1 were utilized to evaluate the activation of Notch signaling. Proliferation and migration of cholangiocytes were assessed by 5-bromodeoxyuridine and transwell assays, respectively. Cholangiocyte proliferation, migration and expression of EMT markers were also evaluated following the inhibition of Notch signaling with N,(N-[3,5-difluorophenacetyl]-L-alanyl)-S-phenylglycine t-butylester (γ-secretase inhibitor) and a Jagged1-neutralizing antibody. Expression of EMT markers by cholangiocytes was observed in liver grafts and isolated cholangiocytes obtained 1 week after transplantation. Inhibition of Notch signaling prevented the expression of EMT markers in bile ducts of liver sections and isolated cholangiocytes. Cholangiocyte proliferative and migratory capacities were also suppressed by the inhibition of Notch signaling. Activation of Notch signaling promotes cholangiocyte proliferation and expression of EMT markers after liver transplantation.

Impact of Hepatic Arterial Reconstruction on Orthotopic Liver Transplantation in the Rat

ABSTRACTOrthotopic liver transplantation (OLT) models in rats have been investigated in many studies, but detailed information on the impact of hepatic artery (HA) reconstruction on postoperative factors remains to be investigated. HA reconstruction also requires advanced skills. The effect of the reconstruction of the HA by a hand-suture technique in rats with a whole-liver syngeneic graft was investigated. Long-term survival, histopathological assessment, immunohistological evaluation, and blood biochemistry were investigated until postoperative day (POD) 28. From the early postoperative period, significant differences between OLTs with or without HA reconstruction were found in graft parenchymal damage, induction of apoptosis, and transaminase levels, though survival curves and the coagulation profile showed no differences. In OLT without HA reconstruction, biliary proliferation was decreased at POD 5–14, and total bilirubin level was increased at PODs 10 and 14. The study indicates that HA reconstruction is required for reliable OLT in rats.

Reduced proteoglycans, cystic disease and primary cilia

Most cells produce proteoglycans. These carbohydrate moieties on these specialized proteins is assembled on specific core protein serines and initiates with a xylose. The enzyme responsible for this is xylosyltransferase and exists in two isoforms. Xylosyltransferase 2 (XylT2) is ubiquitously expressed in many organs suggesting a significant role of XylT2 dependent proteoglycans in those organs. We have shown that XylT2 deficiency causes substantial reduction in liver proteoglycans resulting in cyst development in the biliary tract and in the kidney there is fibrosis and tubule dilation. These findings are similar to those in polycystic kidney disease (PKD). In the liver, biliary epithelial proliferation and fibrosis accompany cyst development. How or if these contribute to cyst formation is unknown, but in other cystic animal models and in patients who suffer from PKD, primary cilia defects occur. Therefore, we determined if primary cilia are abnormal in our cystic model and found that indeed there are cilia abnormalities. These findings provide further evidence illustrating that primary cilia defects can contribute to or may directly cause cystic disease. What is the actual cause of the cilia defects in the Xylt2−/− mice is unknown but is under investigation.

Helicobacter pylori in Thai patients with cholangiocarcinoma and its association with biliary inflammation and proliferation

ObjectivesTo investigate whether Helicobacter spp. infection and the cagA of H. pylori are associated with hepatobiliary pathology, specifically biliary inflammation, cell proliferation and cholangiocarcinoma (CCA). MethodsHelicobacter species including H. pylori, H. bilis and H. hepaticus were detected in the specimens using the polymerase chain reaction (PCR). Biliary inflammation of the liver and gallbladders was semi-quantitatively graded on hematoxylin and eosin (H&E)-stained slides. Biliary proliferation was evaluated by immunohistochemistry using the Ki-67-labelling index. ResultsHelicobacter pylori was found in 66.7%, 41.5% and 25.0% of the patients in the CCA, cholelithiasis and control groups (P < 0.05), respectively. By comparison, H. bilis was found in 14.9% and 9.4% of the patients with CCA and cholelithiasis, respectively (P > 0.05), and was absent in the control group. The cagA gene of H. pylori was detected in 36.2% and 9.1% of the patients with CCA and cholelithiasis, respectively (P < 0.05). Among patients with CCA, cell inflammation and proliferation in the liver and gallbladder were significantly higher among those DNA H. pylori positive than negative. ConclusionsThe present findings suggest that H. pylori, especially the cagA-positive strains, may be involved in the pathogenesis of hepatobiliary diseases, especially CCA through enhanced biliary cell inflammation and proliferation.

Histamine regulation of biliary proliferation

Cholangiocytes, which line the biliary epithelium, are hormone-responsive and responsible for the modification of canalicular bile before reaching the duodenum. Cholangiocytes are the target cells in cholangiopathies such as primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) that are characterized by cholangiocyte proliferation/loss [1]. The biliary epithelium is morphologically and functionally heterogeneous and is lined by small (<15μm in diameter) and large (>15μm in diameter) cholangiocytes lining small and large bile ducts, respectively [2].