Interobserver agreement in contrast harmonic endoscopic ultrasound.

BACKGROUND AND AIM: Contrast harmonic endoscopic ultrasound (CH-EUS) was recently introduced to clinical practice; its reproducibility among endosonographers is unknown. Our aim was to assess the interobserver agreement (IA) in CH-EUS. METHODS: Fifteen endosonographers (eight experienced and seven non-experienced) from 11 Italian EUS centers evaluated 80 video-cases (40 solid pancreatic lesions, 20 pancreatic cystic lesions and 20 submucosal lesions) of CH-EUS, according to the degree of enhancement, the pattern of distribution and the washout of the contrast agent. IA within each group and between the two groups of observers was assessed with the Fleiss kappa statistic. RESULTS: Overall IA was moderate for the uptake and fair for the pattern of distribution and the washout. In solid pancreatic lesions, IA was moderate for the uptake and fair for the pattern and the washout. In cystic pancreatic lesions, IA was uniformly moderate for the assessment of uptake, slight for the pattern and fair for the washout. In submucosal tumors, IA was substantial for the uptake, slight for the pattern and fair for the washout. Non-experienced endosonographers demonstrated, in most cases, comparable IA with the experienced ones. CONCLUSIONS: Interobserver agreement among endosonographers for CH EUS was satisfactory. In particular, overall IA varied from slight to substantial, being fair in the majority of cases. Inherent structural features of the lesions, as well as technical differences between the variables assessed, could have accounted for the fluctuation of the results. Outcomes of IA were reproducible between experienced and non-experienced endosonographers.

Contrast harmonic echo-endoscopic ultrasound improves accuracy in diagnosis of solid pancreatic masses.

BACKGROUND & AIMS: Contrast harmonic echo (CHE) has been developed for endoscopic ultrasound (EUS). This new technique detects echo signals from microbubbles in vessels with very slow flow, without artifacts. We assessed whether CHE-EUS increases the accuracy of diagnosis of pancreatic solid lesions. METHODS: At a tertiary-care EUS center, we examined 90 patients who were suspected of having pancreatic solid neoplasm. Radial and linear echoendoscopes were used with dedicated software for CHE. Sonovue (Bracco International BV, Amsterdam, The Netherlands) uptake, pattern, and washout were studied; data were compared for pancreatic lesions and adjacent parenchyma. The final diagnosis was obtained based on results of surgical pathology and/or EUS-fine needle aspiration (FNA) analyses. RESULTS: The finding of a hypoenhancing mass with an inhomogeneous pattern was a sensitive and accurate identifier of patients with adenocarcinoma (96% and 82%, respectively) (49 of 51 patients with primary pancreatic adenocarcinoma had a hypoenhancing mass that was inhomogeneous and had fast washout). This finding was more accurate in diagnosis than the finding of a hypoechoic lesion using standard EUS (P < .000). Hyperenhancement specifically excluded adenocarcinoma (98%), although sensitivity was low (39%). Of neuroendocrine tumors, 11 of 13 were non-hypo-enhancing (9 hyperenhancing, 2 isoenhancing). Interestingly, CHE-EUS allowed detection of small lesions in 7 patients who had uncertain standard EUS findings because of biliary stents (n = 5) or chronic pancreatitis (n = 2). Targeted EUS-FNA was performed on these lesions. CONCLUSIONS: Detection of a hypoenhancing and inhomogeneous mass accurately identified patients with pancreatic adenocarcinoma. CHE-EUS increased the detection of malignant lesions in difficult cases (patients with chronic pancreatitis or biliary stents) and helped guide EUS-FNA. A hyperenhancing pattern could be used to rule out adenocarcinoma.

New insights on the molecular and cell biology of human cholangiopathies.

Cholangiopathies are diseases of high social impact representing the main indication for liver transplantation in the infanthood and the third in adulthood. Despite the heterogeneous etiology and pathogenesis, cholangiopathies share many different common morphological features and, chronically progress toward a ductupenic condition clinically evidenced by the classical features of a cholestatic syndrome. The primary target of damage in the course of cholangiopathies are cholangiocytes, the epithelia cells lining the biliary tree. A bulk of researches performed in the last decade, highlighted the extraordinary biological properties of cholangiocytes involved in a number of important processes such as bile formation, proliferation, injury repair, fibrosis, angiogenesis and regulation of blood flow. Recent advances on the molecular and cell biology of human cholangiopathies are opening new potential therapeutic perspectives for these diseases.

Serum and biliary insulin-like growth factor I and vascular endothelial growth factor in determining the cause of obstructive cholestasis.

BACKGROUND: Cholangiocarcinoma cells express and secrete insulin-like growth factor I (IGF-I) and vascular endothelial growth factor (VEGF). OBJECTIVE: To measure IGF-I and VEGF in bile and serum of patients with extrahepatic cholangiocarcinoma and to evaluate their performance as diagnostic markers. DESIGN: Cross-sectional study. SETTING: Inpatients at the Division of Gastroenterology, University Hospital, Ancona, Italy. PATIENTS: 73 patients who consecutively had endoscopic retrograde cholangiopancreatography (ERCP), including patients with extrahepatic cholangiocarcinoma (n = 29), pancreatic cancer (n = 19), and benign biliary abnormalities (n = 25; bile duct stones, primary sclerosing cholangitis, and cholangitis). MEASUREMENTS: Diagnosis was based on conventional radiology, ERCP, and follow-up. Insulin-like growth factor I and VEGF were measured by using enzyme-linked immunosorbent assay. RESULTS: The biliary IGF-I concentration was 15- to 20-fold higher (P < 0.001) in extrahepatic cholangiocarcinoma (mean, 84.6 nmol/L [95% CI, 74.0 to 95.2 nmol/L]) than in pancreatic cancer (5.8 nmol/L [CI, 4.0 to 7.5 nmol/L]) or benign biliary abnormalities (4.1 nmol/L [CI, 3.1 to 5.2 nmol/L]). The area under the receiver-operating characteristic curve was 1 when biliary IGF-I values in the extrahepatic cholangiocarcinoma were compared with benign biliary abnormalities or pancreatic cancer. In contrast, biliary VEGF concentration was similar in the 3 groups. Serum IGF-I levels were similar among the groups, whereas serum VEGF levels were higher in the cholangiocarcinoma (0.97 ng/mL [CI, 0.59 to 1.35 ng/mL]; P = 0.0016) and pancreatic cancer groups (0.66 ng/mL [CI, 0.43 to 0.88 ng/mL]; P < 0.001) compared with patients with benign biliary abnormalities (0.28 ng/mL [CI, 0.17 to 0.37 ng/mL]). LIMITATIONS: Data were obtained in a small sample, the study was performed in a single center, and few patients had a tissue diagnosis. CONCLUSIONS: Biliary IGF-I levels in patients undergoing ERCP for biliary obstruction may differentiate extrahepatic cholangiocarcinoma from either pancreatic cancer or benign biliary abnormalities.

Activation of the IGF1 system characterizes cholangiocyte survival during progression of primary biliary cirrhosis.

We evaluated the IGF1 system in cholangiocytes of primay biliary cirrhosis (PBC) patients and investigated the relationships with apoptosis. Biopsies of PBC patients (n=32) and normal subjects (n=5) were investigated by immunohistochemistry for expression in cholangiocytes of IGF1, IGF1-R, pAKT, terminal deoxynucleotide transferase end labeling (TUNEL), Bax (proapoptotic protein), and Bcl2 (antiapoptotic protein). Whereas normal cholangiocytes were almost negative, cholangiocytes of PBC patients showed strong IHC staining for IGF1, IGF1-R, and pAKT, which increases from stage I to stage IV, where >70% of cholangiocytes were positive. Bax/Bcl2 ratio reached the highest value (4.6) in PBC stage III when apoptosis is maximal (24% TUNEL positivity), whereas it declines in stage IV (1.4) when only 7.8% cholangiocytes were TUNEL positive. In PBC stages III and IV, expression of IGF1, IGF1-R, and pAKT in cholangiocytes was directly correlated with the antiapoptotic Bcl2 and inversely correlated with proapoptotic Bax, Bax/Bcl2 ratio, and TUNEL positivity. In conclusion, cholangiocytes of PBC patients showed a marked increase in IGF1, IGF1-R, and pAKT expression involving most cholangiocytes surviving in the terminal ductopenic stage. This was associated and correlated with a balance of pro- and antiapoptotic proteins favoring survival rather than apoptosis, suggesting a major role of IGF1 system in promoting cholangiocyte survival.

[Pathophysiology of cholangiocytes: recent advances]. / Sulla fisiopatologia dei colangiociti. Recenti acquisizioni.

A bulk of researches performed in the last decade highlighted the extraordinary biological properties of cholangiocytes involved in a number of important processes such as bile formation, proliferation, injury repair, fibrosis, angiogenesis and regulation of blood flow. Cholangiocytes proliferation is a key mechanism capable of conditioning the evolution of liver damage. Proliferating cholangiocytes, in fact, acquire the phenotype of neuroendocrine cells and secrete different substances which represent the tools of cross-talk with other hepatic cells. Recent advances on the molecular and cell biology of cholangiocytes are opening new potential therapeutic perspectives for the human chronic liver diseases.

Estrogens and the pathophysiology of the biliary tree.

The scientific framework concerning estrogen effects on different tissues has expanded enormously during the last decades, when estrogen receptor (ER) subtypes were identified. Estrogens are not only essential for the female reproductive system, but they also control fundamental functions in other tissues including the cardiovascular system, bone, brain and liver. Recently, estrogens have been shown to target the biliary tree, where they modulate the proliferative and secretory activities of cholangiocytes, the epithelial cells lining bile ducts. By acting on both estrogen receptors (ER-alpha) and (ER-beta) subtypes, and by activating either genomic or non-genomic pathways, estrogens play a key role in the complex loop of growth factors and cytokines, which modulates the proliferative response of cholangiocytes to damage. Specifically, estrogens activate intracellular signalling cascades [ERK(1/2) (extracellular regulated kinases (1/2), PI3- kinase/AKT (phosphatidylinositol-3' kinase/AKT)] typical of growth factors such as insulin like growth factor (IGF1), nerve growth factor (NGF) and vascular endothelial growth factor (VEGF), thus potentiating their action. In addition, estrogens stimulate the secretion of different growth factors in proliferating cholangiocytes. This review specifically deals with the recent advances related to the role and mechanisms by which estrogens modulate cholangiocyte functions in normal and pathological conditions.

Morphological and functional features of hepatic cyst epithelium in autosomal dominant polycystic kidney disease.

We evaluated the morphological and functional features of hepatic cyst epithelium in adult autosomal dominant polycystic kidney disease (ADPKD). In six ADPKD patients, we investigated the morphology of cyst epithelium apical surface by scanning electron microscopy and the expression of estrogen receptors (ERs), insulin-like growth factor 1 (IGF1), IGF1 receptors (IGF1-R), growth hormone receptor, the proliferation marker proliferating cell nuclear antigen, and pAKT by immunohistochemistry and immunofluorescence. Proliferation of liver cyst-derived epithelial cells was evaluated by both MTS proliferation assay and [(3)H]thymidine incorporation into DNA. The hepatic cyst epithelium displayed heterogeneous features, being normal in small cysts (<1 cm), characterized by rare or shortened cilia in 1- to 3-cm cysts, and exhibiting the absence of both primary cilia and microvilli in large cysts (>3 cm). Cyst epithelium showed marked immunohistochemical expression of ER, growth hormone receptor, IGF1, IGF1-R, proliferating cell nuclear antigen, and pAKT. IGF1 was 10-fold more enriched in the hepatic cyst fluid than in serum. Serum-deprived liver cyst-derived epithelial cells proliferated when exposed to 17beta-estradiol and IGF1 and when exposed to human cyst fluid. ER or IGF1-R antagonists inhibited the proliferative effect of serum readmission, cyst fluid, 17beta-estradiol, and IGF1. Our findings could explain the role of estrogens in accelerating the progression of ADPKD and may suggest a potential benefit of therapeutic strategies based on estrogen antagonism.

Hepatic "stem" cells: state of the art.

As previously showed for the classical self renewing tissues (i.e., bone marrow, gut and skin), rapidly changing concepts about tissue stem cell biology identify the liver as a maturational lineage system capable to generate mature cells (hepatocytes and cholangiocytes) from a resident stem cells compartment localized near the so called Canals of Hering. At present liver transplantation is the only available therapy for end stage liver diseases and there is an ever increasing shortage of donor livers. Thus in the last decades, HPC has becoming the object of many researchers since HPC might offer a new therapeutic approach for controlling the evolution of chronic liver diseases. The aim of the present review is to update readers with the evolving concepts about hepatic stem cells biology, their characterization and isolation methods and finally their therapeutic potential.

Proliferating cholangiocytes: a neuroendocrine compartment in the diseased liver.

In the last 15 years, the intrahepatic biliary tree has become the object of extensive studies, which highlighted the extraordinary biologic properties of cholangiocytes involved in bile formation, proliferation, injury repair, fibrosis, angiogenesis, and regulation of blood flow. Proliferation is a "typical" property of cholangiocytes and is key as a mechanism of repair responsible for maintaining the integrity of the biliary tree. Cholangiocyte proliferation occurs virtually in all pathologic conditions of liver injury where it is associated with inflammation, regeneration, and repair, thus conditioning the evolution of liver damage. Interestingly, proliferating cholangiocytes acquire the phenotype of neuroendocrine cells, and secrete different cytokines, growth factors, neuropeptides, and hormones, which represent potential mechanisms for cross talk with other liver cells. Many studies suggest the generation of a neuroendocrine compartment in the injured liver, mostly constituted by cells with cholangiocyte features, which functionally conditions the progression of liver disease. These insights on cholangiocyte pathophysiology will provide new potential strategies for the management of chronic liver diseases. The purpose of this review is to summarize the recent findings on the mechanisms regulating cholangiocyte proliferation and the significance of the neuroendocrine regulation of cholangiocyte biology.

The alpha2-adrenergic receptor agonist UK 14,304 inhibits secretin-stimulated ductal secretion by downregulation of the cAMP system in bile duct-ligated rats.

Secretin stimulates ductal secretion by activation of cAMP --> PKA --> CFTR --> Cl(-)/HCO(3)(-) exchanger in cholangiocytes. We evaluated the expression of alpha(2A)-, alpha(2B)-, and alpha(2C)-adrenergic receptors in cholangiocytes and the effects of the selective alpha(2)-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)/H(+) exchanger isoform NHE3; and 3) cAMP levels, PKA activity, Cl(-) efflux, and Cl(-)/HCO(3)(-) exchanger activity in purified cholangiocytes. alpha(2)-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, Cl(-) efflux, and Cl(-)/HCO(3)(-) exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. alpha(2)-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases.

Ca2+-dependent cytoprotective effects of ursodeoxycholic and tauroursodeoxycholic acid on the biliary epithelium in a rat model of cholestasis and loss of bile ducts.

Chronic cholestatic liver diseases are characterized by impaired balance between proliferation and death of cholangiocytes, as well as vanishing of bile ducts and liver failure. Ursodeoxycholic acid (UDCA) is a bile acid widely used for the therapy of cholangiopathies. However, little is known of the cytoprotective effects of UDCA on cholangiocytes. Therefore, UDCA and its taurine conjugate tauroursodeoxycholic acid (TUDCA) were administered in vivo to rats simultaneously subjected to bile duct ligation and vagotomy, a model that induces cholestasis and loss of bile ducts by apoptosis of cholangiocytes. Because these two bile acids act through Ca2+ signaling, animals were also treated with BAPTA/AM (an intracellular Ca2+ chelator) or Gö6976 (a Ca2+-dependent protein kinase C-alpha inhibitor). The administration of UDCA or TUDCA prevented the induction of apoptosis and the loss of proliferative and functional responses observed in the bile duct ligation-vagotomized rats. These effects were neutralized by the simultaneous administration of BAPTA/AM or Gö6976. UDCA and TUDCA enhanced intracellular Ca2+ and IP3 levels, together with increased phosphorylation of protein kinase C-alpha. Parallel changes were observed regarding the activation of the MAPK and PI3K pathways, changes that were abolished by addition of BAPTA/AM or Gö6976. These studies provide information that may improve the response of cholangiopathies to medical therapy.

Adrenergic receptor agonists prevent bile duct injury induced by adrenergic denervation by increased cAMP levels and activation of Akt.

Loss of parasympathetic innervation after vagotomy impairs cholangiocyte proliferation, which is associated with depressed cAMP levels, impaired ductal secretion, and enhanced apoptosis. Agonists that elevate cAMP levels prevent cholangiocyte apoptosis and restore cholangiocyte proliferation and ductal secretion. No information exists regarding the role of adrenergic innervation in the regulation of cholangiocyte function. In the present studies, we investigated the role of adrenergic innervation on cholangiocyte proliferative and secretory responses to bile duct ligation (BDL). Adrenergic denervation by treatment with 6-hydroxydopamine (6-OHDA) during BDL decreased cholangiocyte proliferation and secretin-stimulated ductal secretion with concomitant increased apoptosis, which was associated with depressed cholangiocyte cAMP levels. Chronic administration of forskolin (an adenylyl cyclase activator) or beta(1)- and beta(2)-adrenergic receptor agonists (clenbuterol or dobutamine) prevented the decrease in cholangiocyte cAMP levels, maintained cholangiocyte secretory and proliferative activities, and decreased cholangiocyte apoptosis resulting from adrenergic denervation. This was associated with enhanced phosphorylation of Akt. The protective effects of clenbuterol, dobutamine, and forskolin on 6-OHDA-induced changes in cholangiocyte apoptosis and proliferation were partially blocked by chronic in vivo administration of wortmannin. In conclusion, we propose that adrenergic innervation plays a role in the regulation of biliary mass and cholangiocyte functions during BDL by modulating intracellular cAMP levels.

Corticosteroids modulate the secretory processes of the rat intrahepatic biliary epithelium.

BACKGROUND & AIMS: We investigated the expression of glucocorticoid receptors (GcRs) in the intrahepatic biliary epithelium and the role of corticosteroids in the regulation of cholangiocyte secretion. METHODS: GcR was studied by immunohistochemistry, reverse-transcription polymerase chain reaction, and Western blots. The effects of dexamethasone and budesonide on biliary bicarbonate excretion and H+/HCO3- transport processes were investigated in bile fistula rats, isolated intrahepatic bile duct units (IBDUs), and purified cholangiocytes. RESULTS: GcRs were expressed by rat cholangiocytes. Although acute administration of corticosteroids showed no effect, treatment for 2 days with dexamethasone or budesonide increased (P < 0.05) biliary bicarbonate concentration and secretion, which were blocked by the specific GcR antagonist, RU-486. IBDUs isolated from rats treated with dexamethasone or budesonide showed an increased (P < 0.05) activity of the Na+/H+ exchanger (NHE1 isoform) and Cl-/HCO3- exchanger (AE2 member), which was blocked by RU-486. Protein expression of NHE1 and AE2 and messenger RNA for NH1 but not AE2 were increased (P < 0.05) in isolated cholangiocytes by dexamethasone treatment. CONCLUSIONS: The intrahepatic biliary epithelium expresses GcR and responds to corticosteroids by increasing bicarbonate excretion in bile. This is caused by corticosteroid-induced enhanced activities and protein expression of transport processes driving bicarbonate excretion in the biliary epithelium.

Gastrin reverses established cholangiocyte proliferation and enhanced secretin-stimulated ductal secretion of BDL rats by activation of apoptosis through increased expression of Ca2+- dependent PKC isoforms.

We posed these questions: (i) Does administration of gastrin to 1-week bile duct ligation (BDL) rats inhibits established cholangiocyte proliferation and ductal secretion? (ii) Is gastrin inhibition of cholangiocyte proliferation and secretion of BDL rats associated with enhanced apoptosis? (iii) Are gastrin's effects on cholangiocyte function associated with increased expression of protein kinase C (PKC) isoforms; and (iv) Is gastrin stimulation of cholangiocyte apoptosis regulated by the Ca2+-dependent PKC pathway? METHODS: Seven days after BDL, rats were treated with gastrin by minipumps for 14 days. Cholangiocyte proliferation was assessed by measurement of the number of PCNA and CK-19 positive cholangiocytes in sections, and PCNA expression in cholangiocytes. Ductal secretion was determined by measurement of secretin-induced cAMP levels and choleresis. Apoptosis was evaluated by TUNEL analysis in sections and annexin-V staining in cholangiocytes. The expression of PKC isoforms was determined by immunoblots. RESULTS: Gastrin inhibits established cholangiocyte proliferation and enhanced secretin-stimulated ductal secretion of BDL rats.Gastrin's effects on cholangiocyte function were associated with enhanced apoptosis and increased expression of PKC alpha, and beta I and II. Gastrin increases in cholangiocyte apoptosis were blocked by BAPTA/AM and H7. SUMMARY/CONCLUSION: Gastrin inhibits cholangiocyte proliferation and secretin-induced ductal secretion in BDL rats by increasing apoptosis through a PKC-mediated mechanism.

Intracellular pathways mediating estrogen-induced cholangiocyte proliferation in the rat.

The aim of this study was to explore the intracellular signaling pathways involved in the stimulatory effects of estrogens on cholangiocyte proliferation. We investigated the tyrosine kinase-receptor pathway by evaluating the protein expression of total and phosphorylated mitogen-activated protein kinase (MAPK) isoform p44/p42 (e.g., extracellular signal-regulated kinase [ERK]1/2), the steroid-receptor coactivator Src and Shc (Src-homology/collagen protein). The study was performed in 3-week-old bile duct-ligated (BDL) rats, BDL rats treated with the antiestrogens, tamoxifen or Ici 182,780, and normal control rats. Proliferation was also evaluated in normal purified cholangiocytes treated with 17 beta estradiol in the presence or absence of tamoxifen, Ici 182,780, ERK, or Src inhibitors. After bile duct ligation, cholangiocyte proliferation was associated with a marked immunohistochemical nuclear positivity for phosphorylated (p)-ERK1/2, which was inhibited by in vivo treatment with tamoxifen or Ici 182,780. Protein expression of total and p-ERK1/2, and Shc in cholangiocytes isolated from BDL rats was markedly increased compared with controls and was inhibited by in vivo treatment with antiestrogens. In vitro, 17 beta estradiol-induced proliferation of isolated normal cholangiocyte was associated with increased (P <.01) protein expression of p-ERK1/2, Src, and Shc. Specific inhibitors of ER (Ici 182,780), ERK (U0125), and Src (PP2) inhibited in vitro 17 beta estradiol-induced cholangiocyte proliferation. In conclusion, this study showed that estrogens induced cholangiocyte proliferation by activating the Src/Shc/ERK pathway. This might suggest that pharmacologic modulation of ER, ERK, and/or Src could be proposed for the treatment of human pathology characterized by dysregulation of cholangiocyte proliferation.

Nerve growth factor modulates the proliferative capacity of the intrahepatic biliary epithelium in experimental cholestasis.

BACKGROUND & AIMS: We evaluated the expression of neurotrophins in rat cholangiocytes and the role and mechanisms by which nerve growth factor (NGF) modulates cholangiocyte proliferation. METHODS: The expression of neurotrophins and their receptors was investigated by immunohistochemistry in liver sections and reverse-transcription polymerase chain reaction and immunoblots in isolated cholangiocytes. In vitro, the effect of NGF on cholangiocyte proliferation and signal transduction was investigated by immunoblotting for proliferating cell nuclear antigen, phosphorylated AKT (p-AKT), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated c-jun-N-terminal kinase, and phosphorylated p38. In vivo, rats that had undergone bile duct ligation (BDL) were treated with an anti-NGF antibody to immunoneutralize NGF and bile duct mass, proliferation, apoptosis, and inflammation were investigated by immunohistochemistry. RESULTS: NGF and its TrkA receptor were expressed by normal rat cholangiocytes and up-regulated following BDL. Cholangiocytes secrete NGF, and secretion is increased in proliferating BDL cholangiocytes. In vitro, NGF stimulated cholangiocyte proliferation, which was associated with enhanced p-AKT and p-ERK1/2 expression. NGF proliferation in vitro was partially blocked by the MEK inhibitor (UO126) and completely ablated by the phosphatidylinositol 3-kinase inhibitor (wortmannin). In vitro, NGF and estrogens have an additive effect on cholangiocyte proliferation by acting on phosphorylated TrkA and p-ERK1/2. In vivo, immunoneutralization of NGF decreased bile duct mass in BDL rats, which was associated with depressed proliferation and enhanced apoptosis and with increased portal inflammation. CONCLUSIONS: Cholangiocytes secrete NGF and express NGF receptors. NGF induces cholangiocyte proliferation by activating the ERK and, predominantly, the phosphatidylinositol 3-kinase pathway and exerts an additive effect in combination with estrogens on proliferation.

Estrogen receptors in cholangiocytes and the progression of primary biliary cirrhosis.

BACKGROUND/AIMS: Estrogen receptors (ER) in cholangiocytes of primary biliary cirrhosis (PBC) patients and their relationship with cell proliferation and death were evaluated. METHODS: Liver biopsies from PBC patients with different histological stages were investigated by immunohistochemistry for ER-alpha and -beta, cytokeratin-19, proliferating cellular nuclear antigen (PCNA), Fas and terminal deoxynucleotide transferase end labelling (TUNEL). Normal livers and livers from primary sclerosing cholangitis and alcoholic cirrhosis were investigated as controls. RESULTS: ER-alpha and -beta were observed in cholangiocytes of PBC patients but not in normal liver. In PBC, positivity for ER-beta was high (50-65 %) in all histological stages while, positivity for ER-alpha increased from 1% in stage I to 12 % in stage III (positivity correlated and co-localized in the same cell with PCNA). In stage IV of PBC, cholangiocytes were negative for ER-alpha in association with a lower PCNA positivity and with maximal degree of ductopenia. ER-alpha positivity in cholangiocytes of PBC patients was markedly lower than primary sclerosing cholangitis and alcoholic cirrhosis. CONCLUSIONS: ER are expressed in PBC and other pathologies associated with cholangiocyte proliferation but not in normal subjects. The low expression of ER-alpha in PBC and their disappearance in the advanced histological stages suggests that an estrogenic deficiency could favour the evolution of this disease toward ductopenia.

Dopaminergic inhibition of secretin-stimulated choleresis by increased PKC-gamma expression and decrease of PKA activity.

To determine the role and mechanisms of action by which dopaminergic innervation modulates ductal secretion in bile duct-ligated rats, we determined the expression of D1, D2, and D3 dopaminergic receptors in cholangiocytes. We evaluated whether D1, D2 (quinelorane), or D3 dopaminergic receptor agonists influence basal and secretin-stimulated choleresis and lumen expansion in intrahepatic bile duct units (IBDU) and cAMP levels in cholangiocytes in the absence or presence of BAPTA-AM, chelerythrine, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H7), or rottlerin. We evaluated whether 1) quinelorane effects on ductal secretion were associated with increased expression of Ca(2+)-dependent PKC isoforms and 2) increased expression of PKC causes inhibition of PKA activity. Quinelorane inhibited secretin-stimulated choleresis in vivo and IBDU lumen space, cAMP levels, and PKA activity in cholangiocytes. The inhibitory effects of quinelorane on secretin-stimulated ductal secretion and PKA activity were blocked by BAPTA-AM, chelerythrine, and H7. Quinelorane effects on ductal secretion were associated with activation of the Ca(2+)-dependent PKC-gamma but not other PKC isoforms. The dopaminergic nervous system counterregulates secretin-stimulated ductal secretion in experimental cholestasis.