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RESULTS: The resection margins were clearly visualized. The average surgical time of group A (19.2 min) was significantly longer than group B (12.3 min) and C (13.1 min). Hemorrhage volume was less than a gauze. The average AST/ALT level 3-day postoperation were 124.2/107.6, 88.6/73.3 and 76.9/64.1 respectively. The rest of the laboratory data were close. Angiogram showed effective occlusion at the target region after the gel administration. Angiogram and necropsy showed no evidence of abnormities 3 months after the operation.

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3',5'-monophosphate (cAMP), Ca(2+)-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1-H4HRs. H1HR acts by Gαq activating IP(3)/Ca(2+), whereas H2HR activates Gα(s) stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1-H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1-H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP(3) and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1-H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP(3) levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts.

Functional pluripotent characteristics have been observed in specific subpopulations of hepatic cells that express some of the known cholangiocyte markers. Although evidence indicates that specific cytokines, granulocyte macrophage colony-stimulating factors (GM-CSFs), and stem cell factors (SCFs) may be candidate treatments for liver injury, the role of these cytokines in intrahepatic biliary epithelium remodeling is unknown. Thus, our aim was to characterize the specific cytokines that regulate the remodeling potentials of cholangiocytes after 70% partial hepatectomy (PH). The expression of the cytokines and their downstream signaling molecules was studied in rats after 70% PH by immunoblotting and in small and large murine cholangiocyte cultures (SMCCs and LMCCs) by immunocytochemistry and real-time polymerase chain reaction (PCR). There was a significant, stable increase in SCF and GM-CSF levels until 7 days after PH. Real-time PCR analysis revealed significant increases of key remodeling molecules, such as S100 calcium-binding protein A4 (S100A4) and miR-181b, after SCF plus GM-CSF administration in SMCCs. SMCCs produced significant amounts of soluble and bound SCFs and GM-CSFs in response to transforming growth factor-beta (TGF-β). When SMCCs were incubated with TGF-β plus anti-SCF+GM-CSF antibodies, there was a significant decrease in S100A4 expression. Furthermore, treatment of SMCCs with SCF+GM-CSF significantly increased matrix metalloproteinases (MMP-2 and MMP-9) messenger RNA as well as miR-181b expression, along with a reduction of metalloproteinase inhibitor 3. Levels of MMP-2, MMP-9, and miR-181b were also up-regulated in rat liver and isolated cholangiocytes after PH. Conclusion: Our data suggest that altered expression of SCF+GM-CSF after PH can contribute to biliary remodeling (e.g., post-transplantation) by functional deregulation of the activity of key signaling intermediates involved in cell expansion and multipotent differentiation. (HEPATOLOGY 2012;;55:209–221)

Cholangiocarcinoma (CCA) is a devastating biliary cancer. Melatonin is synthesized in the pineal gland and peripheral organs from serotonin by two enzymes, serotonin N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT). Cholangiocytes secrete neuroendocrine factors, including serotonin-regulating CCA growth by autocrine mechanisms. Melatonin exerts its effects by interaction with melatonin receptor type 1A/1B (MT1/MT2) receptors. We propose that 1) in CCA, there is decreased expression of AANAT and ASMT and secretion of melatonin, changes that stimulate CCA growth; and 2) in vitro overexpression of AANAT decreases CCA growth. We evaluated the 1) expression of AANAT, ASMT, melatonin, and MT1/MT2 in human nonmalignant and CCA lines and control and CCA biopsy samples; 2) melatonin levels in nonmalignant and CCA lines, and bile and serum from controls and patients with intrahepatic CCA; 3) effect of melatonin on the growth and expression of AANAT/ASMT and MT1/MT2 in CCA lines implanted into nude mice; and 4) effect of AANAT overexpression on the proliferation, apoptosis, and expression of MT1/MT2 in Mz-ChA-1 cells. The expression of AANAT, ASMT, and melatonin decreased, whereas MT1/MT2 expression increased in CCA lines and biopsy samples. Melatonin secretion decreased in the supernatant of CCA lines and bile of CCA patients. Melatonin decreased xenograft CCA tumor growth in nude mice by increased AANAT/ ASMT and melatonin, along with reduced MT1/MT2 expression. Overexpression of AANAT in Mz-ChA-1 cells inhibited proliferation and MT1/ MT2 expression and increased apoptosis. There is dysregulation of the AANAT/ASMT/melatonin ¡ melatonin receptor axis in CCA, which inhibited melatonin secretion and subsequently enhanced CCA growth. © 2011 the American Physiological Society

Epigenetic changes are associated with the regulation of transcription of key cell regulatory genes [micro RNAs (miRNAs)] during different types of liver injury. This study evaluated the role of methylation-associated miRNA, miR-34a, in alcoholic liver diseases. We identified that ethanol feeding for 4 weeks significantly up-regulated 0.8% of known miRNA compared with controls, including miR-34a. Treatment of normal human hepatocytes (N-Heps) and cholangiocytes [human intrahepatic biliary epithelial cells (HiBECs)] with ethanol and lipopolysaccharide induced a significant increase of miR-34a expression. Overexpression of miR-34a decreased ethanol-induced apoptosis in both N-Heps and HiBECs. In support of the concept that the 5′-promoter region of miR-34a was noted to be embedded within a CpG island, the expression level of miR-34a was significantly increased after demethylation treatment in N-Heps and HiBECs. By methylation-specific PCR, we confirmed that miR-34a activation is associated with ethanol-linked hypomethylation of the miR-34a promoter. A combination of bioinformatics, dual-luciferase reporter assay, mass spectrometry, and Western blot analysis revealed that caspase-2 and sirtuin 1 are the direct targets of miR-34a. Furthermore, modulation of miR-34a also altered expression of matrix metalloproteases 1 and 2, the mediators involved in hepatic remodeling during alcoholic liver fibrosis. These findings provide the basis for an exciting field in which the epigenomic microRNAs of hepatic cells may be manipulated with potential therapeutic benefits in human alcoholic liver diseases.

In bile duct-ligated (BDL) rats, cholangiocyte proliferation is regulated by neuroendocrine factors such as α-calcitonin gene-related peptide (α-CGRP). There is no evidence that the sensory neuropeptide substance P (SP) regulates cholangiocyte hyperplasia. Wild-type (WT,+/+) and NK-1 receptor (NK-1R) knockout (NK-1R−/−) mice underwent sham or BDL for 1 wk. Then we evaluated 1) NK-1R expression, transaminases, and bilirubin serum levels; 2) necrosis, hepatocyte apoptosis and steatosis, and the number of cholangiocytes positive by CK-19 and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling in liver sections; 3) mRNA expression for collagen 1α and α-smooth muscle (α-SMA) actin in total liver samples; and 4) PCNA expression and PKA phosphorylation in cholangiocytes. In cholangiocyte lines, we determined the effects of SP on cAMP and d-myo-inositol 1,4,5-trisphosphate levels, proliferation, and PKA phosphorylation. Cholangiocytes express NK-1R with expression being upregulated following BDL. In normal NK-1R−/−mice, there was higher hepatocyte apoptosis and scattered hepatocyte steatosis compared with controls. In NK-1R−/−BDL mice, there was a decrease in serum transaminases and bilirubin levels and the number of CK-19-positive cholangiocytes and enhanced biliary apoptosis compared with controls. In total liver samples, the expression of collagen 1α and α-SMA increased in BDL compared with normal mice and decreased in BDL NK-1R−/−compared with BDL mice. In cholangiocytes from BDL NK-1R−/−mice there was decreased PCNA expression and PKA phosphorylation. In vitro, SP increased cAMP levels, proliferation, and PKA phosphorylation of cholangiocytes. Targeting of NK-1R may be important in the inhibition of biliary hyperplasia in cholangiopathies.

No information exists on the role of neuropeptide Y (NPY) in cholangiocarcinoma growth. Therefore, we evaluated the expression and secretion of NPY and its subsequent effects on cholangiocarcinoma growth and invasion. Cholangiocarcinoma cell lines and nonmalignant cholangiocytes were used to assess NPY mRNA expression and protein secretion. NPY expression was assessed by immunohistochemistry in human liver biopsies. Cell proliferation and migration were evaluated in vitro by MTS assays and matrigel invasion chambers, respectively, after treatment with NPY or a neutralizing NPY antibody. The effect of NPY or NPY depletion on tumor growth was assessed in vivo after treatment with NPY or the neutralizing NPY antibody in a xenograft model of cholangiocarcinoma. NPY secretion was upregulated in cholangiocarcinoma compared with normal cholangiocytes. Administration of exogenous NPY decreased proliferation and cell invasion in all cholangiocarcinoma cell lines studied and reduced tumor cell growth in vivo. In vitro, the effects of NPY on proliferation were blocked by specific inhibitors for NPY receptor Y2, but not Y1 or Y5, and were associated with an increase in intracellular d- myo-inositol 1,4,5-trisphosphate and PKCα activation. Blocking of NPY activity using a neutralizing antibody promoted cholangiocarcinoma growth in vitro and in vivo and increased the invasiveness of cholangiocarcinoma in vitro. Increased NPY immunoreactivity in human tumor tissue occurred predominantly in the center of the tumor, with less expression toward the invasion front of the tumor. We demonstrated that NPY expression is upregulated in cholangiocarcinoma, which exerts local control on tumor cell proliferation and invasion. Modulation of NPY secretion may be important for the management of cholangiocarcinoma.

Small cholangiocytes proliferate via activation of calcium (Ca2+)-dependent signaling in response to pathological conditions that trigger the damage of large cyclic adenosine monophosphate–dependent cholangiocytes. Although our previous studies suggest that small cholangiocyte proliferation is regulated by the activation of Ca2+-dependent signaling, the intracellular mechanisms regulating small cholangiocyte proliferation are undefined. Therefore, we sought to address the role and mechanisms of action by which phenylephrine, an α1-adrenergic agonist stimulating intracellular D-myo-inositol-1,4,5-triphosphate (IP3)/Ca2+ levels, regulates small cholangiocyte proliferation. Small and large bile ducts and cholangiocytes expressed all AR receptor subtypes. Small (but not large) cholangiocytes respond to phenylephrine with increased proliferation via the activation of IP3/Ca2+-dependent signaling. Phenylephrine stimulated the production of intracellular IP3. The Ca2+-dependent transcription factors, nuclear factor of activated T cells 2 (NFAT2) and NFAT4, were predominantly expressed by small bile ducts and small cholangiocytes. Phenylephrine stimulated the Ca2+-dependent DNA-binding activities of NFAT2, NFAT4, and Sp1 (but not Sp3) and the nuclear translocation of NFAT2 and NFAT4 in small cholangiocytes. To determine the relative roles of NFAT2, NFAT4, or Sp1, we knocked down the expression of these transcription factors with small hairpin RNA. We observed an inhibition of phenylephrine-induced proliferation in small cholangiocytes lacking the expression of NFAT2 or Sp1. Phenylephrine stimulated small cholangiocyte proliferation is regulated by Ca2+-dependent activation of NFAT2 and Sp1. Conclusion: Selective stimulation of Ca2+-dependent small cholangiocyte proliferation may be key to promote the repopulation of the biliary epithelium when large bile ducts are damaged during cholestasis or by toxins. (HEPATOLOGY 2010;53:628-639)

Biliary epithelial cells (BEC) are morphologically and functionally heterogeneous. To investigate the molecular mechanism for their diversities, we test the hypothesis that large and small BEC have disparity in their target gene response to their transcriptional regulator, the biliary cell-enriched hepatocyte nuclear factor HNF6. The expression of the major HNF ( HNF6, OC2, HNF1b, HNF1a, HNF4a, C/EBPb, and Foxa2) and representative biliary transport target genes that are HNF dependent were compared between SV40-transformed BEC derived from large (SV40LG) and small (SV40SM) ducts, before and after treatment with recombinant adenoviral vectors expressing HNF6 (AdHNF6) or control LacZ cDNA (AdLacZ). Large and small BEC were isolated from mouse liver treated with growth hormone, a known transcriptional activator of HNF6, and the effects on selected target genes were examined. Constitutive Foxa2, HNF1a, and HNF4a gene expression were 2.3-, 12.4-, and 2.6-fold, respectively, higher in SV40SM cells. This was associated with 2.7- and 4-fold higher baseline expression of HNF1a- and HNF4a-regulated ntcp and oatp1 genes, respectively. Following AdHNF6 infection, HNF6 gene expression was 1.4-fold higher ( P = 0.02) in AdHNF6 SV40SM relative to AdHNF6 SV40LG cells, with a corresponding higher Foxa2 (4-fold), HNF1a (15-fold), and HNF4a (6-fold) gene expression in AdHNF6-SV40SM over AdHNF6-SV40LG. The net effects were upregulation of HNF6 target gene glucokinase and of Foxa2, HNF1a, and HNF4a target genes oatp1, ntcp, and mrp2 over AdLacZ control in both cells, but with higher levels in AdH6-SV40SM over AdH6-SV40LG of glucokinase, oatp1, ntcp, and mrp2 (by 1.8-, 3.4-, 2.4-, and 2.5-fold, respectively). In vivo, growth hormone-mediated increase in HNF6 expression was associated with similar higher upregulation of glucokinase and mrp2 in cholangiocytes from small vs. large BEC. Small and large BEC have a distinct profile of hepatocyte transcription factor and cognate target gene expression, as well as differential strength of response to transcriptional regulation, thus providing a potential molecular basis for their divergent function.

During bile duct ligation (BDL), the growth of large cholangiocytes is regulated by the cyclic adenosine monophosphate (cAMP)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and is closely associated with increased secretin receptor (SR) expression. Although it has been suggested that SR modulates cholangiocyte growth, direct evidence for secretin-dependent proliferation is lacking. SR wild-type (WT) (SR+/+) or SR knockout (SR-/-) mice underwent sham surgery or BDL for 3 or 7 days. We evaluated SR expression, cholangiocyte proliferation, and apoptosis in liver sections and proliferating cell nuclear antigen (PCNA) protein expression and ERK1/2 phosphorylation in purified large cholangiocytes from WT and -/- BDL mice. Normal WT mice were treated with secretin (2.5 nmoles/kg/day by way of osmotic minipumps for 1 week), and biliary mass was evaluated. Small and large cholangiocytes were used to evaluate the in vitro effect of secretin (100 nM) on proliferation, protein kinase A (PKA) activity, and ERK1/2 phosphorylation. SR expression was also stably knocked down by short hairpin RNA, and basal and secretin-stimulated cAMP levels (a functional index of biliary growth) and proliferation were determined. SR was expressed by large cholangiocytes. Knockout of SR significantly decreased large cholangiocyte growth induced by BDL, which was associated with enhanced apoptosis. PCNA expression and ERK1/2 phosphorylation were decreased in large cholangiocytes from-/- BDL compared with WT BDL mice. In vivo administration of secretin to normal WT mice increased ductal mass. In vitro, secretin increased proliferation, PKA activity, and ERK1/2 phosphorylation of large cholangiocytes that was blocked by PKA and mitogen-activated protein kinase kinase inhibitors. Stable knockdown of SR expression reduced basal cholangiocyte proliferation. SR is an important trophic regulator sustaining biliary growth. Conclusion: The current study provides strong support for the potential use of secretin as a therapy for ductopenic liver diseases. Copyright © 2010 by the American Association for the Study of Liver Diseases., link_to_OA_fulltext

Caffeic acid phenethyl ester (CAPE) inhibits the growth of tumor cells and is a known inhibitor of nuclear factor kappa beta (NF-kappaB), which is constitutively active in cholangiocarcinoma (CCH) cells. We evaluated the effects of CAPE on CCH growth both in vitro and in vivo. Inhibition of NF-kappaB DNA-binding activity was confirmed in nuclear extracts treated with CAPE at 50, 40 and 20 microM. CAPE decreases the expression of NF-kappaB1 (p50) and RelA (p65). CAPE decreased the growth of a number of CCH cells but not normal cholangiocytes. Cell cycle decrease was seen by a decrease in PCNA protein expression and the number of BrdU-positive cells treated with CAPE at 20 microM compared to vehicle. Inhibition of growth and increased cell cycle arrest of Mz-ChA-1 cells by CAPE were coupled with increased apoptosis. Bax expression was increased, whereas Bcl-2 was decreased in cells treated with CAPE compared to vehicle. In vivo studies were performed in BALB/c nude (nu/nu) mice implanted subcutaneously with Mz-ChA-1 cells and treated with daily IP injections of DMSO or CAPE (10 mg/kg body weight in DMSO) for 77 days. Tumor growth was decreased and tumor latency was increased 2-fold in CAPE compared to vehicle-treated nude mice. In tumor samples, decreased CCH growth by CAPE was coupled with increased apoptosis. CAPE both in vivo and in vitro decreases the growth of CCH cells by increasing apoptosis. These results demonstrate that CAPE might be an important therapeutic tool in the treatment of CCH.

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2-4 microm(2), embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl(-)/HCO(3)(-) AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3',5'-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl(-)/HCO(3)(-) exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.

Cholangiocarcinoma is a strongly aggressive malignancy with a very poor prognosis. Effective therapeutic strategies are lacking because molecular mechanisms regulating cholangiocarcinoma cell growth are unknown. Furthermore, experimental in vivo animal models useful to study the pathophysiologic mechanisms of malignant cholangiocytes are lacking. Leptin, the hormone regulating caloric homeostasis, which is increased in obese patients, stimulates the growth of several cancers, such as hepatocellular carcinoma. The aim of this study was to define if leptin stimulates cholangiocarcinoma growth. We determined the expression of leptin receptors in normal and malignant human cholangiocytes. Effects on intrahepatic cholangiocarcinoma (HuH-28) cell proliferation, migration, and apoptosis of the in vitro exposure to leptin, together with the intracellular pathways, were then studied. Moreover, cholangiocarcinoma was experimentally induced in obese fa/fa Zucker rats, a genetically established animal species with faulty leptin receptors, and in their littermates by chronic feeding with thioacetamide, a potent carcinogen. After 24 weeks, the effect of leptin on cholangiocarcinoma development and growth was assessed. Normal and malignant human cholangiocytes express leptin receptors. Leptin increased the proliferation and the metastatic potential of cholangiocarcinoma cells in vitro through a signal transducers and activators of transcription 3–dependent activation of extracellular signal-regulated kinase 1/2. Leptin increased the growth and migration, and was antiapoptotic for cholangiocarcinoma cells. Moreover, the loss of leptin function reduced the development and the growth of cholangiocarcinoma. The experimental carcinogenesis model induced by thioacetamide administration is a valid and reproducible method to study cholangiocarcinoma pathobiology. Modulation of the leptin-mediated signal could be considered a valid tool for the prevention and treatment of cholangiocarcinoma. [Cancer Res 2008;68(16):6752–61]

During cholestatic liver diseases, cholangiocytes express neuroendocrine phenotypes and respond to a number of hormones and neuropeptides by paracrine and autocrine mechanisms. We examined whether the neuroendocrine hormone progesterone is produced by and targeted to cholangiocytes, thereby regulating biliary proliferation during cholestasis. Nuclear (PR-A and PR-B) and membrane (PRGMC1, PRGMC2, and mPRα) progesterone receptor expression was evaluated in liver sections and cholangiocytes from normal and bile duct ligation (BDL) rats, and NRC cells (normal rat cholangiocyte line). In vivo, normal rats were chronically treated with progesterone for 1 wk, or immediately after BDL, rats were treated with a neutralizing progesterone antibody for 1 wk. Cholangiocyte growth was measured by evaluating the number of bile ducts in liver sections. The expression of the progesterone synthesis pathway was evaluated in liver sections, cholangiocytes and NRC. Progesterone secretion was evaluated in supernatants from normal and BDL cholangiocytes and NRC. In vitro, NRC were stimulated with progesterone and cholangiocyte supernatants in the presence or absence of antiprogesterone antibody. Aminoglutethimide was used to block progesterone synthesis. Cholangiocytes and NRC express the PR-B nuclear receptor and PRGMC1, PRGMC2, and mPRα. In vivo, progesterone increased the number of bile ducts of normal rats, whereas antiprogesterone antibody inhibited cholangiocyte growth stimulated by BDL. Normal and BDL cholangiocytes expressed the biosynthetic pathway for and secrete progesterone. In vitro, 1) progesterone increased NRC proliferation; 2) cholangiocyte supernatants increased NRC proliferation, which was partially inhibited by preincubation with antiprogesterone; and 3) inhibition of progesterone steroidogenesis prevented NRC proliferation. In conclusion, progesterone may be an important autocrine/paracrine regulator of cholangiocyte proliferation.

Cholangiopathies are characterized by the heterogeneous proliferation of different-sized cholangiocytes. Large cholangiocytes proliferate by a cAMP-dependent mechanism. The function of small cholangiocytes may depend on the activation of inositol trisphosphate (IP3)/Ca2+-dependent signaling pathways; however, data supporting this speculation are lacking. Four histamine receptors exist (HRH1, HRH2, HRH3, and HRH4). In several cells: 1) activation of HRH1 increases intracellular Ca2+concentration levels; and 2) increased [Ca2+]ilevels are coupled with calmodulin-dependent stimulation of calmodulin-dependent protein kinase (CaMK) and activation of cAMP-response element binding protein (CREB). HRH1 agonists modulate small cholangiocyte proliferation by activation of IP3/Ca2+-dependent CaMK/CREB. We evaluated HRH1 expression in cholangiocytes. Small and large cholangiocytes were stimulated with histamine trifluoromethyl toluidide (HTMT dimaleate; HRH1 agonist) for 24–48 h with/without terfenadine, BAPTA/AM, or W7 before measuring proliferation. Expression of CaMK I, II, and IV was evaluated in small and large cholangiocytes. We measured IP3, Ca2+and cAMP levels, phosphorylation of CaMK I, and activation of CREB (in the absence/presence of W7) in small cholangiocytes treated with HTMT dimaleate. CaMK I knockdown was performed in small cholangiocytes stimulated with HTMT dimaleate before measurement of proliferation and CREB activity. Small and large cholangiocytes express HRH1, CaMK I, and CaMK II. Small (but not large) cholangiocytes proliferate in response to HTMT dimaleate and are blocked by terfenadine (HRH1 antagonist), BAPTA/AM, and W7. In small cholangiocytes, HTMT dimaleate increased IP3/Ca2+levels, CaMK I phosphorylation, and CREB activity. Gene knockdown of CaMK I ablated the effects of HTMT dimaleate on small cholangiocyte proliferation and CREB activation. The IP3/Ca2+/CaMK I/CREB pathway is important in the regulation of small cholangiocyte function.

The endocannabinoid system regulates various aspects of hepatic fibrosis; however, nothing is known about its role in regulating cholangiocyte proliferation and function. We evaluated the effects of anandamide (AEA) on cholangiocyte proliferation and explored the effects of AEA on the thioredoxin 1 (TRX1)/redox factor 1 (Ref1)/activator protein-1 (AP-1) pathway. Mice underwent bile duct ligation (BDL) and were infused with AEA for 3 days postsurgery. Proliferation and apoptosis were evaluated in liver sections. Effects of in vitro AEA treatment on cholangiocyte proliferation and apoptosis were studied in purified cholangiocytes. The relative expression of cannabinoid receptors was also assessed in liver sections and cholangiocytes. mRNA expression of the cannabinoid receptors Cb1 and VR1 was decreased after BDL, whereas there was an upregulation of Cb2 mRNA. AEA decreased cholangiocyte growth and induced accumulation of reactive oxygen species, upregulation of TRX1, Ref1, c-Fos, and c-Jun expression, increased nuclear localization of TRX1, and increased AP-1 transcriptional activity. Specific knockdown of TRX1 or Ref1 expression ablated the AP-1 transcriptional activity and AEA-induced cell death but not expression of c-Fos and c-Jun. Knockdown of c-Fos and c-Jun expression also ablated AEA-induced apoptosis. We conclude that AEA suppresses cholangiocyte proliferation during cholestasis via a Cb2-dependent mechanism. Modulation of the endocannabinoid system may be important in the treatment of cholangiopathies.

The role of sensory innervation in the regulation of liver physiology and the pathogenesis of cholestatic liver disease are undefined. Biliary proliferation has been shown to be coordinately controlled by parasympathetic and sympathetic innervation of the liver. The aim of our study was to address the role of the sensory neuropeptide calcitonin gene-related peptide (alpha-CGRP) in the regulation of cholangiocyte proliferation during cholestasis induced by extrahepatic bile duct obstruction (BDL). Our study utilized a knockout (KO) mouse model, which lacks the sensory neuropeptide alpha-CGRP. Wild-type (WT) and alpha-CGRP KO mice were subjected to sham surgery or BDL for 3 and 7 days. In addition, immediately after BDL, WT and KO mice were administered the CGRP receptor antagonist (CGRP(8-37)) for 3 and 7 days by osmotic minipumps. Liver sections and isolated cholangiocytes were evaluated for proliferation markers. Isolated WT BDL (3 days) cholangiocytes were stimulated with alpha- and beta-CGRP and evaluated for proliferation and cAMP-mediated signaling. Lack of alpha-CGRP inhibits cholangiocyte proliferation induced by BDL at both 3 and 7 days. BDL-induced cholangiocyte proliferation in WT mice was associated with increases of circulating alpha-CGRP levels. In vitro, alpha- and beta-CGRP stimulated proliferation in purified BDL cholangiocytes, induced elevation of cAMP levels, and stimulated the activation of cAMP-dependent protein kinase A and cAMP response element binding protein DNA binding. In conclusion, sensory innervation of the liver and biliary expression of alpha-CGRP play an important role in the regulation of cholangiocyte proliferation during cholestasis.

Cholangiopathies, such as primary biliary cirrhosis and primary sclerosis cholangitis, are characterized at the end stage by ductopenia due to increased cholangiocyte apoptosis and decreased cholangiocyte proliferation. Although cholangiocyte proliferation is associated with an increased number of intra-hepatic bile ducts and secretin-stimulated ductal secretion, ductopenia is coupled with decreased ductal mass and secretin-induced ductal secretory activity. We have shown that a single injection of actinomycin D + tumor necrosis factor-alpha (TNF-alpha ) to bile duct-ligated (BDL) rats induces cholangiocyte injury, which is characterized by loss of cholangiocyte proliferation, and secretory activity and by an increase in cholangiocyte apoptosis. We also have shown that taurocholic acid both in vivo and in vitro stimulates cholangiocyte proliferation. We hypothesize that taurocholic acid feeding protects cholangiocytes against TNF-alpha -induced apoptosis through a phosphatidylinositol-3-kinase (PI3K)-dependent pathway. Immediately after BDL, rats were fed taurocholic acid or control diet in the absence/presence of daily injections of wortmannin for 1 week. Seven days later, control-fed or taurocholic acid-fed rats were treated with a single intraperitoneal injection of actinomycin D + TNF-alpha . Twenty-four hours later we evaluated: (i) cholangiocyte apoptosis and proliferation in liver sections and (ii) basal and secretin-stimulated bile and bicarbonate secretion in bile fistula rats. Taurocholic acid feeding prevented TNF-alpha -induced increases in cholangiocyte apoptosis and decreases in growth and secretin-stimulated bile and bicarbonate secretion, changes that were blocked by PI3K inhibition. The PI3K survival pathway is important in bile acid protection against immune-mediated cholangiocyte injury in cholestatic liver diseases.

Cholangiocarcinomas are devastating cancers of biliary origin with limited treatment options. Modulation of the endocannabinoid system is being targeted to develop possible therapeutic strategies for a number of cancers; therefore, we evaluated the effects of the two major endocannabinoids, anandamide and 2-arachidonylglycerol, on numerous cholangiocarcinoma cell lines. Although anandamide was antiproliferative and proapoptotic, 2-arachidonylglycerol stimulated cholangiocarcinoma cell growth. Specific inhibitors for each of the cannabinoid receptors did not prevent either of these effects nor did pretreatment with pertussis toxin, a G(i/o) protein inhibitor, suggesting that anandamide and 2-arachidonylglycerol did not exert their diametric effects through any known cannabinoid receptor or through any other G(i/o) protein-coupled receptor. Using the lipid raft disruptors methyl-beta-cyclodextrin and filipin, we demonstrated that anandamide, but not 2-arachidonylglycerol, requires lipid raft-mediated events to inhibit cellular proliferation. Closer inspection of the lipid raft structures within the cell membrane revealed that although anandamide treatment had no observable effect 2-arachidonylglycerol treatment effectively dissipated the lipid raft structures and caused the lipid raft-associated proteins lyn and flotillin-1 to disperse into the surrounding membrane. In addition, anandamide, but not 2-arachidonylglycerol, induced an accumulation of ceramide, which was required for anandamide-induced suppression of cell growth. Finally we demonstrated that anandamide and ceramide treatment of cholangiocarcinoma cells recruited Fas and Fas ligand into the lipid rafts, subsequently activating death receptor pathways. These findings suggest that modulation of the endocannabinoid system may be a target for the development of possible therapeutic strategies for the treatment of this devastating cancer.

Secretin stimulates ductal secretion by activation of cAMP → PKA → CFTR → Cl−/HCO3−exchanger in cholangiocytes. We evaluated the expression of α2A-, α2B-, and α2C-adrenergic receptors in cholangiocytes and the effects of the selective α2-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−/HCO3−exchanger activity in purified cholangiocytes. α2-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−/HCO3−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. α2-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases.

A S L D A b st ra ct s BGJ398, a pan-FGFR inhibitor currently being employed in human trials, resulted in a significant increase in cellular apoptosis. Tumor tissue from mice sacrificed 10 weeks after biliary oncogene transduction of AKT and YAP also demonstrated increased expression of FGFR 1-4. BGJ398 treatment resulted in a significant reduction in tumor burden and increase in tumor cell apoptosis as assessed by the TUNEL assay in our mouse model of CCA. In Conclusion, YAP is a critical oncogene in CCA and promotes biliary carcinogenesis, in part, via upregulation of FGFR. In a murine genetic model of CCA, the FGFR specific inhibitor BGJ398 significantly reduces tumor burden by inducing apoptosis. Thus, inhibition of FGFR represents a promising therapeutic approach in YAP-driven human CCA.

A S L D A b st ra ct s GDNF suppresses expression of genes associated with hepatic steatosis Real-time PCR analyses of gene expression in liver from WT and GDNF Tg mice maintained on RD or HFD for 12 weeks. Plotted are means + SEM. *, P 5 mice per group.

A S L D A b st ra ct s BGJ398, a pan-FGFR inhibitor currently being employed in human trials, resulted in a significant increase in cellular apoptosis. Tumor tissue from mice sacrificed 10 weeks after biliary oncogene transduction of AKT and YAP also demonstrated increased expression of FGFR 1-4. BGJ398 treatment resulted in a significant reduction in tumor burden and increase in tumor cell apoptosis as assessed by the TUNEL assay in our mouse model of CCA. In Conclusion, YAP is a critical oncogene in CCA and promotes biliary carcinogenesis, in part, via upregulation of FGFR. In a murine genetic model of CCA, the FGFR specific inhibitor BGJ398 significantly reduces tumor burden by inducing apoptosis. Thus, inhibition of FGFR represents a promising therapeutic approach in YAP-driven human CCA.

Background & Aims: Vascular endothelial growth factor (VEGF) is secreted by several epithelia and modulates cellular functions by autocrine and paracrine mechanisms. The role of VEGF in cholangiocyte pathophysiology is unknown. We evaluated the role of VEGF in the regulation of cholangiocyte proliferation in rats that underwent bile duct ligation. Methods: The expression of VEGF-A and VEGF-C and their receptors in cholangiocytes from normal and BDL rats was evaluated. Normal or BDL rats were treated with recombinant-VEGF-A or recombinant-VEGF-C or anti-VEGF antibodies, and proliferation of cholangiocytes was evaluated in situ by morphometry and in vitro by proliferating cell nuclear antigen immunoblots and MTS assay. In vitro, normal rat cholangiocyte cultures were stimulated with r-VEGF-A or r-VEGF-C and proliferation and signal transduction were evaluated. Results: We found that (1) cholangiocytes express messenger RNA and protein for VEGF-A, VEGF-C, VEGF receptor 2 (VEGFR-2), and VEGF receptor 3 (VEGFR-3) and secrete VEGF; (2) secretion of VEGF and expression of VEGFR-2 and VEGFR-3 increases in BDL cholangiocytes; (3) blocking VEGF in vivo by anti–VEGF-A or anti–VEGF-C antibodies decreases cholangiocyte proliferation; (4) the in vivo administration of r-VEGF-A or r-VEGF-C induces cholangiocyte proliferation in normal rats; and (5) in vitro, VEGF-A increases normal rat cholangiocyte culture proliferation by activation of inositol 1,4,5-triphosphate/Ca 2+ /protein kinase C α and phosphorylation of Src/ERK1/2. Conclusions: Cholangiocytes secrete VEGF and express VEGFR-2 and VEGFR-3, all of which are amplified in BDL cholangiocytes. VEGF induces cholangiocyte proliferation by activation of inositol 1,4,5-triphosphate/[Ca 2+ ] i /protein kinase C α and phosphorylation of Src/ERK1/2. VEGF mediates the adaptive proliferative response of cholangiocytes to cholestasis.

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 β1- and β2-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.

We studied the effect of the inhibitory neurotransmitter, γ-aminobutyric acid (GABA), in the regulation of cholangiocarcinoma growth. We determined the in vitro effect of GABA on the proliferation of the cholangiocarcinoma cell lines (Mz-ChA-1, HuH-28, and TFK-1) and evaluated the intracellular pathways involved. The effect of GABA on migration of Mz-ChA-1 cells was also evaluated. In vivo, Mz-ChA-1 cells were s.c. injected in athymic mice, and the effects of GABA on tumor size, tumor cell proliferation, apoptosis, collagen quantity, and the expression of vascular endothelial growth factor-A (VEGF-A) and VEGF-C (cancer growth regulators) were measured after 82 days. GABA decreased in vitro cholangiocarcinoma growth in a time-dependent and dose-dependent manner, by both cyclic AMP/protein kinase A– and d-myo-inositol-1,4,5-thriphosphate/Ca2+-dependent pathways, leading to down-regulation of extracellular signal-regulated kinase 1/2 phosphorylation. Blocking of GABAA, GABAB, and GABAC receptors prevented GABA inhibition of cholangiocarcinoma proliferation. GABA inhibited Mz-ChA-1 cell migration and, in vivo, significantly decreased tumor volume, tumor cell proliferation, and VEGF-A/C expression whereas increasing apoptosis compared with controls. An increase in collagen was evident in GABA-treated tumors. GABA decreases biliary cancer proliferation and reduces the metastatic potential of cholangiocarcinoma. GABA may represent a therapeutic agent for patients affected by malignancies of the biliary tract. (Cancer Res 2005; 65(24): 11437-46)

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?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.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.Gastrin inhibits cholangiocyte proliferation and secretin-induced ductal secretion in BDL rats by increasing apoptosis through a PKC-mediated mechanism.

Bile acids are cytoprotective in hepatocytes by activating phosphatidylinositol-3-kinase (PI3-K) and its downstream signal AKT. Our aim was to determine whether feeding taurocholate to CCl4-treated rats reduces cholangiocyte apoptosis and whether this cytoprotective effect is dependent on PI3-K. Cholangiocyte proliferation, secretion, and apoptosis were determined in cholangiocytes from bile duct ligation (BDL), CCl4-treated BDL rats, and CCl4-treated taurocholate-fed rats. In vitro, we tested whether CCl4induces apoptosis and whether loss of cholangiocyte proliferation and secretion is dependent on PI3-K. The CCl4-induced cholangiocyte apoptosis and loss of cholangiocyte proliferation and secretion were reduced in CCl4-treated rats fed taurocholate. CCl4-induced cholangiocyte apoptosis, loss of cholangiocytes secretion, and proliferation were prevented by preincubation with taurocholate. Taurocholate cytoprotective effects were ablated by wortmannin. Taurocholate prevented, in vitro, CCl4-induced decrease of phosphorylated AKT protein expression in cholangiocytes. The cytoprotective effects of taurocholate on CCl4effects on cholangiocyte proliferation and secretion were abolished by wortmannin. Taurocholate protects cholangiocytes from CCl4-induced apoptosis by a PI3-K-dependent mechanism. Bile acids are important in the prevention of drug-induced ductopenia in cholangiopathies.

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.

Background & aims: Disruption of circadian rhythm is associated with cancer development and progression. MicroRNAs (miRNAs) are a class of small noncoding RNAs that trigger mRNA translation, repression or degradation. Core clock genes are the molecular basis of circadian rhythms, which include BMAL1, CLOCK, Per1/2/3 and Cry1/2. We have previously demonstrated the downregulation of Per1 expression in human cholangiocarcinoma (CCA) that was predicted as the direct target of miR-34a. Thus, we aimed to evaluate the role of deregulated circadian rhythm and related microRNAs in CCA growth. Methods: Human intraand extrahepatic CCA cells and non-malignant (H69) human cholangiocytes were serum starved for 48 hr before stimulation with 50% serum for 2 hours. The 24-hour rhythmic expression of core clock genes, such as Per1,2/3, CLOCK, BMAL1 and Cry1/2 was evaluated in CCA and H69 cells by real-time PCR. To further evaluate the role of Per1, we overexpressed Per1 by transfecting the Mz-ChA-1 cells with Per1 cDNA and empty vector. In parallel, we also silenced miR-34a expression with lenti-miR-34a ZIP with relative controls. A stably overexpressing Per1 cell line and silencing of miR-34a were established, respectively. Then, wemeasured: (i) cell proliferation byMTS assays and PCNA immunoblots; (ii) cell cycle by BD Cycle test Plus reagent kit; (iii) apoptosis by Annexin V-PE apoptosis detection kit; and (iv) cell migration and invasion by commercially available kits. We utilized luciferase assay to demonstrate that Per1 act as a target of miR-34a. Results: The 24-hour rhythmical expression of Per1 was abolished in all CCA cell lines but not in H69 cells. The rhythmic expression of BMAL1, CLOCK, Per2/3, Cry1/2 was also lost in some of the CCA cell lines tested. After overexpression of Per1, the extrahepatic CCA cell line, Mz-ChA-1, showed: (i) reduced cell proliferation (by MTS assays and PCNA immunoblots); (ii) higher G0/G1 arrest and lower G2/M arrest; (iii) enhanced apoptosis; and (iv) decreased cell invasion and migration compared to control cells. Interestingly, miR-34a was rhythmically expressed in CCA cell lines and H69. The expression level of miR-34a was higher in CCA cell lines compared to H69. Moreover, the inhibition of miR-34a decreased proliferation, migration and invasion in CCA cell lines. Dual luciferase reporter assay demonstrated that miR-34a directly targets Per1. Summary and conclusions: Disruption of circadian rhythms, miR-34a and Per1 expression may contribute to the malignant phenotypes of human cholangiocarcinoma. Our findings suggest that modulation of miR-34a-dependent Per1 expression may represent a novel therapeutic approach for human CCA.