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Double knockout of secretin and secretin receptor exacerbates biliary damage and decreases biliary proliferation and ductal secretion during extrahepatic cholestasis: protective role of bicarbonate secretion during biliary disorders

Shannon S. Glaser1, 2, Fanyin Meng1, 2, Heather L. Francis1, 2, Julie Venter5, Laura Hargrove2, Holly A. Standeford3, Syeda H. Afroze5, Paolo Onori4, Kelly McDaniel3, Micheleine Guerrier5, Eugenio Gaudio4, Gianfranco Alpini1, 2
1Research and Medicine, Central Texas Veterans Health Core System and Texas A&M HSC College of Medicine, Temple, TX; 2Scott & White Digestive Diseases Research Center, Scott & White, Temple, TX; 3Research, Central Texas Veterans Health Care System, Temple, TX; 4Deportment of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Universitá, Sapienza, Rome, Italy; 5Medicine, Texas A&M HSC COM, Temple, TX

Secretin (SEC) stimulates ductal secretion by interaction with its receptor (SR) by activation of the SR[RIGHTWARDS ARROW]CFTR[RIGHTWARDS ARROW]Cl-/HC〇3- AE2 exchanger pathway. Cholangiocyte proliferation/loss and changes in ductal secretion are key aspects of cholangiopathies. We have shown that activation of the SEC/SR axis sustains biliary proliferation in bile duct ligated (BDL) rats. The knockdown of either SEC or SR results in only partial inhibition of biliary proliferation and slight liver damage, which may be due to confounding effects of individual knockdown of SEC and SR. To pinpoint the role of the SEC/SR axis in regulating biliary proliferation and function, we established a double knockout mouse model for both SEC and SR (SEC/SR KO). Our hypothesis is that the lack of a protective bicarbonate umbrella (provided by the SEC/SR axis) in bile ducts leads to increased hepatobiliary damage. The aim of our study was to evaluate cholangiocyte proliferation and function in SEC/SR K〇 with extrahepatic cholestasis. Methods: Studies were performed in normal and BDL (7 day) SEC KO, SR KO and double SEC/SR KO mouse models. Biliary proliferation was assessed by measurement of: (i) intrahepatic bile duct mass (IBDM) in liver sections; and (ii) PCNA expression in isolated cholangiocytes by real-time PCR/immunoblots. Necrosis and inflammation were evaluated in H&E-stained liver sections. Biliary function was evaluated by measurement of the expression of CFTR and Cl-/HC〇3 - AE2.We also evaluated the expression of Bax in all experimental groups. Results: There was an increased reduction in IBDM and PCNA expression in BDL SEC/SR KO mice compared to BDL SEC or SR KO mice. There was more periportal necrosis and increased portal inflammation in BDL SEC/SR KO mice compared to BDL SEC or SR KO mice. We observed spontaneous damage in the liver sections from normal SEC/SR KO, which was associated with a significant increase in the expression of the pro-apoptotic protein Bax. In BDL SEC/SR KO mice, there was a greater reduction in both CFTR and Cl-/HCO3 - AE2 expression (compared to BDL SEC or SR KO mice) indicating a reduction of biliary growth and enhanced biliary damage due to lack of bicarbonate secretion. Summary/Conclusion: Knockdown of the SEC/SR axis down-regulates both biliary proliferation and the expression of CFTR and Cl-/HCO3 - AE2, which is associated with increased periportal necrosis, portal inflammation and biliary damage. These findings suggest that the complete inhibition of the SEC/SR axis triggers increased hepatic damage due to a lack of a: (i) functional SEC/SR/CFTR/Cl-/HC〇3 - AE2 system; and (ii) protective bicarbonate umbrella during extrahepatic cholestasis.

Disclosures:

The following people have nothing to disclose: Shannon S. Glaser, Fanyin Meng, Heather L. Francis, Julie Venter, Laura Hargrove, Holly A. Standeford, Syeda H. Afroze, Paolo Onori, Kelly McDaniel, Micheleine Guerrier, Eugenio Gaudio, Gianfranco Alpini

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Knockout of the histidine decarboxylase (HDC) gene reduces biliary hyperplasia in cholestatic bile duct ligated (BDL) mice

Laura Hargrove2, Hiroshi Ohtsu5, Taylor Francis3, Yoshiyuki Ueno4, Lindsey Kennedy2, Kyle M. Hodges2, Allyson B. Graf1, John F. Greene2, Heather L. Francis1, 2
1Central Texas Veterans Health Care System, Temple, TX; 2Scott and White Memorial Hospital, Temple, TX; 3Texas A&M Health Science Center, Temple, TX; 4Yamagata University, Yamagata, Japan; 5Tohoku University, Sendai, Japan

Background: Histamine is formed by the conversion of histidine by the enzyme, 丨-histidine decarboxylase (HDC). Cholangiocytes (i) express HDC and histamine receptors, H1-H4HRs; (ii) secrete histamine; and (iii) proliferate after histamine treatment via increased phosphorylation of ERK1 /2.In cholestatic rodents, there is enhanced biliary hyperplasia, HDC expression and histamine secretion. Inhibition of HDC decreases cholangiocarcinoma growth by decreasing VEGF. Aim: to demonstrate that knockdown of HDC in vivo and in vitro inhibits biliary proliferation. Methods: HDC-/- mice backcrossed with C57Bl6 mice as well as matching wild-type (WT) were subjected to sham or bile duct ligation (BDL). After 1 wk, serum, liver blocks and cholangiocytes were collected. Immunohistochemistry was performed for evaluating (i) intrahepatic bile duct mass (IBDM) by CK-19; (ii) PCNA-positive cholangiocytes; and (iii) HDC biliary expression in liver sections. We measured: (i) serum histamine levels by EIA; and (ii) lobular damage, necrosis, inflammation (H&E) and fibrosis (Sirius Red staining) in liver sections. In cholangiocytes, we studied: (i) the expression of HDC, H1-H4HRs, VEGF, PCNA, and the mast cell stem factor receptor, c-kit by q PCR; and (ii) PCNA protein expression and pERK1/2 by immunoblotting. Histamine levels were assessed by EIA in cholangiocyte supernatants. In vitro, cholangiocyte cultures were stably transfected with shRNA-HDC plasmids (or control) to knockdown HDC expression. After transfection we evaluated: HDC, H1-H4HRs, VEGF expression by qPCR, histamine release by EIA and pERK1/2 and cholangiocyte proliferation by immunoblots. Results: In BDL HDC-- mice, there was decreased IBDM, PCNA-positive cholangiocytes and HDC expression compared to BDL WT mice. Histamine levels were decreased in BDL HDC٪. BDL HDC٪ livers were void of necrosis and inflammation compared to WT BDL. In BDL HDC٪ cholangiocyfes, there was a reduction of HDC, H1-H4HR, VEGF, PCNA and c-kit expression compared to BDL WT. PCNA and pERK1/2 protein expression (increased in BDL WT) was lower in BDL HDC٪ cholangiocytes. In vitro, knockdown of HDC (85%) decreased HDC and H1-H4HR expression, proliferation, histamine secretion and VEGF expression. Protein expression of PCNA and ERK1/2 phosphorylation was decreased in shHDC-transfected cholangiocytes compared to control. Conclusion: Loss of HDC decreases BDL-induced biliary mass and VEGF expression via pERK1/2 signaling. Our data suggests that HDC is a key regulator of biliary proliferation and may be a potential target for the treatment of cholangiopathies.

Disclosures:

Yoshiyuki Ueno - Advisory Committees or Review Panels: Jansen

The following people have nothing to disclose: Laura Hargrove, Hiroshi Ohtsu, Taylor Francis, Lindsey Kennedy, Kyle M. Hodges, Allyson B. Graf, John F. Greene, Heather L. Francis

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Epigenetic regulation of definitive endoderm markers in biliary-committed progenitor cells during cholestatic liver injury

Kelly McDaniel4, Julie Venter2, Heather L. Francis1, 5, Yuyan Han2, Taylor Francis2, Jia-ming Lai3, Li Huang3, Debolina Ray2, Shannon S. Glaser1, 5, Gianfranco Alpini1,5,Fanyin Meng1,5
1Research and Medicine, Central Texas Veterans Health Care System and Texas A&M HSC COM, Temple, TX; 2Medicine, Texas A&M HSC COM, Temple, TX; 3Cell, Department of Hepatobiliary SurgeryBiology and Physiology, First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China; 4Research, Central Texas Veterans Health Care System, Temple, TX; 5Scott & White Digestive Diseases Research Center, Scott & White, Temple, TX

BACKGROUND & AIMS: Biliary-committed progenitor cells (small cholangiocytes) are more resistant to hepatobiliary injury than large cholangiocytes (LCCs). The definitive endoderm markers, FoxA2 and Sox17, are the key transcriptional factors that regulate cell differentiation and tissue regeneration. Our aim was to characterize the functional roles of epigenetic-regulated definitive endoderm markers in biliary progenitor cells during cholestatic liver injury. METHODS: Murine biliary committed progenitors (BCPs) and control LCCs were isolated from mice livers based on size distribution by counterflow elutriation. mRNA expression in BCPs, LCCs and human cholangiocytes (H69) was assessed by microarray analysis; the expression of selected mRNAs was further evaluated by real-time PCR assay. Bile duct ligation (BDL) and MDR2 knockout (MDR2-/-) mice were used as the animal model of cholestatic liver injury. RESULTS: We identified definitive endoderm markers including FoxA2, Sox17 as well as Notch1 that are differentially expressed in BCPs when compared to control LCCs by microArray analysis. Foxa2, Sox17 and Notch1 were notably more enhanced in murine liver progenitor cells compared with BCPs and LCCs. Transient transfection of siRNAs to FoxA2, Sox17 and Notch 1 simultaneously in BCPs for 7 days significantly reduced the nucleus to cytoplasm ratio, changed cell morphology from cuboid to columnar, and decreased levels of the antiapoptotic proteins, annexin-V and Bcl2.The combined siRNA treatment also induced a differentiation switch from N-Cadherin to E-Cadherin and promoted mesenchymal-epithelial transition (MET) events in biliary-committed progenitors, as well as significantly enlarged the size of BCPs derived from small bile ducts. Individual silencing of these three genes also reduced annexinV and Bcl2 levels without altering the morphological characteristics of BCPs. The 5'-promoter regions of FoxA2 and Sox17 were CpG island enriched. The expressions of FoxA2 ,and Sox17 were considerably increased after 5-aza-2'-deoxycytidine treatment in LCCs but not in BCPs cells. Methylation-specific PCR detected that FoxA2 and Sox17 promoters were hypermethylated in LCCs, but hypomethylated in BCPs. The activated definitive endoderm differentiation markers FoxA2 and Sox17 were also observed in murine small bile ducts in BDL and MDR2-/- mouse liver, suggesting that they are important mediators for biliary differentiation/remodeling during cholestatic liver injury. CONCLUSION: Epigenetic enzymes can regulate cell differentiation and survival by modulating the expression of definitive endoderm markers, the known positive regulators of biliary development and injury.

Disclosures:

The following people have nothing to disclose: Kelly McDaniel, Julie Venter, Heather L. Francis, Yuyan Han, Taylor Francis, Jia-ming Lai, Li Huang, Debolina Ray, Shannon S. Glaser, Gianfranco Alpini, Fanyin Meng

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MicroRNAs Dysregulation Induces HDAC6 Overexpression in Cholangiocarcinoma

Sergio A. Gradilone1,Brynn N. Radtke1, Gabriella Gajdos B1, Christy E. Trussoni1,Justin L. Mott2, Nicholas F. LaRusso1
1Center for Cell Signaling in Gastroenterology, Division of Gastroenterology and Hepatology, Mayo Clinic,Rochester, MN; 2Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE

PURPOSE: Cholangiocarcinoma (CCA) is a malignancy arising from cholangiocytes. While cholangiocytes normally express cilia, we showed that cilia are absent in cholangiocytes in CCA; the mechanism involves overexpression of histone deacetylase 6 (HDAC6), a molecule that deacetylates the ciliary axoneme promoting ciliary disassembly. Despite HDAC6 protein overexpression in CCA, we found no differences in its messanger RNA (mRNA) level, suggesting a post-transcriptional mechanism like microRNA (miRNA) regulation. Thus, we hypothesized decreased levels of specific miRNAs induce overexpression of HDAC6 in CCA. METHODS AND RESULTS: We compared miRNA levels between normal (H69) and CCA (KMCH) cholangiocyte cell lines by microarray and assessed results by in silico analysis. We found three miRNAs potentially targeting HDAC6 are downregulated (40-55% decrease) in CCA: miR-433, miR-22, and miR-200a. Experimental overexpression of miR-433, miR-22, or miR-200a in H69 cells decreased HDAC6 expression by 82%, 33%, and 39%, respectively. We selected miR-433 for further analysis; the downregulation of miR-433 was confirmed by qRT-PCR of nor-.mal and two different CCA cell lines (KMCH and HuCCT-1), and by in situ hybridization on control and CCA human liver samples (n=4 in each). Transfection of H69 cells with anti-miR-increased HDAC6 protein by 40%, while transfection with miR-433 decreased HDAC6 expression by 50% compared to scrambled controls. Also, miR-433 decreased (50%) luciferase activity of a reporter containing the HDAC6 3'UTR, consistent with binding of miR-433 to HDAC6 mRNA. In contrast to decreased levels of mature miR-433 in malignant cells, we found increased levels (4-fold) of its precursor (pre-miR-433) form by qRT-PCR. In situ hybridization showed that pre-miR-433 was sequestered in the nucleus of malignant cholangiocytes. Since the data suggested potential impairment of nuclear export of pre-miR-433, we assessed expression of exportin-5, a protein responsible for transport of pre-miRNAs from the nucleus. By western blotting, we found that exportin-5 is decreased by 50% in HuCCT-1 and KMCH CCA cell lines compared to normal cholangiocyte cell lines. We confirmed downregulation of exportin-5 protein by immunofluorescence of normal and CCA human liver samples (n=4 in each). CONCLUSIONS: Our data suggest that downregulation of exportin 5 prevents transport of pre-mir-433 from the nucleus into the cytoplasm thus limiting the ability of mature miR-433 to inhibit translation of HDAC6 mRNA. Thus, our work clarifies the molecular mechanisms accounting for the absence of cilia in CCA and also identifies multiple potential molecules for targeted therapies.

Disclosures:

The following people have nothing to disclose: Sergio A. Gradilone, Brynn N. Radtke, Gabriella Gajdos B, Christy E. Trussoni, Justin L. Mott, Nicholas F. LaRusso

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Cholangiocytes present antigens to NKT cells

Elisabeth Schrumpf1, 2, Tom H. Karlsen1, 3, Sebastian Zeissig4, Mark A.Exley5, Richard S. Blumberg5, Espen Melum1, 2
1Norwegian PSC Research Center, Division of Cancer, Surgery and Transplantation, Oslo University Hospital, Rikshospitalet, Oslo, Norway; 2K. G. Jebsen Inflammation Research Centre, Research Institute of Internal Medicine, Oslo University Hospital, Rikshospitalet, Oslo, Norway; 3Division of Gastroenterology, Department of Clinical Medicine, University of Bergen, Bergen, Norway; 4Deportment of Internal Medicine, University Hospital Schleswig-Holstein, Kiel, Germany; 5Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine, Brigham and Women΄s Hospital, Harvard Medical School, Thorn 1405, 75 Francis St, Boston, MA

Purpose: Natural killer T (NKT) cells are lymphocytes reacting to lipid antigens presented by Cd1d molecules and constitute up to 50% of the lymphocyte pool in the liver of mice. This study aimed to determine whether cholangiocytes express Cd1d, and to examine whether they present antigens to and activate NKT cells. Methods: Cd1d expression on immortalized murine small and large cholangiocytes was determined by flow cytometry. The same cholangiocyte cell-lines were cultured overnight with 7 different NKT cell hybridomas (DN32, 24.7, 24.8, 14s6, 14s7, 14s10 and 14s15) as well as primary NKT cells extracted from the livers of C57BL/6 mice. Cytokine secretion was measured in the culture supernatants with ELISA. Exogenous antigen presentation was evaluated by loading cholangiocytes with the model antigen a-GalCer for 4 hours while for endogenous antigen presentation no pre-incubation was performed. In some experiments Cd1d was blocked with a monoclonal anti-Cd1d antibody (19G11). Results: Flow cytometry demonstrated that both small and large cholangiocytes express Cd1d. When the cholangiocytes were cultured together with NKT hybridomas 2 out of 7 (DN32 and 24.7) of the hybridomas secreted IL-2 when the cholangiocytes had been incubated with a-GalCer (example in Figure 1A). The cholangiocytes presented endogenous antigens to 3 out of 7 hybridomas (14s6, 14s15 and 24.8) (example in Figure 1 B). Blocking with an anti-Cd1d antibody confirmed that the antigen presentation was Cd1d restricted (Figure 1A and 1B). Activation by both exogenous and endogenous antigens was also seen when primary NKT cells were cultured together with cholangiocytes (Figure 1C). Conclusions: Our results demonstrate that murine cholangiocytes express Cd1d and present antigens to NKT cell lines and primary NKT cells. The findings implicate that cholangiocytes can regulate this important lymphocyte subset of the liver and potentially bile duct inflammation in the context of various cholestatic liver diseases.

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Disclosures:

Mark A. Exley – Stock Shareholder : NKT Therapautics Inc.

The following people have nothing to disclose: Elisabeth Schrumpf, Tom H.Karlsen, Sebastian Zeissig, Richard S. Blumberg, Espen Melum

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Development and characterization of an extraheptic cholangicyte culture system from the rat common bile duct

Julie Venter4, Laura Hargrove5, Sharon DeMorrow4, 5, Kelly McDaniel6, Micheleine Guerrier4, Marco Marzioni2, Gabriel A. Frampton4, Holly A. Standeford4, Eugenio Gaudio3, Paolo Onori3, Debolina Ray4, Shannon S. Glaser1, 5, Fanyin Meng1, 5, Heather L. Francis1, 5, Gianfranco Alpini1, 5
1Research and Medicine, Central Texas Veterans Health Care System, Texas A&M HSC College of Medicine, Temple, TX; 2Gastroenterology, Universita Politecnica delle Marche, Ancona, Italy; 3Deportment of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Universita΄ Sapienza, Rome, Italy; 4Medicine, Texas A&M HSC COM, Temple, TX; 5Scoff & White Digestive Diseases Research center, Scott& White, Temple, TX; 6Research, Central Texas Veterans Health Care System, Temple, TX

Cholangiocytes, which line the intrahepatic and extrahepatic bile ducts, secrete bile and proliferate in response to gastrointestinal hormones such as secretin and somatostatin. While secretin stimulates bile secretion by interacting with secretin receptor (SR) by activating the cAMP═›PKA═›CFTR═›Cl-/HC〇3-_ AE2 pathway, somatostatin inhibits secretin-stimulated choleresis by acting on SSTR2 receptors. Numerous in vitro models are available for studying the function of rodent and human intrahepatic cholangiocytes and gallbladder epithelial cells. However, limited in vitro tools exist for evaluating the pathophysiology of extrahepatic common bile ducts (ECBDs). Aim: To develop a culture system from ECBDs from normal rats. Methods: ECBDs were dissected from normal male Fischer 344 rats, cut in half length-wise and cultured on collagen-I coated plates containing DMEM/F12 media with 5% of FBS and 24 ng/ml of EGF. TransEpithelial Electrical Resistance (TEER, an index of cell confluence across the epithelial monolayer) was determined with an EV〇M2 epithelial volt-ohm meter. At ∼85% confluency, cells were trypsinized before measuring: (i) cell size and distribution by a Cellometer, and expression for CK-19, secretin, SR, SSTR2, CFTR, Cl-/HC〇3 - AE2, albumin, VEGF-A/C and NGF (key trophic factors in cholangiocytes) by real-time PCR and/or immunoblots, and immunofluorescence in cell smears; and (ii) the effect of secretin, somatostatin and secretin + somatostatin (all at 100 nM) on cAMP levels by EIA and proliferation (by MTS assays, 100 nM, 1-7 days). Results: ECBDs composed entirely of CK-19-positive cholangiocytes, cultured for 6 passages formed a monolayer of epithelial cells. Passaged cultured ECBDs formed spherical cysts, an indication of water secretion. Cholangiocytes proliferated to confluency over a 2-wk period, as indicated by the TEER value of 195.8±3.54 ohms/cm2.The size of ECBDs ranged from 8 to 26 μm, mean diameter ∼16 μm. ECBDs were positive for secretin, SR, SSTR2, CFTR, Cl-/HC〇3 - AE2, VEGF-A/C and NGF but negative for albumin, a marker of mature hepatocytes. Secretin increased cAMP levels and cell mitosis, increases that were prevented by pre-incubation with somatostatin. Summary/Conclusion. We have developed a culture system of polarized ECBDs displaying morphological, phenotypical and functional features of normal cholangiocytes. This culture system may be key for a better understanding of diseases targeting extrahepatic bile ducts such as primary sclerosing cholangitis and biliary atresia.

Disclosures:

The following people have nothing to disclose: Julie Venter, Laura Hargrove, Sharon DeMorrow, Kelly McDaniel, Micheleine Guerrier, Marco Marzioni, Gabriel A. Frampton, Holly A. Standeford, Eugenio Gaudio, Paolo Onori, Debolina Ray, Shannon S. Glaser, Fanyin Meng, Heather L. Francis, Gianfranco Alpini