AA

SL

DA

bst

ract

s829

Cysteine Sulfinic Acid Decarboxylase Regulation by Bile Acids: A Role forFXR and SHP in Hepatic Taurine MetabolismThomas A. Kerr, Yuri Matsumoto, Hitoshi Matsumoto, Yan Xie, SayeepriyadarshiniAnakk, Susan Kennedy, David D. Moore, Nicholas O. Davidson

Background and Hypothesis: Bile acid synthesis is tightly regulated by nuclear receptorsfarnesoid X receptor (FXR) and short heterodimer partner (SHP), and by fibroblast growthfactor-15 (FGF15) to protect the liver from bile acid toxicity. Since bile acids regulate bothcholate synthesis and conjugation to taurine, we hypothesized that hepatic synthesis oftaurine is also tightly regulated. Little is known about transcriptional regulation of the rate-limiting enzyme in hepatic taurine synthesis, cysteine sulfinic acid decarboxylase (CSAD).We reasoned that hepatic CSAD mRNA expression is controlled by bile acids in a feedbackfashion to coordinate taurine and cholate availability and that CSAD gene expression utilizesregulatory mechanisms shared with cholesterol 7-alpha-hydroxylase. Methods and Results:We found that CSAD mRNA was widely expressed in C57BL/6 mice with highest levels inliver, kidney, white adipose tissue and lung. We next examined dietary, pharmacologic, andgenetic control of hepatic CSAD mRNA expression. Mice (n=5/group) were fed chow aloneor with 0.25% cholate, 0.5% cholate, or 2% cholestyramine for one week and fasted for 4hours prior to sacrificed for tissue analysis. Dietary cholate feeding resulted in a dose-dependent suppression of hepatic CSAD mRNA levels. 0.25% and 0.5% cholate feedingresulting in 77% (±4%, p=0.003) and 87% (±2%, p=0.001) decrease in CSAD mRNA levelsrespectively while inducing SHP mRNA expression 2.3 fold (±0.25, p=0.002 for 0.25%cholate feeding). By contrast, 2% cholestyramine feeding resulted in a 2.2 fold (±0.28, p=0.006) increase in CSAD mRNA levels compared to chow-fed control mice, suggesting basalphysiologic suppression of CSAD by enteric bile acids. We next examined FXR regulationof CSAD in chow-fed mice (n=4/group) gavaged twice daily with the FXR agonist GW4064or vehicle for 4 days with the last treatment 2 hours prior to sacrifice. FXR activation byGW4064 resulted in 75% suppression of CSAD mRNA (±1.1%, p=0.021). We then turnedto examine SHP regulation of CSAD expression in chow-fed WT and Shp-/- mice (n=6/group). We found that CSAD mRNA expression was increased 8.49 fold (±0.25, p<0.0001)in Shp-/- mice compared to WT controls. Conclusions: Hepatic CSAD mRNA levels arephysiologically regulated by bile acids in a feedback fashion. The mechanism of CSADsuppression involves the nuclear receptors SHP and FXR. Bile acid regulation of CSADexpressionmay allow for coordinated availability of cholate and taurine preventing hepatotox-icity. These data implicate bile acids as potential regulators of hepatic taurine metabolismvia mechanisms shared with cholesterol 7-alpha-hydroxylase. These findings may promotegreater understanding of cholestatic liver conditions and a better understanding of pharmacol-ogic FXR agonists now in clinical development.

830

Cysteine 266 of Rat NTCP is Necessary for Optimal Taurocholate Uptake andMethylthiosulfonate Inhibition but is Not Solely Responsible for Inhibition byNitric OxideUmadevi Ramasamy, Mohammed S. Anwer, Christopher M. Schonhoff

BACKGROUND:We have previously shown that nitric oxide (NO) non-competitively inhibitstaurocholate (TC) uptake by the human sodium dependent taurocholate co-transportingpolypeptide (NTCP). NO causes a decrease in the amount of NTCP present at the membraneand NO treatment results in S-nitrosylation of NTCP under these conditions. However,which of the 8 cysteine residues of NTCP is/are nitrosylated by NO is not known. It hasbeen reported that membrane impermeant methylthiosulfonates (a cysteine labeling agent)can inhibit NTCP function and this effect was reversed when cysteine 266 of NTCP ismutated to alanine (C266A NTCP). These results may suggest that NO inhibits TC uptakeby S-nitrosylating cysteine 266 of NTCP. AIM: The aim of the present study was to determinewhether NO targets cysteine 266 of rat Ntcp resulting in an inhibition of TC uptake.METHODS: Studies were conducted in a human hepatoma cell line, HuH7, that weretransiently transfected with wild type and C266A Ntcp constructs. Cells were treated withthe membrane impermeant methylthiosulfonate (MTSET) or with the NO donor S-nitroso-cysteine (CysNO). TC uptake was measured using 3H-taurocholate. Total levels of Ntcpand plasma membrane levels of Ntcp were measured by immunoblot and by selectivebiotinylation of plasma membrane bound proteins. RESULTS: Transient transfections withwild type and C266A Ntcp were confirmed by immunoblotting. Taurocholate uptake bythe C266A mutant decreased significantly as compared to wild type Ntcp. Mutation of ratNtcp cysteine 266 to alanine did not affect the transfection efficiency or plasma membranelocalization of Ntcp indicating that cysteine 266 is required for optimal TC uptake by Ntcp.Treatment with the membrane impermeant methylthiosulfonate MTSET (1 mM), inhibiteduptake by wild type Ntcp but did not inhibit uptake by C266A Ntcp. However, 10 mMMTSET inhibited TC uptake by both wild type and C266A Ntcp, indicating that cysteineresidues other than C266 may be involved in TC uptake by Ntcp. This is further supportedby results that TC uptake by C266A Ntcp was also inhibited by the NO donor, CysNO(0.2 - 1 mM). CONCLUSION: Taken together, these results suggest that cysteine 266 ofrat Ntcp is necessary for optimal TC uptake and the inhibition of TC uptake by NO mayin part be due to S-nitrosylation of C266. Whether inhibition of TC uptake is due to S-nitrosylation of other cysteine residues is currently under investigation.

831

The Sonic Hedgehog (SHH) Signaling Pathway: A Novel Player and PotentialTherapeutic Target for Biliary ObstructionEleonora Gaetani, Daniela Pugliese, Franco Scaldaferri, Mariachiara Campanale, SilviaPecere, Fabrizio Forte, Gian Ludovico Rapaccini, Antonio Gasbarrini, Roberto Pola

Background and Aim: Sonic Hedgehog (Shh) is a morphogen with crucial regulatory functionsduring embryogenesis of the liver. This developmental pathway is recapitulated in post-natal life in the liver in response to biliary obstruction. In this study we investigated thefunctional role and the potential therapeutic utility of Shh in the setting of cholestatic liver

S-930AASLD Abstracts

diseases. We also assessed the relationship between the Shh pathway and the angiogenicfactor VEGF, which is known to be upregulated in the liver after BDL and is a mediator ofShh function in other organs and tissues. Material and Methods : Liver cholestasis wascreated using an established model of bile duct ligation (BDL) in mice. Expression levels ofShh and VEGF were assessed by real-time PCR. Inhibition of Shh was obtained by systemictreatment with cyclopamine. Shh gene therapy was performed using the phShh, a plasmidthat encodes the human Shh gene. VEGF inhibition was obtained using the sFlt1 plasmid.Results: BDL induced significant increase of ALT and AST plasma levels, liver fibrosis, andnecrosis, along with reactivation of the Shh signaling pathway and significant upregulationof VEGF. Inhibition of Shh significantly increased cholestasis (in terms of ALT and ASTplasma levels) and enhanced liver fibrosis and necrosis (P<0.01). Shh inhibition also reducedupregulation of VEGF after BDL (P<0.01). In contrast, in mice treated with phShh, we founda significant reduction of ALT and AST plasma levels and amelioration of liver fibrosis andnecrosis (P<0.01). These phShh-induced beneficial effects were inhibited by combinedadministration of the sFlt1 plasmid, which inhibits VEGF signaling (P<0.01). Conclusions:These data demonstrate that inhibition of the Shh pathway increases liver damage after BDL,while treatment with Shh diminishes cholestasis and reduces liver fibrosis and necrosis. Thebeneficial effects of Shh gene therapy are reduced by inhibition of VEGF, thus indicatingthat they are VEGF-dependent.

832

Increased Synthesis of Melatonin From Pineal Gland and Cholangiocytes (byProlonged Exposure of Cholestatic Rats to Complete Dark) Leads to Inhibitionof Biliary Hyperplasia by Autocrine/Paracrine MechanismsGiuseppina Dusio, Anastasia Renzi, Fanyin Meng, Sharon DeMorrow, Julie Venter,Mellanie White, Heather L. Francis, Yuyan Han, Paolo Onori, Kimberly K. Baker, WendyButler, Pietro Invernizzi, Eugenio Gaudio, Gianfranco Alpini, Shannon Glaser

In cholestatic bile duct ligated (BDL) rats, cAMP-dependent biliary hyperplasia is regulatedby several neuroendocrine autocrine/paracrine factors including serotonin and melatoninthat are secreted by cholangiocytes. Melatonin is formed from L-tryptophan by the activityof the enzymes, serotonin N-acetyltransferase (AANAT, expressed in liver only by cholangi-ocytes), and N-acetylserotonin O-methyltransferase, and is produced by the pineal gland aswell as small intestine and liver. Melatonin (whose synthesis is higher at dark) is a keycircadian timing signal regulating the expression of clock genes and function in a numberof cells. We have previously shown that: (i) chronic administration of melatonin increasedmelatonin serum levels and inhibits cholangiocyte hyperplasia in BDL rats by downregulationof the clock genes, CLOCK, BMAL1, CRY1, and PER1; and (ii) downregulation of AANATbiliary expression (by Vivo-morpholino) increases cholangiocyte hyperplasia in rats. Noinformation exists regarding possible effects of dark therapy in the regulation of biliaryhyperplasia. We tested the hypothesis that prolonged exposure to complete dark increasesmelatonin synthesis by the pineal gland and cholangiocytes leading to inhibition of cholangi-ocyte growth during cholestasis by both autocrine/pathways mechanisms. Methods: Normaland BDL (immediately after surgery) rats were housed for 12:12 hr light/dark cycles orcomplete dark for 1 week before evaluating: (i) serum levels of melatonin, bilirubin andtransaminases; (ii) intrahepatic bile duct mass (IBDM) in liver sections and secretin effectson bile secretion (a functional index of biliary growth); (iii) the mRNA expression of AANATin pineal gland, small intestine and cholangiocytes, and the expression of PCNA and clockgenes in cholangiocytes by qPCR; and (iv) the levels of melatonin in the medium of short-term cultures of cholangiocytes. Results: In BDL rats exposed to continuous dark there was:(i) enhanced melatonin serum levels and reduced levels of bilirubin and transaminases; (ii)IBDM and lack of choleretic response to secretin; (iii) enhanced AANAT expression mostlyin pineal gland and cholangiocytes and reduced expression of PCNA and clock genes incholangiocytes; and (iv) increased secretion of melatonin in cholangiocyte medium comparedto rats exposed to light/dark cycles. No significant effects were observed in normal rats.Summary/conclusion: Exposure of cholestatic rats to prolonged dark increases the synthesisof melatonin from pineal gland and cholangiocytes leading to inhibition of biliary hyperplasialikely by both autocrine/paracrine mechanisms. These findings highlight a critical need forthe further evaluation of alterations of the dark/light cycle in the progression of cholestaticliver injury and as a concomitant therapeutic approach for patients with liver diseases.

833

Distribution Dynamics of Radixin and NHERF-1 on Regulation of mrp-2Trafficking in HepatocytesJo Suda, Lixin Zhu, Serhan Karvar

Radixin is a member of ezrin, radixin, moesin (ERM) protein family that links F-actin tomembranes. The NH2- and COOH (N-C)-terminal association domains of ERM proteinsparticipate in interactions with membrane proteins and F-actin, and molecular interactionswithin ERM. Radixin has been reported to selectively tether mrp-2 to the canalicular mem-brane. We have examined the dynamic distribution of radixin and NHERF-1 in live hepato-cytes using fluorescence-tagged constructs. Cyan fluorescent protein (CFP)-tagged Radixin,yellow fluorescent protein (YFP)-tagged NHERF-1 wild type and mutant adenoviral con-structs were used. Functional analyses were characterized quantitatively using CMFDA. Themolecular (N-C) binding of radixin was visualized using fluorescence resonance energytransfer (FRET). Live fluorescence imaging showed that wild type radixin and NHERF-1 islocalized to the canalicular membrane along with the mrp-2 in dual infected cells. CFP-Radixin-T564A is a non phosphorylated mutant that does not bind to F-Actin. Similar toendogenous radixin, wild type and CFP-radixin T564A localized to canalicular membrane.Interestingly, mrp-2 was incorporated into the canalicular membranes along with wild typeand CFP-radixin T564Amutant radixin. CFP-Radixin T564Dmutant, which mimics constantphosphorylation, was more typically localized to the basolateral membrane, often associatedwith long spikes and fingerlike projections. Mrp-2 distribution was detected throughout thecytoplasm in CFP-radixin T564D infected cells. The T564D mutant infected WIF-B cellswere devoid of a secretory response. We have also observed cytoplasmic distribution ofNHERF-1 F355R (radixin binding site mutated) where as wild type HNERF-1 was localizedat the canalicular memebrane. Mrp-2 distribution was detected throughout the cytoplasm

in NHERF-1 F355R infected cells. FRET was observed in wild type and T564A YFP-radixin-CFP constructs, indicating molecular (N-C)-terminal binding. No significant FRET wasdetected in T564D YFP-radixin-CFP. In Vitro FRET analysis and actin binding binding assaydemonstrated that Thr564 phosphorylation opens the (N-C)-terminal interaction and theopened phosphorylated form of radixin more readily cosediments with F-actin and bindsto membrane. In addition, we have characterized of NHERF1 as a binding partner of Mrp-2. We identified the presence of radixin and NHERF-1 in hepatocytes and our data stronglyimplicate that radixin and NHERF-1 are essential for maintaining the polarized targetingand retaining of canalicular transporters and is a critical determinant of the overall structureand function of the canalicular membrane of hepatocytes.

834

StarD5, an Intracellular Cholesterol Transport Protein, is Regulated by the ER-Stress Activated Transcription Factor XBP-1: A Possible Mechanism to ReduceER Free Cholesterol AccumulationMaria Calderon-Dominguez, William M. Pandak, Gregorio Gil, Shunlin Ren, DanielRodriguez-Agudo

StarD5 belongs to the StarD4 subfamily of START (steroidogenic acute regulatory lipidtransfer) domain proteins. StarD5 is found in the cytosol and maintains a loose associationwith the golgi. Like StarD1 and StarD4, StarD5 is known to bind cholesterol. Although ithas been hypothesized that StarD5 plays a role in non-vesicular intracellular lipid transportand homeostasis, the true function of StarD5 remains poorly defined. Recently, it has beenshown that its mRNA expression is induced in response to Endoplasmic Reticulum (ER)stress. The objective of this study was to characterize StarD5 expression under ER-stressconditions in order to pursue its function. Methods: All the experiments were performedin 3T3-L1 cells. Relative levels of mRNAs were measured by qRT-PCR following transfectionof cells with three different ER-stress transcription factors (XBP-1(s), ATF-6 (1-373), andATF4), or following ER-stress induction by Thapsigargin (Tg; ER calcium ATPase inhibitor);to simulate the calcium ATPase disruption seen with ER free cholesterol accumulation.Filiping staining was used to determine accumulation of free cholesterol within the cells.Studies were also performed to determine the presence of potential ER-stress sequenceelements in the StarD5 -2000 5'-flanking region, and the stability of the StarD5 mRNAunder ER-stress conditions. Results: When cells were incubated in the presence of Tg, StarD5mRNA expression and ER free cholesterol increased. Time course studies showed that StarD5mRNA peaked 6 hours after Tg treatment, correlating with an increase in free cholesterolaccumulation as determined by filiping staining. Cells transfected with the transcriptionfactors involved in ER stress showed that only XBP-1(s) induced StarD5 mRNA expression.When cells were transfected with an expression vector encoding the ER resident proteinBiP (believe to protect the cells from ER stress), Tg-mediated upregulation of StarD5 mRNAlevels was markedly repressed as compared with non-transfected cells. The promoter studiesdid not show the presence of any ER stress responsive element within the first 2000 basesupstream of the transcriptional initiation site. However, additional upstream regulatoryelements cannot be excluded. In contrast, induction of ER-stress stabilized StarD5 mRNA.Conclusions: The ability of StarD5 to bind cholesterol coupled with its responsiveness to ER-stress, allow us to hypothesize a potential role for StarD5 in the maintenance of intracellularcholesterol levels under ER-stress conditions. More specifically, StarD5 mRNA stabilizationand up-regulation by the transcription factor XBP-1(s) during the cell protective phase ofthe ER stress, suggests a potential role for StarD5 in the transport of cholesterol from theER to other membranes in order to lower the amount of cholesterol that accumulates inthe ER during ER-stress.

835

Exposure-Response Relationships in Telaprevir Combination Therapy inTreatment-NaïVE Genotype 1 Chronic HCV PatientsStuart C. Gordon, Andrew J. Muir, Varun Garg, Joshua W. Henshaw

Introduction: Telaprevir (TVR) in combination with peginterferon alfa-2a (Peg-IFN) andribavirin (RBV) was recently approved in the United States, Canada, Europe, and Japan forthe treatment of genotype 1 chronic HCV infection. The relationship between drug exposureand safety and efficacy parameters is unclear. We evaluated relationships between efficacyand safety and concentrations of TVR, RBV, and Peg-IFN.Methods: Treatment-naïve patients(N=472, ADVANCE/ILLUMINATE) who were assigned 12-week TVR combination therapywith 24- or 48-week total PR and had PK data available were included. Using generalizedlinear modeling (logistic regression) and receiver operating characteristic (ROC) analyses,we investigated associations between drug exposures, eRVR, SVR, anorectal symptoms, andcertain subsets of anemia (defined by DAIDS toxicity grading) and rash. Population PKmodel-predicted average steady-state TVR concentrations, the observed Week-4 RBV (plasma)concentrations, and observed Week-4 Peg-IFN (serum) concentrations were representativeexposure measures. Results: eRVR and SVR weakly correlated with TVR, RBV, and Peg-IFN exposure. With increasing exposure, each drug exhibited a shallow, non-significantincrease in eRVR and SVR probability. Anemia incidence during the TVR-phase correlatedwith TVR, RBV and Peg-IFN exposure, and correlated most strongly with RBV exposure.No drug exposures strongly correlated with rash or anorectal symptoms during the TVR-phase. Conclusions: Within the observed concentration ranges, TVR, Peg-IFN, and RBVexposures had non-significant associations with eRVR and SVR, suggesting that increasingexposure is unlikely to significantly impact eRVR or SVR rates. Treatment-emergent anemiacorrelated with exposure to TVR, RBV, and Peg-IFN, most strongly with RBV exposure,supporting RBV dose reduction in treatment-emergent anemia management. The absenceof correlation between drug exposure and rash or anorectal symptom occurrence or severitysuggests that dose adjustment is unlikely to significantly impact these parametersTable: ROC area under the curve

S-931 AASLD Abstracts

†extended Rapid Virologic Response: undetectable HCV RNA at weeks 4 and 12 * SustainedVirologic Response

836

Sustained Virologic Response (SVR) in Prior Peginterferon/Ribavirin (PR)Treatment Failures After Retreatment With Boceprevir (BOC) + PR: theProvide Study Interim ResultsJohn M. Vierling, Mitchell N. Davis, Steven L. Flamm, Stuart C. Gordon, Eric J. Lawitz,Eric M. Yoshida, Joseph Galati, Velimir A. Luketic, Jonathan McCone, Ira M. Jacobson,Patrick Marcellin, Andrew J. Muir, Fred Poordad, Lisa D. Pedicone, Weiping Deng,Michelle A. Treitel, Janice Wahl, Jean-Pierre Bronowicki

Background: Patients in the PR control arms of BOC Phase 2/3 studies who did not achieveSVR could enroll in PROVIDE and receive BOC + PR. This interim analysis examines thepreliminary efficacy and safety of BOC + PR in patients who failed prior treatment with PR.Methods: BOC (800 mg TID with food) was given with P 1.5 μg/kg/week and weight-basedR (600-1400 mg/day) BID for up to 44 weeks. If >2 weeks had elapsed since end of treatmentin the previous study, PR was given for 4 weeks before adding BOC. Protocol specifiedanalyses include patients who received at least one dose of BOC. Denominators for on-treatment response include patients who reached the specific time point or discontinued.The denominators for SVR include all patients who reached end of follow-up, discontinued,or were treatment failures. Results: Characteristics of 168 enrolled patients were: 67% male,84% Caucasian, mean age 52 years, mean BMI 27.9 kg/m2, 77% high viral load (>800,000IU/mL; mean log10 6.26); 10% cirrhotic; 61% subtype 1a. Table shows the proportion ofBOC-treated patients with undetectable HCV RNA at tested time points. SVR was achievedin 40% of prior null responders (<2 log10 decline in HCV RNA at TW12 in prior study)and 68% of prior partial responders/relapsers; 78% (38/49) of prior null responders and24% (26/107) of prior partial responders/relapsers had <1 log10 decline in HCV RNA afterthe PR lead in. Overall, SVR was 47% in patients with <1 log10 decline with lower SVRrates in prior null responders (36%) vs. prior partial responders/relapsers (65%). 68% ofpatients with >1 log decline achieved SVR (55% prior null responders; 70% prior partialresponders/relapsers). Seven percent of patients discontinued due to AEs, while 48% experi-enced anemia, 34% dysgeusia, and 22% neutropenia. Conclusions: BOC + PR achievedhigh SVR rates regardless of prior response to PR. The degree of interferon responsivenessafter PR lead in correlates with prior response and can help predict SVR for prior nullresponders. The safety profile is comparable to that previously reported for BOC + PR.

837

Early Virologic Response in Patients With HCV Recurrence After LiverTransplant Treated With Triple Therapy: A Single Center ExperienceParvez S. Mantry, Abdullah Mubarak, Jeffrey S. Weinstein, Bahar Madani, Hector E.Nazario, Alejandro Mejia, Tiffany Anthony, Steve Cheng

Background: Telaprevir (TVR) is currently not FDA approved in post transplant patients.We attempted treatment on 9 patients with Hepatitis C (HCV) recurrence after liver transplantusing triple therapy with Interferon, Telaprevir (TVR) and Ribavirin (RBV) with judiciousadjustment of their immunosuppressive regimen and report our early experience. Methods:9patients with documented genotype 1 HCV infection post liver transplant were started oncombination therapy with pegylated interferon alpha 2a or 2b, RBV, and TVR. All patientswere on tacrolimus(TAC) at a stable dose prior to start of the antiviral regimen. At the startof therapy, the TAC dose was reduced to approximately half the pre-treatment dose andthe frequency was reduced to once a week. Trough TAC levels were checked intensivelyduring the TVR therapy period. After completing TVR, TAC was re-introduced slowly topre-TVR dosing. CBC, Chemistry, HCV RNA (by TMA) and clinic evaluation of patientswas performed once a week during the first 4 weeks of Rx and every 2 weeks there after.Results: There were 6 male and 3 female patients in the cohort who were from 6 monthsto 6 years out post transplant. Ethnicity was 6 caucasian, 2 hispanic and 1 Asian. Treatmentcharacteristics are described in table I. In patient no.2 TVR was added due to lack of adequateresponse to PEG/RBV at week 12. The remaining patients started all three drugs at the sametime. of the 9 patients, 6 had rapid virologic response (RVR) and extended (e)RVR; Twopatients became aviremic at 33 and 34 days and had complete early virologic response. Onepatient was a non responder and treatment was stopped. In terms of tolerability, the mainadverse effect was severe anemia with 6 patients requiring blood transfusions. Dehydration,renal insufficiency and infections were amongst the other prominent adverse events- includingone patient who had very severe recurrence with ascites and allograft failure due to HCVrecurrence- severely decompensated during therapy with pneumonia, ARDS and died ofsepsis. There was one documented episode of moderate rejection in a patient which was

AA

SL

DA

bst

ract

s