- Email: [email protected]
Adenoviral gene transfer of lipopolysaccharide binding protein (LBP) results in increased acetaminophen-induced hepatotoxicity
mice and least in MCD mice; EtOH mice produced intermediate levels. Oxidativ'e DNA damage was increased significantly in all fatty liver groups. DNA damage and inhibited DNA synthesis in mature hepatocytes paralleled H202 productiorl (ob/ob > EtOH > MCD > control mice). At baseline, mice with fatty livers had significantly more oval cells than controls (controls 3 _+1 vs. MCD diet fed 14 -+4; ob/ob 21 -+9; EtOH-fed 31 -+9 O r - 6 + cells/field, p -<0.001 controls vs. others). PH and tumor-promoting drags generally increased oval cells, but fatty livers always had 2-4 told more owal ceils than similarly-treated controls. Ethionine-treated ob/ob mice had the most oval cells ( 1 7 0 +_ 10 ceils/field; p -.<0001 vs others). Oval cells were also increased significantly in patients with NAFLD and ALD and the numbers strongly correlated with fibrosis stage. Conclusions: Fatty livers produce excess H202 and have oxidative DNA damage and inhibited prolif~:ration of mature hepatcx:ytes, This is known to induce compensatory' hepatic accumulation of progenitor cells and, thus, provides a mechanism for increased oval cells in fatty livers. Given evidence that oval cell accumulation precedes HCC, fatty liver may be a pre-amlignam condition.
1 Cathepsin B Inactivation Attenuates Hepatic Apoptosis and Fibrosis During Cholestasis Aft Can/Jay, Ha}line Hignchi, Maria E Guicciardi, Steven F Bronk Ariel Feldstein, Makiko Taniai, Robert M Rydzewski, Gregory J Gures Hepatocyte apoptosis and liver fibrosis are key teamres of cholestatic liver injury. Emerging data implicate a lysosolnal, cathepsin B-dependent pathway of apoptosis in bepatocytes. In this pathway, catbepsin B is released from lysosomes and tuggers mitochondrial dyshmction l%wever, the contribution of this pathway to hver injury and the relationship ot apoptosis to fibrosis remains unclear. Our Aim was to ascertain if inactivation of cathepsin B attenuates hvcr injury and fibrogenesis dunng extrahepatic cholestasis METHODS: Wild type (catB *s ~), cathepsin B-deticient (knockout) (catB~); and R 3032 (a catbepsin B inhibitor) treated catB */* mice undement bile duct ligation, Liver injury was examined by quantitating hepatocyte apopmsis with the TUNEL assay and determining serum ALT values, mRNA expression iur markers of stellate cell activation, a4MA, and fibrogenic actiwty, TGF-b and collagen I, was quantitated using real time PCR technolo~'. Liver fibrosis was assessed by digital image analysis ot Sirras red stained sections RESULTS: TUNEL positive hepamcytes and serum All- values were significandy reduced in cattY: and R 3032-treated catB +j+ 3-day BDL mice vs, catB +~ BDL mice, Consistent with these observations, mitochondrial cytochn)me c release, a marker for the mitochondrial pathway of apoptosis, was reduced in catB e and in R 3032-treated catB++ compared to catB ++ BDL mice, Stellate cell activation, as assessed by a-SMA was 14.0-fold greater in catB j+ vs catB v and 3-fold greater than in R 3032treated cazB +j+ BDL mice. Collagen al(I) was 102-told greater in catB ~+ vs. catB / BDL mice and 76-told greater in catB ~ ~ vs R 3032-treated ca~B +~ BDL mice. Serum bile acid and bilimbin values were mrtually identical in nil three experimental groups indicating that the above diliFrences could not be ascribed to difterences in cholestasis. Finally, in 2 week BDL mice, Sirius red staining for hepatic collagen deposition was significantly reduced in catB ~ mice compared to wdd type animals In CONCLUSION these findings not only support a prminnent tom tor the lysosomal pathway of apoptosis in cholestatic liver injury, but also link hepatocyte apoptosis to liver fibrogenesis These observations suggest cathepsin B inactivation or inhibition may be a therapeutic antifibrogenic strategy" in cholestatic liver diseases.
4 Bile Acid Induced Negative Feedback Regulation of the Human Ileal Bile Acid Transporter is Mediated by the Farnesoid X-Receptor (FXR), the Short Heterodimer Partner (SHP), and the Retinoid X Receptor (RAR/RXR) Ezequlel Neimark, Frank Chen, Benjamin Shneider Introduction: Ileal expression of the apical sodium dependent bile acids transporter (ASBT) in the mouse but not rat is under negative-feedback regulation by bile salts (Chen J. Biol Chem. in press). This species-specific difle:rence is secondary to the presence or absence of LRH-1 cis-elements and trans-acting factors. The bile acid responsiveness of human ASBT is unknown. Methods: 1.8 kb of the human 5' flanking region ot ASBT was subcloned into a luciferase reporter vector. Transient tmnsfection analyses were perfi)rmed in Caco-2 cells using expression plasmids thr FXR, SHP, LRH and RAR/RXR.Bile acid response was measured after treating the cells with varying concentrations (5 to 100 b~M) of CDCA Results: CDCA treatment of Caco-2 cells led to a 75% reduction in steady-state ASBT mRNA levels. Activity of the human ASBT prmnoter was reduced in a dose-dependent t~tshion by increasing concentrations of CDCA. Co-tr_anst;ection of FXR enhanced the bile acid mediated repression of ASBT promoter activity, while co-transfection of a dominant negative FXR abrogated the inhibitory" effect of CDCA. Site directed mutagenesis of an RAR/RXRcis-element in the ASBT promoter, reduced the basal activity' of the promoter by 9096 and eliminated the bite acid response, 9-cis retinoic acid activated the human ASBT promoter seven fold, while LRH-1 had no effect. SHP repressed the basal activity of' the ASBT promoter and sigmficandy reduced its activation by 9-cis retinoic acid. Conclusion: The human ileal bile acid transporter is positively regnlated by 9-cis retmoic acid. Bile acids induce a negativedeedback regulation of ASBT via an FXR-mediated, SHP-dependent effect upon ILA.D'I~XiRactivation ot ASBT.
2 Ca2+ in the Nucleus ls Independently Regulated and Has Distinct Effects on Protein Ydnase C (PKC) in a Liver Cell Line Mateus "iF Guerra, Maria de Fatima keite, Wihelma Echevarria Michael Nathanson Cytosohc Ca* regulates a wide range of fnnctions in hepatocytes. Ca > signals also occur iu the nucleus of hepatocytes, but it is unckar how they are regulated or whether they have distinct etfects on cell tunction. To investigate these questions, we examined Caz§ release mechainsms in the nucleus and the eltects of nuclear and cy'losolic Ca2~ signals on PKC translocation in the 5kHepl liver cell line. Contocal imnmnoBuorescence of the ER-resident protein calreticulin revel(led a reticular netwoik extending from the ER and nuclear envelope into the intenor of the nucleus. Confocal nucroscopic examination of live cells loaded vdth the low aflimty Ca2' dye umg-fluo-4 revealed sinlilar intranuclear structures, Fluorescence recovery after photobleaching of mag-fluo-4 in the nucleus (0.32 +- 0.09 sec~; n = 14) and cytosol (0,40 _'2'.0 0 8 sec; n = 19) showed similar recovery rates (p>0,4), providing complementary evidence that C a t stores within these nuclear structures are continuous with those in the ER, In order to determine the physiological role of these intranuclear Ca2+ stores, caged inositoI 1 4,5-trisphosphate (InsP3) was photoreleased selectively within the cytosol or nncleus using two-photon excitation. Release of lnsP3 witbm the cytosol resuhed in localized c)~osohc Ca" * pulls, wbereas release of InsP3 within tire nucleus resulted in localized intranuclear increases in Ca"* Stimnlation of cells with bepatocyte growth factor (50 ng/ml) increased Ca + within both the nucleus and cytosot (n= 51), but the nuclear Ca> signal preceded the" sigual in the cytosol by 2,63 • 0.25 sec (p<0 000l) in 21 of these cells. Finally, to determine whether nuclear Ca ~' signals play"a unique thnctional role, cells were t~nstkcted with GFP-tagged PKC7 that retained only the Ca~+-sensitive (C2) regulatory domain. Two-photon flash photolysis ot caged Ca~* in the nucleus resulted in transiocation ol PKC from the nuclear interior to the nuclear envelope (n = 8), while photorelease of C a * within the cytosol instead resuhed in transIocation of cytosofic PKC to the plasma membrane (n = 5) Together these results provide evidence that the nucleus of liver cells contains lnsP3-sensitive Ca ~+ stores that are preterentiaIly mobilized by growth factors aud that can locally activate PKC The presence of such machinery provides a novel signal transdnction system that can locally regulate events within the nncleus
5 Isolation of Alpha-l-antitrypsin Mutant Z Protein Polymers from Liver and Their Role in Cellular Injury Jefh'ey H Teckman, Keith Blomenkamp, ] Air Alpha-l-antitrypsin (alAT) deficiency is the most common cause of genetic fiver disease in children and an important cause of chronic liver injury, cin'hosis and bepatc.cellular earcinonla in adults. The a 1AT mutant Z gene directs the synthesis of a mutant protein molecule, which accumulates within hepatocytes and is tbought to be toxic to liver cells. In some hepatocytes the accumulations of alAT mutant Z protein are large enough to be seen by light microscopy as the Periodic Acid-Schiff (PAS) positive, diastase digestion resistant globules classically described in this disease, l~is mutant Z molecule has a unique tendency to adopt a highly stable polymer conformation, although the proportion of molecules in the polymerized conformation in v,ivo has until now been unknown. Furthermore, the direct relatiouship between Z protein polymerization, accumulation, globule formation and cellular injury is also unclear. In the. present study we report three new findings that directly address these questions. First, we have developed the only" biochemical assay available to quantitatively isolate alAT mutant Z protein polymers as insoh/ble protein aggregates from human and transgenic mouse liver. ~cond, we have isolated individual PAS positive globules from liver by laser capture-microdissection and shown that they are entirely alAT mutant Z protein in the polymerized conformation. Finally, using these techniques we have shown that some parameters of liver cell injury directly correlate to the total quantity of Z protein accumulated and to the fraction present as polymers. Furthermore, survival of aIAT mutant Z transgenic mine subjected to hepatic stress, including nutrient deprivation or fever-range whole body hyperthermia is decreased when the hepatic content of aIAT Z polymers is increased. These data provide the first direct evidence that polymerization of alAT mutant Z is directly related to disease patbogenesis in ~ivo and provide the tools needed to begin to evaluate anti-polymerogenic therapeutic strategies.
3 Oxidative Stress and Oval Cell Aecunmlation in Mice and Humans with Fatty Liver Disease Ayman Koteish, Tania Roskams Sfnqi "fang, Jiawen Huang, Dumez Anne, Rita Devos, Chris Verslype, Anna Mae Oiehl
6 Adenoviral Gene Transfer of Lipopolysaccharide Binding Protein (LBP) Results in Increased Acetaminophen-lnduced Hepatotoxicity Ke Qin Gong, Ming Hui Fan, Jian Min Sun, Stewart C. Wang, Grace L. Su
Why bepatocellular carcinoma (HCC) risk is increased ill alcoholic liver disease (ALD) and noi~lcolldic fatty liver disease (NAILD) is unki:.)wn, in animals, oxidative liver damage and inhibited hepatocyte proliferation promote liver progenitor (i,e., oval cell) accumulation bebre HCC develops Mice with ALD or NAFLD have inhibited hepatocyte DNA synthesis. Our goal was to @termine it"oxidative DNA damage and oval cell accumulation also occur. 3 mouse models of fatty liver (and their respective controls) were studied: ethanol (EtOH)ted mice, niethionine-choline deticietrt (MCD) diet-fi:d mice, and obese ob/ob iince. Mitochondrial H202 production and oxidative DNA damage were assessed, Using immunobistochemistry tor Or-6, an oval cell rnarker, tmpatic progenitors were quantified at baseline and alter hepatocyte turnover was stimulated by partial hepatectomy (PH) or tumor protnoting drags (TCPOBOP or ethionine) Liver biopsies from patients with NAFLD (n = 14), ALD (n = 25), and controls (it = 3) were also evaluated Compared to controls, all mice with fatty livers produced excessive H 2 0 2 a known cell cycle inhibitor This was greatest in ob/ob
Background: Acetaminopben bepatotoxicity remains the main cause of acute liver failure in the United States and Europe. Although the majority of cases result from intentional overdose, a significant number occur because of "therapeutic misadventures" where relatively low doses of acetaminopben have resulted in liver toxicity Some of these variations in clinical outcome are due to differences acetaminophen metabolism. However, we have prewiously sbown that LBP, which tacilitates cellular interactions with LPS, may play-a role in determining susceptibility to acetaminopben toxicity. Our previous work demonstrated that LBP deficient mice are protected from acetaminophen-induced hepatotoxicity. In order determine if the protection found in LBP deficient mice is specifically" due to IBP, we sought to reconstitute LBP deficient mice with LBP using adenoviral gene transfi~r. Method: OUT recombinant
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AASLD Abstracts
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adenoviru was constructed with a N-terrninus it*st*dine tagNed recombinant IBP seqnence. In vito, inh'ctinn with our ader~oviral constrncl resnhed in the expression of a 60 kDa protein with I.BP biological activity In viva, adenovirus containing either a ccmtro] irrelevant protein (Adot~ga])or LgP (Adl BP) was green three days pnor to acetaminophen challenge in 1BP dehdent mice Liver injury was assessed by plasma traasaminase levels and histology at 6 m d 24 bnurs after acetaminophen Results: In viva imramuseular deliveU of Ad-LBP n'sul ed i~ plasma s levels sligh/y above that bund normal mouse serum Immunoblot confirmed b e presence o[ a 60 kDa protein in the Ad-LBP treated mice As expected, no detectable plasma ls was fPur,d in tha LBP deficient animals given Ad-13gal Plasma ALT and AST levels i.creased at 6 hams aud peaked at 24 hours alter acetanrinophen m kBP dehcient mice gwe* dlher Adts m Ad-~gaL However, signihcantly higher AST and AIT levels a 24 hours af'.er acetam ~mphen were bund in the Ad-LBP compared to Ad-,ggal neared race (~ = 5/gr(mp, p < 0001) }tistolog*caIanalysis at the fiver showed the classic ten lobnlar necrosis in both groups ot mice: however, quantitative' analysis revealed sign*itcarny mnre r:ectosis i r e A d I s treated mice (p= 00094 by ANOVA) Conclusion: Our restil5 shnw that leuans~ uriah of LBP in LBP deficieru mice resulted in increased hepa oloxici y fore acetanfirnphen These finding support the hypothesis that LBP is a critical meda or of acelantinopher nduced hepatotox/city
ls Obesity" a Risk Factol/ for Cirrhosis-Related Death or Hospitalization? A Population-Based Cohort Study George N loammu, Nod S Weiss, Kris V Kowdley Jason A Dominitz Context: Obesity has been associated wilh the presence of advanced fibrosis in patients with chronic liver disease Objective: To determine whether increased body mass index (BM[) in the general population is associated with cirrhosis-related death or hospitalization Design: Prospect*re cohort study. Setting: The first National Health and Nutrition Examination Survey and Epidemiologic Follow-up Study, a nationally representative survey" Participants: 11,465 persons aged 25-74 years witliout evidence of cirrhosis at entry into the study, or during the first 5 years of tallow-up, were subsequendy tbllowed tar a mean of 129 years Measurements: The BMI measured at baseline was used to categorize parlicipams into normal-weight (BMI < 25 kg/m~, N = 5,752), overweight (BM125 to < 30 k g / m N = 3,774) and obese (BMI > = 30 kg/m~ N = 1,939), Deaths and hospitalizations due to cirrhosis were identified from death certificates and hospitalization records. Results: Cirrhosis resulted in death or hospitalization of 89 participants during a total of 150,233 person-years of f011ow-np (059/1000 person-years) Cirrhosis-selated deaths or hospitalizations were more common in obese persons (0 81/1000 person-years, adjusted hazard ratio 169, 95% CI I0-3.0) and m overwdght persons (071/1000 person-years, actinsted hazard ratio 1.16, 95% C[ 0 7-1 9) compared to normal-weight persons (0 45/1000 person-years). Among persons who did not consume alcohol, them was a particularly strong association between obesity (adjusted hazard ratio 4 l, 95% Ct l 4-1 i 4) or overweight (adjusted hazard ratio 193.95% C107-53) and cirrhosis-related death or hospitalization. In contrast, this association was weaker among persons who cousnmed up to 03 alcoholic drinks per day (adjusted hazard ratio 248, 95% CI 0.7-8 4 for obesily, and adjusted hazard ratio 131, 95% CI 0442 thr ov'erweigbt) and no association was identified among those who consumed more than 03 alcoholic drinks/day (adjusted hazard ratio 080, 95% CI 0 3 - 2 1 tor obesity, and adjusted hazard ratio 097, 95% CI 05-1 8 for over weight). Conclusions: Obesity appears to be a risk tactor tar cirrhosis-related death or hospitalization among persons who consume fitde or no alcohol
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Non-transterrin Bound Iron Uptake By ttepatocytes Is Increased In a Hie Knockout Mouse Model Of ttereditary Hemochromatosis A:ua C (hua, John K Ol nyk, Deborah 7rinder Hereditary hemochmmatosis (HH) i~ a~ antosnmal recessive disorder of iron metabolism b a ca *sesran ore and lu runs HH patients, the genetic deft:el is due to a C282Y mutation m the fqFE gene HI} knoc~..o ~t mice have rnany c)aracteristics of the human disease, accumulating e,~cess iou i~ h e live, as a res*r increased i o n absorption by the duoden u m [~ Htf. pasma non- ransk~r n bonnd iron (NTB[) levels ate increased and NTBI is /amnd mainly by ci rate ghe a m o ths study was to examine the importance of iron citrate r) fie pa hog,'nesis ul bepa ic n e loading in the Hfi"knockout mouse Hepatocytes were iso/aled tom ~fi~ knock(n and control (57BL/6] mice and
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Changing Epidemiology of Hepatitis B in a U.S. Community.: A Preliminary Report from the Olmsted County Hepatitis B Registry Kiran Bambha, Joanne T Benson, W Ray Kim Background: Recent data indicate that mortality related to hepatitis B (HBV) Juliet*ion increased in the past 20 years predominantI? among non-white, non-black men (Hepatology 2002;222A) Aims:el)To estatafish a population-based registry of commumty residents with HBV and (2)To describe demographic and serologm characteristics of the registry members Method: Population-based epidemiologic research can be conducted in Olmsted CounV, MN, because medical care is virtually sell-contained and heakhcare delivered by the by, providers in the community is tracked by the Rochester Epidemiology Proiect(REP). The REP database has been used to study the epidemiology of diseases such as primary biliab cirrhosis (Gastro 2000;I631) All Olmsted County residents diagnosed with I-fBV were identified using the REP database Complete in- and out-patient medical records were revsewed to verily residency slants and HBV diagnosis and to extract demographic, clinical and laboratory' data Results: To date, 172 community residents diagnosed with hepatitis B or had positive HBsAg dunng 1999 and 2000 have been included in the registry. O[ those 27, including 18 who had a clinical diagnosis of acute hepatitis B, had a subsequent negative HBsAg, while in the remaining 145 patients the most recent HBsAg was positive The mean age of the 145 patients as of 1/1/2000 was 38 years. Asian/Pacitlc Islander accounted thr 51%, followed by' African (31%) and Caucasian (I4%) races in the table, the vast majont} of subjects of non-white race were immigrants The most common origin elf the immigrants were Somailia(n = 36) and Cambodia(n = 23) Race-specific prevalence was much higher for not>whites than whites. There was a substantial difference m the proportions of replicative disease and transaminase activity by race Conclusions An initial cohort of a communibbased hepantis B reSiStU suggests that patients with chronic HBV intection in a Midwestem US comnmnity are mostly- immigrants lrom endemic countries Seroiogm and biochemical characteristics in these individuals were distinct from Caucasians. The registry will fie instrumental in studying the natural histou, virologic profile and treatment response in this population.
8 "lhc Incidence of ttepatocellular Carcinoma in the Umted States; Is It Still Rising? tiashe n B El-Scrag jessca Davtla, Nancy Petersen P,ackgm nn~: Increasing incidence rares [or fiepatocellutar carcinoma (HCC) were previously inputted in the U ~ited States possibly due o chronic HCV inti'ction However, ahernate ~'xplanations were diag ~ostic and,'or reclassification bias, and changes in the demographics ~f the general popn/ation {he purpose l this study is to ~*pdate recenl trends in HCC incide ~ce hnmgh 9 9 8 and to exarrine t mporal changes in incidence while adjusting for changes in age, gender e hnicty wthin g~'ograpbic regions Methods: Using inti,rmation )am ~e S)rvei/lance, pidemiol gb and laud Results (SEER) program, we identified all histologically c n[ *~ ed cases of primary HCC dining 1975-98 Age-adinsted incidence rates (AIR) were calculated k~r consecutive 3-yer per ads during 1975-98 We used Hierarchical ?nisson mnlt valia e regress*or: o examine temporal trends in HCC commI/ing for changes m age ger;der arat race a m n g h(Xi cases and in the underlying population at risk, while acc(ml ~g ha po ential cIustering ot persons with similar characteristics within geographic ~egions Resuts: Ifhe ovetall AIR ncreased from 14/100000 in 1975-77 to 3W100,000 n 1996-98:114% overall increase There was a 25% increase over the last 3 years of the study T~e pl >par o~s of patie ~ts with liver cancer undergoing m~croscnpic confirnmtion remained relative y sta)le over i m e Phe in*re'ase in HCC afiected most age groups above '40, wth tie grea est increase between ages 45 and 49 For instance, there was a striking 110% i~ crease dv~ri~:gtie [990s ~ wtim men (including Hispanic) between 45-49 The AIRs were 2 tnlds greater in blacks than whites and 2-folds greater in Asians than blacks However, w-fi~e ~:e~ and black women had the g~eatest percentage increase (31%) in the last ime period (996-98) as compared to 1993-95 Tha Poisson hierehichal regression model conhrmed an almost two thld increase in HCC incidence between 1975 and 1998. A signiheant ~ teraction between line of diagnosis and race such as that a greater increase iu H(C c i d e n c e ace.fred n whites compared to blacks and !Mians during more recent nine periods ( onclusio ~s T~e incidence of HCC continues to rapidly increase in the United States Axhough lis increase has atiected most age, gender, ethnic groups the [fastestrising rates have been obselved n whir* men between the ages at 45 and 55. "lhese findings are cons strut wit* a ~ue increase in HCC and could be explained by consequences of HCV acquired earlier n lde duing the 1960s and 1970s
AASLD Abstracts
Race
n
Age
Male
A~an African Whites OL~/Llnknown
74 ~5 20 5
38 35 44 3?
53% 53% 65% 100%
~ 99% 91% 15% I0(~%
P,ac~ HBe~ Abnom'~ Treabne~ Npciflc or DNAr Te'~msam~eeee ~revaleece 13% 35% ~% 12% 3.6% 10% 26% 2% 0.02% 25% 50% 5% 0.2% 25% 80% 0%
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Model tor End Stage Liver Disease (MELD) Scores Predict Mortality Across a Broad Spectrum of Liver Disease in a Us Tertiary"Care-Setting Adnan Said, Jeremy P. Holden, John B Williams, A.lexandru Musat, Rafael C, Botero, Michael R. Lucey Background: Although MELD scores are proposed as a predictor of 3-month mortality m severe liver disease, their accuracy have been tested intYequent/y among patients referred to a US tertiary care liver center We studied risk factors for mortality in 1611 consecutwe chronic liver disease patients presenting to a university-based hepatology service (1994 2001) and esmnated the predictive ability of MELD for 3 and 12-month mortality, Methods: 75 % were seen in the ompatlent clinic, 25% as inpatients, Survival data were censored at transplant and median survival fi'om ~terral was calculated for each diagnosis Cox proportional hazards were' used to model risk factors for mortality'. MELD scores were compared with observed 3- and 12-month sut'vival for the cohort and ROC curves analyzed to evaluate model validity (concordance and c-statistic) Next, we analyzed MELD in predicting 1-year
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1 Cathepsin B Inactivation Attenuates Hepatic Apoptosis and Fibrosis During Cholestasis Aft Can/Jay, Ha}line Hignchi, Maria E Guicciardi, Steven F Bronk Ariel Feldstein, Makiko Taniai, Robert M Rydzewski, Gregory J Gures Hepatocyte apoptosis and liver fibrosis are key teamres of cholestatic liver injury. Emerging data implicate a lysosolnal, cathepsin B-dependent pathway of apoptosis in bepatocytes. In this pathway, catbepsin B is released from lysosomes and tuggers mitochondrial dyshmction l%wever, the contribution of this pathway to hver injury and the relationship ot apoptosis to fibrosis remains unclear. Our Aim was to ascertain if inactivation of cathepsin B attenuates hvcr injury and fibrogenesis dunng extrahepatic cholestasis METHODS: Wild type (catB *s ~), cathepsin B-deticient (knockout) (catB~); and R 3032 (a catbepsin B inhibitor) treated catB */* mice undement bile duct ligation, Liver injury was examined by quantitating hepatocyte apopmsis with the TUNEL assay and determining serum ALT values, mRNA expression iur markers of stellate cell activation, a4MA, and fibrogenic actiwty, TGF-b and collagen I, was quantitated using real time PCR technolo~'. Liver fibrosis was assessed by digital image analysis ot Sirras red stained sections RESULTS: TUNEL positive hepamcytes and serum All- values were significandy reduced in cattY: and R 3032-treated catB +j+ 3-day BDL mice vs, catB +~ BDL mice, Consistent with these observations, mitochondrial cytochn)me c release, a marker for the mitochondrial pathway of apoptosis, was reduced in catB e and in R 3032-treated catB++ compared to catB ++ BDL mice, Stellate cell activation, as assessed by a-SMA was 14.0-fold greater in catB j+ vs catB v and 3-fold greater than in R 3032treated cazB +j+ BDL mice. Collagen al(I) was 102-told greater in catB ~+ vs. catB / BDL mice and 76-told greater in catB ~ ~ vs R 3032-treated ca~B +~ BDL mice. Serum bile acid and bilimbin values were mrtually identical in nil three experimental groups indicating that the above diliFrences could not be ascribed to difterences in cholestasis. Finally, in 2 week BDL mice, Sirius red staining for hepatic collagen deposition was significantly reduced in catB ~ mice compared to wdd type animals In CONCLUSION these findings not only support a prminnent tom tor the lysosomal pathway of apoptosis in cholestatic liver injury, but also link hepatocyte apoptosis to liver fibrogenesis These observations suggest cathepsin B inactivation or inhibition may be a therapeutic antifibrogenic strategy" in cholestatic liver diseases.
4 Bile Acid Induced Negative Feedback Regulation of the Human Ileal Bile Acid Transporter is Mediated by the Farnesoid X-Receptor (FXR), the Short Heterodimer Partner (SHP), and the Retinoid X Receptor (RAR/RXR) Ezequlel Neimark, Frank Chen, Benjamin Shneider Introduction: Ileal expression of the apical sodium dependent bile acids transporter (ASBT) in the mouse but not rat is under negative-feedback regulation by bile salts (Chen J. Biol Chem. in press). This species-specific difle:rence is secondary to the presence or absence of LRH-1 cis-elements and trans-acting factors. The bile acid responsiveness of human ASBT is unknown. Methods: 1.8 kb of the human 5' flanking region ot ASBT was subcloned into a luciferase reporter vector. Transient tmnsfection analyses were perfi)rmed in Caco-2 cells using expression plasmids thr FXR, SHP, LRH and RAR/RXR.Bile acid response was measured after treating the cells with varying concentrations (5 to 100 b~M) of CDCA Results: CDCA treatment of Caco-2 cells led to a 75% reduction in steady-state ASBT mRNA levels. Activity of the human ASBT prmnoter was reduced in a dose-dependent t~tshion by increasing concentrations of CDCA. Co-tr_anst;ection of FXR enhanced the bile acid mediated repression of ASBT promoter activity, while co-transfection of a dominant negative FXR abrogated the inhibitory" effect of CDCA. Site directed mutagenesis of an RAR/RXRcis-element in the ASBT promoter, reduced the basal activity' of the promoter by 9096 and eliminated the bite acid response, 9-cis retinoic acid activated the human ASBT promoter seven fold, while LRH-1 had no effect. SHP repressed the basal activity of' the ASBT promoter and sigmficandy reduced its activation by 9-cis retinoic acid. Conclusion: The human ileal bile acid transporter is positively regnlated by 9-cis retmoic acid. Bile acids induce a negativedeedback regulation of ASBT via an FXR-mediated, SHP-dependent effect upon ILA.D'I~XiRactivation ot ASBT.
2 Ca2+ in the Nucleus ls Independently Regulated and Has Distinct Effects on Protein Ydnase C (PKC) in a Liver Cell Line Mateus "iF Guerra, Maria de Fatima keite, Wihelma Echevarria Michael Nathanson Cytosohc Ca* regulates a wide range of fnnctions in hepatocytes. Ca > signals also occur iu the nucleus of hepatocytes, but it is unckar how they are regulated or whether they have distinct etfects on cell tunction. To investigate these questions, we examined Caz§ release mechainsms in the nucleus and the eltects of nuclear and cy'losolic Ca2~ signals on PKC translocation in the 5kHepl liver cell line. Contocal imnmnoBuorescence of the ER-resident protein calreticulin revel(led a reticular netwoik extending from the ER and nuclear envelope into the intenor of the nucleus. Confocal nucroscopic examination of live cells loaded vdth the low aflimty Ca2' dye umg-fluo-4 revealed sinlilar intranuclear structures, Fluorescence recovery after photobleaching of mag-fluo-4 in the nucleus (0.32 +- 0.09 sec~; n = 14) and cytosol (0,40 _'2'.0 0 8 sec; n = 19) showed similar recovery rates (p>0,4), providing complementary evidence that C a t stores within these nuclear structures are continuous with those in the ER, In order to determine the physiological role of these intranuclear Ca2+ stores, caged inositoI 1 4,5-trisphosphate (InsP3) was photoreleased selectively within the cytosol or nncleus using two-photon excitation. Release of lnsP3 witbm the cytosol resuhed in localized c)~osohc Ca" * pulls, wbereas release of InsP3 within tire nucleus resulted in localized intranuclear increases in Ca"* Stimnlation of cells with bepatocyte growth factor (50 ng/ml) increased Ca + within both the nucleus and cytosot (n= 51), but the nuclear Ca> signal preceded the" sigual in the cytosol by 2,63 • 0.25 sec (p<0 000l) in 21 of these cells. Finally, to determine whether nuclear Ca ~' signals play"a unique thnctional role, cells were t~nstkcted with GFP-tagged PKC7 that retained only the Ca~+-sensitive (C2) regulatory domain. Two-photon flash photolysis ot caged Ca~* in the nucleus resulted in transiocation ol PKC from the nuclear interior to the nuclear envelope (n = 8), while photorelease of C a * within the cytosol instead resuhed in transIocation of cytosofic PKC to the plasma membrane (n = 5) Together these results provide evidence that the nucleus of liver cells contains lnsP3-sensitive Ca ~+ stores that are preterentiaIly mobilized by growth factors aud that can locally activate PKC The presence of such machinery provides a novel signal transdnction system that can locally regulate events within the nncleus
5 Isolation of Alpha-l-antitrypsin Mutant Z Protein Polymers from Liver and Their Role in Cellular Injury Jefh'ey H Teckman, Keith Blomenkamp, ] Air Alpha-l-antitrypsin (alAT) deficiency is the most common cause of genetic fiver disease in children and an important cause of chronic liver injury, cin'hosis and bepatc.cellular earcinonla in adults. The a 1AT mutant Z gene directs the synthesis of a mutant protein molecule, which accumulates within hepatocytes and is tbought to be toxic to liver cells. In some hepatocytes the accumulations of alAT mutant Z protein are large enough to be seen by light microscopy as the Periodic Acid-Schiff (PAS) positive, diastase digestion resistant globules classically described in this disease, l~is mutant Z molecule has a unique tendency to adopt a highly stable polymer conformation, although the proportion of molecules in the polymerized conformation in v,ivo has until now been unknown. Furthermore, the direct relatiouship between Z protein polymerization, accumulation, globule formation and cellular injury is also unclear. In the. present study we report three new findings that directly address these questions. First, we have developed the only" biochemical assay available to quantitatively isolate alAT mutant Z protein polymers as insoh/ble protein aggregates from human and transgenic mouse liver. ~cond, we have isolated individual PAS positive globules from liver by laser capture-microdissection and shown that they are entirely alAT mutant Z protein in the polymerized conformation. Finally, using these techniques we have shown that some parameters of liver cell injury directly correlate to the total quantity of Z protein accumulated and to the fraction present as polymers. Furthermore, survival of aIAT mutant Z transgenic mine subjected to hepatic stress, including nutrient deprivation or fever-range whole body hyperthermia is decreased when the hepatic content of aIAT Z polymers is increased. These data provide the first direct evidence that polymerization of alAT mutant Z is directly related to disease patbogenesis in ~ivo and provide the tools needed to begin to evaluate anti-polymerogenic therapeutic strategies.
3 Oxidative Stress and Oval Cell Aecunmlation in Mice and Humans with Fatty Liver Disease Ayman Koteish, Tania Roskams Sfnqi "fang, Jiawen Huang, Dumez Anne, Rita Devos, Chris Verslype, Anna Mae Oiehl
6 Adenoviral Gene Transfer of Lipopolysaccharide Binding Protein (LBP) Results in Increased Acetaminophen-lnduced Hepatotoxicity Ke Qin Gong, Ming Hui Fan, Jian Min Sun, Stewart C. Wang, Grace L. Su
Why bepatocellular carcinoma (HCC) risk is increased ill alcoholic liver disease (ALD) and noi~lcolldic fatty liver disease (NAILD) is unki:.)wn, in animals, oxidative liver damage and inhibited hepatocyte proliferation promote liver progenitor (i,e., oval cell) accumulation bebre HCC develops Mice with ALD or NAFLD have inhibited hepatocyte DNA synthesis. Our goal was to @termine it"oxidative DNA damage and oval cell accumulation also occur. 3 mouse models of fatty liver (and their respective controls) were studied: ethanol (EtOH)ted mice, niethionine-choline deticietrt (MCD) diet-fi:d mice, and obese ob/ob iince. Mitochondrial H202 production and oxidative DNA damage were assessed, Using immunobistochemistry tor Or-6, an oval cell rnarker, tmpatic progenitors were quantified at baseline and alter hepatocyte turnover was stimulated by partial hepatectomy (PH) or tumor protnoting drags (TCPOBOP or ethionine) Liver biopsies from patients with NAFLD (n = 14), ALD (n = 25), and controls (it = 3) were also evaluated Compared to controls, all mice with fatty livers produced excessive H 2 0 2 a known cell cycle inhibitor This was greatest in ob/ob
Background: Acetaminopben bepatotoxicity remains the main cause of acute liver failure in the United States and Europe. Although the majority of cases result from intentional overdose, a significant number occur because of "therapeutic misadventures" where relatively low doses of acetaminopben have resulted in liver toxicity Some of these variations in clinical outcome are due to differences acetaminophen metabolism. However, we have prewiously sbown that LBP, which tacilitates cellular interactions with LPS, may play-a role in determining susceptibility to acetaminopben toxicity. Our previous work demonstrated that LBP deficient mice are protected from acetaminophen-induced hepatotoxicity. In order determine if the protection found in LBP deficient mice is specifically" due to IBP, we sought to reconstitute LBP deficient mice with LBP using adenoviral gene transfi~r. Method: OUT recombinant
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adenoviru was constructed with a N-terrninus it*st*dine tagNed recombinant IBP seqnence. In vito, inh'ctinn with our ader~oviral constrncl resnhed in the expression of a 60 kDa protein with I.BP biological activity In viva, adenovirus containing either a ccmtro] irrelevant protein (Adot~ga])or LgP (Adl BP) was green three days pnor to acetaminophen challenge in 1BP dehdent mice Liver injury was assessed by plasma traasaminase levels and histology at 6 m d 24 bnurs after acetaminophen Results: In viva imramuseular deliveU of Ad-LBP n'sul ed i~ plasma s levels sligh/y above that bund normal mouse serum Immunoblot confirmed b e presence o[ a 60 kDa protein in the Ad-LBP treated mice As expected, no detectable plasma ls was fPur,d in tha LBP deficient animals given Ad-13gal Plasma ALT and AST levels i.creased at 6 hams aud peaked at 24 hours alter acetanrinophen m kBP dehcient mice gwe* dlher Adts m Ad-~gaL However, signihcantly higher AST and AIT levels a 24 hours af'.er acetam ~mphen were bund in the Ad-LBP compared to Ad-,ggal neared race (~ = 5/gr(mp, p < 0001) }tistolog*caIanalysis at the fiver showed the classic ten lobnlar necrosis in both groups ot mice: however, quantitative' analysis revealed sign*itcarny mnre r:ectosis i r e A d I s treated mice (p= 00094 by ANOVA) Conclusion: Our restil5 shnw that leuans~ uriah of LBP in LBP deficieru mice resulted in increased hepa oloxici y fore acetanfirnphen These finding support the hypothesis that LBP is a critical meda or of acelantinopher nduced hepatotox/city
ls Obesity" a Risk Factol/ for Cirrhosis-Related Death or Hospitalization? A Population-Based Cohort Study George N loammu, Nod S Weiss, Kris V Kowdley Jason A Dominitz Context: Obesity has been associated wilh the presence of advanced fibrosis in patients with chronic liver disease Objective: To determine whether increased body mass index (BM[) in the general population is associated with cirrhosis-related death or hospitalization Design: Prospect*re cohort study. Setting: The first National Health and Nutrition Examination Survey and Epidemiologic Follow-up Study, a nationally representative survey" Participants: 11,465 persons aged 25-74 years witliout evidence of cirrhosis at entry into the study, or during the first 5 years of tallow-up, were subsequendy tbllowed tar a mean of 129 years Measurements: The BMI measured at baseline was used to categorize parlicipams into normal-weight (BMI < 25 kg/m~, N = 5,752), overweight (BM125 to < 30 k g / m N = 3,774) and obese (BMI > = 30 kg/m~ N = 1,939), Deaths and hospitalizations due to cirrhosis were identified from death certificates and hospitalization records. Results: Cirrhosis resulted in death or hospitalization of 89 participants during a total of 150,233 person-years of f011ow-np (059/1000 person-years) Cirrhosis-selated deaths or hospitalizations were more common in obese persons (0 81/1000 person-years, adjusted hazard ratio 169, 95% CI I0-3.0) and m overwdght persons (071/1000 person-years, actinsted hazard ratio 1.16, 95% C[ 0 7-1 9) compared to normal-weight persons (0 45/1000 person-years). Among persons who did not consume alcohol, them was a particularly strong association between obesity (adjusted hazard ratio 4 l, 95% Ct l 4-1 i 4) or overweight (adjusted hazard ratio 193.95% C107-53) and cirrhosis-related death or hospitalization. In contrast, this association was weaker among persons who cousnmed up to 03 alcoholic drinks per day (adjusted hazard ratio 248, 95% CI 0.7-8 4 for obesily, and adjusted hazard ratio 131, 95% CI 0442 thr ov'erweigbt) and no association was identified among those who consumed more than 03 alcoholic drinks/day (adjusted hazard ratio 080, 95% CI 0 3 - 2 1 tor obesity, and adjusted hazard ratio 097, 95% CI 05-1 8 for over weight). Conclusions: Obesity appears to be a risk tactor tar cirrhosis-related death or hospitalization among persons who consume fitde or no alcohol
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Non-transterrin Bound Iron Uptake By ttepatocytes Is Increased In a Hie Knockout Mouse Model Of ttereditary Hemochromatosis A:ua C (hua, John K Ol nyk, Deborah 7rinder Hereditary hemochmmatosis (HH) i~ a~ antosnmal recessive disorder of iron metabolism b a ca *sesran ore and lu runs HH patients, the genetic deft:el is due to a C282Y mutation m the fqFE gene HI} knoc~..o ~t mice have rnany c)aracteristics of the human disease, accumulating e,~cess iou i~ h e live, as a res*r increased i o n absorption by the duoden u m [~ Htf. pasma non- ransk~r n bonnd iron (NTB[) levels ate increased and NTBI is /amnd mainly by ci rate ghe a m o ths study was to examine the importance of iron citrate r) fie pa hog,'nesis ul bepa ic n e loading in the Hfi"knockout mouse Hepatocytes were iso/aled tom ~fi~ knock(n and control (57BL/6] mice and
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Changing Epidemiology of Hepatitis B in a U.S. Community.: A Preliminary Report from the Olmsted County Hepatitis B Registry Kiran Bambha, Joanne T Benson, W Ray Kim Background: Recent data indicate that mortality related to hepatitis B (HBV) Juliet*ion increased in the past 20 years predominantI? among non-white, non-black men (Hepatology 2002;222A) Aims:el)To estatafish a population-based registry of commumty residents with HBV and (2)To describe demographic and serologm characteristics of the registry members Method: Population-based epidemiologic research can be conducted in Olmsted CounV, MN, because medical care is virtually sell-contained and heakhcare delivered by the by, providers in the community is tracked by the Rochester Epidemiology Proiect(REP). The REP database has been used to study the epidemiology of diseases such as primary biliab cirrhosis (Gastro 2000;I631) All Olmsted County residents diagnosed with I-fBV were identified using the REP database Complete in- and out-patient medical records were revsewed to verily residency slants and HBV diagnosis and to extract demographic, clinical and laboratory' data Results: To date, 172 community residents diagnosed with hepatitis B or had positive HBsAg dunng 1999 and 2000 have been included in the registry. O[ those 27, including 18 who had a clinical diagnosis of acute hepatitis B, had a subsequent negative HBsAg, while in the remaining 145 patients the most recent HBsAg was positive The mean age of the 145 patients as of 1/1/2000 was 38 years. Asian/Pacitlc Islander accounted thr 51%, followed by' African (31%) and Caucasian (I4%) races in the table, the vast majont} of subjects of non-white race were immigrants The most common origin elf the immigrants were Somailia(n = 36) and Cambodia(n = 23) Race-specific prevalence was much higher for not>whites than whites. There was a substantial difference m the proportions of replicative disease and transaminase activity by race Conclusions An initial cohort of a communibbased hepantis B reSiStU suggests that patients with chronic HBV intection in a Midwestem US comnmnity are mostly- immigrants lrom endemic countries Seroiogm and biochemical characteristics in these individuals were distinct from Caucasians. The registry will fie instrumental in studying the natural histou, virologic profile and treatment response in this population.
8 "lhc Incidence of ttepatocellular Carcinoma in the Umted States; Is It Still Rising? tiashe n B El-Scrag jessca Davtla, Nancy Petersen P,ackgm nn~: Increasing incidence rares [or fiepatocellutar carcinoma (HCC) were previously inputted in the U ~ited States possibly due o chronic HCV inti'ction However, ahernate ~'xplanations were diag ~ostic and,'or reclassification bias, and changes in the demographics ~f the general popn/ation {he purpose l this study is to ~*pdate recenl trends in HCC incide ~ce hnmgh 9 9 8 and to exarrine t mporal changes in incidence while adjusting for changes in age, gender e hnicty wthin g~'ograpbic regions Methods: Using inti,rmation )am ~e S)rvei/lance, pidemiol gb and laud Results (SEER) program, we identified all histologically c n[ *~ ed cases of primary HCC dining 1975-98 Age-adinsted incidence rates (AIR) were calculated k~r consecutive 3-yer per ads during 1975-98 We used Hierarchical ?nisson mnlt valia e regress*or: o examine temporal trends in HCC commI/ing for changes m age ger;der arat race a m n g h(Xi cases and in the underlying population at risk, while acc(ml ~g ha po ential cIustering ot persons with similar characteristics within geographic ~egions Resuts: Ifhe ovetall AIR ncreased from 14/100000 in 1975-77 to 3W100,000 n 1996-98:114% overall increase There was a 25% increase over the last 3 years of the study T~e pl >par o~s of patie ~ts with liver cancer undergoing m~croscnpic confirnmtion remained relative y sta)le over i m e Phe in*re'ase in HCC afiected most age groups above '40, wth tie grea est increase between ages 45 and 49 For instance, there was a striking 110% i~ crease dv~ri~:gtie [990s ~ wtim men (including Hispanic) between 45-49 The AIRs were 2 tnlds greater in blacks than whites and 2-folds greater in Asians than blacks However, w-fi~e ~:e~ and black women had the g~eatest percentage increase (31%) in the last ime period (996-98) as compared to 1993-95 Tha Poisson hierehichal regression model conhrmed an almost two thld increase in HCC incidence between 1975 and 1998. A signiheant ~ teraction between line of diagnosis and race such as that a greater increase iu H(C c i d e n c e ace.fred n whites compared to blacks and !Mians during more recent nine periods ( onclusio ~s T~e incidence of HCC continues to rapidly increase in the United States Axhough lis increase has atiected most age, gender, ethnic groups the [fastestrising rates have been obselved n whir* men between the ages at 45 and 55. "lhese findings are cons strut wit* a ~ue increase in HCC and could be explained by consequences of HCV acquired earlier n lde duing the 1960s and 1970s
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Race
n
Age
Male
A~an African Whites OL~/Llnknown
74 ~5 20 5
38 35 44 3?
53% 53% 65% 100%
~ 99% 91% 15% I0(~%
P,ac~ HBe~ Abnom'~ Treabne~ Npciflc or DNAr Te'~msam~eeee ~revaleece 13% 35% ~% 12% 3.6% 10% 26% 2% 0.02% 25% 50% 5% 0.2% 25% 80% 0%
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Model tor End Stage Liver Disease (MELD) Scores Predict Mortality Across a Broad Spectrum of Liver Disease in a Us Tertiary"Care-Setting Adnan Said, Jeremy P. Holden, John B Williams, A.lexandru Musat, Rafael C, Botero, Michael R. Lucey Background: Although MELD scores are proposed as a predictor of 3-month mortality m severe liver disease, their accuracy have been tested intYequent/y among patients referred to a US tertiary care liver center We studied risk factors for mortality in 1611 consecutwe chronic liver disease patients presenting to a university-based hepatology service (1994 2001) and esmnated the predictive ability of MELD for 3 and 12-month mortality, Methods: 75 % were seen in the ompatlent clinic, 25% as inpatients, Survival data were censored at transplant and median survival fi'om ~terral was calculated for each diagnosis Cox proportional hazards were' used to model risk factors for mortality'. MELD scores were compared with observed 3- and 12-month sut'vival for the cohort and ROC curves analyzed to evaluate model validity (concordance and c-statistic) Next, we analyzed MELD in predicting 1-year
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