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sfusion. Further, clonal cell culture allows certain identification of the cell responsible andlarge quantity MSC infusion appears to obviate the need for prior irradiation. This modelwill serve as a useful platform for studying repair of injury, and fate of fused hepatocytes.

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Dysfunction of Hepatocyte Nuclear Factor-4α Contributes to Alcohol-InducedIntestinal Barrier DisruptionHai Zhong, Yantao Zhao, Craig J. McClain, Y. James Kang, Zhanxiang Zhou

Background: Alcohol consumption causes intestinal barrier disruption, leading to endotoxe-mia and alcoholic liver disease. However, the mechanisms by which alcohol interferes withintestinal barrier have not been fully defined. Hepatocyte nuclear factor-4α (HNF-4α) is anuclear receptor and critically regulates intestinal gene expression and function. Aims: Thepresent study was undertaken to determine (1) if alcohol affects intestinal HNF-4α; and (2)the link between HNF-4α and intestinal barrier structure and function. Methods: Theassociation of HNF-4α dysfunction with alcohol-induced intestinal barrier disruption wasdetermined in a mouse model of alcoholic liver disease. Mice were pair-fed a modifiedLieber-DeCarli liquid diet containing ethanol or isocaloric maltose dextrin for 4 weeks. Thelink between HNF-4α and epithelial barrier was studied in Caco-2 cell culture. Results:Alcohol exposure caused endotoxemia and hepatitis as indicated by elevated blood endotoxinlevels and ALT activities and neutrophil infiltration in the liver. The intestinal protein leveland DNA binding activity of HNF-4α were reduced by alcohol exposure. Intestinal barrierfunction was estimated by ex vivo assay of the permeability of ileum to FITC-dextran, andalcohol exposure remarkably increased the ileal permeability. Accumulation of reactiveoxygen species in the intestinal epithelial cells of alcohol-fed mice was detected by dihydro-ethidium fluorescence microscopy. Alcohol exposure also increased the intracellular freezinc as indicated by increase in Zinquin (a free zinc indicator) fluorescence. The link betweenzinc coordination and HNF-4α function was studies by zinc deprivation in Caco-2 cells.Zinc deprivation suppressed the DNA binding activity of HNF-4α in a dose-dependentmanner. The link between HNF-4α and epithelial barrier was determined by silencing HNF-4α. HNF-4α siRNA transfection in Caco-2 cells resulted in disruption of the epithelial barrierfunction in association with disassembly of junction proteins. Conclusions: These resultssuggest that dysfunction of HNF-4α is involved in alcohol-induced disruption of intestinalbarrier function, and decrease in zinc coordination accounts for alcohol-induced reductionof the DNA binding activity of HNF-4α. (Supported in part by the National Institutes ofHealth and Office of Dietary Supplements grants and the Veterans Administration).

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Nucleoplasmic Calcium Regulates Proliferation of Hepatocytes ThroughLegumain and Reticulon4Viviane A. Andrade, Camila A. Jardim, Flávia M. Melo, Wilson A. Silva Jr, Michael H.Nathanson, J Miguel Ortega, Maria F. Leite

Liver regeneration depends upon growth factors such as HGF and insulin that induce Ca2+signals in the nucleus, and nucleoplasmic Ca2+ signals are necessary for proliferation ofcells. The mechanism by which nuclear Ca2+ regulates liver regeneration is not known. Weexamined whether nuclear Ca2+ modulates expression of genes involved in cell proliferation.To investigate this question we used Rapid Subtraction Hybridization (RaSH) to subtractgenes expressed in SkHep1 liver cells infected with an adenoviral construct encoding theCa2+ buffer protein parvalbumin (PV) targeted to the nucleus with a nuclear localizationsignal (Ad-PV-NLS), from genes expressed in SkHep1 cells infected with a mutated formof PV-NLS (PV-NLS-CD), which has one Ca2+ binding sites inactivated. The subtractionpermitted selection of genes whose expression was affected by a small alteration in nuclearCa2+ concentration. A BLAST search identified the genes of legumain (LGMN), reticulon 4(RTN4) and transforming growth factor beta regulator 4 (TBRG4) among the selected clones.LGMN is an endopeptidase highly expressed in several types of tumors, including colorectalcancer. RTN4 interacts with the neurotrophin receptor and induces differentiation of hepato-cyte growth factor-secreting cells to promote hepatocyte proliferation. RTN4 is also a criticalinhibitory axonal growth and regeneration. TBRG4 acts as a regulator of growth factors andcell proliferation. The differential expression of these genes was validated by semi quantitativeRT PCR and by Real Time PCR. When Ca2+ was buffered in the nucleus of SkHep1 cells,LGMN and TBRG4 mRNA was decreased by approximately 80% (p<0.0001) while RTN4expression was increased by 90% (p<0.0001). These results were confirmed at the proteinlevel by western blot and immunofluorescence. To determine the influence of the selectedgenes in the proliferative response, siRNA was used to knockdown expression of each ofthese proteins. The BrdU assay showed that, when LGMN was silenced, the proliferationof SkHep1 cells decreased by 60% compared to control cells (p<0.05). Knockdown ofTBRG4 resulted in a small but not statistically significant reduction in cell proliferation. Incontrast, knockdown of RTN4 increased cell proliferation by 70% compared to controls(p<0.05). Together, these results suggest that nuclear Ca2+ signals regulate proliferation ofhepatocytes through modulation of LGMN and RTN4. This in turn reveals an importantnew signaling pathway that may be exploited for the regulation of hepatocyte proliferationin normal conditions such as liver regeneration and abnormal conditions such as hepatocellu-lar carcinoma.

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TGF-β Signaling Modulates Hepatocyte Phenotype in Liver RegenerationFollowing Partial HepatectomyArun Thenappan, Majed El Zouhairi, Kirti Shetty, Lynt Johnson, Lopa Mishra

Liver injury induces a synchronized sequence of events that proceed in an orderly mannerwith hepatocyte proliferation advancing from the periportal to pericentral areas of the lobule.In contrast, liver repopulation after acute liver failure depends on the differentiation ofprogenitor cells. We have previously demonstrated small groups of histone positive cells

A-792AASLD Abstracts

that express stem cell markers Oct3/4, Nanog, and Stat3 as well as TGF-β members, TGF-β receptor type 2 (TBR2) and ELF, at the end of human liver regeneration at 4 months.Surprisingly, these cells dramatically lose their phenotype to express Oct3/4, Nanog, andStat3 but not TBR2 or ELF in hepatocellular cancer (HCC) (PNAS 2008; 105:2445-2450).Aims:These observations led us to pursue the role of ELF, other TGF-β signaling components,and stem cell proteins in liver regeneration. Methods and Results: We initially examinedtissue biopsy specimens in 10 living donor liver transplant recipients at one week, six weeks,and three months post-transplant. Using immunohistochemical and confocal immunofluo-rescent analysis, we (1) demonstrate that Oct3/4 and Nanog are expressed in a contiguousstreaking manner, strongest in the periportal region and diminishing through the midzoneof the liver, one week post transplant. At six weeks and three months post-transplant,however, we observe a progressive decrease in Oct3/4 and Nanog labeling, finally localizingto small clusters of 2-4 cells per 30,000-50,000 cells surrounding the portal tract. (2) Thesecells consistently label for histone, albumin, and CK19 indicating that they are hepatic stem/progenitor cells. (3) TBR2 and ELF co-localize with Oct3/4 and Nanog in a linear pattern,but progressively increase in intensity and breadth by 3 months post-transplant, implyingthat TBR2 and ELF modulate the transition from a stem cell to differentiated phenotype.We then analyzed regenerating liver in elf+/- mice following 2/3 partial hepatectomy anddemonstrate (4) an expanded population of Oct3/4 expressing cells in a periportal band 24hours post-hepatectomy, which persists as a pericentral band 72 hours post-hepatectomy,when compared to wild type mice. Conclusions: Here, we report that (1) normal humanadult hepatocytes express a transient stem cell phenotype early in regeneration in recipientsof living donor liver transplants. (2) Moreover, TGF-β signaling components, in particularELF, appears to play a role in modulation of stem cell phenotype. These experimentsreveal an important role for TGF-β signaling in liver regeneration and modulation of stemcell phenotype.

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Sustained Activation of RAC1 Enhances Hedgehog-Mediated Epithelial-Mesenchymal Transition in Mice with Chronic Liver InjurySteve S. Choi, Rafal P. Witek, Alessia Omenetti, Liu Yang, Wing-Kin Syn, Youngmi Jung,Jason K. Sicklick, Anna Mae Diehl

Background & Aim: Liver injury induces reparative responses, including activation of quiescenthepatic stellate cells (Q-HSC) to a mesenchymal phenotype that promotes fibrosis. Weshowed that sustained activation of the small GTPase, Rac1, in myofibroblastic (MF) HSCexacerbated liver fibrosis caused by carbon tetrachloride (CCl4) injury. We used adenoviralvectors to manipulate Rac1 activity in primary rat HSC and demonstrated that Rac1 stimulatesQ-HSC to become MF-HSC by repressing Hedgehog-interacting protein (Hip, a Hh pathwayinhibitor), thereby activating the Hedgehog (Hh) signaling pathway. Pharmacologic inhibitionof Hh activity proved that Hh signaling inducedQ-HSC to undergo epithelial-to-mesenchymaltransition (EMT), and promoted their proliferation and viability. Our Aim was to determineif Rac1 activation exerted similar effects on Hh signaling and EMT in mouse models ofchronic liver injury. Methods: We injected C57Bl/6 mice with adenoviral vectors containingconstitutively active Rac1 (RacV12), dominant-negative Rac1 (RacN17), or null (ø) vector.After injection, bile duct ligation (BDL) or sham surgery was performed. After 10 days, micewere sacrificed; serum and liver were harvested. Fibrosis was assessed by morphometry andmeasurement of hydroxyproline. We assessed expression of mesenchymal and epithelialmarkers, as well as Hh-target genes by QRT-PCR analysis of whole liver RNA. Similar analysiswas performed on CCl4-treated Rac transgenic mice that overexpress constitutively activehuman Rac1, and their littermate controls. Results: Compared to BDL mice treated with øvector, RacV12-treated mice demonstrated greater fibrosis post-BDL by both morphometryand hydroxyproline content. RacV12-treated BDL mice had greater inhibition of Hip, andsignificantly greater induction of Sonic hedgehog (Shh) ligand, Gli2 (a Hh-target gene), andmesenchymal markers, includingαSMA, Col1αI, and S100A4 (a marker of fibroblasts derivedfrom epithelial cells). In contrast, expression of BMP7, an EMT inhibitor, and epithelialmarkers (e.g., desmoplakin) was significantly more repressed in these mice (all P<0.05 v.ø-injected mice). CCl4-treated Rac transgenic mice had significantly greater induction of Hhsignaling and mesenchymal markers, and more repression of BMP7 and epithelial genes,than littermate controls. RacN17-treated mice did not survive beyond 3 days post-BDL.Conclusions: Rac1 activation induces Hh signaling and promotes EMT, leading to fibrosis ininjured livers. However, Rac1 activation is also necessary for survival during chronic liverinjury. Therefore, Hh pathway activation and EMT may be essential components of liverrepair.

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Proliferative Advantage of Transplanted Wildtype Hepatocytes Results inSpontaneous Repopulation of the Liver of a Mouse Model of Human Alpha-1-Antitrypsin DiseaseJianqiang Ding, Govardhana R. Yannam, Tunda Hidvegi, Xia Wang, Chandan Guha,Namita Roy-Chowdhury, David H. Perlmutter, Ira J. Fox, Jayanta Roy-Chowdhury

Introduction: Human alpha-1-antitrypsin (AAT) disease has both pulmonary and hepaticmanifestations. The most common disease-causing allele, PiZ results in the accumulation ofmisfolded mutant AAT globules in hepatocytes. The deficiency of circulating functional AATresults in pulmonary emphysema, while the effect of the accumulated PiZ in hepatocytesmay range from mild, subclinical liver disorder to cirrhosis and hepatoma. We hypothesizethat the stress of accumulation of misfolded PiZ in host hepatocytes should confer a proliferat-ive advantage to transplanted wildtype donor hepatocytes. To test this hypothesis, wetransplanted hepatocytes from LacZ-transgenic ROSA26(C57/Bl6) mice into the liver ofrecipient mice transgenic for human PiZ. Methods: PiZ expressing mice were identified bymeasuring PiZ in plasma by ELISA. Liver biopsies were performed to identify mice with alarge number of diastase-resistant PAS-positive PiZ globules in hepatocytes. Hepatocytesisolated from the livers of ROSA26 mice (5x105 to 1x106) were transplanted by intrasplenicinjection into the livers of PiZ mice exhibiting a large number of PiZ globules in hepatocytes.For some experiments, ROSA26 hepatocytes were transduced with a lentiviral vector,expressing firefly luciferase (F-luc) at MOI 30 (transduction efficiency 30%). One group of