Analysis of gene expression using Cdna microarray in the liver of patients with chronic hepatitis C during interferon therapy

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alpha-tetoprotein, connexin 43, c-kit, musashi-l, a marker for neural stem sells, negative for albumin, transferrin, cytokeratin 19, gamma-GTP. W~en HSLCs were co-cultured with HSCs, HSLCs express albumin, transferrin, HNF3 alpha, gamma-GTP. Among them, antiepimorphin antibody inhibited albumin and transterrin expression. Morphologicany, HSLCs piled up with cuboidal formation on the HSCs adhered to dish surface Tight junctions and bile-canalicui-like structures were observed between HSLCs co-cultured with HSCs. Antiepimorphin antibody inhibited the piling up of HSLCs. Epimorphin induced tyrosine aminotransferase, CJEBP beta expression in addition to positive markers induced by HSCs such as albumin and transferrin. Expression of c-kit and mnsashi- 1 decreased with differentiation of I-ISLCs. HSLCs became rich in mitochondria and rough endoplasmic reticulum. Conclusions: HSLCs are blpotential cells that can difl~remiate into both hepatocytes and cholangiocytes Epimorphin is a key molecule that induces a differentiation of HSLCs.

Ett~:ct of VEGF and Endostatin Gene Transfer on the Time Course Expression of Endoglin After Partial Hepatectomy in a Mouse Model David ~mela, Ciaudio Redaelli, Verena Schneider, Franc*he Carrick, Juri Kovac, Monika Ledemrann, Jean-Francois Dufour, Bernhard V. Sauter Hepatic regeneration aher partial hepatectomy is dependent upon angiogenesis. Endoghn, a homodimer*c membrane glycoprotein and co-*~ceptor of transforming growth tactor-beta (TGF-beta), plays an important role in angiogenesis and is regulated by hypoxia-inducible l~:ctor (HIE)- 1 Endoglin, which is mutated m patients suffering from hereditary hemorrhagic tdangiectasia, is expressed mainly by vascular endothchum Nothing is known about the role: of endoglin d~aring liver regeneration We studied the expression of end@in in mice atter 2/3 hepatectomy alone and in con:bloat*on with adenovirus-mediated gene transt}r of pro-angiogenic vascular e/dothelial growth lactor (VEGF) and anti-angiogenic endc:statin. Methods Recombiuant adenovirus pkDV.endostatin pADV.VEGF165 and pADV.beta-Gal (control adenoviros) were administered by tail vein injection 2 days before hepatectomy in :::ale C57B16 :nice NaCI treated animals served as additional controls Three and 6 days after hepatectomy, livers of the mice were harvested. Reahime quantitative PCR of homogeuated liver tissue f*~rendoglin was done after hepatectomy and harvest 18s RNA was used as an interual connol Resuks are given as mean +/-SD. Results In all tour groups, endoglin mRNA levels were lower 3 days at:er hepatectomy (16.8 +/-1) compared to the mRNA level at time of hepatectomy (163 +/-1 cycles) Endoglin levels, however, increased 6 days alier hcpatectomy in all groups (154 +/-1 cycles) even beyond basal levels. At both time-points, '~vben the tour groups were compared, VEGF-treated mice (n= 13) showed highest levels of endoglin and the endostatin-treated *nice (n = 10) had the lowest endoglin levels while the control vector-treated mice (n = 7) or NaCl-treated mice (n = 5) ranged in the middle. Couchlsion Hepatic expression of endoglin mRNA is regulated during liver regeneration. It reaches highest levels in :~sponse to VEGF. Of note, the initial decrease of endoglin levels is in contrast to the otherwise observed increase ot angiogenesis related factors (e.g VEGF lvceptors, HIE-1 etc.) in liver regeneration. The' mechanisms responsible for this regulation, the association with the expression of ang:ogenesis modulators, and the role of endoglin in live:' regeneration remain to be studied.

$917 Cell Aggregates Formation Method Enables to Enrich Hepatic Progenitor Cells from Adult Mouse Liver Hisaya Azuma, Tetsuro Hirose, Hideaki Fu}ii, Kentaro Yasuchika, Masato Naito, Toshitaka Hoppo, Shinji Baba, Takafumi Machimoto, Yoshio Yamaoka Hepatic progenitor cells (HPCs) have been well characterized as oval cells in several liver injury models or as hepatoblasts in fetal liver. On the other hand, the properties of HPCs in normal adult liver have not been fully clarified, because of thier small population in noninjured liver. In an atempt to resolve this, we developed a new enrichment system for HPCs. Nonparenchyanal cells derived from enzynratically-digested liver ceils in normal adult mouse liver were treated in a two-hour hypoxic suspension culture under constant shaking condition. This procedure resulted in cell aggregates formation and complete elimination of :nature hepatocytes. Cell aggregates were formed only in calcium-containing medium, suggesting cadherin-depender:t homophilic cell-cell adhesion. In the cell aggregates, about 5% consisted of small epithelial cells, whereas remaining 95% consisted of vascular endothelial cells. The small epithelial cells rapidly" proliterated in our standard medium and express alphafetoprotein, albumin, and E-cadherin, but not cytokeratin-19, VE
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Analysis of Gene ExpresS!on Using Cdna Microarray in The Liver of Patients with Chronic Hepatitis C During Interferon Therapy Furuihiko Komine Hitoshi C)kubo, Masaki Slmnojima, Toshiki Yamamoto, Terunobu lshiga:ni, Yasuyuki Arakawa Purpose: The combination of interferon (IFN) therapy with ribavirin has been proving to be more efhcacious than IFN mono-therapy m treating chronic hepatitis C (CHC). But the reason for its effectiveness has :~ot been clear. Moreover only viral factors, such as g e n o t ~ and viral load were utilized to assume IFN efficacy before therapy. However, more hostside intormation is required for new evidence-based strategies tbr therapy of HCV-infi:cted patients. Ttmrefore, we analyzed gene expression in tire liver of HCV infected patients during IFN thm~py using cDNA microarray. Materials and methods We investigated 16 patients with CHHCduring 1FN therapy The combination therapy group (group A) included 11 patients undel\going treatnmnt with IFN-alpha 2b (i0 million units six times weekly for tbur weeks, and three times weekly for 20 weeks: Total 840 mega units) plus ribavirin (0.60 8g/day) fbr CHC (genotypelB:4, 2A:3, 2B:4 cases). The mono-therapy group (group B) included five patients undergoing the same IFN treatment without ribavirin (genotypelB: 1, 2A12, 2B:2 cases). All underwent needle biopsy of the liver bdbre therapies, and re-biopsy was pertbm~ed after four weeks. With tim two .samples, we analyzed mRNAs expression in the liver of patients using eDNA microarray We have utilized 294 cDNAs on the chip for expression analysis Of the 294 cDNAs, there were 26 kinds of lFN-related genes, 27 kinds cy~okine-lymphokine related genes, etc. Resuhs: We translated the values of the enhanced mP,NAs expression as atier / bdbre therapy rate Alter comparing the gene expression pmtiles between groups A and B, group A showed a higher enhance than gnrup B in 2-5AS, lL2, et al. and in about ten kinds of genes as well as 3SD Compariso~ between genotype 1B, 2A, and 2B groups and between the effects of anti-virus CRs and NRs, some gene expression ddiered as well as 3SD not only in samples taken bdore therapy but also in the after / before therapy rate. Conclusions: Our results suggest that combination therapy IFN with nbavmn enhances son:e IFN-rdated genes and cytokine expression in liver. Furthermore our resuhs suggest that there are difterences of gene expressimrs among different HCV genotypes infected patron*s. Our observations seen: m correlate with the efficacy of IFN treatment T~se results may be of significance as host-side iniormation for new evidencebased strategies for therapy oi HCV-iniected patients.

$918 Cross-Talk Between Protein Kinase C-zeta and Protein Kinase B in cAMPMediated Stimulation of Hepatic Na +/Tanrocholate Cotransport Marie E. McConkey, Cynthia R. Webster, Mohammed S. Anwer Cyclic AMP stimulates Na +/taurocholate (TC) cotransport and Ntcp translocation in hepatocytes via the phosphoinositide-3-kinase/protein kinase B (PI3K/PKB) signaling pathway 0BC 277: 28578, 2002). Another downstream mediator of PI3K signaling pathway is protein kinase C-zeta (PKC-zeta) and our preliminary studies with non-specific inhibitors suggested a role for PKC-zeta in cAMP-mediated Na+FfC cotransport. The am: of the present study was to turther characterize the role of PKC-zeta by using a specific inhibitor of PKCzeta, myristoylated PKC-zeta pseudosubstarte (PKC-zeta-PS), in hepatocytes and transient transfections with wild type (v~q'-PKC-zeta) and dominant negative (DN-PKC-zeta) PKCzeta in HUH-7 cell stably transfected with rat liver Ntcp (HuH-Ntcp ceils). Hepatoc)~es were pretreated (90 rain) with 10 or 100~M PKC-zeta-PS and then with 10 I.LMchlorophenylthiocAMP (CPT-cAMP) for 15 rain followed by determination of TC uptake, Ntcp translocation, the activities of PKC-zeta and PKB activity. HUH-Ntcp cells were transiently transfected with WT-PKC-zeta, DN-PKC-zeta or both with studies conducted 48hrs after transfection. RESULTS: 100 ~M, but not 10 ~M, PKC-zeta-PS inhibited cAMP-induced increases in TC uptake and Ntcp translocation by 70% and 80%, respectively, and PKC-zeta activity by 50%. In Huh-Ntcp cells, transient transtectinn with WT-PKC-zeta (0.5 big) resulted in 2fold increase in PKC-zeta activity and in 34% increase in TC uptake. These effects of WTPKC-zeta were completely inhibited when HuH-Ntcp cells were co-transtected wit}: 10 fold excess DN-PKC-zeta. 100 ~M PKC-zeta-PS also inhibited cAMP-stimulated PKB activity by 80% in hepatocytes; PKC-zeta-PS does not inhibit PKB directly" (JBC 272: 30075, 1997). In HuH-Ntcp cells, PKB activity was inhibited by 40% when transfected with DN-PKC-zeta, although PKB activity was not stimulated when transfected with WT-PKC-zeta. CONCLUSIONS: Taken together, these results suggest that 1) PKC-zeta is involved in cAMP-mediated stimulation of hepatic TC uptake, 2) cAMP-mediated activation of PKB activity in hepatocytes is dependent on PKC-zeta activity and 3) PKC-zeta may be an intermediate mediator in cAMP-mediated stimulation of hepatic TC uptake via the PI3K/PKB signaling pathway.

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Epimorphin, a Novel Stellate Cell-Derived Protein, Induces Not Only HepatoD, tic Differentiation But Also Cholangiocytic Differentiation of Rat Hepatic Stem-Like Cells l
$919 The Transcriptional Activation of Liver-Specific Organic Anion Transporter-2 Gene Is Regulated by Three Liver Enriched Transcriptional Factors; Hnfla, Hnf3b, and Fxr Hideo Ohtsuka, Michiaki Unno, Takaaki Abe, Yu Katayose, Toshiki Rikiyama, He*go Takeuchi, Tohru Onogawa, Takeaki Satoh, Seiki Matsuno

Background: Epimorphin, a mesenchymal protein, was originally identii~ed as a morphogen (Cell 69:471-481,1992). We have reported that this protein, detected on hepatic stellate cells (HSCs), induces an albumin expression of hepatic stemdike cells (HSLCs) of *~ts (Gastroenterol%7/ 122: P23, 2002) Detailed hepatocyBc and cholangiocytic markers in HSLCs were investigated in this study. Materials and Methods: HSLCs and HSCs were isolated horn normal SD rats, age at 6 to 9 weeks, as previously reported (BBRC 293:14201425,2002) HSLCs were co-cultured w:th HSCs in the presence o[ anti-epimorphin antibody. HSLCs were' also cultured in the presence of epimorphin Morphologic changes were observed with a phase contrast microscopy and an electron microscopy. Hepatex:ytic and cholangincytic markers were investigated by Western blot or RT-PCR. Results: HSLCs were positive for

AASLD Abstracts

The basolateral membrane of the hepatocyte is characterized by various transporters, one of which are responsible for the uptake of organic anions substances, including conjugated bilimbin, bile acids, steroids and some kinds of drugs from the blood into the hepatoc~'te. In 1999, we have isolated human liver-specific organic anion transporter, LST-2 (SLC21A8, OATP-8), which is exclusively expressed in the basolateral membrane of the hepatocyte. We demonstrated that LST-2 transports chenodeoxycholate(CDCA), which is known as a ligand for FXR. in this study, we analyzed the transcriptional regulation of LST-2 gene in

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