S1571 PI3 Kinase/AKT Signaling Mediates Epithelial-Mesenchymal Transition in Hypoxic Hepatocellular...
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AASLD Abstracts from NHBD. Rat livers were explanted immediately after 30 min of aorta clamping, stored in UW solution at 0-1 o C for 4 h, and then implanted. 1400W, a specific iNOS inhibitor, was added to the storage solution and the lactated Ringer's post-storage rinse solution at a concentration of 5 μM. Hepatic iNOS expression was detected by Western blotting. iNOS was barely detectable in livers from untreated rats and in cold-stored, unimplanted livers from NHBD. After transplantation, iNOS increased slightly in grafts from heart beating donors (HBD). By contrast, iNOS increased markedly in grafts from NHBD. Serum nitrite and nitrate levels were ~3-fold higher in rats received grafts from NHBD than from HBD, indicating formation of nitric oxide. Similarly, hepatic 3-nitrotyrosine adducts, an indicator of peroxynitrite formation, was substantially higher in grafts from NHBD than those from HBD after transplantation. Production of reactive nitrogen species (RNS) was largely blocked by 1400W. Alanine aminotransferase (ALT) release, serum bilirubin, and apoptosis were 2.7-, 10.0-, and 3.1-fold higher after transplantation of grafts from NHBD than grafts from HBD, indicating more severe liver injury in grafts from NHBD. Cleaved caspase-3 detected by Western blotting was significantly higher in grafts from NHBD than grafts from HBD after transplantation, further conforming the occurrence of apoptosis. 1400W almost totally blocked these alterations. Survival decreased from 100% after transplantation of grafts from HBD to 33% after transplantation of grafts from NHBD. 1400W increased survival of grafts from NHBD to 80%. Previous studies showed that activation of c-Jun-N-terminal kinase (JNK) caused mitochondrial dysfunction and cell death after liver transplantation. Phos- phorylated JNK and phosphorylated c-Jun increased substantially in grafts from NHBD after transplantation, and these effects were blocked by 1400W. In summary, hepatic iNOS increased substantially after non-heart beating liver transplantation, which leads to over- production of RNS, JNK activation, and more severe graft injury. Inhibitors of iNOS are suggested as an effective therapy to improve the outcome of non-heart beating liver trans- plantation (NIDDK). S1570 RAC1, Caveolin-1 and VEGF Mediated Liver Sinusoidal Endothelial Cell Angiogenesis Hiroaki Yokomori, Masaya Oda, Fumihiko Kaneko, Toshifumi Hibi Background and Aims: Rho GTPases are major regulators of cell migration. We investigated the roles of cytoskeleton and Rho GTPases during cell migration and morphogenesis processes in isolated rat liver sinusoidal endothelial cell (LSEC)s cultured on a Matrigel matrix when stimulated by vascular endothelial growth factor (VEGF). Materials and Methods: LSECs obtained by collagenase infusion of a rat liver were cultured on Matrigel for 5-17 hours to obtain primary monolayers, in the presence or absence of VEGF. The morphology of sinus- oidal endothelial fenestrae (SEF) was observed by scanning and transmission electron micro- scopy. RhoA, Rac1 and phosphorylated myosin light chain kinase (P-MLC), Rhotekin- RBD and PAK-PBD were analyzed by Western blotting. Results: LSECs were morphology characterized by the formation of dynamic cellular protrusions and by the assembly of discrete aggregates or cords of aligned cells resembling primitive capillary-like structures, concomitant with a decrease in number of characteristic endothelial pores. In this process, time course analysis of RhoA and Rac1 activation matched specific morphological changes. Activated RhoA is markedly elevated at 5 h, although the level is declined at 7h, activation increases again at 17 h. Activated Rac1 shows marked increase at 7 hr, then decreases to lower than the basal level at 17 h. In cells cultured in the presence of VEGF, there were progressively increased at 17 h. The levels of endogenous CAV-1 expression increased in a time-dependent manner when endothelial cells underwent proliferation, reaching a peak at 7 h. CAV-1 expression occurred just prior to the formation of capillary-like tube. Moreover, by treatment with VEGF, relatively down-regulates CAV-1 expression in LSECs. Conclusions: Spatial activation of Rac1 and RhoA is considered to be involved in the formation of capillary- like tubular network accompanying fenestral contraction in LSECs, implying that endothelial migration and adhesion are essential for sinusoidal angiogenesis in the liver. Taken together, the above results indicate that CAV-1 may play an important positive role in the regulation of LSECs proliferation as a prerequisite step in the process of angiogenesis. S1571 PI3 Kinase/AKT Signaling Mediates Epithelial-Mesenchymal Transition in Hypoxic Hepatocellular Carcinoma Cells Wei Yan, Fu Yu, Jiazhi Liao, Mei Liu, Bo Wang, Dean Tian Background: Hypoxia is a hallmark of diverse human malignancies, including liver cancer, breast cancer and prostate cancer. Hypoxia activates genetic programs that facilitate cell survival; however it may promote invasion and metastasis in cancer. Although the exact mechanisms driving hypoxia-induced invasion and metastasis remain elusive, we hypothes- ized that epithelial-mesenchymal transition (EMT) may play a major role. Methods: In study, two hepatocellular carcinoma cells, HepG2 and Huh7, were cultured in 21% O2 (normoxia) or 1.0% O2 (hypoxia) for 72 h. After the hypoxia treatment, cell images were captured by phase-contrast microscopy. F-actin cytoskeletal arrangement was examined by confocal microscopy. Cell migration and invasion induced by hypoxia were ananlyed. Expression of the epithelia-specific marker E-cadherin and the myofibroblast-specific marker vimentin were examined by western blot. We also analysed the expression of p-Akt by Immunofluorescent microscopy and western blot. PI3K inhibitor LY294002 was used to show whether it could inhibit hypoxia-induced EMT. Results: After cultured in 1.0% O2, the cells exhibited some morphological changes including cell elongation (with a large degree of detachment) and junctional disruption. Under normoxic conditions, F-actin fibers were densely arranged just inside the cell periphery. Slim central fibers were also visible. In cells exposed to 1.0% O2, the reorganization of the F-actin cytoskeleton could be seen. The densely arranged peripheral F-actin was redistributed into strong central fibers termed stress fibers. The stress fibers were aligned in parallel and could be clearly seen to project out of the cell at higher magnification. F-actin fibers arranged in parallel contribute to the elongated shape of a cell, which is a feature of cells with front end-back end polarity like myofibroblast. Moreover, expression of E-cadherin was decreased and expression of vimentin was detected in the treated cells (1.0% O2). Migration of HepG2 cells was increased 2.33 ± 0.21 fold (P < 0.01; n = 5) in 1.0% O2. Similarly, invasion of HepG2 cells through the ECM gel matrices was A-816 AASLD Abstracts increased 1.86 ± 0.17-fold (P < 0.01; n = 5) in 1.0% O2. These finding were consistent with events observed during EMT. Hypoxia-induced EMT is accompanied by increased phosphorylation, activation of Akt. The downstream signaling Twist and nuclear NF-κB expression also were increased. Hypoxia-induced EMT was blocked by LY294002. Conclu- sions: The results suggest that hypoxia could induce EMT in hepatocellular carcinoma cells. The PI3k/Akt-dependent signaling pathways serve to regulate hypoxia-induced EMT of hepatocellular carcinoma cells. S1572 Epimorphin Protects Hepatocytes from Oxidative Stress By Inhibiting Mitochondrial Injury Nobukatsu Kinoshita, Yasuo Horie, Shigetoshi Ohshima, Takahiro Dohmen, Mario Jin, Hirohide Ohnishi BACKGROUND AND AIM: Epimorphin, a mesenchymal protein, functions as a morphogen in various organs and cells. In addition, Epimorphin exerts cell protection effects in some types of cells. Although Epimorphin is expressed in hepatic stellate cells and induces hepatocyte aggregates into spheroids In Vitro, its detailed function remains unclear. In this study, we hypothesized that Epimorphin may have hepatocytes protective function. We thus examined whether Epimorphin protected primarily cultured hepatocytes from oxidative stress. METHODS: Hepatocytes were isolated from mice using two-step collagenase perfusion method and cultured in Waymouth's medium overnight. Primarily cultured hepatocytes were incubated in Krebs-Ringer HEPES (KRH) buffer at pH 7.4 for 1 hour with Epimorphin (20 μg/ml) or medium only, and then cells were exposed to 0.5 mM hydrogen peroxide. Cell viability was determined by propidium iodide assay. The intracellular levels of reactive oxygen species (ROS) were determined by measurering fluorescence of the dichlorodihydro- fluoresceindiacetate. The mitochondrial injury of hepatocytes such as mitochondrial per- meability transition (MPT) and mitochondrial depolarization were observed under confocal microscopy with 1 μM calcein and 300 nM TMRM, respectively. Effects of Epimorphin on JNK phosphorylation was examined using Western blot analysis. RESULT: One hour after exposure to hydrogen peroxide, cell viability was 36 % and 77 % of primarily cultured hepatocytes preincubated with Epimorphin or medium only, respectively. On the other hand, 1 hour after exposure to hydrogen peroxide, the intracellular levels of ROS were elevated in both hepatocytes preincubated with Epimorphin or medium only (2.6 and 2.3 times over those in cells without exposure to hydrogen peroxide, respectively). These findings suggest that Epimorphin protects primarily cultured hepatocyte from oxidative stress inde- pendently of intracellular ROS levels. Although the onset of MPT and mitochondrial depolar- ization were observed in cells without Epimorphin at 30 minutes after exposure to hydrogen peroxide, those were not observed in cells with Epimorphin until 1 hour after exposure to hydrogen peroxide. In addition, Western blot analysis revealed that Epimorphin attenuated hydrogen peroxide-induced phosphorylation of JNK, a death signal mediator molecule, in primarily cultured hepatocytes. CONCLUSION: Epimorphin protected primarily cultured hepatocytes from oxidative stress by inhibiting mitochondrial injury independently of intracellular ROS levels. JNK phosphorylation was possibly involved in its mechanism. S1573 Expression of YAP Protein and Evaluation of the YAP Genomic Loci in Human Hepatocellular Carcinoma Haibo Bai, Robert A. Anders Introduction: The Hippo signaling pathway is a newly recognized, potent regulator of cell growth, death and size. The YAP protein is the nuclear effector of Hippo signaling pathway. Overexpression of YAP in rodent liver results in an enlarged liver and malignant transforma- tion to hepatocellular carcinoma (HCC). In humans, amplification of the chromosome region containing the YAP gene (11q22) has been observed in several tumor types, and an elevated YAP expression level has been reported in multiple carcinomas types. Aim: The aims of this study are to determine if YAP expression is elevated in human HCC tissues, and whether the overexpression of YAP in the tumor tissues correlates with gene amplification. Method: We performed immunohistochemical staining on 81 patients with HCC and scored the YAP expression level (absent, present) and distribution (cytoplasmic, nuclear) from the paired HCC tumor and non-tumor liver tissue. We also used real time PCR to determined genomic copy number of the YAP loci on a subset of 17 human HCCs. Greater than 2 genomic copies of the YAP loci, relative to 2 unrelated loci on the same chromosome, were considered evidence of a YAP amplification. Results: We found significantly more nuclear expression of YAP in HCC compared to non-tumor tissue, however, there was no difference in the cytoplasmic expression of YAP (Table 1). Genomic DNA amplification of the YAP loci is a fairly frequent event (4/17) in HCCs, however, it did not correlate with YAP expression in the nucleus. Conclusions: Our findings suggest that dysregulation of the Hippo signaling pathway is a common event in hepatocellular carcinogenesis, however, genomic amplification does not account for the increased nuclear expression of YAP in HCC. Table 1
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sfrom NHBD. Rat livers were explanted immediately after 30 min of aorta clamping, storedin UW solution at 0-1oC for 4 h, and then implanted. 1400W, a specific iNOS inhibitor,was added to the storage solution and the lactated Ringer's post-storage rinse solution at aconcentration of 5 μM. Hepatic iNOS expression was detected by Western blotting. iNOSwas barely detectable in livers from untreated rats and in cold-stored, unimplanted liversfrom NHBD. After transplantation, iNOS increased slightly in grafts from heart beatingdonors (HBD). By contrast, iNOS increased markedly in grafts from NHBD. Serum nitriteand nitrate levels were ~3-fold higher in rats received grafts from NHBD than from HBD,indicating formation of nitric oxide. Similarly, hepatic 3-nitrotyrosine adducts, an indicatorof peroxynitrite formation, was substantially higher in grafts from NHBD than those fromHBD after transplantation. Production of reactive nitrogen species (RNS) was largely blockedby 1400W. Alanine aminotransferase (ALT) release, serum bilirubin, and apoptosis were2.7-, 10.0-, and 3.1-fold higher after transplantation of grafts from NHBD than grafts fromHBD, indicating more severe liver injury in grafts from NHBD. Cleaved caspase-3 detectedby Western blotting was significantly higher in grafts from NHBD than grafts from HBDafter transplantation, further conforming the occurrence of apoptosis. 1400W almost totallyblocked these alterations. Survival decreased from 100% after transplantation of grafts fromHBD to 33% after transplantation of grafts from NHBD. 1400W increased survival of graftsfrom NHBD to 80%. Previous studies showed that activation of c-Jun-N-terminal kinase(JNK) caused mitochondrial dysfunction and cell death after liver transplantation. Phos-phorylated JNK and phosphorylated c-Jun increased substantially in grafts from NHBD aftertransplantation, and these effects were blocked by 1400W. In summary, hepatic iNOSincreased substantially after non-heart beating liver transplantation, which leads to over-production of RNS, JNK activation, and more severe graft injury. Inhibitors of iNOS aresuggested as an effective therapy to improve the outcome of non-heart beating liver trans-plantation (NIDDK).
S1570
RAC1, Caveolin-1 and VEGF Mediated Liver Sinusoidal Endothelial CellAngiogenesisHiroaki Yokomori, Masaya Oda, Fumihiko Kaneko, Toshifumi Hibi
Background and Aims: Rho GTPases are major regulators of cell migration. We investigatedthe roles of cytoskeleton and Rho GTPases during cell migration andmorphogenesis processesin isolated rat liver sinusoidal endothelial cell (LSEC)s cultured on a Matrigel matrix whenstimulated by vascular endothelial growth factor (VEGF). Materials and Methods: LSECsobtained by collagenase infusion of a rat liver were cultured on Matrigel for 5-17 hours toobtain primary monolayers, in the presence or absence of VEGF. The morphology of sinus-oidal endothelial fenestrae (SEF) was observed by scanning and transmission electron micro-scopy. RhoA, Rac1 and phosphorylated myosin light chain kinase (P-MLC), Rhotekin-RBD and PAK-PBD were analyzed by Western blotting. Results: LSECs were morphologycharacterized by the formation of dynamic cellular protrusions and by the assembly ofdiscrete aggregates or cords of aligned cells resembling primitive capillary-like structures,concomitant with a decrease in number of characteristic endothelial pores. In this process,time course analysis of RhoA and Rac1 activation matched specific morphological changes.Activated RhoA is markedly elevated at 5 h, although the level is declined at 7h, activationincreases again at 17 h. Activated Rac1 shows marked increase at 7 hr, then decreases tolower than the basal level at 17 h. In cells cultured in the presence of VEGF, there wereprogressively increased at 17 h. The levels of endogenous CAV-1 expression increased in atime-dependent manner when endothelial cells underwent proliferation, reaching a peak at7 h. CAV-1 expression occurred just prior to the formation of capillary-like tube. Moreover,by treatment with VEGF, relatively down-regulates CAV-1 expression in LSECs. Conclusions:Spatial activation of Rac1 and RhoA is considered to be involved in the formation of capillary-like tubular network accompanying fenestral contraction in LSECs, implying that endothelialmigration and adhesion are essential for sinusoidal angiogenesis in the liver. Taken together,the above results indicate that CAV-1 may play an important positive role in the regulationof LSECs proliferation as a prerequisite step in the process of angiogenesis.
S1571
PI3 Kinase/AKT Signaling Mediates Epithelial-Mesenchymal Transition inHypoxic Hepatocellular Carcinoma CellsWei Yan, Fu Yu, Jiazhi Liao, Mei Liu, Bo Wang, Dean Tian
Background: Hypoxia is a hallmark of diverse human malignancies, including liver cancer,breast cancer and prostate cancer. Hypoxia activates genetic programs that facilitate cellsurvival; however it may promote invasion and metastasis in cancer. Although the exactmechanisms driving hypoxia-induced invasion and metastasis remain elusive, we hypothes-ized that epithelial-mesenchymal transition (EMT) may play a major role. Methods: In study,two hepatocellular carcinoma cells, HepG2 and Huh7, were cultured in 21% O2 (normoxia)or 1.0% O2 (hypoxia) for 72 h. After the hypoxia treatment, cell images were captured byphase-contrast microscopy. F-actin cytoskeletal arrangement was examined by confocalmicroscopy. Cell migration and invasion induced by hypoxia were ananlyed. Expression ofthe epithelia-specific marker E-cadherin and themyofibroblast-specific marker vimentin wereexamined by western blot. We also analysed the expression of p-Akt by Immunofluorescentmicroscopy and western blot. PI3K inhibitor LY294002 was used to show whether it couldinhibit hypoxia-induced EMT. Results: After cultured in 1.0% O2, the cells exhibited somemorphological changes including cell elongation (with a large degree of detachment) andjunctional disruption. Under normoxic conditions, F-actin fibers were densely arranged justinside the cell periphery. Slim central fibers were also visible. In cells exposed to 1.0% O2,the reorganization of the F-actin cytoskeleton could be seen. The densely arranged peripheralF-actin was redistributed into strong central fibers termed stress fibers. The stress fiberswere aligned in parallel and could be clearly seen to project out of the cell at highermagnification. F-actin fibers arranged in parallel contribute to the elongated shape of a cell,which is a feature of cells with front end-back end polarity like myofibroblast. Moreover,expression of E-cadherin was decreased and expression of vimentin was detected in thetreated cells (1.0% O2). Migration of HepG2 cells was increased 2.33 ± 0.21 fold (P < 0.01;n = 5) in 1.0% O2. Similarly, invasion of HepG2 cells through the ECM gel matrices was
A-816AASLD Abstracts
increased 1.86 ± 0.17-fold (P < 0.01; n = 5) in 1.0% O2. These finding were consistentwith events observed during EMT. Hypoxia-induced EMT is accompanied by increasedphosphorylation, activation of Akt. The downstream signaling Twist and nuclear NF-κBexpression also were increased. Hypoxia-induced EMT was blocked by LY294002. Conclu-sions: The results suggest that hypoxia could induce EMT in hepatocellular carcinoma cells.The PI3k/Akt-dependent signaling pathways serve to regulate hypoxia-induced EMT ofhepatocellular carcinoma cells.
S1572
Epimorphin Protects Hepatocytes from Oxidative Stress By InhibitingMitochondrial InjuryNobukatsu Kinoshita, Yasuo Horie, Shigetoshi Ohshima, Takahiro Dohmen, Mario Jin,Hirohide Ohnishi
BACKGROUND AND AIM: Epimorphin, a mesenchymal protein, functions as a morphogenin various organs and cells. In addition, Epimorphin exerts cell protection effects in some typesof cells. Although Epimorphin is expressed in hepatic stellate cells and induces hepatocyteaggregates into spheroids In Vitro, its detailed function remains unclear. In this study, wehypothesized that Epimorphin may have hepatocytes protective function. We thus examinedwhether Epimorphin protected primarily cultured hepatocytes from oxidative stress.METHODS: Hepatocytes were isolated from mice using two-step collagenase perfusionmethod and cultured in Waymouth's medium overnight. Primarily cultured hepatocyteswere incubated in Krebs-Ringer HEPES (KRH) buffer at pH 7.4 for 1 hour with Epimorphin(20 μg/ml) or medium only, and then cells were exposed to 0.5 mM hydrogen peroxide.Cell viability was determined by propidium iodide assay. The intracellular levels of reactiveoxygen species (ROS) were determined by measurering fluorescence of the dichlorodihydro-fluoresceindiacetate. The mitochondrial injury of hepatocytes such as mitochondrial per-meability transition (MPT) and mitochondrial depolarization were observed under confocalmicroscopy with 1 μM calcein and 300 nM TMRM, respectively. Effects of Epimorphin onJNK phosphorylation was examined using Western blot analysis. RESULT: One hour afterexposure to hydrogen peroxide, cell viability was 36 % and 77 % of primarily culturedhepatocytes preincubated with Epimorphin or medium only, respectively. On the otherhand, 1 hour after exposure to hydrogen peroxide, the intracellular levels of ROS wereelevated in both hepatocytes preincubated with Epimorphin or medium only (2.6 and 2.3times over those in cells without exposure to hydrogen peroxide, respectively). These findingssuggest that Epimorphin protects primarily cultured hepatocyte from oxidative stress inde-pendently of intracellular ROS levels. Although the onset of MPT and mitochondrial depolar-ization were observed in cells without Epimorphin at 30 minutes after exposure to hydrogenperoxide, those were not observed in cells with Epimorphin until 1 hour after exposure tohydrogen peroxide. In addition, Western blot analysis revealed that Epimorphin attenuatedhydrogen peroxide-induced phosphorylation of JNK, a death signal mediator molecule, inprimarily cultured hepatocytes. CONCLUSION: Epimorphin protected primarily culturedhepatocytes from oxidative stress by inhibiting mitochondrial injury independently ofintracellular ROS levels. JNK phosphorylation was possibly involved in its mechanism.
S1573
Expression of YAP Protein and Evaluation of the YAP Genomic Loci in HumanHepatocellular CarcinomaHaibo Bai, Robert A. Anders
Introduction: The Hippo signaling pathway is a newly recognized, potent regulator of cellgrowth, death and size. The YAP protein is the nuclear effector of Hippo signaling pathway.Overexpression of YAP in rodent liver results in an enlarged liver and malignant transforma-tion to hepatocellular carcinoma (HCC). In humans, amplification of the chromosome regioncontaining the YAP gene (11q22) has been observed in several tumor types, and an elevatedYAP expression level has been reported in multiple carcinomas types. Aim: The aims of thisstudy are to determine if YAP expression is elevated in human HCC tissues, and whetherthe overexpression of YAP in the tumor tissues correlates with gene amplification. Method:We performed immunohistochemical staining on 81 patients with HCC and scored the YAPexpression level (absent, present) and distribution (cytoplasmic, nuclear) from the pairedHCC tumor and non-tumor liver tissue. We also used real time PCR to determined genomiccopy number of the YAP loci on a subset of 17 human HCCs. Greater than 2 genomiccopies of the YAP loci, relative to 2 unrelated loci on the same chromosome, were consideredevidence of a YAP amplification. Results: We found significantly more nuclear expressionof YAP in HCC compared to non-tumor tissue, however, there was no difference in thecytoplasmic expression of YAP (Table 1). Genomic DNA amplification of the YAP loci is afairly frequent event (4/17) in HCCs, however, it did not correlate with YAP expression inthe nucleus. Conclusions: Our findings suggest that dysregulation of the Hippo signalingpathway is a common event in hepatocellular carcinogenesis, however, genomic amplificationdoes not account for the increased nuclear expression of YAP in HCC.Table 1
