- Email: [email protected]
170 Hemodynamic Effects of Propofol Sedation During Hepatic Venous Pressure Gradient Measurement
recipients was injected with an adenoviral vector expressing human hepatocyte growth factor (Ad-HGF 1x1011 particles i.v.). Tacrolimus was injected (2mg/kg sc daily, beginning 7 days before transplantation). Engraftment and repopulation were assessed by bioluminescence imaging, and by histochemical staining of liver sections for bacterial β-glucuronidase activity. Results: Conventional liver serological tests (serum albumin, bilirubin and alanine aminotransferase) were normal in the PiZ mice. Large number of PiZ globules were found in the hepatocytes of ~25% of female 75% of male PiZ mice. Non-invasive IVIS imaging showed progressive repopulation of the liver by F-luc-transduced donor hepatocytes. Approximately 5%, 10% and 20% of the hepatocyte mass consisted of donor-derived β-glucuronidasepositive hepatocytes, 1, 2 and 3 months after transplantation, respectively. In the group that received Ad-HGF, 25% liver repopulation was observed in 3 months. Conclusion: Spontaneous repopulation of the PiZ mouse liver with wildtype donor hepatocytes indicate a proliferative advantage of the donor cells against the recipient hepatocytes. This is in contrast to findings in most liver-based metabolic disorders, where the host hepatocytes are not under stress. These findings may have therapeutic implications for patients with AAT disease.
169 Neutral Endopeptidase (EC 3.4.24.11) Inhibition Blunts Endothelin-1 Production in Cirrhotic Rat Livers: Physiopathological Implications Giovanni G. Sansoe, Manuela Aragno, Floriano Rosina, Antonina Smedile, Maurizio Parola
167 Single Agent Activity of Obatoclax (Gx15-070) in a Rat Cholangiocarcinoma Model Rory Smoot, Boris Blechacz, Alphonse E. Sirica, Gregory J. Gores Background: Cholangiocarcinoma is a devastating cancer with limited treatment options. Direct induction of apoptosis by BH3-only protein mimetics is an emerging therapeutic strategy for human cancer and as such our aim was to examine the single agent activity of Obatoclax (GX15-070) a novel, small molecule BH3 mimetic using a previously validated rat cholangiocarcinoma model. (Hepatology. 2008 Apr;47(4):1178-90.) Methods: The rat cholangiocarcinoma cell line BDEneu was used for these studies. Real-time PCR and immunoblot analysis were employed to profile Bcl-2 proteins in treated and untreated BDEneu cells. In Vitro dose response to Obatoclax was determined via clonogenic assays. Bax cellular compartmentalization was assessed by immunofluorescence. Mitochondria were isolated from cultured cells by nitrogen cavitation and Bax protein was assayed by immunoblot analysis. Isolated mitochondria were subjected to bicarbonate treatment or cross-linking with 2mM disuccinimidyl suberate (DSS) and bis[sulfosuccinimidyl] suberate (BS3) followed by immunoblot analysis to examine Bax protein membrane insertion and oligomerization. Results: No change in mRNA or protein expression for BCL-2 (Mcl-1, Bcl-2, Bcl-Xl, Bak, Bax) or BH3-only (Bid, Bim, Noxa, Puma) proteins occurred following treatment of BDEneu cells with 100nM Obatoclax for 24 hours. Clonogenic assays demonstrated a dose response to Obatoclax with 43% inhibition of colony formation at 10 nM and 90% inhibition of colony formation in 100nM Obatoclax. Immunofluorescense demonstrated Bax translocation from the cytosol to mitochondria in Obatoclax treated cells. In mitochondria isolated from Obatoclax treated BDEneu cells, total Bax protein levels, membrane-integrated Bax protein levels, and Bax oligomers were increased. Conclusions: In conclusion, these data suggest single agent activity of Obatoclax in this model of cholangiocarcinoma. The mechanism of cell death involves activation of Bax, with mitochondrial membrane-integration and oligomerization. Based on these data Obatoclax, which is in phase II human trials for hematologic malignancies, warrants exploration for therapy in human cholangiocarcinoma.
170 Hemodynamic Effects of Propofol Sedation During Hepatic Venous Pressure Gradient Measurement Mona Y. Hellstern, Ludwig T. Heuss, Markus H. Heim Background: Hepatic venous pressure gradient (HVPG) measurements provide reliable information about the portal venous pressure. Recently it could be shown that HVPG can provide prognostic information about the risk of variceal bleeding and efficacy of pharmacological treatment of portal hypertension. For many patients, the procedure causes some degree of discomfort and sometimes pain. Sedation during the procedure would increase patient comfort. For more than 6 years propofol has routinely and safely been used in our department for sedation during endoscopies. Propofol can lead to a lowering of the arterial blood pressure and therefore may lead to an incorrect result of the HVPG measurement in sedated patients. Aim: The aim of the present study was to investigate if sedation of patients with propofol has a significant effect on HVPG measurements. Methods: After informed consent, HVPG measurements were performed in 18 cirrhotic patients with liver disease before and during propofol sedation. Results: All patients had portal hypertension. The mean HVPG before sedation was 17.4 mmHg (SD 4.4). The mean decrease of HVPG after sedation was 1.9 mmHg (p=0.0003), the mean decrease of the mean arterial pressure (MAP) was 8 mmHg (p=0.0014). There was no significant correlation between decrease in MAP and decrease in HVPG. In 2 of the 18 patients HVPG decreased to a value less than 12 mmHg and in 3 patients out of 18 the decrease was more than 20%. Conclusion: Propofol sedation is safe and increases patient comfort during HVPG measurements. However, propofol sedation significantly (>20%) reduced HVPG in 17% of the patients. HVPG values obtained during propofol sedation have to be interpreted with caution, especially when they are close to the clinically important value of 12mm Hg or when used for monitoring of therapy with non-selective beta blockers.
168 SULF2 Promotes Tumorigenesis of Hepatocellular Carcinoma By Activating the Wnt/β-Catenin Pathway Jinping Lai, Chunrong Yu, Catherine D. Moser, Hajime Isomoto, Ju-Seog Lee, Alex A. Adjei, Snorri Thorgeirsson, Schuyler Sanderson, Lewis R. Roberts Background: It is estimated that 626,000 patients are diagnosed with hepatocellular carcinoma (HCC) each year and it is the third most common cause of cancer related death, accounting for 598,000 deaths annually worldwide. We have found that the heparin-degrading endosulfatase, SULF2, increases HCC growth and predicts a worse prognosis. Heparindegrading endosulfatases have been implicated in regulation of Wnt signaling during embryonic development. The Wnt signaling pathway is important in the growth of a proportion of HCCs. Our Aim was to determine if the tumorigenic effect of SULF2 in HCCs is mediated through the Wnt/β-catenin pathway. Methods: We evaluated the oncogenic effect of SULF2 in activation of the Wnt/β-catenin pathway In Vitro and In Vivo using Western blotting, immunocytochemistry and confocal microscopy. The effect of SULF2 on TCF transcriptional activity was evaluated by measuring luciferase activity after transfection of the TOPflash and FOPflash TCF-responsive and control plasmid vectors. Results: SULF2 expression was increased in 79 (57%) of 139 HCCs and 9 (82%) of 11 HCC cell lines. HCCs with high SULF2 expression have a worse prognosis. SULF2 up-regulated phosphorylation of Erk44/ 42 and AKT ser473 and increased HCC cell growth and migration In Vitro, and enhanced tumorigenesis of nude mouse xenografts. Down regulation of SULF2 abrogated HCC cell proliferation and migration. SULF2-mediated cell growth was associated with increased expression of glypican 3 (GPC 3) at the cell surface, increased GSK3β phosphorylation, inhibition of β-catenin degradation, and activation of TCF and cyclin D1, suggesting that it is mediated through the Wnt signaling pathway. Conclusions: These data suggest that SULF2 contributes to hepatocarcinogenesis by up-regulating cell surface GPC3 and enhancing Wnt signaling. (Supported by NIH Grants CA82862 and CA100882).
171 Outcomes of Digital Image Analysis (DIA) and Fluorescence in Situ Hybridization (FISH) Test in Primary Sclerosing Cholangitis (PSC) Sanjay Bangarulingam, Einar Bjornsson, Keith D. Lindor Background: Patients with PSC are at increased risk for developing cholangiocarcinoma (CCA). DIA and FISH are advanced cytological tests to enhance early diagnosis of CCA. We analyzed the long term outcomes of PSC patients with positive DIA and FISH tests. Methods: All PSC patients with at least one DIA or FISH test between 2003 and 2008 were identified. Patients were defined to have CCA if they had a positive tissue biopsy, positive cytology or evidence of cancer in the explant following liver transplantation. Results: A total of 236 PSC patients had at least one DIA or FISH test performed during the study period. A total of 56 patients had CCA on histopathology (n=34) and cytology (n=22). Overall 119/ 236 (50 %) of PSC patients tested for FISH were positive but only 40/119 (34 %) had CCA. Of the 51 patients with positive polysomy FISH test, 28 (55%) had CCA whereas 68 patients with positive trisomy 7 or 3 and tetrasomy, only 12 (18%) had CCA. There were 79/232 (34 %) with positive DIA; 39 % (31/79) had CCA. Overall 54/79 had aneuploidy out of which 22 (41%) had an identifiable CCA. Tetraploidy was found in 25/79 and 9 (36%) had CCA. The sensitivity, specificity, PPV and NPV for FISH were (71%, 56%, 34%, 86%), for FISH trisomy 7 or 3 (21%, 69%,18%, 74%), for FISH polysomy (50%, 87%, 55%, 85%) for DIA they were (60%, 73%, 39%, 86%), DIA aneuploidy (42%, 82%,41%, 83%), DIA tetraploidy (17%, 91%, 36%,79%), respectively. Male patients more often had CCA (84% vs 16%; p= 0.01). Symptoms of weight loss (48% vs 16%; p < 0.0001), jaundice (57% vs 38%; p= 0.01) and smoking (37% vs 25%: p= 0.04) were associated with CCA. CA19-9 and total bilirubin (TB) were higher in those with CCA, 71 units/ml (20-566) vs 28 (1599), p=0.0004) and 4 mg/dL (0.9-11) vs 2 (0.9-5), p= 0.03, respectively. Overall 8 patients with FISH polysomy were followed for 23 months (13-40) without CCA development. These patients in comparison with those with positive FISH polysomy and CCA (n=28) had lower TB, 1 (0.5-2.2) vs. 6 (1.6-12), p=0.006, lower CA19-9, 27 (17-223) vs 90 (21-978), p=0.1, lower Mayo risk score, 0.3 (-0.3-1.1) vs. 1.9 (0.07-2.2), p=0.02 and lower occurrence of dominant strictures, 1/8 (13%) vs. 21/28 (75%), p=0.001. Conclusion: Only one third of
A-793
AASLD Abstracts
AASLD Abstracts
The membrane-bound neutral metalloendopeptidase EC 3.4.24.11 (NEP) degrades atrial natriuretic peptide (ANP) and bradykinin (BK) and generates endothelin-1 (ET-1) from bigendothelin, leading to vasoconstriction and fibrogenesis. Transforming growth factor-β (TGFβ) enhances NEP expression in fibroblasts, bronchial and kidney tubular epithelial cells In Vitro. In cirrhotic animals, the NEP inhibitor candoxatrilat decreases intrahepatic vascular resistance and portal pressure (1). Aims of this study are to evaluate in cirrhotic rat livers: a) the role of NEP in ANP, BK and ET-1 handling; b) NEP protein concentration and immunostaining; c) the degree of expression of TGF-β. We studied two groups of 5 control rats injected with vehicle or 10 mg/Kg b.w. candoxatrilat, and two groups of 10 rats with CCl4-induced liver cirrhosis receiving vehicle alone or 10 mg/Kg b.w. candoxatrilat. In cirrhotic rats, candoxatrilat significantly decreased hepatic venous concentrations of ET-1 (P<0.01) and increased systemic plasma levels of ANP (P<0.05), whilst in controls ANP, BK and ET-1 handling seemed unaffected by candoxatrilat. In cirrhotic livers, western blot analysis revealed 400% increase in TGF-β protein content in the whole homogenate and 300% increase in NEP content in the cytosol fraction with respect to controls (both P<0.001). NEP was strictly localized in desmin- and smooth muscle actin-positive activated stellate (SCs) of fibrous septa. In conclusion, overproduced TGF-β in cirrhotic liver enhances NEP expression in activated SCs of fibrous septa. This leads to contraction of activated SCs and hence increased intrahepatic portal vascular resistance through local generation of ET-1. 1) J Hepatol 43: 791-8, 2005.
169 Neutral Endopeptidase (EC 3.4.24.11) Inhibition Blunts Endothelin-1 Production in Cirrhotic Rat Livers: Physiopathological Implications Giovanni G. Sansoe, Manuela Aragno, Floriano Rosina, Antonina Smedile, Maurizio Parola
167 Single Agent Activity of Obatoclax (Gx15-070) in a Rat Cholangiocarcinoma Model Rory Smoot, Boris Blechacz, Alphonse E. Sirica, Gregory J. Gores Background: Cholangiocarcinoma is a devastating cancer with limited treatment options. Direct induction of apoptosis by BH3-only protein mimetics is an emerging therapeutic strategy for human cancer and as such our aim was to examine the single agent activity of Obatoclax (GX15-070) a novel, small molecule BH3 mimetic using a previously validated rat cholangiocarcinoma model. (Hepatology. 2008 Apr;47(4):1178-90.) Methods: The rat cholangiocarcinoma cell line BDEneu was used for these studies. Real-time PCR and immunoblot analysis were employed to profile Bcl-2 proteins in treated and untreated BDEneu cells. In Vitro dose response to Obatoclax was determined via clonogenic assays. Bax cellular compartmentalization was assessed by immunofluorescence. Mitochondria were isolated from cultured cells by nitrogen cavitation and Bax protein was assayed by immunoblot analysis. Isolated mitochondria were subjected to bicarbonate treatment or cross-linking with 2mM disuccinimidyl suberate (DSS) and bis[sulfosuccinimidyl] suberate (BS3) followed by immunoblot analysis to examine Bax protein membrane insertion and oligomerization. Results: No change in mRNA or protein expression for BCL-2 (Mcl-1, Bcl-2, Bcl-Xl, Bak, Bax) or BH3-only (Bid, Bim, Noxa, Puma) proteins occurred following treatment of BDEneu cells with 100nM Obatoclax for 24 hours. Clonogenic assays demonstrated a dose response to Obatoclax with 43% inhibition of colony formation at 10 nM and 90% inhibition of colony formation in 100nM Obatoclax. Immunofluorescense demonstrated Bax translocation from the cytosol to mitochondria in Obatoclax treated cells. In mitochondria isolated from Obatoclax treated BDEneu cells, total Bax protein levels, membrane-integrated Bax protein levels, and Bax oligomers were increased. Conclusions: In conclusion, these data suggest single agent activity of Obatoclax in this model of cholangiocarcinoma. The mechanism of cell death involves activation of Bax, with mitochondrial membrane-integration and oligomerization. Based on these data Obatoclax, which is in phase II human trials for hematologic malignancies, warrants exploration for therapy in human cholangiocarcinoma.
170 Hemodynamic Effects of Propofol Sedation During Hepatic Venous Pressure Gradient Measurement Mona Y. Hellstern, Ludwig T. Heuss, Markus H. Heim Background: Hepatic venous pressure gradient (HVPG) measurements provide reliable information about the portal venous pressure. Recently it could be shown that HVPG can provide prognostic information about the risk of variceal bleeding and efficacy of pharmacological treatment of portal hypertension. For many patients, the procedure causes some degree of discomfort and sometimes pain. Sedation during the procedure would increase patient comfort. For more than 6 years propofol has routinely and safely been used in our department for sedation during endoscopies. Propofol can lead to a lowering of the arterial blood pressure and therefore may lead to an incorrect result of the HVPG measurement in sedated patients. Aim: The aim of the present study was to investigate if sedation of patients with propofol has a significant effect on HVPG measurements. Methods: After informed consent, HVPG measurements were performed in 18 cirrhotic patients with liver disease before and during propofol sedation. Results: All patients had portal hypertension. The mean HVPG before sedation was 17.4 mmHg (SD 4.4). The mean decrease of HVPG after sedation was 1.9 mmHg (p=0.0003), the mean decrease of the mean arterial pressure (MAP) was 8 mmHg (p=0.0014). There was no significant correlation between decrease in MAP and decrease in HVPG. In 2 of the 18 patients HVPG decreased to a value less than 12 mmHg and in 3 patients out of 18 the decrease was more than 20%. Conclusion: Propofol sedation is safe and increases patient comfort during HVPG measurements. However, propofol sedation significantly (>20%) reduced HVPG in 17% of the patients. HVPG values obtained during propofol sedation have to be interpreted with caution, especially when they are close to the clinically important value of 12mm Hg or when used for monitoring of therapy with non-selective beta blockers.
168 SULF2 Promotes Tumorigenesis of Hepatocellular Carcinoma By Activating the Wnt/β-Catenin Pathway Jinping Lai, Chunrong Yu, Catherine D. Moser, Hajime Isomoto, Ju-Seog Lee, Alex A. Adjei, Snorri Thorgeirsson, Schuyler Sanderson, Lewis R. Roberts Background: It is estimated that 626,000 patients are diagnosed with hepatocellular carcinoma (HCC) each year and it is the third most common cause of cancer related death, accounting for 598,000 deaths annually worldwide. We have found that the heparin-degrading endosulfatase, SULF2, increases HCC growth and predicts a worse prognosis. Heparindegrading endosulfatases have been implicated in regulation of Wnt signaling during embryonic development. The Wnt signaling pathway is important in the growth of a proportion of HCCs. Our Aim was to determine if the tumorigenic effect of SULF2 in HCCs is mediated through the Wnt/β-catenin pathway. Methods: We evaluated the oncogenic effect of SULF2 in activation of the Wnt/β-catenin pathway In Vitro and In Vivo using Western blotting, immunocytochemistry and confocal microscopy. The effect of SULF2 on TCF transcriptional activity was evaluated by measuring luciferase activity after transfection of the TOPflash and FOPflash TCF-responsive and control plasmid vectors. Results: SULF2 expression was increased in 79 (57%) of 139 HCCs and 9 (82%) of 11 HCC cell lines. HCCs with high SULF2 expression have a worse prognosis. SULF2 up-regulated phosphorylation of Erk44/ 42 and AKT ser473 and increased HCC cell growth and migration In Vitro, and enhanced tumorigenesis of nude mouse xenografts. Down regulation of SULF2 abrogated HCC cell proliferation and migration. SULF2-mediated cell growth was associated with increased expression of glypican 3 (GPC 3) at the cell surface, increased GSK3β phosphorylation, inhibition of β-catenin degradation, and activation of TCF and cyclin D1, suggesting that it is mediated through the Wnt signaling pathway. Conclusions: These data suggest that SULF2 contributes to hepatocarcinogenesis by up-regulating cell surface GPC3 and enhancing Wnt signaling. (Supported by NIH Grants CA82862 and CA100882).
171 Outcomes of Digital Image Analysis (DIA) and Fluorescence in Situ Hybridization (FISH) Test in Primary Sclerosing Cholangitis (PSC) Sanjay Bangarulingam, Einar Bjornsson, Keith D. Lindor Background: Patients with PSC are at increased risk for developing cholangiocarcinoma (CCA). DIA and FISH are advanced cytological tests to enhance early diagnosis of CCA. We analyzed the long term outcomes of PSC patients with positive DIA and FISH tests. Methods: All PSC patients with at least one DIA or FISH test between 2003 and 2008 were identified. Patients were defined to have CCA if they had a positive tissue biopsy, positive cytology or evidence of cancer in the explant following liver transplantation. Results: A total of 236 PSC patients had at least one DIA or FISH test performed during the study period. A total of 56 patients had CCA on histopathology (n=34) and cytology (n=22). Overall 119/ 236 (50 %) of PSC patients tested for FISH were positive but only 40/119 (34 %) had CCA. Of the 51 patients with positive polysomy FISH test, 28 (55%) had CCA whereas 68 patients with positive trisomy 7 or 3 and tetrasomy, only 12 (18%) had CCA. There were 79/232 (34 %) with positive DIA; 39 % (31/79) had CCA. Overall 54/79 had aneuploidy out of which 22 (41%) had an identifiable CCA. Tetraploidy was found in 25/79 and 9 (36%) had CCA. The sensitivity, specificity, PPV and NPV for FISH were (71%, 56%, 34%, 86%), for FISH trisomy 7 or 3 (21%, 69%,18%, 74%), for FISH polysomy (50%, 87%, 55%, 85%) for DIA they were (60%, 73%, 39%, 86%), DIA aneuploidy (42%, 82%,41%, 83%), DIA tetraploidy (17%, 91%, 36%,79%), respectively. Male patients more often had CCA (84% vs 16%; p= 0.01). Symptoms of weight loss (48% vs 16%; p < 0.0001), jaundice (57% vs 38%; p= 0.01) and smoking (37% vs 25%: p= 0.04) were associated with CCA. CA19-9 and total bilirubin (TB) were higher in those with CCA, 71 units/ml (20-566) vs 28 (1599), p=0.0004) and 4 mg/dL (0.9-11) vs 2 (0.9-5), p= 0.03, respectively. Overall 8 patients with FISH polysomy were followed for 23 months (13-40) without CCA development. These patients in comparison with those with positive FISH polysomy and CCA (n=28) had lower TB, 1 (0.5-2.2) vs. 6 (1.6-12), p=0.006, lower CA19-9, 27 (17-223) vs 90 (21-978), p=0.1, lower Mayo risk score, 0.3 (-0.3-1.1) vs. 1.9 (0.07-2.2), p=0.02 and lower occurrence of dominant strictures, 1/8 (13%) vs. 21/28 (75%), p=0.001. Conclusion: Only one third of
A-793
AASLD Abstracts
AASLD Abstracts
The membrane-bound neutral metalloendopeptidase EC 3.4.24.11 (NEP) degrades atrial natriuretic peptide (ANP) and bradykinin (BK) and generates endothelin-1 (ET-1) from bigendothelin, leading to vasoconstriction and fibrogenesis. Transforming growth factor-β (TGFβ) enhances NEP expression in fibroblasts, bronchial and kidney tubular epithelial cells In Vitro. In cirrhotic animals, the NEP inhibitor candoxatrilat decreases intrahepatic vascular resistance and portal pressure (1). Aims of this study are to evaluate in cirrhotic rat livers: a) the role of NEP in ANP, BK and ET-1 handling; b) NEP protein concentration and immunostaining; c) the degree of expression of TGF-β. We studied two groups of 5 control rats injected with vehicle or 10 mg/Kg b.w. candoxatrilat, and two groups of 10 rats with CCl4-induced liver cirrhosis receiving vehicle alone or 10 mg/Kg b.w. candoxatrilat. In cirrhotic rats, candoxatrilat significantly decreased hepatic venous concentrations of ET-1 (P<0.01) and increased systemic plasma levels of ANP (P<0.05), whilst in controls ANP, BK and ET-1 handling seemed unaffected by candoxatrilat. In cirrhotic livers, western blot analysis revealed 400% increase in TGF-β protein content in the whole homogenate and 300% increase in NEP content in the cytosol fraction with respect to controls (both P<0.001). NEP was strictly localized in desmin- and smooth muscle actin-positive activated stellate (SCs) of fibrous septa. In conclusion, overproduced TGF-β in cirrhotic liver enhances NEP expression in activated SCs of fibrous septa. This leads to contraction of activated SCs and hence increased intrahepatic portal vascular resistance through local generation of ET-1. 1) J Hepatol 43: 791-8, 2005.