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OXYSTEROLS PLASMA LEVELS CORRELATE WITH CHRONIC LIVER DISEASES
S42
Oral Communications / European Journal of Internal Medicine 19S (2008), S1–S59
Friday, 9 May 2008, 14.30–15.45
Room D
Gastroenterology FR-47 HEPATIC EXPRESSION OF CYCLOOXYGENASE-2 IN NON-ALCOHOLIC FATTY LIVER DISEASE L. Giannitrapani, S. Ingrao, M. Soresi, S. Petta, A.M. Florena, V. Di Marco, M. Cervello, G. Montalto. University of Palermo Background and aims: Non alcoholic fatty liver disease (NAFLD) is a spectrum of disorders ranging from steatosis to steatohepatitis (NASH), which in turn can progress to cirrhosis, but the molecular mechanisms influencing the disease progression are still poorly understood. Ciclooxygenase-2 (COX-2), the inducible isoform of the enzymes that catalyse the prostaglandin synthesis from arachidonic acid, has been described to be overexpressed in the livers of mice with methionine- and choline-deficient (MCD) diet-induced steatohepatitis and in patients with chronic hepatitis C associated steatosis. The aim of this study was to evaluate the expression of COX-2 in a population of subjects with NAFLD/NASH to confirm its possible role as pro-inflammatory mediator in metabolic forms of fatty liver disease. Methods: We used 47 formalin-fixed, paraffin-embedded liver tissue samples obtained by needle biopsies from patients with NAFLD/NASH (10F/37M), scored for hepatic steatosis, grading and staging according to Brunt. Five histologically normal livers obtained during surgery of biliary tracts, after receiving written informed consent from the patient, were used as controls. The expression of COX-2 was investigated by immunohistochemistry. The positive signals for COX-2 were observed in the cytoplasm of hepatocytes and scored on the basis of: 1) maximum intensity (i.e. the maximum level among all positive cells); 2) dominant intensity (i.e. the level observed in the majority of positive cells); and 3) extensiveness (by percentage population) of positively stained cells. The score of each specimen was the sum of the three parameters (sum of the scores). Results: No hepatic expression of COX-2 was shown in the healthy liver. A positive staining for COX-2, ranging from slight to more intense, was shown in almost all the pathologic livers. A significant correlation between higher hepatic expression of COX-2 (evaluated as sum of the scores) and higher percentage of histological steatosis (rho=0.31; p<0.05) and with ALT levels (rho=0.48, p<0.001), was found. No significant correlation was observed between the sum of the scores and grading (rho= 0.29, p=0.056), staging, total and HDL-cholesterol, triglycerides, BMI and HOMA. Conclusions: The positive correlation between the expression of COX-2 and ALT levels, even if not paralleled by a similar relationship with histological grading, let us hypothesize a possible role of COX-2 as pro-inflammatory mediator in NAFLD/NASH which may deserve further studies for its use as a marker of disease progression
FR-48 OXYSTEROLS PLASMA LEVELS CORRELATE WITH CHRONIC LIVER DISEASES E. Mici, S. Petta, V. Diurni, A. Craxì, C. Balsano. Università "Sapienza" Roma, Fondazione A.Cesalpino ed Università di Medicina Interna dell’Aquila. Background and Aims: Hepatic steatosis is a characteristic histological feature of patients with chronic hepatitis C. Steatosis is observed in about 70% of patients with chronic hepatitis C. Several studies confirm the existence of a relationship between chronic hepatitis C and steatosis and suggest a direct role of the HCV core protein in inducing liver steatosis. Increasing experimental data suggest an important role of oxidative stress in chronic liver diseases. HCV infection is characterized by increased markers of oxidative stress, such as oxysterols, which could be involved in the evolution of liver damage. Oxysterols, generated from peroxidation of cholesterol are sensitive and specific markers of enhanced oxidative stress. Oxysterols bind to orphan nuclear receptors like Liver X Receptors (LXR) alpha (is expressed at particularly high levels in liver) and beta. These receptors form heterodimers with the Retinoid X Receptor (RXR), thus regulating lipid and lipoprotein metabolism. In the liver, LXR-alpha regulate expression of a number of proteins involved in cholesterol and fatty acid metabolism, including CYP7A and sterol regulatory binding element protein 1c (SREBP-1c). In addition, LXR-alpha controls the
transcription of several genes involved in cellular cholesterol efflux including ATP-binding cassette (ABC)A1, ABC(G1), and apolipoprotein E. We investigate the relationship between HCV-related steatosis and oxysterol plasma levels, using a recently developed specific and sensitive mass spectrometry assay to measure oxysterols (7-beta-hydroxycholesterol and 7-ketocholesterol) in patients with HCV infection and steatosis. Patients with HBV infection and liver steatosis have been considered as controls. Methods: A Gas-Chromatography/Mass-Spectrometry technique was used for the dosage of oxysterol plasma concentration in 30 patients affected by HCV or HBV infection or NAFLD (10 patients for each group). Results: HCV infection causes a significant increase in plasma oxysterol concentration as compared with controls. Plasma oxysterol concentration correlates with liver ecogenicity (evaluated by ultrasound) and grading and staging of liver fibrosis (revealed by biopsies), in HCV-positive patients independently of sex, age and BMI. Conclusions: The study of the relationship between HCV and oxidative stress will improve our knowledge on the progression of chronic liver disease, indicating new parameters to be considered when HCV patients undergo antiviral therapy
FR-49 EMODIN INHIBITS CELL INVASIVENESS INDUCED BY HCV B. Buchetti, A. Spaziani, A. Alisi, S. Anticoli, C. Balsano. Università "Sapienza" Roma, Fondazione A.Cesalpino ed Università di Medicina Interna dell’Aquila. Background and Aims: there are several clinical and basic reports about intrahepatic spread of transformed cells during hepatocellular carcinoma course (HCC). Hepatitis C virus (HCV) is the major causative viral agent of cirrhosis and hepatocarcinoma. Systemic chemotherapy for unresectable or metastatic HCC are quite ineffective. The metastatic capability is related to the modulation of cell adhesion and motility and since the molecular and cellular mechanisms controlling these events are not completely understood we decided to analye them in depth in HCV-related HCC. Methods: Cells invasiveness was examined by a migration and adhesion assay. We performed western blotting, immunoprecipitation and immunofluorescence assays, silencing gene expression with specific siRNA molecules for HCV core protein and FAK. Emodin, the cancer cell migration and invasion inhibitor, was purchased by Sigma. Results: HepG2 cells were used to establish HCV-wt (entire genome) and HCV core stable polyclones. Both HCV-wt and HCV core protein induced paxillin and beta1-integrin expression levels increase; no changes were observed in alpha-actinin expression. IF assay showed a spread beta1-integrin distribution in the cytosol and alpha actinin delocalization from the cytosol to the perinuclear region, in HCV-wt polyclones. HCV core protein silencing by a specific siRNA completely abrogated HCV-related effects. In addition, Focal Adhesion Kinases (FAK) expression and activity was increased in HCV wt policlones and HCV core protein; HCV core siRNA re-estabilished FAK basal levels in these cells. Interestingly, FAK siRNA targeting only partially reverted the HCV focal adhesion deregulation, whereas emodin completely abrogated it. Conclusions: Emodin counteracting HCV related adhesion molecules alterations could be considered as a valid alternative to the therapeutic approaches to date available.
FR-50 CLINICAL COURSE OF NIMESULIDE-INDUCED LIVER INJURY A. Licata 1 , V. Calvaruso 1 , M. Cappello 1 , D. Cabibi 1 , V. Di Marco 1 , C. Randazzo 1 , A. Tuttolomondo 2 , P.L. Almasio 2 , G. Licata 1 , A. Craxì. 1 Gastroenterology and Hepatology Unit; 2 Internal Medicine, Di.Bi.M.I.S., University of Palermo, Italy Background & Aim: Nimesulide has been shown to cause a wide range of liver injuries, from mildly abnormal liver tests to severe liver damage, including fulminant hepatic failure. We report the clinical course of patients with nimesulide-induced liver injury and evaluate predictors of susceptibility or hypersensivity to the drug. Methods: We reviewed all clinical records of patients with diagnosis of druginduced liver injury (DILI) admitted to our Department from January 2001 to October 2005, and constructed a database of data on age, sex, clinical features at onset, biochemistry and follow-up. Diagnosis of DILI was based on the presence of at least three of the International Consensus Criteria (J. Hepatol,
Oral Communications / European Journal of Internal Medicine 19S (2008), S1–S59
Friday, 9 May 2008, 14.30–15.45
Room D
Gastroenterology FR-47 HEPATIC EXPRESSION OF CYCLOOXYGENASE-2 IN NON-ALCOHOLIC FATTY LIVER DISEASE L. Giannitrapani, S. Ingrao, M. Soresi, S. Petta, A.M. Florena, V. Di Marco, M. Cervello, G. Montalto. University of Palermo Background and aims: Non alcoholic fatty liver disease (NAFLD) is a spectrum of disorders ranging from steatosis to steatohepatitis (NASH), which in turn can progress to cirrhosis, but the molecular mechanisms influencing the disease progression are still poorly understood. Ciclooxygenase-2 (COX-2), the inducible isoform of the enzymes that catalyse the prostaglandin synthesis from arachidonic acid, has been described to be overexpressed in the livers of mice with methionine- and choline-deficient (MCD) diet-induced steatohepatitis and in patients with chronic hepatitis C associated steatosis. The aim of this study was to evaluate the expression of COX-2 in a population of subjects with NAFLD/NASH to confirm its possible role as pro-inflammatory mediator in metabolic forms of fatty liver disease. Methods: We used 47 formalin-fixed, paraffin-embedded liver tissue samples obtained by needle biopsies from patients with NAFLD/NASH (10F/37M), scored for hepatic steatosis, grading and staging according to Brunt. Five histologically normal livers obtained during surgery of biliary tracts, after receiving written informed consent from the patient, were used as controls. The expression of COX-2 was investigated by immunohistochemistry. The positive signals for COX-2 were observed in the cytoplasm of hepatocytes and scored on the basis of: 1) maximum intensity (i.e. the maximum level among all positive cells); 2) dominant intensity (i.e. the level observed in the majority of positive cells); and 3) extensiveness (by percentage population) of positively stained cells. The score of each specimen was the sum of the three parameters (sum of the scores). Results: No hepatic expression of COX-2 was shown in the healthy liver. A positive staining for COX-2, ranging from slight to more intense, was shown in almost all the pathologic livers. A significant correlation between higher hepatic expression of COX-2 (evaluated as sum of the scores) and higher percentage of histological steatosis (rho=0.31; p<0.05) and with ALT levels (rho=0.48, p<0.001), was found. No significant correlation was observed between the sum of the scores and grading (rho= 0.29, p=0.056), staging, total and HDL-cholesterol, triglycerides, BMI and HOMA. Conclusions: The positive correlation between the expression of COX-2 and ALT levels, even if not paralleled by a similar relationship with histological grading, let us hypothesize a possible role of COX-2 as pro-inflammatory mediator in NAFLD/NASH which may deserve further studies for its use as a marker of disease progression
FR-48 OXYSTEROLS PLASMA LEVELS CORRELATE WITH CHRONIC LIVER DISEASES E. Mici, S. Petta, V. Diurni, A. Craxì, C. Balsano. Università "Sapienza" Roma, Fondazione A.Cesalpino ed Università di Medicina Interna dell’Aquila. Background and Aims: Hepatic steatosis is a characteristic histological feature of patients with chronic hepatitis C. Steatosis is observed in about 70% of patients with chronic hepatitis C. Several studies confirm the existence of a relationship between chronic hepatitis C and steatosis and suggest a direct role of the HCV core protein in inducing liver steatosis. Increasing experimental data suggest an important role of oxidative stress in chronic liver diseases. HCV infection is characterized by increased markers of oxidative stress, such as oxysterols, which could be involved in the evolution of liver damage. Oxysterols, generated from peroxidation of cholesterol are sensitive and specific markers of enhanced oxidative stress. Oxysterols bind to orphan nuclear receptors like Liver X Receptors (LXR) alpha (is expressed at particularly high levels in liver) and beta. These receptors form heterodimers with the Retinoid X Receptor (RXR), thus regulating lipid and lipoprotein metabolism. In the liver, LXR-alpha regulate expression of a number of proteins involved in cholesterol and fatty acid metabolism, including CYP7A and sterol regulatory binding element protein 1c (SREBP-1c). In addition, LXR-alpha controls the
transcription of several genes involved in cellular cholesterol efflux including ATP-binding cassette (ABC)A1, ABC(G1), and apolipoprotein E. We investigate the relationship between HCV-related steatosis and oxysterol plasma levels, using a recently developed specific and sensitive mass spectrometry assay to measure oxysterols (7-beta-hydroxycholesterol and 7-ketocholesterol) in patients with HCV infection and steatosis. Patients with HBV infection and liver steatosis have been considered as controls. Methods: A Gas-Chromatography/Mass-Spectrometry technique was used for the dosage of oxysterol plasma concentration in 30 patients affected by HCV or HBV infection or NAFLD (10 patients for each group). Results: HCV infection causes a significant increase in plasma oxysterol concentration as compared with controls. Plasma oxysterol concentration correlates with liver ecogenicity (evaluated by ultrasound) and grading and staging of liver fibrosis (revealed by biopsies), in HCV-positive patients independently of sex, age and BMI. Conclusions: The study of the relationship between HCV and oxidative stress will improve our knowledge on the progression of chronic liver disease, indicating new parameters to be considered when HCV patients undergo antiviral therapy
FR-49 EMODIN INHIBITS CELL INVASIVENESS INDUCED BY HCV B. Buchetti, A. Spaziani, A. Alisi, S. Anticoli, C. Balsano. Università "Sapienza" Roma, Fondazione A.Cesalpino ed Università di Medicina Interna dell’Aquila. Background and Aims: there are several clinical and basic reports about intrahepatic spread of transformed cells during hepatocellular carcinoma course (HCC). Hepatitis C virus (HCV) is the major causative viral agent of cirrhosis and hepatocarcinoma. Systemic chemotherapy for unresectable or metastatic HCC are quite ineffective. The metastatic capability is related to the modulation of cell adhesion and motility and since the molecular and cellular mechanisms controlling these events are not completely understood we decided to analye them in depth in HCV-related HCC. Methods: Cells invasiveness was examined by a migration and adhesion assay. We performed western blotting, immunoprecipitation and immunofluorescence assays, silencing gene expression with specific siRNA molecules for HCV core protein and FAK. Emodin, the cancer cell migration and invasion inhibitor, was purchased by Sigma. Results: HepG2 cells were used to establish HCV-wt (entire genome) and HCV core stable polyclones. Both HCV-wt and HCV core protein induced paxillin and beta1-integrin expression levels increase; no changes were observed in alpha-actinin expression. IF assay showed a spread beta1-integrin distribution in the cytosol and alpha actinin delocalization from the cytosol to the perinuclear region, in HCV-wt polyclones. HCV core protein silencing by a specific siRNA completely abrogated HCV-related effects. In addition, Focal Adhesion Kinases (FAK) expression and activity was increased in HCV wt policlones and HCV core protein; HCV core siRNA re-estabilished FAK basal levels in these cells. Interestingly, FAK siRNA targeting only partially reverted the HCV focal adhesion deregulation, whereas emodin completely abrogated it. Conclusions: Emodin counteracting HCV related adhesion molecules alterations could be considered as a valid alternative to the therapeutic approaches to date available.
FR-50 CLINICAL COURSE OF NIMESULIDE-INDUCED LIVER INJURY A. Licata 1 , V. Calvaruso 1 , M. Cappello 1 , D. Cabibi 1 , V. Di Marco 1 , C. Randazzo 1 , A. Tuttolomondo 2 , P.L. Almasio 2 , G. Licata 1 , A. Craxì. 1 Gastroenterology and Hepatology Unit; 2 Internal Medicine, Di.Bi.M.I.S., University of Palermo, Italy Background & Aim: Nimesulide has been shown to cause a wide range of liver injuries, from mildly abnormal liver tests to severe liver damage, including fulminant hepatic failure. We report the clinical course of patients with nimesulide-induced liver injury and evaluate predictors of susceptibility or hypersensivity to the drug. Methods: We reviewed all clinical records of patients with diagnosis of druginduced liver injury (DILI) admitted to our Department from January 2001 to October 2005, and constructed a database of data on age, sex, clinical features at onset, biochemistry and follow-up. Diagnosis of DILI was based on the presence of at least three of the International Consensus Criteria (J. Hepatol,