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Extracellular matrix combinations differentially modulate hepatic stellate cell biology
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Abstracts / Digestive and Liver Disease 40 (2008) A119–A137
present versus absent on the basis of our routinary follow-up evaluations. Results. Median liver stiffness score was 5,1 kPa (range: 3.2–8.7). Patient with an 8.7 value has an echographic pattern of steatosis and a APRI value of 0.16. Median APRI value was 0.26 (range: 0.14–0.53). Patient with 0.53 has a liver stiffness score of 3.4. There are no significant statistical correlations between APRI score, liver stiffness and visceral organ involvement. Discussion. Absence of significant liver fibrosis has been demonstrated with non-invasive methods in SSc patients with no clinical evidence of hepatic disease and with multiorgan visceral involvement, as liver could be less prone to SScspecific pathophysiologic events. doi:10.1016/j.dld.2008.07.162 TRANSIENT ELASTOGRAPHY (TE) DETECTS GRAFT DAMAGE IN LIVER TRANSPLANTED (LT) PATIENTS WITH AETIOLOGIES OTHER THAN HCV C. Rigamonti a , M.F. Donato a , M. Fraquelli b , F. Agnelli a , P. Reggiani c , G. Rossi c , M. Colombo a a
First Division of Gastroenterology IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy b Second Division of Gastroenterology IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy c Liver Transplant Unit, IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy Background and aims. TE reliably predicts severity of graft damage in LT patients with recurrent hepatitis C; its accuracy in other aetiologies is not validated. We aimed at evaluating TE in LT patients with graft damage due to other than HCV causes. Methods. 69 recipients were studied (41 males, median age 51 years): 37 hepatitis B/delta recurrence-free, 20 autoimmune/cholestatic disease (8 primary biliary cirrhosis, PBC, 7 primary sclerosing cholangitis, PSC, 5 autoimmune hepatitis, AIH), 6 alcoholic liver disease (ALD), 6 mixed. All underwent protocol/on demand liver biopsy (LB) and concomitant TE examination between Sept 2005 and May 2008. Histological diagnosis of graft disease was based on criteria defined by Banff Working Group (Hepatology 2006; 44: 489–501). Patients were divided according to the histological diagnosis into 2 groups, i.e. with or without graft damage. TE was considered adequate if ≥10 valid measurements for each patient were obtained with a >65% success rate. Results. In 4 patients (6%) TE examination was unsuccessful, thus the number of patients investigated was 65 (40 males). Median time from LT was 18 months (range 6–251). Median LB length was 35 mm (range 16–50). LB showed presence of graft damage in 28 patients (43%). Among 13
hepatitis B/delta with graft damage, 10 showed idiopathic chronic hepatitis, 1 steatohepatitis, 1 rejection and 1 cholangitis. Among autoimmune/cholestatic disease, 9 had recurrence (3 PBC, 3 PSC, 3 AIH). Among ALD, 2 had steatohepatitis, 1 rejection and 1 cholangitis. One further idiopathic hepatitis (fulminant hepatic failure at LT) and 1 rejection (Budd-Chiari syndrome at LT) were diagnosed. The 28 patients with graft damage compared to the 37 without had significantly higher serum liver enzymes and TE results (median 7.8 kPa; range 5.4–27.4 vs. median 5.3 kPa; range 3.1–7.4, respectively; p < 0.0001). AUROC for detection of graft damage was 0.93 (95%CI 0.84–0.98) with optimal TE cut-off of 6.1 kPa (89% sensitivity, 87% specificity, 91% negative predictive value). Conclusions. The fact that TE accurately predicts nonHCV related graft damage too extends the applicability of this procedure in the management of LT patients. doi:10.1016/j.dld.2008.07.163 EXTRACELLULAR MATRIX COMBINATIONS DIFFERENTIALLY MODULATE HEPATIC STELLATE CELL BIOLOGY S. De Minicis a , D.A. Brafman b , M. Marzioni a , E. Seki b , K. Willert b , S. Chien b , A. Benedetti a , David A. Brenner b a
Department Gastroenterology, Polytechnic University of Marche, Ancona, Italy b UCSD University of California, San Diego, USA Background. Extracellular matrix proteins (ECMP), such as collagen, laminin or fibronectin are fundamental components of every tissue. However, excessive and modified deposition of ECMP observed in tissue injury and subsequent fibrosis suggest that ECMP may play a role in both physiological and pathological status. Recent evidence also shows that in vitro-cultured hepatic stellate cells (HSC), the main ECMP producing-cells in the liver, require a specific environment with Kupffer cells (KC) and endotoxins to resemble the characteristics of in vivo-activated HSCs (Gastroenterology 2007; 132:1937). Moreover, the spontaneous activation of HSC cultured on plastic limits the in vitro study on quiescent HSCs. Thus, ECMP may represent an additional factor regulating HSC status. Aim. To simultaneously investigate whether different ECMP environments modulate HSCs status. Methods. HSCs were isolated from transgenic mice expressing GFP under a Collagen ␣1(I) promoter/enhancer and plated on a chip with an array of combinations of ECMP. The chip was specifically engineered with a microarray spotter plating ECMP on a normal glass-slide covered with an inert acrylamide gel pad. The system simultaneously tested 80 different combinations of ECMP containing collagen I, III, IV, V, laminin, fibronectin. The Activation Index (AI), calculated as GFP fluorescence expression normalized to DNA content, was monitored for each condition in real time for 6 days. Full factorial and cluster analyses were performed on 4
Abstracts / Digestive and Liver Disease 40 (2008) A119–A137
arrays. Specific ECMP combinations were coated on plastic dishes and HSCs cultured for 6 days. mRNA levels of Collagen ␣1(I), ␣SMA and TIMP-1 were compared to quiescent HSCs (1-day cultured). Results. Statistical analysis of the 80 ECMP combinations revealed different HSC activation status. ECMP combinations of collagen I + IV, IV + V or I + IV + fibronectin showed low AI, while other combinations such as collagen I + fibronectin or collagen III + V + fibronectin showed high AI. Concordantly, mRNA levels of HSCs cultured on plastic dishes coated with collagen I + IV, sample of low AI, showed an increase in collagen ␣1(I), ␣SMA and Timp-1, respectively, of 10.5, 7.0 and 1.1, while the combination collagen I + fibronectin, sample of high AI, showed respectively an increase of 16.1, 12.7 and 1.8, compared to quiescent HSCs. Conclusion. ECMP modulate the HSC phenotype. Specific ECMP combinations may be used, in vitro, to study characteristics of quiescent HSCs, or conversely to provide a proper environment for an in vivo-like HSC activation. The knowledge of the ECMP combinations able to maintain quiescence or promote activation will provide new techniques in understanding HSC biology. doi:10.1016/j.dld.2008.07.164 PHAGOCYTIC AND NON-PHAGOCYTIC NADPHOXIDASE ISOFORMS DIFFERENTIALLY REGULATE FIBROSIS BUT NOT STEATOSIS IN THE LIVER S. De Minicis a , E. Seki b , M. Marzioni a , C.H. b b c Osterreicher , Y. Kodama , J. Kluwe , R.F. Schwabe c , A. Benedetti a , D.A. Brenner b a
Department of Gastroenterology, Polytechnic University of Marche, Ancona, Italy b UCSD University of California, San Diego, USA c Columbia University, New York, USA Background. Functional NADPH oxidase is expressed in several cell types in the liver and is required for hepatic fibrosis in mouse models. Phagocytic NADPH oxidase is expressed in Kupffer cells (KC) and hepatocytes (HEP), while a non-phagocytic NADPH oxidase isoform in hepatic stellate cells (HSC) mediates intracellular signalling and fibrogenic pathways. However, the cell-specific contribution of NADPH oxidase to liver fibrosis in vivo is unknown. Aims. To investigate the differential contribution of NADPH oxidase in KCs, HSCs and HEPs to liver fibrosis. Methods. Hepatic fibrosis was induced by bile duct ligation (BDL) for 21 days or by methionine-choline deficient (MCD) diet for 10 weeks in wild-type (WT) mice and mice deficient in p47phox (p47KO), a component of both NADPH oxidase isoforms. Hepatic fibrosis was evaluated by Sirius red staining, Western blot and immunohistochemistry for ␣SMA, hepatic hydroxyproline content and mRNA levels of collagen␣1(I), ␣SMA, TGF and TIMP-1. Hepatic steato-
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sis was determined by oil red-O staining, hepatic triglyceride measurement and mRNA levels of SERBP-1c. Hepatocellular injury was assessed by serum ALT and AST levels. p47KO chimeric mice were generated by the combination of liposomal clodronate, irradiation and bone marrow (BM) transplantation of p47KO BM into WT recipients and vice versa. Hepatocytes were isolated from WT and p47KO mice and plated on collagen-coated dishes. Results. Upon BDL, the chimeric mice with p47KO KCs and WT HSCs showed 25% reduction of fibrosis, while the chimeric mice with WT KCs and p47KO HSCs displayed 60% reduction of fibrosis, compared to WT mice. In addition, p47KO mice compared to WT mice treated with an MCD diet showed no significant changes in steatosis and hepatocellular injury but a 50% reduction in fibrosis. Primary cultures of WT and p47KO HEPs treated with free fatty acids (FFA) showed a similar increase in fat deposition, as assessed by oil red-O staining. Moreover, FFA promote a 1.5-fold increase in reactive oxygen species (ROS) production both in p47KO and in WT HEPs pre-treated with the specific NADPH oxidase inhibitor (DPI), indicating that ROS production by FFA in HEPs is NADPH oxidase-independent. Conclusion. NADPH oxidase isoforms from both KC and HSC stimulate liver fibrosis; however, the non-phagocytic NADPH oxidase in HSCs is the major contributor. HEP steatosis induced by MCD diet in vivo, or by FFA in vitro, does not involve NADPH oxidase. Thus, selective inhibition of non-phagocytic NADPH oxidase has antifibrotic activity on HSCs, but does not affect HEP biology or host defences mediated by KCs. doi:10.1016/j.dld.2008.07.165 C-ABL ACTIVITY IS REQUIRED FOR THE FIBROGENETIC BEHAVIOUR OF HEPATIC STELLATE CELLS C. Candelaresi a , G. Svegliati Baroni a , S. Saccomanno a , F. Marra b , S. De Minicis a , L. Agostinelli a , A. Benedetti a a
Institute of Gastroenterology, Polytechnic University of Marche, Ancona, Italy b Institute Internal Medicine, University of Florence, Florence, Italy Background. Hepatic fibrosis represents the final event in all forms of chronic liver injury, independently from aetiological agent. Currently, no therapies are available for hepatic fibrosis. Hepatic stellate cells (HSCs) are the main hepatic phenotypes involved in extracellular matrix synthesis and their fibrogenetic behaviour is based on ligand binding to specific receptor kinase. c-ABL is a target of activated receptor tyrosine kinases that regulates DNA synthesis, and can be specifically inhibited by drugs used for several chronic human diseases. By their specific receptor binding, PDGF and TGF1 represent respectively the most potent proliferative and fibrogenetic stimuli for HSCs.
Abstracts / Digestive and Liver Disease 40 (2008) A119–A137
present versus absent on the basis of our routinary follow-up evaluations. Results. Median liver stiffness score was 5,1 kPa (range: 3.2–8.7). Patient with an 8.7 value has an echographic pattern of steatosis and a APRI value of 0.16. Median APRI value was 0.26 (range: 0.14–0.53). Patient with 0.53 has a liver stiffness score of 3.4. There are no significant statistical correlations between APRI score, liver stiffness and visceral organ involvement. Discussion. Absence of significant liver fibrosis has been demonstrated with non-invasive methods in SSc patients with no clinical evidence of hepatic disease and with multiorgan visceral involvement, as liver could be less prone to SScspecific pathophysiologic events. doi:10.1016/j.dld.2008.07.162 TRANSIENT ELASTOGRAPHY (TE) DETECTS GRAFT DAMAGE IN LIVER TRANSPLANTED (LT) PATIENTS WITH AETIOLOGIES OTHER THAN HCV C. Rigamonti a , M.F. Donato a , M. Fraquelli b , F. Agnelli a , P. Reggiani c , G. Rossi c , M. Colombo a a
First Division of Gastroenterology IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy b Second Division of Gastroenterology IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy c Liver Transplant Unit, IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milano, Italy Background and aims. TE reliably predicts severity of graft damage in LT patients with recurrent hepatitis C; its accuracy in other aetiologies is not validated. We aimed at evaluating TE in LT patients with graft damage due to other than HCV causes. Methods. 69 recipients were studied (41 males, median age 51 years): 37 hepatitis B/delta recurrence-free, 20 autoimmune/cholestatic disease (8 primary biliary cirrhosis, PBC, 7 primary sclerosing cholangitis, PSC, 5 autoimmune hepatitis, AIH), 6 alcoholic liver disease (ALD), 6 mixed. All underwent protocol/on demand liver biopsy (LB) and concomitant TE examination between Sept 2005 and May 2008. Histological diagnosis of graft disease was based on criteria defined by Banff Working Group (Hepatology 2006; 44: 489–501). Patients were divided according to the histological diagnosis into 2 groups, i.e. with or without graft damage. TE was considered adequate if ≥10 valid measurements for each patient were obtained with a >65% success rate. Results. In 4 patients (6%) TE examination was unsuccessful, thus the number of patients investigated was 65 (40 males). Median time from LT was 18 months (range 6–251). Median LB length was 35 mm (range 16–50). LB showed presence of graft damage in 28 patients (43%). Among 13
hepatitis B/delta with graft damage, 10 showed idiopathic chronic hepatitis, 1 steatohepatitis, 1 rejection and 1 cholangitis. Among autoimmune/cholestatic disease, 9 had recurrence (3 PBC, 3 PSC, 3 AIH). Among ALD, 2 had steatohepatitis, 1 rejection and 1 cholangitis. One further idiopathic hepatitis (fulminant hepatic failure at LT) and 1 rejection (Budd-Chiari syndrome at LT) were diagnosed. The 28 patients with graft damage compared to the 37 without had significantly higher serum liver enzymes and TE results (median 7.8 kPa; range 5.4–27.4 vs. median 5.3 kPa; range 3.1–7.4, respectively; p < 0.0001). AUROC for detection of graft damage was 0.93 (95%CI 0.84–0.98) with optimal TE cut-off of 6.1 kPa (89% sensitivity, 87% specificity, 91% negative predictive value). Conclusions. The fact that TE accurately predicts nonHCV related graft damage too extends the applicability of this procedure in the management of LT patients. doi:10.1016/j.dld.2008.07.163 EXTRACELLULAR MATRIX COMBINATIONS DIFFERENTIALLY MODULATE HEPATIC STELLATE CELL BIOLOGY S. De Minicis a , D.A. Brafman b , M. Marzioni a , E. Seki b , K. Willert b , S. Chien b , A. Benedetti a , David A. Brenner b a
Department Gastroenterology, Polytechnic University of Marche, Ancona, Italy b UCSD University of California, San Diego, USA Background. Extracellular matrix proteins (ECMP), such as collagen, laminin or fibronectin are fundamental components of every tissue. However, excessive and modified deposition of ECMP observed in tissue injury and subsequent fibrosis suggest that ECMP may play a role in both physiological and pathological status. Recent evidence also shows that in vitro-cultured hepatic stellate cells (HSC), the main ECMP producing-cells in the liver, require a specific environment with Kupffer cells (KC) and endotoxins to resemble the characteristics of in vivo-activated HSCs (Gastroenterology 2007; 132:1937). Moreover, the spontaneous activation of HSC cultured on plastic limits the in vitro study on quiescent HSCs. Thus, ECMP may represent an additional factor regulating HSC status. Aim. To simultaneously investigate whether different ECMP environments modulate HSCs status. Methods. HSCs were isolated from transgenic mice expressing GFP under a Collagen ␣1(I) promoter/enhancer and plated on a chip with an array of combinations of ECMP. The chip was specifically engineered with a microarray spotter plating ECMP on a normal glass-slide covered with an inert acrylamide gel pad. The system simultaneously tested 80 different combinations of ECMP containing collagen I, III, IV, V, laminin, fibronectin. The Activation Index (AI), calculated as GFP fluorescence expression normalized to DNA content, was monitored for each condition in real time for 6 days. Full factorial and cluster analyses were performed on 4
Abstracts / Digestive and Liver Disease 40 (2008) A119–A137
arrays. Specific ECMP combinations were coated on plastic dishes and HSCs cultured for 6 days. mRNA levels of Collagen ␣1(I), ␣SMA and TIMP-1 were compared to quiescent HSCs (1-day cultured). Results. Statistical analysis of the 80 ECMP combinations revealed different HSC activation status. ECMP combinations of collagen I + IV, IV + V or I + IV + fibronectin showed low AI, while other combinations such as collagen I + fibronectin or collagen III + V + fibronectin showed high AI. Concordantly, mRNA levels of HSCs cultured on plastic dishes coated with collagen I + IV, sample of low AI, showed an increase in collagen ␣1(I), ␣SMA and Timp-1, respectively, of 10.5, 7.0 and 1.1, while the combination collagen I + fibronectin, sample of high AI, showed respectively an increase of 16.1, 12.7 and 1.8, compared to quiescent HSCs. Conclusion. ECMP modulate the HSC phenotype. Specific ECMP combinations may be used, in vitro, to study characteristics of quiescent HSCs, or conversely to provide a proper environment for an in vivo-like HSC activation. The knowledge of the ECMP combinations able to maintain quiescence or promote activation will provide new techniques in understanding HSC biology. doi:10.1016/j.dld.2008.07.164 PHAGOCYTIC AND NON-PHAGOCYTIC NADPHOXIDASE ISOFORMS DIFFERENTIALLY REGULATE FIBROSIS BUT NOT STEATOSIS IN THE LIVER S. De Minicis a , E. Seki b , M. Marzioni a , C.H. b b c Osterreicher , Y. Kodama , J. Kluwe , R.F. Schwabe c , A. Benedetti a , D.A. Brenner b a
Department of Gastroenterology, Polytechnic University of Marche, Ancona, Italy b UCSD University of California, San Diego, USA c Columbia University, New York, USA Background. Functional NADPH oxidase is expressed in several cell types in the liver and is required for hepatic fibrosis in mouse models. Phagocytic NADPH oxidase is expressed in Kupffer cells (KC) and hepatocytes (HEP), while a non-phagocytic NADPH oxidase isoform in hepatic stellate cells (HSC) mediates intracellular signalling and fibrogenic pathways. However, the cell-specific contribution of NADPH oxidase to liver fibrosis in vivo is unknown. Aims. To investigate the differential contribution of NADPH oxidase in KCs, HSCs and HEPs to liver fibrosis. Methods. Hepatic fibrosis was induced by bile duct ligation (BDL) for 21 days or by methionine-choline deficient (MCD) diet for 10 weeks in wild-type (WT) mice and mice deficient in p47phox (p47KO), a component of both NADPH oxidase isoforms. Hepatic fibrosis was evaluated by Sirius red staining, Western blot and immunohistochemistry for ␣SMA, hepatic hydroxyproline content and mRNA levels of collagen␣1(I), ␣SMA, TGF and TIMP-1. Hepatic steato-
A133
sis was determined by oil red-O staining, hepatic triglyceride measurement and mRNA levels of SERBP-1c. Hepatocellular injury was assessed by serum ALT and AST levels. p47KO chimeric mice were generated by the combination of liposomal clodronate, irradiation and bone marrow (BM) transplantation of p47KO BM into WT recipients and vice versa. Hepatocytes were isolated from WT and p47KO mice and plated on collagen-coated dishes. Results. Upon BDL, the chimeric mice with p47KO KCs and WT HSCs showed 25% reduction of fibrosis, while the chimeric mice with WT KCs and p47KO HSCs displayed 60% reduction of fibrosis, compared to WT mice. In addition, p47KO mice compared to WT mice treated with an MCD diet showed no significant changes in steatosis and hepatocellular injury but a 50% reduction in fibrosis. Primary cultures of WT and p47KO HEPs treated with free fatty acids (FFA) showed a similar increase in fat deposition, as assessed by oil red-O staining. Moreover, FFA promote a 1.5-fold increase in reactive oxygen species (ROS) production both in p47KO and in WT HEPs pre-treated with the specific NADPH oxidase inhibitor (DPI), indicating that ROS production by FFA in HEPs is NADPH oxidase-independent. Conclusion. NADPH oxidase isoforms from both KC and HSC stimulate liver fibrosis; however, the non-phagocytic NADPH oxidase in HSCs is the major contributor. HEP steatosis induced by MCD diet in vivo, or by FFA in vitro, does not involve NADPH oxidase. Thus, selective inhibition of non-phagocytic NADPH oxidase has antifibrotic activity on HSCs, but does not affect HEP biology or host defences mediated by KCs. doi:10.1016/j.dld.2008.07.165 C-ABL ACTIVITY IS REQUIRED FOR THE FIBROGENETIC BEHAVIOUR OF HEPATIC STELLATE CELLS C. Candelaresi a , G. Svegliati Baroni a , S. Saccomanno a , F. Marra b , S. De Minicis a , L. Agostinelli a , A. Benedetti a a
Institute of Gastroenterology, Polytechnic University of Marche, Ancona, Italy b Institute Internal Medicine, University of Florence, Florence, Italy Background. Hepatic fibrosis represents the final event in all forms of chronic liver injury, independently from aetiological agent. Currently, no therapies are available for hepatic fibrosis. Hepatic stellate cells (HSCs) are the main hepatic phenotypes involved in extracellular matrix synthesis and their fibrogenetic behaviour is based on ligand binding to specific receptor kinase. c-ABL is a target of activated receptor tyrosine kinases that regulates DNA synthesis, and can be specifically inhibited by drugs used for several chronic human diseases. By their specific receptor binding, PDGF and TGF1 represent respectively the most potent proliferative and fibrogenetic stimuli for HSCs.