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P624 CANONICAL VS NON-CANONICAL Wnt SIGNALLING
POSTERS [3] Schieweck et al. (2009) [4] Platt et al. (2012) [5] Kong et al. In revision
P624 CANONICAL VS NON-CANONICAL Wnt SIGNALLING L. Corbett, J. Mann, D.A. Mann. Newcastle University, Newcastle upon Tyne, United Kingdom E-mail: [email protected] Background and Aims: Hepatic stellate cells (HSCs) are key regulators in the onset and progressionof liver fibrosis, activated upon injury, transdifferentiating to a fibrogenic, myofibroblast like phenotype. Understanding the mechanisms underlying this activation is necessary for advancement of fibrosis treatment. Perturbations in Wnt signalling can predispose to disease as Wnts are critical in regulating cell differentiation and regeneration. Recent studies suggest increased Wnt signalling may contribute to HSC activation. Wnts signal either through canonical/b-catenin associated or non-canonical pathways. The functional pathway in HSCs has yet to be resolved. Methods: Wnt pathway expression was assessed in rat HSCs by qRT-PCR and Western Blot. Wnt signalling was simulated either by overexpression in the LX2 cell line or treatment of primary HSCs with Wnt containing conditioned medium. A TCF luciferase reporter (TOPFLASH) was used to measure canonical/b-catenin activity. Noncanonical activity was assessed by measuring downstream effectors such as JNK by Western Blot or luciferase reporter. Results: Transdifferentiation of HSCs was associated with upregulated expression of noncanonical Wnts and extracellular modulators. Canonical Wnt expression was not detected in quiescent or activated HSCs. Endogenous b-catenin activity was limited with no increase observed under Wnt stimulated conditions, either through treatment with Wnt conditioned medium or transfection with an active b-catenin construct. Increased JNK phosphorylation, a non-canonical effector, occurred with Wnt treatment. Conclusions: Canonical signalling does not appear to influence HSC transdifferentiation as ligand expression and b-catenin activity is limited. Increased expression of non-canonical ligands and effectors suggest that instead non-canonical Wnt may contribute to HSC activation. P625 HEPATIC STELLATE CELLS ARE THE MAIN SOURCE OF MYOFIBROBLASTS IN A MOUSE MODEL OF CHRONIC CHOLESTATIC LIVER DISEASE 1 1 ¨ U.J. Lemberger1 , M. Trauner2 , C.H. Osterreicher . Medical University of Vienna, Institute of Pharmacology, 2 Medical University of Vienna, Department of Gastroenterology and Hepatology, Vienna, Austria E-mail: [email protected] Background and Aims: The cellular source of collagen in chronic liver disease has been discussed controversially in the recent past. Hepatic stellate cells are the major source of collagen in toxic liver injury. Portal myofibroblasts, bone marrow and epithelial cells (hepatocytes and cholangiocytes) have been suggested as alternative sources of myofibroblasts in chronic liver disease. Mice with genetic disruption of the ATP-binding cassette transporter B4 (abcb4) lack phosphatidylcholine excretion into bile associated with accumulation of toxic bile acids. Abcb4 KO mice mimic sclerosing cholangitis in humans and represent a valuable model to investigate the progression of cholestatic liver disease and the development of liver cancer in a fibrotic and inflammatory environment. The aim of this study was to characterize collagen-producing myofibroblasts in a mouse model of sclerosing cholangitis. Methods: Coll-GFP reporter mice in which the collagen a1(I) promoter drives expression of a green fluorescent protein
(GFP) were crossed to abcb4 KO mice and were analyzed for expression of myofibroblastic and cell type specific markers by immunofluorescence and confocal microscopy. GFP+ cells were sorted from livers of Coll-GFP/abcb4 KO mice at different time points and gene expression was analyzed by microarray technology. Results: GFP+ cells lacked expression of epithelial cell markers keratin 8 and 19, Kupffer cell marker F4/80 and the endothelial cell marker CD31. In contrast, 80.3% of collagen producing cells stained positive for desmin, a marker typical for hepatic stellate cells. Conclusions: Hepatic stellate cells are the major source of collagen producing cells in abcb4 KO mice. P626 PROCESSING OF miR17–92 CLUSTER IN HEPATIC STELLATE CELLS PROMOTES HEPATIC FIBROGENESIS E. Brandon-Warner, C.O. Field, C.R. Culberson, I.H. McKillop, L.W. Schrum. Carolinas Medical Center, Charlotte, NC, United States E-mail: [email protected] Background and Aims: Hepatic stellate cell (HSC) activation is a key event in hepatic fibrogenesis, which is mediated by transforming growth factor beta (TGFb). MicroRNA17–92 (miR17–92) cluster, consisting of miR17a, 18a, 19a, 20a, 19b, and 92, modulates TGFb signaling. Individual miRs of this cluster were expressed differently during HSC activation; therefore, we hypothesize that differential expression of miR17–92 family members is regulated post-transcriptionally, promoting fibrogenesis. Methods: Primary rat HSCs were isolated from male SpragueDawley rats. Culture-activated (day 10) and freshly isolated (day 0) cells were used. HSCs were treated with a pan-HDAC (trichostatin, TSA), HDAC I (valproic acid) or DNMT (5-aza-2 deoxycytadine, 5AZA) inhibitor or transfected with miR19b plasmid. Pair-matched sera and livers were isolated from fibrotic/injured (bile duct ligation, ethanol-drinking water, ethanol-LPS) and control rat livers. Exosomes were isolated from media or sera; pri-miR17–92 and individual miR and profibrotic gene expression were measured by qRT-PCR during HSC activation and liver pathologies. Results: Pri-miR17–92 cluster expression was up-regulated in activated HSCs, and inhibited by TSA, 5AZA, and reintroduction of miR19b. miR17a and 18a were significantly elevated in fibrotic/injured livers and in activated HSCs compared to controls. Conversely, expression of miR19a, 19b and 92 were significantly down-regulated. miR19b and 92 were significantly elevated in exosomes from media of activated HSCs and sera of fibrotic/injured rats, while miR17a, 18a, and 19a were undetected. Conclusions: Expression/regulation of miR17–92 cluster is linked to HSC activation, and differently expressed individual miRs may potentiate HSC activation. Modulating this cluster may prove to be a potential therapy for hepatic fibrogenesis. P627 ANALYSIS OF METABOLIC ACTIVITIES IN FETAL AND ADULT HUMAN HEPATOCYTES FOR CLINICAL THERAPY R. Gramignoli, S.C. Strom. Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden E-mail: [email protected] Background and Aims: Fetal hepatocytes have been proposed as a source of cells for clinical hepatocyte transplants. However, to be useful, sufficient metabolic activities must be present. Aim: To assess the viability and metabolic activities in isolated fetal human hepatocytes as compared to pediatric and adult hepatocytes. Methods: Hepatocytes were isolated from 254 fetal livers, 11–24 weeks gestation and 229 post-natal tissues (3 months-85 years). Cell viability, apoptosis and Cytochrome P450 (CYP) activities, phase II-conjugation and ammonia metabolism were analyzed.
Journal of Hepatology 2014 vol. 60 | S215–S359
S277
P624 CANONICAL VS NON-CANONICAL Wnt SIGNALLING L. Corbett, J. Mann, D.A. Mann. Newcastle University, Newcastle upon Tyne, United Kingdom E-mail: [email protected] Background and Aims: Hepatic stellate cells (HSCs) are key regulators in the onset and progressionof liver fibrosis, activated upon injury, transdifferentiating to a fibrogenic, myofibroblast like phenotype. Understanding the mechanisms underlying this activation is necessary for advancement of fibrosis treatment. Perturbations in Wnt signalling can predispose to disease as Wnts are critical in regulating cell differentiation and regeneration. Recent studies suggest increased Wnt signalling may contribute to HSC activation. Wnts signal either through canonical/b-catenin associated or non-canonical pathways. The functional pathway in HSCs has yet to be resolved. Methods: Wnt pathway expression was assessed in rat HSCs by qRT-PCR and Western Blot. Wnt signalling was simulated either by overexpression in the LX2 cell line or treatment of primary HSCs with Wnt containing conditioned medium. A TCF luciferase reporter (TOPFLASH) was used to measure canonical/b-catenin activity. Noncanonical activity was assessed by measuring downstream effectors such as JNK by Western Blot or luciferase reporter. Results: Transdifferentiation of HSCs was associated with upregulated expression of noncanonical Wnts and extracellular modulators. Canonical Wnt expression was not detected in quiescent or activated HSCs. Endogenous b-catenin activity was limited with no increase observed under Wnt stimulated conditions, either through treatment with Wnt conditioned medium or transfection with an active b-catenin construct. Increased JNK phosphorylation, a non-canonical effector, occurred with Wnt treatment. Conclusions: Canonical signalling does not appear to influence HSC transdifferentiation as ligand expression and b-catenin activity is limited. Increased expression of non-canonical ligands and effectors suggest that instead non-canonical Wnt may contribute to HSC activation. P625 HEPATIC STELLATE CELLS ARE THE MAIN SOURCE OF MYOFIBROBLASTS IN A MOUSE MODEL OF CHRONIC CHOLESTATIC LIVER DISEASE 1 1 ¨ U.J. Lemberger1 , M. Trauner2 , C.H. Osterreicher . Medical University of Vienna, Institute of Pharmacology, 2 Medical University of Vienna, Department of Gastroenterology and Hepatology, Vienna, Austria E-mail: [email protected] Background and Aims: The cellular source of collagen in chronic liver disease has been discussed controversially in the recent past. Hepatic stellate cells are the major source of collagen in toxic liver injury. Portal myofibroblasts, bone marrow and epithelial cells (hepatocytes and cholangiocytes) have been suggested as alternative sources of myofibroblasts in chronic liver disease. Mice with genetic disruption of the ATP-binding cassette transporter B4 (abcb4) lack phosphatidylcholine excretion into bile associated with accumulation of toxic bile acids. Abcb4 KO mice mimic sclerosing cholangitis in humans and represent a valuable model to investigate the progression of cholestatic liver disease and the development of liver cancer in a fibrotic and inflammatory environment. The aim of this study was to characterize collagen-producing myofibroblasts in a mouse model of sclerosing cholangitis. Methods: Coll-GFP reporter mice in which the collagen a1(I) promoter drives expression of a green fluorescent protein
(GFP) were crossed to abcb4 KO mice and were analyzed for expression of myofibroblastic and cell type specific markers by immunofluorescence and confocal microscopy. GFP+ cells were sorted from livers of Coll-GFP/abcb4 KO mice at different time points and gene expression was analyzed by microarray technology. Results: GFP+ cells lacked expression of epithelial cell markers keratin 8 and 19, Kupffer cell marker F4/80 and the endothelial cell marker CD31. In contrast, 80.3% of collagen producing cells stained positive for desmin, a marker typical for hepatic stellate cells. Conclusions: Hepatic stellate cells are the major source of collagen producing cells in abcb4 KO mice. P626 PROCESSING OF miR17–92 CLUSTER IN HEPATIC STELLATE CELLS PROMOTES HEPATIC FIBROGENESIS E. Brandon-Warner, C.O. Field, C.R. Culberson, I.H. McKillop, L.W. Schrum. Carolinas Medical Center, Charlotte, NC, United States E-mail: [email protected] Background and Aims: Hepatic stellate cell (HSC) activation is a key event in hepatic fibrogenesis, which is mediated by transforming growth factor beta (TGFb). MicroRNA17–92 (miR17–92) cluster, consisting of miR17a, 18a, 19a, 20a, 19b, and 92, modulates TGFb signaling. Individual miRs of this cluster were expressed differently during HSC activation; therefore, we hypothesize that differential expression of miR17–92 family members is regulated post-transcriptionally, promoting fibrogenesis. Methods: Primary rat HSCs were isolated from male SpragueDawley rats. Culture-activated (day 10) and freshly isolated (day 0) cells were used. HSCs were treated with a pan-HDAC (trichostatin, TSA), HDAC I (valproic acid) or DNMT (5-aza-2 deoxycytadine, 5AZA) inhibitor or transfected with miR19b plasmid. Pair-matched sera and livers were isolated from fibrotic/injured (bile duct ligation, ethanol-drinking water, ethanol-LPS) and control rat livers. Exosomes were isolated from media or sera; pri-miR17–92 and individual miR and profibrotic gene expression were measured by qRT-PCR during HSC activation and liver pathologies. Results: Pri-miR17–92 cluster expression was up-regulated in activated HSCs, and inhibited by TSA, 5AZA, and reintroduction of miR19b. miR17a and 18a were significantly elevated in fibrotic/injured livers and in activated HSCs compared to controls. Conversely, expression of miR19a, 19b and 92 were significantly down-regulated. miR19b and 92 were significantly elevated in exosomes from media of activated HSCs and sera of fibrotic/injured rats, while miR17a, 18a, and 19a were undetected. Conclusions: Expression/regulation of miR17–92 cluster is linked to HSC activation, and differently expressed individual miRs may potentiate HSC activation. Modulating this cluster may prove to be a potential therapy for hepatic fibrogenesis. P627 ANALYSIS OF METABOLIC ACTIVITIES IN FETAL AND ADULT HUMAN HEPATOCYTES FOR CLINICAL THERAPY R. Gramignoli, S.C. Strom. Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm, Sweden E-mail: [email protected] Background and Aims: Fetal hepatocytes have been proposed as a source of cells for clinical hepatocyte transplants. However, to be useful, sufficient metabolic activities must be present. Aim: To assess the viability and metabolic activities in isolated fetal human hepatocytes as compared to pediatric and adult hepatocytes. Methods: Hepatocytes were isolated from 254 fetal livers, 11–24 weeks gestation and 229 post-natal tissues (3 months-85 years). Cell viability, apoptosis and Cytochrome P450 (CYP) activities, phase II-conjugation and ammonia metabolism were analyzed.
Journal of Hepatology 2014 vol. 60 | S215–S359
S277