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981 EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) IN CHOLANGIOCYTES DOES NOT CONTRIBUTE TO THE POPULATION OF α-SMA+ MYOFIBROBLASTS IN EXPERIMENTAL MODELS OF LIVER FIBROSIS
POSTERS a standardised meal, to reduce the variation in dietary iron, along with ferrous sulphate 200 mg tablet. Blood was collected at 1, 2, 3, 4 and 24 hrs afteringestion of the iron tablet. Serum ferritin, iron and transferrin saturation were measured using standard methods. Serum hepcidin was measured using radio-immuno assay. Results: The mean hepcidin levels increased in a step wise fashion in response to the oral iron challenge. This mirrored an increase in transferrin saturation and serum iron levels although serum ferritin and total iron binding capacity did not follow any pattern.
Time (hrs)
Hepcidin (ng/ml)
Transferrin saturation %
Serum iron (mmol/L)
Ferritin (mg/L)
TIBC
0 1 2 3 4 24
35.2 40.2 57.3 66.2 72.1 31.6
22.5 27.1 41.8 49.9 45.8 23.9
13.3 15.7 22.4 31.2 31.8 15.5
51.3 43.6 44.3 44.8 42.5 32.1
61.7 61.6 59.5 61.3 62.3 57.8
Graph1: Relationship between hepcidin and Transferrin Saturation. Conclusion: This in vivo study confirms that in normal iron homeostasis, hepcidin increases in response to tranferrin saturation. Iron sensors in hepatocytes appear to be exquisitively sensitive to changes in transferrin saturation resulting in modulation of hepcidin synthesis. 981 EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) IN CHOLANGIOCYTES DOES NOT CONTRIBUTE TO THE POPULATION OF a-SMA+ MYOFIBROBLASTS IN EXPERIMENTAL MODELS OF LIVER FIBROSIS 2 ¨ D. Scholten1,2 , A. Scholten2 , C. Ostereicher , D.A. Brenner2 , T. Kisseleva2 . 1 Department of Internal Medicine III, RWTH Aachen, Aachen, Germany; 2 Department of Medicine, University of California, San Diego, CA, USA E-mail: [email protected] Background: Recent studies have demonstrated that chronic injury causes an epithelial-to-mesenchymal-transition (EMT), a process when fully differentiated epithelial cells undergo phenotypic transition to fully differentiated mesenchymal cells. An opposite process, mesenchymal-to-epithelial-transition (MET) can be also result from sustained injury. While contribution of EMT and MET to embryogenesis and renal fibrosis is well documented, the role of EMT for liver fibrosis remains unclear.
Aim: To determine if CK19+ cholangiocytes give rise to SMA+ FSP+ myofibroblasts (EMT); and to determine if GFAP+ hepatic stellate cells (HSCs) upregulate epithelial cell markers (CK19, E-Cadherin) in response to liver injury (MET). Methods: EMT and MET was studied using the Cre-LoxP system to map the cell fate of CK19+ cholangiocytes or GFAP+ HSCs. For this purpose, cholangiocyte-specific tamoxifen inducible CK19CreERT mice were crossed with Rosa26EYFP/EYFP reporter mice to generate CK19YFP mice, in which YFP labeled 40±12% of CK19+ cholangiocytes upon tamoxifen administration. FSP1Cre mice were crossed to Rosa26EYFP/EYFP mice to label cells for the expression of the EMT-specific marker FSP1. Mice were subjected to CCl4 - or bile duct ligation (BDL) liver injury and liver tissues were analyzed by hydroxyproline assay, Sirius Red staining and immunofluorescence for upregulation of mesenchymal markers in cholangiocytes undergoing EMT. To study MET, GFAPCre mice were crossed to Rosa26mT/mG -reporter mice, subjected to liver injury and analyzed for expression of epithelial and oval cell markers in HSCs. Results: CCl4 and BDL induced liver fibrosis and upregulated collagen-a1(I) and a-SMA mRNA. However, specific imunostaining revealed no co-expression of a-SMA, desmin, or FSP1 (an established marker of EMT) in CK19YFP -labeled cholangiocytes. Moreover, FSP1Cre -labeled cells did not co-express cholangiocyte markers CK19 or E-cadherin. Co-localization of a-SMA was detected in CK19YFP labeled cells during embryogenesis, confirming the role of EMT in development. Quiescent GFAPCre -labeled HSCs or activated Collagen-a1(I)Cre -labeled HSCs showed no co-expression of epithelial markers or phenotypical changes in HSCs undergoing MET into hepatocytes, cholangiocytes or oval cells. Conclusions: In response two models of chronic liver injury, cholangiocytes do not undergo EMT. Quiescent and activated HSCs do not upregulate MET markers or exhibit progenitor properties. 982 BMI1 IS A MARKER OF ACTIVATED LIVER PROGENITOR CELLS B.A. Schotanus1 , B. Spee1,2 , H.S. Kruitwagen1 , B.B. Brinkhof1 , T.S.G.A.M. van den Ingh3 , L.C. Penning1 , T. Roskams2 . 1 Clinical Sciences of Companion Animals, Utrecht University, Utrecht, The Netherlands; 2 Department of Morphology and Molecular Pathology, University Hospitals Leuven, Leuven, Belgium; 3 TCCI Consultancy, Utrecht, The Netherlands E-mail: [email protected] Background and Aims: The polycomb-group gene BMI1 is essential in self-renewal of adult hematopoietic, neuronal, mammary, and intestinal progenitor cells. Forced over-expression of BMI1 in the fetal liver enhances self-renewal of hepatoblasts. The expression and role of BMI1 in adult liver progenitor cells has however not been studied extensively. This prompted us to investigate BMI1 expression in a wide range of liver pathologies with various stages of progenitor cell activation in a large animal model (dog), and confirm the findings in man. Methods: Whole tissue gene-expression analysis was performed in acute hepatitis (AH), chronic hepatitis (CH), lobular dissecting hepatitis (LDH), and healthy liver tissue from dogs. Laser microdissection of the activated liver progenitor cell (LPC) niche was performed on canine LDH, characterized by massive LPCactivation, and on AH, CH, and primary biliary cirrhosis (PBC) in man. Immunohistochemistry for BMI1 was performed on all disease groups and healthy tissue. Results: Gene expression analysis on whole canine liver tissue showed no different expression for BMI1 in diseased liver (AH, CH) when compared to healthy livers, except for a six fold induction in LDH (Mann Whitney U test, p < 0.01). Gene-expression analysis from the laser microdissected tissue from canine LDH and human AH, CH, and PBC showed a high expression of BMI1 in the activated LPC niches in all diseases when compared to the quiescent LPC
Journal of Hepatology 2010 vol. 52 | S319–S457
S379
Time (hrs)
Hepcidin (ng/ml)
Transferrin saturation %
Serum iron (mmol/L)
Ferritin (mg/L)
TIBC
0 1 2 3 4 24
35.2 40.2 57.3 66.2 72.1 31.6
22.5 27.1 41.8 49.9 45.8 23.9
13.3 15.7 22.4 31.2 31.8 15.5
51.3 43.6 44.3 44.8 42.5 32.1
61.7 61.6 59.5 61.3 62.3 57.8
Graph1: Relationship between hepcidin and Transferrin Saturation. Conclusion: This in vivo study confirms that in normal iron homeostasis, hepcidin increases in response to tranferrin saturation. Iron sensors in hepatocytes appear to be exquisitively sensitive to changes in transferrin saturation resulting in modulation of hepcidin synthesis. 981 EPITHELIAL-TO-MESENCHYMAL TRANSITION (EMT) IN CHOLANGIOCYTES DOES NOT CONTRIBUTE TO THE POPULATION OF a-SMA+ MYOFIBROBLASTS IN EXPERIMENTAL MODELS OF LIVER FIBROSIS 2 ¨ D. Scholten1,2 , A. Scholten2 , C. Ostereicher , D.A. Brenner2 , T. Kisseleva2 . 1 Department of Internal Medicine III, RWTH Aachen, Aachen, Germany; 2 Department of Medicine, University of California, San Diego, CA, USA E-mail: [email protected] Background: Recent studies have demonstrated that chronic injury causes an epithelial-to-mesenchymal-transition (EMT), a process when fully differentiated epithelial cells undergo phenotypic transition to fully differentiated mesenchymal cells. An opposite process, mesenchymal-to-epithelial-transition (MET) can be also result from sustained injury. While contribution of EMT and MET to embryogenesis and renal fibrosis is well documented, the role of EMT for liver fibrosis remains unclear.
Aim: To determine if CK19+ cholangiocytes give rise to SMA+ FSP+ myofibroblasts (EMT); and to determine if GFAP+ hepatic stellate cells (HSCs) upregulate epithelial cell markers (CK19, E-Cadherin) in response to liver injury (MET). Methods: EMT and MET was studied using the Cre-LoxP system to map the cell fate of CK19+ cholangiocytes or GFAP+ HSCs. For this purpose, cholangiocyte-specific tamoxifen inducible CK19CreERT mice were crossed with Rosa26EYFP/EYFP reporter mice to generate CK19YFP mice, in which YFP labeled 40±12% of CK19+ cholangiocytes upon tamoxifen administration. FSP1Cre mice were crossed to Rosa26EYFP/EYFP mice to label cells for the expression of the EMT-specific marker FSP1. Mice were subjected to CCl4 - or bile duct ligation (BDL) liver injury and liver tissues were analyzed by hydroxyproline assay, Sirius Red staining and immunofluorescence for upregulation of mesenchymal markers in cholangiocytes undergoing EMT. To study MET, GFAPCre mice were crossed to Rosa26mT/mG -reporter mice, subjected to liver injury and analyzed for expression of epithelial and oval cell markers in HSCs. Results: CCl4 and BDL induced liver fibrosis and upregulated collagen-a1(I) and a-SMA mRNA. However, specific imunostaining revealed no co-expression of a-SMA, desmin, or FSP1 (an established marker of EMT) in CK19YFP -labeled cholangiocytes. Moreover, FSP1Cre -labeled cells did not co-express cholangiocyte markers CK19 or E-cadherin. Co-localization of a-SMA was detected in CK19YFP labeled cells during embryogenesis, confirming the role of EMT in development. Quiescent GFAPCre -labeled HSCs or activated Collagen-a1(I)Cre -labeled HSCs showed no co-expression of epithelial markers or phenotypical changes in HSCs undergoing MET into hepatocytes, cholangiocytes or oval cells. Conclusions: In response two models of chronic liver injury, cholangiocytes do not undergo EMT. Quiescent and activated HSCs do not upregulate MET markers or exhibit progenitor properties. 982 BMI1 IS A MARKER OF ACTIVATED LIVER PROGENITOR CELLS B.A. Schotanus1 , B. Spee1,2 , H.S. Kruitwagen1 , B.B. Brinkhof1 , T.S.G.A.M. van den Ingh3 , L.C. Penning1 , T. Roskams2 . 1 Clinical Sciences of Companion Animals, Utrecht University, Utrecht, The Netherlands; 2 Department of Morphology and Molecular Pathology, University Hospitals Leuven, Leuven, Belgium; 3 TCCI Consultancy, Utrecht, The Netherlands E-mail: [email protected] Background and Aims: The polycomb-group gene BMI1 is essential in self-renewal of adult hematopoietic, neuronal, mammary, and intestinal progenitor cells. Forced over-expression of BMI1 in the fetal liver enhances self-renewal of hepatoblasts. The expression and role of BMI1 in adult liver progenitor cells has however not been studied extensively. This prompted us to investigate BMI1 expression in a wide range of liver pathologies with various stages of progenitor cell activation in a large animal model (dog), and confirm the findings in man. Methods: Whole tissue gene-expression analysis was performed in acute hepatitis (AH), chronic hepatitis (CH), lobular dissecting hepatitis (LDH), and healthy liver tissue from dogs. Laser microdissection of the activated liver progenitor cell (LPC) niche was performed on canine LDH, characterized by massive LPCactivation, and on AH, CH, and primary biliary cirrhosis (PBC) in man. Immunohistochemistry for BMI1 was performed on all disease groups and healthy tissue. Results: Gene expression analysis on whole canine liver tissue showed no different expression for BMI1 in diseased liver (AH, CH) when compared to healthy livers, except for a six fold induction in LDH (Mann Whitney U test, p < 0.01). Gene-expression analysis from the laser microdissected tissue from canine LDH and human AH, CH, and PBC showed a high expression of BMI1 in the activated LPC niches in all diseases when compared to the quiescent LPC
Journal of Hepatology 2010 vol. 52 | S319–S457
S379