- Email: [email protected]
138 CROSSTALK BETWEEN c-myc OVEREXPRESSING HEPATOCYTES AND HEPATIC STELLATE CELLS FACILITATES THE ONSET OF LIVER FIBROSIS
ORAL PRESENTATIONS Hence, we investigated primary HSCs from Jnk1−/− livers showing reduced transdifferentiation compared with WT and Jnk1Dhepa derived HSCs. Conclusion: Jnk1 in HSCs, but not in hepatocytes, plays a crucial role in the development of liver fibrosis, thus we identify Jnk1 in HSCs as a pro-fibrotic kinase and a promising cell-directed target for treatment of fibrotic patients. 136 ENDOGLIN DEFICIENCY IN HEPATIC STELLATE CELLS HAS A DIFFERENTIAL EFFECT ON LIVER FIBROSIS AND TGF-b SIGNALLING IN TWO EXPERIMENTAL MODELS OF MURINE LIVER FIBROSIS M. Alsamman1 , R. Weiskirchen1 , H. Wasmuth1 , D. Brenner2 , C. Trautwein1 , D. Scholten1 . 1 RWTH Aachen University Hospital, Aachen, Germany; 2 University of California San Diego, San Diego, CA, USA E-mail: [email protected] Background: Hepatic stellate cells (HSCs) are the major source for extracellular matrix (ECM) production in liver fibrosis. Endoglin (ENG) is a type III auxiliary receptor for TGF-b that is expressed on proliferating endothelial cells and HSCs. TGF-b is the most profibrotic cytokine expressed in response to liver injury. This study analyzes the role of ENG and TGF-b signalling in two models of experimental liver fibrosis by cell line specific Endoglin deletion in HSCs. Methods: Using the Cre-LoxP genetic recombination system we created HSC specific ENG−/− mice by crossing GFAPCre to ENGflox/flox mice. Mice were subjected to liver injury by CCl4 treatment (8 weeks) or bile duct ligation (BDL) (21 days). Liver fibrosis was analyzed by hydroxyproline measurement and Sirius red staining for collagen fibers. For in vitro analysis HSCs from ENGflox/flox mice were isolated and treated with adenoviral CMVCre -RSVGFP expressing vector. TGF-b signalling was analyzed by western blots and qPCR. Results: In response to CCl4 treatment livers of GFAPCre(+) Engd/d mice (n = 8) showed 18.2% less hydroxyproline content compared to GFAPCre(−) Engf /f litter mates reflecting less severe fibrosis. Sirius red stainings underlined these findings. Interestingly BDL inflicted injury showed contrasting results. Compared to GFAPCre(−) Engf /f mice GFAPCre(+) Engd/d litter mates (n = 8) showed 193.3% (p = 0.02) more ECM deposition. Western blot analysis of TGF-b signalling in vitro using adenoviral Cre treated ENGflox/flox primary HSCs demonstrated a differential effect of ENG on TGF-b signalling. In the absence of ENG activation of Smad1/5/8 was downregulated. Moreover, the phosphorylation of ERK1/2 was decreased and the expression of a-smooth muscle actin and connective tissue growth factor was downregulated. Conclusion: Endoglin deficiency in hepatic stellate cells has a differential effect on liver fibrosis depending on the cause of injury. Hepatocyte necrosis due to CCl4 treatment leads to strong TGF-b expression Smad signal transduction. Endoglin deficiency leads to amelioration of liver fibrosis, underlined by in vitro results. In contrast cholestatic liver injury is aggravated by endoglin deficiency in HSCs, reflecting a complex interplay of cholestasis, inflammation and toxic injury. Endoglin obviously modulates TGF-b signalling in this periportal-centered injury differentially, leading to significantly more fibrotic changes.
137 CXCL1 AND CXCL10 SECRETION BY FIBROCYSTIN-DEFECTIVE CHOLANGIOCYTES RECRUITS PERIBILIARY FIBROCYTES IN CONGENITAL HEPATIC FIBROSIS L. Fabris1 , L. Locatelli2 , D. Vigano` 2 , M. De Matteis1 , R. Fiorotto3 , M. Cadamuro1 , C. Spirlì3 , M. Strazzabosco2,3 . 1 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, 2 Department of Surgery and Interdisciplinary Medicine, University of Milano-Bicocca, Monza, Italy; 3 Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, USA E-mail: [email protected] Introduction: Congenital Hepatic Fibrosis (CHF) is a genetic disease caused by mutations in PKHD1, the gene encoding for Fibrocystin, a ciliary protein expressed by ductal epithelia. CHF is characterized by biliary dysgenesia associated with progressive portal fibrosis, ultimately leading to portal hypertension. In CHF, mechanisms of portal fibrosis are unknown. Fibrocytes, monocyte-derived cells bearing features of both macrophages and fibroblasts, are involved in several fibrosing conditions, but their role in liver scarring is controversial. High levels of CD45+ cells are present in the peribiliary fibrotic areas of Fibrocystin-defective mice, indicating the possible involvement of fibrocytes. Methods: Using a transgenic mouse model of CHF (Pkhd1del4/del4 ), we investigated: (a) the immunohistochemical expression of CD45, Collagen type I (COL1) (fibrocyte markers) and a-SMA (myofibroblast marker) in the portal inflammatory cells, and their correlation with the extension of portal fibrosis (Sirius-red) in liver samples from different ages (1–12 months); (b) a panel of 32 cyto/chemokines in both apical and basolateral medium of cultured polarized cholangiocytes (Luminex); )c) the effects of cholangiocyte conditioned media (CM) and cytokines on WEHI265.1-monocyte proliferation (MTS), chemoattraction (Boyden chamber) and transdifferentiation into fibrocytes (RT-PCR for COL1(A1)). WT littermates served as controls. Results: In Pkhd1-KO mice, in spite of progressive fibrosis, portal accumulation of a-SMA+ cells (portal myofibroblasts) was evident only after 9th months. In contrast, we observed an early and important peribiliary recruitment of CD45+ cells. The number of cells co-expressing CD45/COL1 correlated positively with the Sirius-red area (r = 0.89, p < 0.01) reaching 54% of the COL1+ cell population by 12 months. Cholangiocytes isolated from Pkhd1-KO mice secreted significantly higher levels of CXCL1 and CXCL10 at their basolateral side, as compared to WT cholangiocytes. Conditioned medium from Pkhd1-KO cholangiocytes, as well as CXCL1 and CXCL10 stimulated the proliferation and migration of WEHI265.1-monocytes, as well as their expression of COL1(A1) mRNA. Conclusions: In Pkhd1-KO mice, progressive portal accumulation of CD45+/COL1+ cells (fibrocytes), rather than of myofibroblasts, correlates with portal fibrosis. Pkhd1-KO cholangiocytes secrete increased amounts of CXCL1 and CXCL10 on the basolateral side, a mechanism that may underlay the recruitment of monocytes and their transdifferentiation into fibrocytes. 138 CROSSTALK BETWEEN c-myc OVEREXPRESSING HEPATOCYTES AND HEPATIC STELLATE CELLS FACILITATES THE ONSET OF LIVER FIBROSIS Y.A. Nevzorova1 , W. Hu1 , U. Haas1 , J. Freimuth2 , F. Tacke1 , C. Trautwein1 , C. Liedtke1 . 1 Department of Medicine III, University Hospital Aachen, Aachen, Germany; 2 UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA, USA E-mail: [email protected] Background and Aims: C-myc is a widely accepted oncogene as its overexpression or deregulation leads to a variety of cancers. In mice, transgenic overexpression of c-myc in hepatocytes (alb-myctg )
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ORAL PRESENTATIONS is sufficient to initiate hepatocellular carcinoma (HCC). However, the biologic significance of c-myc in precursor stages such as liver fibrosis is less defined. Liver fibrogenesis involves cell proliferation of hepatic cell populations such as hepatocytes and hepatic stellate cells (HSC). Here, we aimed to determine the potential role of c-myc in this process. Methods: Expression of c-myc was measured in biopsies of patients with liver fibrosis of different etiologies by qPCR and immunohistochemistry. Liver fibrosis in wildtype (WT) and albmyctg mice was induced by periodic CCl4 treatment. Primary HSC were isolated from WT and alb-myctg mice and investigated for markers of cell cycle progression and fibrosis by qPCR and immunofluorescence microscopy. Results: In patients with advanced (F3) liver fibrosis, hepatic c-myc was tenfold upregulated compared to healthy controls. Immunohistochemistry revealed an accumulation of c-mycexpressing cells in areas of fiber formation. Similarly, c-myc was also induced in murine WT liver during liver fibrogenesis. In turn, overexpression of c-myc in alb-myctg mice resulted in increased collagen deposition and induction of a-smooth-muscleactin (a-SMA) expression in liver over time. Most strikingly, primary HSC derived from alb-myctg mice showed: i. increased basal expression of a-SMA directly after isolation, ii. premature a-SMA induction, and iii. enhanced proliferation and accelerated transdifferentiation into myofibroblasts in comparison to WT HSC. Importantly, c-myc expression in HSC of both groups was only transiently detected. We hypothesized that c-myc overexpressing hepatocytes promote fibrosis by triggering the activation of HSC. In agreement with this idea, fibrosis initiation after chronic CCl4 treatment was detected significantly earlier in alb-myctg mice compared to controls, which was associated with increased expression of pro-fibrotic markers and strong induction of hepatocytes proliferation. Conclusion: Here we provide in vitro and in vivo evidence that c-myc overexpression in hepatocytes is a molecular trigger of HSC activation and predisposes to liver fibrosis. We propose that high c-myc expression in liver of patients could be a useful predictive fibrosis marker.
Journal of Hepatology 2013 vol. 58 | S45–S61
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137 CXCL1 AND CXCL10 SECRETION BY FIBROCYSTIN-DEFECTIVE CHOLANGIOCYTES RECRUITS PERIBILIARY FIBROCYTES IN CONGENITAL HEPATIC FIBROSIS L. Fabris1 , L. Locatelli2 , D. Vigano` 2 , M. De Matteis1 , R. Fiorotto3 , M. Cadamuro1 , C. Spirlì3 , M. Strazzabosco2,3 . 1 Department of Surgery, Oncology and Gastroenterology, University of Padova, Padova, 2 Department of Surgery and Interdisciplinary Medicine, University of Milano-Bicocca, Monza, Italy; 3 Section of Digestive Diseases, Yale University School of Medicine, New Haven, CT, USA E-mail: [email protected] Introduction: Congenital Hepatic Fibrosis (CHF) is a genetic disease caused by mutations in PKHD1, the gene encoding for Fibrocystin, a ciliary protein expressed by ductal epithelia. CHF is characterized by biliary dysgenesia associated with progressive portal fibrosis, ultimately leading to portal hypertension. In CHF, mechanisms of portal fibrosis are unknown. Fibrocytes, monocyte-derived cells bearing features of both macrophages and fibroblasts, are involved in several fibrosing conditions, but their role in liver scarring is controversial. High levels of CD45+ cells are present in the peribiliary fibrotic areas of Fibrocystin-defective mice, indicating the possible involvement of fibrocytes. Methods: Using a transgenic mouse model of CHF (Pkhd1del4/del4 ), we investigated: (a) the immunohistochemical expression of CD45, Collagen type I (COL1) (fibrocyte markers) and a-SMA (myofibroblast marker) in the portal inflammatory cells, and their correlation with the extension of portal fibrosis (Sirius-red) in liver samples from different ages (1–12 months); (b) a panel of 32 cyto/chemokines in both apical and basolateral medium of cultured polarized cholangiocytes (Luminex); )c) the effects of cholangiocyte conditioned media (CM) and cytokines on WEHI265.1-monocyte proliferation (MTS), chemoattraction (Boyden chamber) and transdifferentiation into fibrocytes (RT-PCR for COL1(A1)). WT littermates served as controls. Results: In Pkhd1-KO mice, in spite of progressive fibrosis, portal accumulation of a-SMA+ cells (portal myofibroblasts) was evident only after 9th months. In contrast, we observed an early and important peribiliary recruitment of CD45+ cells. The number of cells co-expressing CD45/COL1 correlated positively with the Sirius-red area (r = 0.89, p < 0.01) reaching 54% of the COL1+ cell population by 12 months. Cholangiocytes isolated from Pkhd1-KO mice secreted significantly higher levels of CXCL1 and CXCL10 at their basolateral side, as compared to WT cholangiocytes. Conditioned medium from Pkhd1-KO cholangiocytes, as well as CXCL1 and CXCL10 stimulated the proliferation and migration of WEHI265.1-monocytes, as well as their expression of COL1(A1) mRNA. Conclusions: In Pkhd1-KO mice, progressive portal accumulation of CD45+/COL1+ cells (fibrocytes), rather than of myofibroblasts, correlates with portal fibrosis. Pkhd1-KO cholangiocytes secrete increased amounts of CXCL1 and CXCL10 on the basolateral side, a mechanism that may underlay the recruitment of monocytes and their transdifferentiation into fibrocytes. 138 CROSSTALK BETWEEN c-myc OVEREXPRESSING HEPATOCYTES AND HEPATIC STELLATE CELLS FACILITATES THE ONSET OF LIVER FIBROSIS Y.A. Nevzorova1 , W. Hu1 , U. Haas1 , J. Freimuth2 , F. Tacke1 , C. Trautwein1 , C. Liedtke1 . 1 Department of Medicine III, University Hospital Aachen, Aachen, Germany; 2 UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, CA, USA E-mail: [email protected] Background and Aims: C-myc is a widely accepted oncogene as its overexpression or deregulation leads to a variety of cancers. In mice, transgenic overexpression of c-myc in hepatocytes (alb-myctg )
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Journal of Hepatology 2013 vol. 58 | S45–S61
ORAL PRESENTATIONS is sufficient to initiate hepatocellular carcinoma (HCC). However, the biologic significance of c-myc in precursor stages such as liver fibrosis is less defined. Liver fibrogenesis involves cell proliferation of hepatic cell populations such as hepatocytes and hepatic stellate cells (HSC). Here, we aimed to determine the potential role of c-myc in this process. Methods: Expression of c-myc was measured in biopsies of patients with liver fibrosis of different etiologies by qPCR and immunohistochemistry. Liver fibrosis in wildtype (WT) and albmyctg mice was induced by periodic CCl4 treatment. Primary HSC were isolated from WT and alb-myctg mice and investigated for markers of cell cycle progression and fibrosis by qPCR and immunofluorescence microscopy. Results: In patients with advanced (F3) liver fibrosis, hepatic c-myc was tenfold upregulated compared to healthy controls. Immunohistochemistry revealed an accumulation of c-mycexpressing cells in areas of fiber formation. Similarly, c-myc was also induced in murine WT liver during liver fibrogenesis. In turn, overexpression of c-myc in alb-myctg mice resulted in increased collagen deposition and induction of a-smooth-muscleactin (a-SMA) expression in liver over time. Most strikingly, primary HSC derived from alb-myctg mice showed: i. increased basal expression of a-SMA directly after isolation, ii. premature a-SMA induction, and iii. enhanced proliferation and accelerated transdifferentiation into myofibroblasts in comparison to WT HSC. Importantly, c-myc expression in HSC of both groups was only transiently detected. We hypothesized that c-myc overexpressing hepatocytes promote fibrosis by triggering the activation of HSC. In agreement with this idea, fibrosis initiation after chronic CCl4 treatment was detected significantly earlier in alb-myctg mice compared to controls, which was associated with increased expression of pro-fibrotic markers and strong induction of hepatocytes proliferation. Conclusion: Here we provide in vitro and in vivo evidence that c-myc overexpression in hepatocytes is a molecular trigger of HSC activation and predisposes to liver fibrosis. We propose that high c-myc expression in liver of patients could be a useful predictive fibrosis marker.
Journal of Hepatology 2013 vol. 58 | S45–S61
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