- Email: [email protected]
MYOFIBROBLASTS IN HUMAN LIVER DISEASES IN RELATION TO DIFFERENTIATION AND FIBROSIS PATTERNS
04C. MOLECULAR AND CELLULAR BIOLOGY control levels (HSC with L..obtusilobaalone). One XAD resolved fraction showed antifibrotic activity. t-butylhydroperoxide-inducedROS production in HSC was neutralized by the addition of non-toxic concentration of the L.obtusiloba extract. Conclusions: Aqueous extracts of L. obtusiloba exhibit direct anti-fibrotic but no toxic or apoptotic effects by inhibiting proliferation and collagen synthesis as well as ROS production of activated HSC. Our data suggest that L.obtusiloba may directly target TGF-beta induced fibrosis.
13391 THE ENDOCANNABINOID 2-ARACHIDONOYL GLYCEROL INDUCES APOPTOSIS IN PRIMARY HEPATIC STELLATE CELLS VIA MEMBRANE CHOLESTEROL AND MITOCHONDRIAL ROS PRODUCTION INDEPENDENTLY OF CANNABINOID-RECEPTORS OR NADPH OXIDASE ACTIVATION S.V. Siegmund’,2, T. Qian3, S. de Minicis2, J. Harvey-White4, G. Kunos4, K.Y. Vinod’, B. Hungund’, D.A. Brenner’, R.F. Schwabe’. ‘Department of Medicine I1 (Gutroenterology and Heputology), University Hospital
Mannheim, Uniuer~xit?,of Heidelberg, Mannheim, Germuny; 2Depurtment of Medicine, College of Phyxicians und Surgeons, Columbia Uniuersity, N n v York, N F ”Department of‘ Neuroscience and Cell Biology, liniversity of Texus Medical Brunch, Galveston, TX; 4Nutional Institute on Alcohol Ahuse und Alcoholism, Nutional Institute of Heulth, Bethesda, MD; ”Dqurtnzent of’ Psychiutv, College of’ Physicians and Snrgeons, Colunzbiu liniversity, N n v York, NY, USA E-mail: [email protected] Background: We have previously shown that the anti-inflammatory endocannabinoid 2-arachidonoyl glycerol (2-AG) is upregulated during hepatic fibrogenesis in vivo and induces cell death in hepatic stellate cells (HSCs), but not in hepatocytes. Aim: To determine the mechanism of cell death by 2-AG in HSCs. Methods: Primary HSCs from rats, humans and wildtype or p47phox’and (lacking hnctional NADPH oxidase), cannabinoid receptor (CB) 1 CB2’- mice were isolated from healthy livers by pronase/collagenase perfusion. 2-AG-induced cell death was analyzed by LDH assay and western blot for caspase 3-, PARP-cleavage and cytochrome C. Reactive oxygen species (ROS) formation was monitored by DCFDA fluorescence and confocal microscopy. Membrane cholesterol was depleted by methyl(i-cyclodextrin (MCD). Expression of the cannabinoid receptors CB 1 and CB2 was measured by real time PCR. Results: Treatment of primary activated human and rat HSCs with 2-AG dose-dependently induced apoptosis (starting from 1 pM and 70‘X after 24h at IOpM), accompanied by PARP- caspase 3-cleavage and cytochrome C release from mitochondria. 2-AG induced an excessive increase in ROS production in HSCs starting at nanomolar concentrations. The antioxidants GSH-ethylester or Trolox reduced 2-AG-induced cell death in HSCs, whereas GSH depletion increased 2-AG-induced death. The NADPH oxidase inhibitor DPT did not significantly reduce 2-AG-induced ROS production or cell death. HSCs from p47phox’- mice displayed equal generation of ROS and susceptibility towards 2-AG-induced death compared to wildtype HSCs, therefore excluding NADPH oxidase as the source of 2-AG-mediated ROS generation. Confocal fluorescence microscopy clearly revealed mitochondria as the site of ROS production. CB1 and CB2 were faintly expressed in HSCs. The CB1 and CB2 receptor antagonists SR 141716 and SR 144628 failed to block 2-AGinduced cell death. Moreover, HSCs from CBI-’- or CB2”- mice were not resistant against 2-AG-mediated apoptosis. Conversely, membrane cholesterol depletion with MCD efficiently prevented 2-AG-mediated ROS formation and cell death in HSCs. Conclusions: 2-AG induces apoptosis in HSCs in an ROS- and membrane cholesterol-dependent manner and is clearly independent from cannabinoid receptors or NADPH oxidase activation. Thus, increasing the levels of 2-AG in vivo may serve as a novel strategy for antifibrotic therapy through promotion of HSC apoptosis.
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C) HSCs AND FIBROSIS
S133
13401 HUMAN PROGENITOR CELLS ARE SURROUNDED BY DIFFERENT SUBTYPES OF HEPATIC STELLATE CELLSlMYOFlBROBLASTS IN HUMAN LIVER DISEASES IN RELATION TO DIFFERENTIATION AND FIBROSIS PATTERNS B. Spee, A. Katoonizadeh, T.A. Roskams. Depurtment of Morphology and Molecular Pathology, linioersiiy Hospitals Leuven, Lenoen, Belgium E-mail: [email protected] Background and Aims: Liver progenitor cells (PCs) are activated in a variety of human liver diseases (e.g. biliary diseases, hepatitis, alcoholic and non-alcoholic steatohepatitis). They play a role in regeneration and can differentiate into hepatocytes and cholangiocytes, a process which is influenced by the microenvironment. Since hepatic stellate cellsImyotibroblasts (HSCIMF) co-proliferate with PCs, we investigated the different subtypes of HSC/MF in different human liver diseases. Methods: lmmunohistochemistry for markers of lobular HSC {neurotrophin 3+ (NT3), neural cell adhesion molecule+ (NCAM), glial fibrillary acidic protein+ (GFAP)}, and for activated portal/septal MFs {a-smooth muscle actin (a-SMA)+, NCAM-, NT3-} was used on human specimens of different stages of chronic hepatitis ( n = 30), alcoholic liver disease (n = 20), non-alcoholic steatohepatitis (n = 27), primary biliary cirrhosis (n = 30). The stainings were blindly, semiquantitatively assessed at the different locations: in the parenchyma, fibrotic septa, portal tracts, and at the interface. Chi-square statistical test was used. Results: In all human liver diseases, the number of NT3, GFAP and a-SMA-reactive HSCiMFs correlated positively with the stage of fibrosis. In chronic hepatitis, the number of lobular phenotype HSCiMFs at the interface of portal tracts and septa, and around the PCs, was significantly higher than in (N)ASH and biliary diseases. In the parenchyma there was no significant difference of lobular phenotype HSC/MFs between chronic hepatitis and (N)ASH. However both diseases showed significantly more lobular phenotype HSC/MF activation compared to biliary diseases. Biliary diseases showed significantly more portal phenotype HSC/MF activation at the interface surrounding PCs in portal tracts and in septa compared to chronic hepatitis and (N)ASH. Conclusions: (N)ASH and chronic hepatitis are characterized by activation of lobular HSCiMF in the parenchyma, while this is only minimally present in biliary diseases. In chronic hepatitis lobular types of HSCiMF are mobilized at the interface and surrounding PCs. In contrast, in biliary diseases, portal subtypes of MFs are present at the interface and surrounding PCs, related to the different fibrosis and differentiation pattern (related to ductular reaction) in biliary diseases.
13411 INHIBITION OF BETA-ADRENORECEPTORS RESULTS IN DELAYED FIBROSIS IN Abcb4 KNOCKOUT MICE 1. Strack’, K. Petmecky’ , M. Scheffler’, S. Schulte2, F. Lammert3, H. Varnholt’, H.P. Dienes’ , M. Odenthal’ . ‘Institute j i w Puthology;
’Depwtment of‘ Gastroenterology and Heputhology, linioersiiy Hospital o f Cologne, Cologne; 3Department qf Internal Medicine I, U n i u e r s i ~ ~ Hospitul Bonn, Bonn, Germuny E-mail: [email protected]
Background: Transdifferentiation of hepatic stellate cells (HSC) into myofibroblastic cells is one ofthe central mechanisms of liver fibrogenesis. Activated myotibroblastic cells are the main producers of extracellular matrix. Recent reports have shown that HSC express neurotrophic mediators after myotibroblastic activation and that blockade of the sympathetic signalling reduces the fibrogenic activity of HSC in vitro and in experimental fibrosis. In the presented study we investigated the role of the sympathetic innervation in the Abcb4 (MDR2) knockout model, developing periportal, cholestatic fibrosis thereby mimicking primary sclerosing cholangitis (PSC).
13391 THE ENDOCANNABINOID 2-ARACHIDONOYL GLYCEROL INDUCES APOPTOSIS IN PRIMARY HEPATIC STELLATE CELLS VIA MEMBRANE CHOLESTEROL AND MITOCHONDRIAL ROS PRODUCTION INDEPENDENTLY OF CANNABINOID-RECEPTORS OR NADPH OXIDASE ACTIVATION S.V. Siegmund’,2, T. Qian3, S. de Minicis2, J. Harvey-White4, G. Kunos4, K.Y. Vinod’, B. Hungund’, D.A. Brenner’, R.F. Schwabe’. ‘Department of Medicine I1 (Gutroenterology and Heputology), University Hospital
Mannheim, Uniuer~xit?,of Heidelberg, Mannheim, Germuny; 2Depurtment of Medicine, College of Phyxicians und Surgeons, Columbia Uniuersity, N n v York, N F ”Department of‘ Neuroscience and Cell Biology, liniversity of Texus Medical Brunch, Galveston, TX; 4Nutional Institute on Alcohol Ahuse und Alcoholism, Nutional Institute of Heulth, Bethesda, MD; ”Dqurtnzent of’ Psychiutv, College of’ Physicians and Snrgeons, Colunzbiu liniversity, N n v York, NY, USA E-mail: [email protected] Background: We have previously shown that the anti-inflammatory endocannabinoid 2-arachidonoyl glycerol (2-AG) is upregulated during hepatic fibrogenesis in vivo and induces cell death in hepatic stellate cells (HSCs), but not in hepatocytes. Aim: To determine the mechanism of cell death by 2-AG in HSCs. Methods: Primary HSCs from rats, humans and wildtype or p47phox’and (lacking hnctional NADPH oxidase), cannabinoid receptor (CB) 1 CB2’- mice were isolated from healthy livers by pronase/collagenase perfusion. 2-AG-induced cell death was analyzed by LDH assay and western blot for caspase 3-, PARP-cleavage and cytochrome C. Reactive oxygen species (ROS) formation was monitored by DCFDA fluorescence and confocal microscopy. Membrane cholesterol was depleted by methyl(i-cyclodextrin (MCD). Expression of the cannabinoid receptors CB 1 and CB2 was measured by real time PCR. Results: Treatment of primary activated human and rat HSCs with 2-AG dose-dependently induced apoptosis (starting from 1 pM and 70‘X after 24h at IOpM), accompanied by PARP- caspase 3-cleavage and cytochrome C release from mitochondria. 2-AG induced an excessive increase in ROS production in HSCs starting at nanomolar concentrations. The antioxidants GSH-ethylester or Trolox reduced 2-AG-induced cell death in HSCs, whereas GSH depletion increased 2-AG-induced death. The NADPH oxidase inhibitor DPT did not significantly reduce 2-AG-induced ROS production or cell death. HSCs from p47phox’- mice displayed equal generation of ROS and susceptibility towards 2-AG-induced death compared to wildtype HSCs, therefore excluding NADPH oxidase as the source of 2-AG-mediated ROS generation. Confocal fluorescence microscopy clearly revealed mitochondria as the site of ROS production. CB1 and CB2 were faintly expressed in HSCs. The CB1 and CB2 receptor antagonists SR 141716 and SR 144628 failed to block 2-AGinduced cell death. Moreover, HSCs from CBI-’- or CB2”- mice were not resistant against 2-AG-mediated apoptosis. Conversely, membrane cholesterol depletion with MCD efficiently prevented 2-AG-mediated ROS formation and cell death in HSCs. Conclusions: 2-AG induces apoptosis in HSCs in an ROS- and membrane cholesterol-dependent manner and is clearly independent from cannabinoid receptors or NADPH oxidase activation. Thus, increasing the levels of 2-AG in vivo may serve as a novel strategy for antifibrotic therapy through promotion of HSC apoptosis.
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C) HSCs AND FIBROSIS
S133
13401 HUMAN PROGENITOR CELLS ARE SURROUNDED BY DIFFERENT SUBTYPES OF HEPATIC STELLATE CELLSlMYOFlBROBLASTS IN HUMAN LIVER DISEASES IN RELATION TO DIFFERENTIATION AND FIBROSIS PATTERNS B. Spee, A. Katoonizadeh, T.A. Roskams. Depurtment of Morphology and Molecular Pathology, linioersiiy Hospitals Leuven, Lenoen, Belgium E-mail: [email protected] Background and Aims: Liver progenitor cells (PCs) are activated in a variety of human liver diseases (e.g. biliary diseases, hepatitis, alcoholic and non-alcoholic steatohepatitis). They play a role in regeneration and can differentiate into hepatocytes and cholangiocytes, a process which is influenced by the microenvironment. Since hepatic stellate cellsImyotibroblasts (HSCIMF) co-proliferate with PCs, we investigated the different subtypes of HSC/MF in different human liver diseases. Methods: lmmunohistochemistry for markers of lobular HSC {neurotrophin 3+ (NT3), neural cell adhesion molecule+ (NCAM), glial fibrillary acidic protein+ (GFAP)}, and for activated portal/septal MFs {a-smooth muscle actin (a-SMA)+, NCAM-, NT3-} was used on human specimens of different stages of chronic hepatitis ( n = 30), alcoholic liver disease (n = 20), non-alcoholic steatohepatitis (n = 27), primary biliary cirrhosis (n = 30). The stainings were blindly, semiquantitatively assessed at the different locations: in the parenchyma, fibrotic septa, portal tracts, and at the interface. Chi-square statistical test was used. Results: In all human liver diseases, the number of NT3, GFAP and a-SMA-reactive HSCiMFs correlated positively with the stage of fibrosis. In chronic hepatitis, the number of lobular phenotype HSCiMFs at the interface of portal tracts and septa, and around the PCs, was significantly higher than in (N)ASH and biliary diseases. In the parenchyma there was no significant difference of lobular phenotype HSC/MFs between chronic hepatitis and (N)ASH. However both diseases showed significantly more lobular phenotype HSC/MF activation compared to biliary diseases. Biliary diseases showed significantly more portal phenotype HSC/MF activation at the interface surrounding PCs in portal tracts and in septa compared to chronic hepatitis and (N)ASH. Conclusions: (N)ASH and chronic hepatitis are characterized by activation of lobular HSCiMF in the parenchyma, while this is only minimally present in biliary diseases. In chronic hepatitis lobular types of HSCiMF are mobilized at the interface and surrounding PCs. In contrast, in biliary diseases, portal subtypes of MFs are present at the interface and surrounding PCs, related to the different fibrosis and differentiation pattern (related to ductular reaction) in biliary diseases.
13411 INHIBITION OF BETA-ADRENORECEPTORS RESULTS IN DELAYED FIBROSIS IN Abcb4 KNOCKOUT MICE 1. Strack’, K. Petmecky’ , M. Scheffler’, S. Schulte2, F. Lammert3, H. Varnholt’, H.P. Dienes’ , M. Odenthal’ . ‘Institute j i w Puthology;
’Depwtment of‘ Gastroenterology and Heputhology, linioersiiy Hospital o f Cologne, Cologne; 3Department qf Internal Medicine I, U n i u e r s i ~ ~ Hospitul Bonn, Bonn, Germuny E-mail: [email protected]
Background: Transdifferentiation of hepatic stellate cells (HSC) into myofibroblastic cells is one ofthe central mechanisms of liver fibrogenesis. Activated myotibroblastic cells are the main producers of extracellular matrix. Recent reports have shown that HSC express neurotrophic mediators after myotibroblastic activation and that blockade of the sympathetic signalling reduces the fibrogenic activity of HSC in vitro and in experimental fibrosis. In the presented study we investigated the role of the sympathetic innervation in the Abcb4 (MDR2) knockout model, developing periportal, cholestatic fibrosis thereby mimicking primary sclerosing cholangitis (PSC).