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Treatment with human metalloproteinase-8 gene delivery ameliorates experimental rat liver cirrhosis resembling alcoholic human disease
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Saturday, April 20, 2002 -] TRUNCATED TYPE II TGF-BETA RECEPTOR PROMOTES LIVER REGENERATION IN DIMETYLNITROSAMIN-TREATED CIRROHOTIC RATS
Tom Nakamura 1,2, Ryuichiro Sakata 1,2, Masaharu Sakamoto 1,2, Takuji Torimura 1,2 Takato Ueno 1,2, Michio Sata L2 1Liver Division,
Research Centerfor Innovative Cancer Therapy, Kurume University Kurume; 22nd Department of lnternal Medicine, Kurume UniversityScool of Medicine, Kurume, Japan We investigated whether anti-TGF-beta intervention can enhance liver regeneration or not. To block TGF-beta action, we prepared an adenovirus expressing a truncated type II TGF-beta receptor (AdTbeta-TR). We also used an adenovirus expressing bacterial beta-galactosidase (AdLacZ). Rats were first treated with dimethylnitrosamine (DMN) for 3 weeks. Then, vectors or saline were intravenously applied only at once from the tail vein. Following the gene transfer, DMN administration was continued for additional 3 weeks. The liver tissues were extracted at 4 and 6 weeks after DMN administration, and liver regeneration was confirmed. Namely, proliferating cells were semiquantitated by immunohistochemistry using an anti-Ki-67 antibody, and conversely apoptosis of the hepatocyte was measured by immunohistochemistry using an anti-digoxygenin antibody. Biochemical parameters were measured by standard methods, and body weight and survival rate were monitored dally. All AdTbeta-TR-treated rats remained alive, whereas almost all the DMN-treated rats infused either AdLacZ or saline died. Moreover, body weights of rats treated with AdTbeta-TR were not decreased compared with either AdLacZ or saline. Serum total bilirubin, total protein, and albumin levels in the AdThetaTR-treated rats were preserved at the level in rats treated with DMN for only 3 weeks. Ki-67 labeling index in AdTheta-TR-treated livers (10.1 42.4%) increased compared with that in AdLacZ-treated livers (2.3 4- 1.0%) at 6 weeks after DMN administration. In contrast, apoptosis in AdTbetaTR-treated livers was inhibited at 4 weeks after DMN administration. Our results suggest that anti-TGF-beta intervention facilitates liver regeneration in DMN-treated rats.
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TREATMENT WITH HUMAN METALLOPROTEINASE-8 GENE DELIVERY AMELIORATES EXPERIMENTAL RAT LIVER CIRRHOSIS RESEMBLING ALCOHOLIC HUMAN DISEASE
Fernando Siller I , Silvia Salgado l, Jesus Garcia 1, Jose M. Vera ] , Alejandra Miranda 1, Guillermo Grijalva 1, Javier Galvez 1, Estuardo Aguilar-Cordova 2, Juan Arrnendariz-Borunda l . llnstituto de
Biologia Moleculary Terapia Gdnica, CUCS, Universidad de Guadalajara, Guadalajara, Mexico; 2Harvard Gene Therapy Initiative, Boston MA, USA Hepatic cirrhosis is a widespread Public Health problem. Represents the uniform response of the liver to toxic or infectious agents and is characterized by an increase in synthesis and deposition of extracellular matrix proteins (ECM) mainly collagen type I. Physiologically, collagen degradation is performed by interstitial collagenases, so-called metalloproteases. An extrahepatic human neutrophil collagenase eDNA (MMP-8) cloned in an Ad-vector was used as therapeutic agent. A high titer of clinicalgrade AdMMP-8 was obtained from 293 cells. HeLa cells transduced with AdMMP-8 expressed recombinant MMP-8 mRNA. MMP-8 protein was detected by ELISA in cell lysate and culture supernatant and was increased 1,300-fold as compared with mock-transduced control cells. Also, MMP-8 in culture sups showed enzymatic activity against native type I collagen which was inhibited by EDTA, 1,10-phenanthroline and TIMP-1. In vivo studies to establish efficacy of this characterized vector were carded out in severely CCl4-cirrhotic rats. A variable but consistent response in fibrosis reversal (30-60%) with 1 x 1011 vp/kg, correlated with improvement in ascitis, functional hepatic tests and gastric varices indicating a diminished intrahepatic blood pressure in animals injected with AdMMP-8. Therefore, therapy with MMP-8 gene possess promise to be used in a clinical setting.
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ESTROGENS INDUCE CHOLANGIOCYTE PROLIFERATION THROUGH THE ACTIVATION OF THE ERK1/2 SYSTEM AND THE ADAPTER PROTEINS Sch, AND Src
Domenico Alvaro 1, Gianfranco Alpini3, Paolo Onori2, Antonio Franchitto 2, Shannon Glaser 3, Gene Le Sage 3, Alessandro Gigliozzi 1, Adolfo Attili 1, Gianluca Svegliati Baronigaudio 4, Veronica Drudi Metalli 1, Maria Grazia Mancino 1, Eugenio Gaudio 3.
1Department of Clinical Medicine Division of Gastroenterology University of Rome La Sapienza, Rome; 2Departmentof Anatomy University of Rome La Sapienza, Rome; 4Chairof Gastroenterology, University of Ancona, Ancona, Italy; 3Dept. of lnternal Medicine, Medical Physiology, Scott & WhiteHospital and The TexasA&M University System HSC, COM and Central Texas VeteransHealth Care System, Temple, Texas, USA We have recently shown that the intrahepatic rat cholangiocytes express estrogen receptors and that estrogens stimulate in vitro cholangiocyte proliferation. The AIM of this study was to investigate the intracellular signaling pathways and, specifically, the involvement of tyrosine kinase receptor pathways. We evaluated the protein expression of total and phosphorylated MAP kinase isoform ERKI/2 and the adapter proteins She and Src. The study was performed in bile duct ligated (BDL) rats, BDL rats treated with the antiestrogen, tamoxifen and normal control rats. Immunohistochemistry and western blot analyses were performed by using a tyrosinethreonine diphosphorylated or total ERK antibody and a polyclonal She and Src antibodies. Results: Cholangiocyte proliferation induced by BDL was associated with a marked immunohistochemical nuclear positivity for phosphorylated ERK1/2. The protein expression (western blot) of total and phosphorylated ERK1/2, Sch and Src in cholangiocytes isolated from BDL rats was markedly increased compared to controls, but was inhibited by tamoxifen. In vitro, cholangiocyte proliferation induced by beta-estradiol was associated with increased protein expression of phosphorylated ERK1/2, She and Src. Inhibitors of estrogen receptors, of Src and MAP kinases blocked the cholangiocyte proliferation induced in vitro by beta-estradiol. In conclusion, the tyrosine kinase receptor pathway is activated during cholangiocyte proliferation induced by BDL and is inhibited by anti-estrogens. This might suggests that estrogens stimulate cholangiocyte proliferation by synergizing the effects of growth factors and by acting through similar intracellular pathways.
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ALTERATIONS OF HEPATOBILIARY TRANSPORTER EXPRESSION IN PRIMARY BILIARY CIRRHOSIS (PBC)
Gernot Zollner 1, Peter Fickert 1, Dagmar Silbert 1, Andrea Fuchsbichler 2, Conny Stumptner 2, James Neuberger 3, Kurt Zatloukal 2, Helmut Denk 2, Michael Trauner 1. ~Division of Gastroenterology and Hepatology,
Department of Medicine, Karl-Franzens University Graz; 2Institute of Pathology, Karl-Franzens University Graz, Austria; 3Liver Unit, Queen Elisabeth Hospital, Birmingham, UK Background: Little is known about alterations of transporter expression in different stages of PBC. Such data could provide further insights in the pathogenesis of human cholestatic liver diseases. Aim: To determine hepatobiliary transporter expression in various stages of PBC. Materials & Methods: Liver tissue was obtained from patients with PBC histological (Ludwig) stage II (n = 3), III (n = 3) and IV (n = 7) and from controls (n = 15). Transporter expression was assessed by Western analysis and competitive RT-PCR. Results: OATP2 protein expression was reduced in PBC II (61%, p < 0.01), III (59%, p < 0.01) and IV (54%, p < 0.01), whereas reduction of NTCP protein occurred only in PBC IV (48%, p < 0.01). P-glycoprotein expression was up-regulated in PBC II (337%, p < 0.001) and PBC III (275%, p < 0.01). A trend towards increased expression was also observed in PBC IV. MRP3 protein levels were elevated in PBC III and IV (292%,
Saturday, April 20, 2002 -] TRUNCATED TYPE II TGF-BETA RECEPTOR PROMOTES LIVER REGENERATION IN DIMETYLNITROSAMIN-TREATED CIRROHOTIC RATS
Tom Nakamura 1,2, Ryuichiro Sakata 1,2, Masaharu Sakamoto 1,2, Takuji Torimura 1,2 Takato Ueno 1,2, Michio Sata L2 1Liver Division,
Research Centerfor Innovative Cancer Therapy, Kurume University Kurume; 22nd Department of lnternal Medicine, Kurume UniversityScool of Medicine, Kurume, Japan We investigated whether anti-TGF-beta intervention can enhance liver regeneration or not. To block TGF-beta action, we prepared an adenovirus expressing a truncated type II TGF-beta receptor (AdTbeta-TR). We also used an adenovirus expressing bacterial beta-galactosidase (AdLacZ). Rats were first treated with dimethylnitrosamine (DMN) for 3 weeks. Then, vectors or saline were intravenously applied only at once from the tail vein. Following the gene transfer, DMN administration was continued for additional 3 weeks. The liver tissues were extracted at 4 and 6 weeks after DMN administration, and liver regeneration was confirmed. Namely, proliferating cells were semiquantitated by immunohistochemistry using an anti-Ki-67 antibody, and conversely apoptosis of the hepatocyte was measured by immunohistochemistry using an anti-digoxygenin antibody. Biochemical parameters were measured by standard methods, and body weight and survival rate were monitored dally. All AdTbeta-TR-treated rats remained alive, whereas almost all the DMN-treated rats infused either AdLacZ or saline died. Moreover, body weights of rats treated with AdTbeta-TR were not decreased compared with either AdLacZ or saline. Serum total bilirubin, total protein, and albumin levels in the AdThetaTR-treated rats were preserved at the level in rats treated with DMN for only 3 weeks. Ki-67 labeling index in AdTheta-TR-treated livers (10.1 42.4%) increased compared with that in AdLacZ-treated livers (2.3 4- 1.0%) at 6 weeks after DMN administration. In contrast, apoptosis in AdTbetaTR-treated livers was inhibited at 4 weeks after DMN administration. Our results suggest that anti-TGF-beta intervention facilitates liver regeneration in DMN-treated rats.
~-]
TREATMENT WITH HUMAN METALLOPROTEINASE-8 GENE DELIVERY AMELIORATES EXPERIMENTAL RAT LIVER CIRRHOSIS RESEMBLING ALCOHOLIC HUMAN DISEASE
Fernando Siller I , Silvia Salgado l, Jesus Garcia 1, Jose M. Vera ] , Alejandra Miranda 1, Guillermo Grijalva 1, Javier Galvez 1, Estuardo Aguilar-Cordova 2, Juan Arrnendariz-Borunda l . llnstituto de
Biologia Moleculary Terapia Gdnica, CUCS, Universidad de Guadalajara, Guadalajara, Mexico; 2Harvard Gene Therapy Initiative, Boston MA, USA Hepatic cirrhosis is a widespread Public Health problem. Represents the uniform response of the liver to toxic or infectious agents and is characterized by an increase in synthesis and deposition of extracellular matrix proteins (ECM) mainly collagen type I. Physiologically, collagen degradation is performed by interstitial collagenases, so-called metalloproteases. An extrahepatic human neutrophil collagenase eDNA (MMP-8) cloned in an Ad-vector was used as therapeutic agent. A high titer of clinicalgrade AdMMP-8 was obtained from 293 cells. HeLa cells transduced with AdMMP-8 expressed recombinant MMP-8 mRNA. MMP-8 protein was detected by ELISA in cell lysate and culture supernatant and was increased 1,300-fold as compared with mock-transduced control cells. Also, MMP-8 in culture sups showed enzymatic activity against native type I collagen which was inhibited by EDTA, 1,10-phenanthroline and TIMP-1. In vivo studies to establish efficacy of this characterized vector were carded out in severely CCl4-cirrhotic rats. A variable but consistent response in fibrosis reversal (30-60%) with 1 x 1011 vp/kg, correlated with improvement in ascitis, functional hepatic tests and gastric varices indicating a diminished intrahepatic blood pressure in animals injected with AdMMP-8. Therefore, therapy with MMP-8 gene possess promise to be used in a clinical setting.
~
ESTROGENS INDUCE CHOLANGIOCYTE PROLIFERATION THROUGH THE ACTIVATION OF THE ERK1/2 SYSTEM AND THE ADAPTER PROTEINS Sch, AND Src
Domenico Alvaro 1, Gianfranco Alpini3, Paolo Onori2, Antonio Franchitto 2, Shannon Glaser 3, Gene Le Sage 3, Alessandro Gigliozzi 1, Adolfo Attili 1, Gianluca Svegliati Baronigaudio 4, Veronica Drudi Metalli 1, Maria Grazia Mancino 1, Eugenio Gaudio 3.
1Department of Clinical Medicine Division of Gastroenterology University of Rome La Sapienza, Rome; 2Departmentof Anatomy University of Rome La Sapienza, Rome; 4Chairof Gastroenterology, University of Ancona, Ancona, Italy; 3Dept. of lnternal Medicine, Medical Physiology, Scott & WhiteHospital and The TexasA&M University System HSC, COM and Central Texas VeteransHealth Care System, Temple, Texas, USA We have recently shown that the intrahepatic rat cholangiocytes express estrogen receptors and that estrogens stimulate in vitro cholangiocyte proliferation. The AIM of this study was to investigate the intracellular signaling pathways and, specifically, the involvement of tyrosine kinase receptor pathways. We evaluated the protein expression of total and phosphorylated MAP kinase isoform ERKI/2 and the adapter proteins She and Src. The study was performed in bile duct ligated (BDL) rats, BDL rats treated with the antiestrogen, tamoxifen and normal control rats. Immunohistochemistry and western blot analyses were performed by using a tyrosinethreonine diphosphorylated or total ERK antibody and a polyclonal She and Src antibodies. Results: Cholangiocyte proliferation induced by BDL was associated with a marked immunohistochemical nuclear positivity for phosphorylated ERK1/2. The protein expression (western blot) of total and phosphorylated ERK1/2, Sch and Src in cholangiocytes isolated from BDL rats was markedly increased compared to controls, but was inhibited by tamoxifen. In vitro, cholangiocyte proliferation induced by beta-estradiol was associated with increased protein expression of phosphorylated ERK1/2, She and Src. Inhibitors of estrogen receptors, of Src and MAP kinases blocked the cholangiocyte proliferation induced in vitro by beta-estradiol. In conclusion, the tyrosine kinase receptor pathway is activated during cholangiocyte proliferation induced by BDL and is inhibited by anti-estrogens. This might suggests that estrogens stimulate cholangiocyte proliferation by synergizing the effects of growth factors and by acting through similar intracellular pathways.
[-~
ALTERATIONS OF HEPATOBILIARY TRANSPORTER EXPRESSION IN PRIMARY BILIARY CIRRHOSIS (PBC)
Gernot Zollner 1, Peter Fickert 1, Dagmar Silbert 1, Andrea Fuchsbichler 2, Conny Stumptner 2, James Neuberger 3, Kurt Zatloukal 2, Helmut Denk 2, Michael Trauner 1. ~Division of Gastroenterology and Hepatology,
Department of Medicine, Karl-Franzens University Graz; 2Institute of Pathology, Karl-Franzens University Graz, Austria; 3Liver Unit, Queen Elisabeth Hospital, Birmingham, UK Background: Little is known about alterations of transporter expression in different stages of PBC. Such data could provide further insights in the pathogenesis of human cholestatic liver diseases. Aim: To determine hepatobiliary transporter expression in various stages of PBC. Materials & Methods: Liver tissue was obtained from patients with PBC histological (Ludwig) stage II (n = 3), III (n = 3) and IV (n = 7) and from controls (n = 15). Transporter expression was assessed by Western analysis and competitive RT-PCR. Results: OATP2 protein expression was reduced in PBC II (61%, p < 0.01), III (59%, p < 0.01) and IV (54%, p < 0.01), whereas reduction of NTCP protein occurred only in PBC IV (48%, p < 0.01). P-glycoprotein expression was up-regulated in PBC II (337%, p < 0.001) and PBC III (275%, p < 0.01). A trend towards increased expression was also observed in PBC IV. MRP3 protein levels were elevated in PBC III and IV (292%,