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protein expression in the liver in patients with chronic diseases
418A
AASLD ABSTRACTS
HEPATOLOGYOctober 1995
1245 CHARACTERIZATION OF TGF-fll OVEREXPRESSING RAT
1246 SERUM LEVEL OF THROMBOMODULIN ('I'M) AND MRNA/PROTEIN
HEPATOMA CELLS. E. Fuchs. A.M. Crressner and O.H. Wciuer. Dept. of Clinical Chemistry and Central Laboratory, Philipps-University, 35033 Marburg, F.R.G.
EXPRESSION IN ~ LIVER IN PATIENTS WITH CHRONIC LIVER DISEASES. H Fukata, Y Aizawa. H Takahashi, F Watanabe, M Zeni~'a and G Toda. Internal medicine(I), The JIKEI University Sebool of Medicine, Tokyo
The transforming growth factor-51 (TGF-I~1) is involved in the transformation of hepatic stellate cells into myofibroblasts and induction of hepatoeyte apoptosis. Although genc expression of TGF-131 and 2 is detectable in isolated rat fiver hepatoeytes only little is known about the protein expression and the resulting functional consequences for this call type. Therefore we overexpress TGF-B1 in rat hepatoma ceils (FAO) and analyse the effects. Methods: FAO ceUs were transfeeted with an ¢pisomal expression vector harbouring the complete human TGF-fll coding sequence under the control of a CMVpromoter. TGF-I~ I overproducing cell lines were obtained.after hygromycin B seleetinn and analyzed by R.T-PCR, ELISA, Mvl-Lu-cell bioassay and immanfluorescence analysis. As a control untrausfected and moc& transfected cells were used. Results: TGF-131 overproducing cell lines expressed large amounts o f TGF-131 mRNA, whereas the message was only barely detectable in antransfected cells. The generated TGF-fll mRNA is translated and the protein is released into the culture medium. The culture medium of the transfected cells contained 6 ng/rrd (controls: 0 to 5 pg/ml) nonactivated, intent and 40-60 pg/ml (controls: 0 pg/ml) activated TGF&I. The latent TGF-131 was funetioanlly active ai~.er acidificarion. Cellbiolngieal eharaoterization of the transfectants showed that the proliferation rate is reduced by about 30%. They have reduced TGF-132 and TGF-I~ type II rocesRor mRNA levels, whereas the amount of TGF-B type IIl r e c i t e r mRNA (betaglycan) was elevated. A characteristic feature of the TGF-IH overproducing cell line was their altered aggregation behavior, because the transfected calls had a strong tendency to form aggregates which was not observed in control cells. Conclusion: We could show that TGF-B1 overproduetien in rat hepatoma calls alters the expression of TGF-13 isoforms, TGF-I~ recqymrs and call-cell or ceil-matrix adhesion molecules.
Background: Thrombomodulin (TM) is a thrombin-binding protein which suppress the coagulatory action of thrombin. Serum level of TM is reported to be elevated in various collagen diseases and an indicator of endothelial cell injury. However, the serum TM level and the modulation of TM expression in the liver in chronic liver diseases have not been reported. Aim: To clarify the involvment of TM in pathophysiology of chronic liver diseases, we examined the serum level and gene/protein expression of TM in patients with chronic liver diseases. Subjects and methods: Subjects were 102 cases of chronic viral hepatitis (CH),12 cases of liver eirthosis (LC) and 8 cases of hepatocellular carcinoma (HCC): Serum level of TM was analysed by ELISA (Mitobishi-gas;Japan). The TM-mRNA expression was examined by northern hybridization. 10,o.g each of total RNA was electrophoresed and transfered to nylon membrane. After hybridization, the radioactivity was quantified by image analyzer (BAS 2000, Fuji; Japan). The quanlity of TM-mRNA was expressed as the ratio of radioactivity of normal liver. As a control, the level of GAPDH-mRNA was used. The TM-protein expression in the liver tissue was examined histuebemically. The TM-protein expression was estimated semiquantitativaly as - to 3+ according to the intensity of the staining. Results: SertmaTM is significantly higher in patients with CH (26.0+10.6ng/ml: n=64) and LC( 44.75: 10.lng/ml: n= 12) than in normal control (19.6 5: 3.5ng/ml: n=9; P<0.001). The expression of TM-mRNA was significantly higher hi LC group than in CH group (3.65:1.3:n=4 vs 1.5 5:1.6:n=16; P<0.05), and the level of control GAPDH-mRNA was almost the same. ]'he TM-protein was found mainly in sinusoidal endothelial cell in the liver tissue. Strong (3+) expression of TM-protein in sinusoidal endothelial call was observed in 35 out of 102 patients with CH and in one out of 8 patients with LC. In addition, weak (1+) expression of TM in the cytosol of tumor cell was observed in 2 out of 8 patients with HCC. There was no relation between the serum TM and ALT. Moreover, neither the intensity of TM staining nor the serum level of TM correlated with the histological grading of inflammation. Conalusian: Serum level of 'I'M increases according to the progression of liver disease with no relation to the inflammatory activity . Increased expression of TM-mRNA and TM-protein in chronic liver diseases may indicate that the serum TM is originated at least partially by the enhanced production of TM in sinusoidal endothelial cell.
1247 Liver transplantation for Budd-Chiari syndrome: current survival and incidence of recurrence. H Furukawa. S Todo. JJ Fun~. TE Starzl. Pittsburgh Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA. From 1974 to 1994, 46 patients underwent liver transplantation for the Budd-Chiari syndrome (BCS). The diagnosis was confirmed by post resection pathologic examination in all patients. The age at the time of transplantation ranged from 4 month to 70 years with a median age of 30 years. Thirteen patients were male and 33 were female. The major underlying etiologies included myeloproliferative disorder (12), paroxysmal nocturnal hemoglobinuria (2), oral contraceptive (8), Lupus anticoagulants (3). The cause in the remaining 21 (45%) was cryptoganie. One-, 3-, and 5year actuarial patient survival was 77.8%, 65.6%, and 62.8%, respectively. Main causes of death were sepsis, multiorgan failure and pneumonia. One-, 3-,and 5-year actuarial graft survival was 63.2%, 53.6% and 49.3%, respectively. Main reason for retransplantation were hepatic artery thrombosis, primary non-function and recurrence of BcS. Recurrence of BCS was found in 5 patients (11%). Two patients had an episode of subtherapeutic level of anticoagulation and one patient discontinued anticoagulation. Recurrence occurred 1 t o 6 years (median: 3 years) after liver transplantation. Two patients died, 2 patients had retransplantation and 1 patient has been on palliative therapy. Liver transplantation is the most effective treatment for the patients with end stage liver disease secondary to BCS. Anticoagulation is essential to avoid the recurrence of BCS.
1248 EXPRESSION OF METALLOPANSTIMULIN IN LIVER NEOPLASIA. DR GANGER'. DJ SANTA CRUZz. PD HAMILTON3. DJ KLOS3~.__G_G KOUKOULIS4 and JA FERNANDEZ POL~ ~Laboratory of Molecular Oncology, DVA Medical Center, St. Louis University, 2 Dept of Pathology, Saint John's Mercy Medical Menter, St. Louis, MO, ~Section of Hepatology and 4Dept of Pathology, Rush Medical College, Chicago, IL. MetaUopansfimulin (MPS) is a 10-kDal "zinc fmger"protein which is expressed in a wide variety of actively proliferating cells and tumor tissues (Cell Growth and Diff. 5:811,1994). Obleetlve: To study the distribution of MPS in normal fiver, fiver of patients with chronic fiver disease and hepatoeefiular carcinoma (HCC). Materinl and Methods: Tissues were obtained at the time of liver resection, biopsy or transplantation. MPS protein antigen was detected with anti-MPS antibodies using the Avidiue/Biotiue/Chromogen technique. Results: Normal hepatecytes show weak immuanstaining. In regenerating fiver nodules there is a gradient of MPS expression with pronounced staining at the periphery. Hepatocytes in the eentrilobular areas show little staining. While some nodules stain intensely, others have variable expression of MPS antigen. This may correlate with intralesional transformation. The staining pattern of HCC shows intense staining with perinnclear distribution of MPS in most instances studied. Summar3,: MPS is expressed in normal fiver. Interestingly, MPS expression is enhanced in regenerating cirrhotic nodules and is expressed very strongly in HCC. Condusion: MPS is expressed in areas of active proliferation and with remarkable intensity in HCC.
AASLD ABSTRACTS
HEPATOLOGYOctober 1995
1245 CHARACTERIZATION OF TGF-fll OVEREXPRESSING RAT
1246 SERUM LEVEL OF THROMBOMODULIN ('I'M) AND MRNA/PROTEIN
HEPATOMA CELLS. E. Fuchs. A.M. Crressner and O.H. Wciuer. Dept. of Clinical Chemistry and Central Laboratory, Philipps-University, 35033 Marburg, F.R.G.
EXPRESSION IN ~ LIVER IN PATIENTS WITH CHRONIC LIVER DISEASES. H Fukata, Y Aizawa. H Takahashi, F Watanabe, M Zeni~'a and G Toda. Internal medicine(I), The JIKEI University Sebool of Medicine, Tokyo
The transforming growth factor-51 (TGF-I~1) is involved in the transformation of hepatic stellate cells into myofibroblasts and induction of hepatoeyte apoptosis. Although genc expression of TGF-131 and 2 is detectable in isolated rat fiver hepatoeytes only little is known about the protein expression and the resulting functional consequences for this call type. Therefore we overexpress TGF-B1 in rat hepatoma ceils (FAO) and analyse the effects. Methods: FAO ceUs were transfeeted with an ¢pisomal expression vector harbouring the complete human TGF-fll coding sequence under the control of a CMVpromoter. TGF-I~ I overproducing cell lines were obtained.after hygromycin B seleetinn and analyzed by R.T-PCR, ELISA, Mvl-Lu-cell bioassay and immanfluorescence analysis. As a control untrausfected and moc& transfected cells were used. Results: TGF-131 overproducing cell lines expressed large amounts o f TGF-131 mRNA, whereas the message was only barely detectable in antransfected cells. The generated TGF-fll mRNA is translated and the protein is released into the culture medium. The culture medium of the transfected cells contained 6 ng/rrd (controls: 0 to 5 pg/ml) nonactivated, intent and 40-60 pg/ml (controls: 0 pg/ml) activated TGF&I. The latent TGF-131 was funetioanlly active ai~.er acidificarion. Cellbiolngieal eharaoterization of the transfectants showed that the proliferation rate is reduced by about 30%. They have reduced TGF-132 and TGF-I~ type II rocesRor mRNA levels, whereas the amount of TGF-B type IIl r e c i t e r mRNA (betaglycan) was elevated. A characteristic feature of the TGF-IH overproducing cell line was their altered aggregation behavior, because the transfected calls had a strong tendency to form aggregates which was not observed in control cells. Conclusion: We could show that TGF-B1 overproduetien in rat hepatoma calls alters the expression of TGF-13 isoforms, TGF-I~ recqymrs and call-cell or ceil-matrix adhesion molecules.
Background: Thrombomodulin (TM) is a thrombin-binding protein which suppress the coagulatory action of thrombin. Serum level of TM is reported to be elevated in various collagen diseases and an indicator of endothelial cell injury. However, the serum TM level and the modulation of TM expression in the liver in chronic liver diseases have not been reported. Aim: To clarify the involvment of TM in pathophysiology of chronic liver diseases, we examined the serum level and gene/protein expression of TM in patients with chronic liver diseases. Subjects and methods: Subjects were 102 cases of chronic viral hepatitis (CH),12 cases of liver eirthosis (LC) and 8 cases of hepatocellular carcinoma (HCC): Serum level of TM was analysed by ELISA (Mitobishi-gas;Japan). The TM-mRNA expression was examined by northern hybridization. 10,o.g each of total RNA was electrophoresed and transfered to nylon membrane. After hybridization, the radioactivity was quantified by image analyzer (BAS 2000, Fuji; Japan). The quanlity of TM-mRNA was expressed as the ratio of radioactivity of normal liver. As a control, the level of GAPDH-mRNA was used. The TM-protein expression in the liver tissue was examined histuebemically. The TM-protein expression was estimated semiquantitativaly as - to 3+ according to the intensity of the staining. Results: SertmaTM is significantly higher in patients with CH (26.0+10.6ng/ml: n=64) and LC( 44.75: 10.lng/ml: n= 12) than in normal control (19.6 5: 3.5ng/ml: n=9; P<0.001). The expression of TM-mRNA was significantly higher hi LC group than in CH group (3.65:1.3:n=4 vs 1.5 5:1.6:n=16; P<0.05), and the level of control GAPDH-mRNA was almost the same. ]'he TM-protein was found mainly in sinusoidal endothelial cell in the liver tissue. Strong (3+) expression of TM-protein in sinusoidal endothelial call was observed in 35 out of 102 patients with CH and in one out of 8 patients with LC. In addition, weak (1+) expression of TM in the cytosol of tumor cell was observed in 2 out of 8 patients with HCC. There was no relation between the serum TM and ALT. Moreover, neither the intensity of TM staining nor the serum level of TM correlated with the histological grading of inflammation. Conalusian: Serum level of 'I'M increases according to the progression of liver disease with no relation to the inflammatory activity . Increased expression of TM-mRNA and TM-protein in chronic liver diseases may indicate that the serum TM is originated at least partially by the enhanced production of TM in sinusoidal endothelial cell.
1247 Liver transplantation for Budd-Chiari syndrome: current survival and incidence of recurrence. H Furukawa. S Todo. JJ Fun~. TE Starzl. Pittsburgh Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA. From 1974 to 1994, 46 patients underwent liver transplantation for the Budd-Chiari syndrome (BCS). The diagnosis was confirmed by post resection pathologic examination in all patients. The age at the time of transplantation ranged from 4 month to 70 years with a median age of 30 years. Thirteen patients were male and 33 were female. The major underlying etiologies included myeloproliferative disorder (12), paroxysmal nocturnal hemoglobinuria (2), oral contraceptive (8), Lupus anticoagulants (3). The cause in the remaining 21 (45%) was cryptoganie. One-, 3-, and 5year actuarial patient survival was 77.8%, 65.6%, and 62.8%, respectively. Main causes of death were sepsis, multiorgan failure and pneumonia. One-, 3-,and 5-year actuarial graft survival was 63.2%, 53.6% and 49.3%, respectively. Main reason for retransplantation were hepatic artery thrombosis, primary non-function and recurrence of BcS. Recurrence of BCS was found in 5 patients (11%). Two patients had an episode of subtherapeutic level of anticoagulation and one patient discontinued anticoagulation. Recurrence occurred 1 t o 6 years (median: 3 years) after liver transplantation. Two patients died, 2 patients had retransplantation and 1 patient has been on palliative therapy. Liver transplantation is the most effective treatment for the patients with end stage liver disease secondary to BCS. Anticoagulation is essential to avoid the recurrence of BCS.
1248 EXPRESSION OF METALLOPANSTIMULIN IN LIVER NEOPLASIA. DR GANGER'. DJ SANTA CRUZz. PD HAMILTON3. DJ KLOS3~.__G_G KOUKOULIS4 and JA FERNANDEZ POL~ ~Laboratory of Molecular Oncology, DVA Medical Center, St. Louis University, 2 Dept of Pathology, Saint John's Mercy Medical Menter, St. Louis, MO, ~Section of Hepatology and 4Dept of Pathology, Rush Medical College, Chicago, IL. MetaUopansfimulin (MPS) is a 10-kDal "zinc fmger"protein which is expressed in a wide variety of actively proliferating cells and tumor tissues (Cell Growth and Diff. 5:811,1994). Obleetlve: To study the distribution of MPS in normal fiver, fiver of patients with chronic fiver disease and hepatoeefiular carcinoma (HCC). Materinl and Methods: Tissues were obtained at the time of liver resection, biopsy or transplantation. MPS protein antigen was detected with anti-MPS antibodies using the Avidiue/Biotiue/Chromogen technique. Results: Normal hepatecytes show weak immuanstaining. In regenerating fiver nodules there is a gradient of MPS expression with pronounced staining at the periphery. Hepatocytes in the eentrilobular areas show little staining. While some nodules stain intensely, others have variable expression of MPS antigen. This may correlate with intralesional transformation. The staining pattern of HCC shows intense staining with perinnclear distribution of MPS in most instances studied. Summar3,: MPS is expressed in normal fiver. Interestingly, MPS expression is enhanced in regenerating cirrhotic nodules and is expressed very strongly in HCC. Condusion: MPS is expressed in areas of active proliferation and with remarkable intensity in HCC.