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P-165 Expression of hepatocyte growth factor (HGF) and HGF activator in carbon tetrachloride-induced acute hepatic injury
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Posters/International Hepatoiogy Communications 3 SuppL (1995) $37-S169
P-165 EXPRESSION OF HEPATOCYTE GROWTH FACTOR (HGF) AND HGF ACTIVATOR IN CARBON TETRACHLORIDE-INDUCED ACUTE HEPATIC INJURY Y.Ishinoda,1 R.Matsushita,1 T.Etoh, l S.Hirono,1 K.KomuraJ T.Kitamura,1 K.Hayashi,1 T.Maruyama,] K.Miyazawa,2 N.Kitamura,2 H.Tsubouchfl 12nd Dept. of Internal Medicine, Miyazaki Medical College, Miyazaki, and 2Institute for Liver Research, Kansai Medical University, Osaka, Japan Recently, an HGF activator which processes HGF activation from the HGF precurssor (pro-HGF) to mature HGF has been purified. Herein, we examined the mRNA levels of HGF and HGF activator in various organs in normal and carbon tetrachloride (CC14)-treated Japanese monkeys by Northern blot analysis. Serum HGF levels of CCl4-treated monkeys markedly increased 12 hours after CC14 administration and reached a maximum at 24 hours. HGF protein and HGF mRNA levels were markedly increased only in the liver and spleen in parallel with the changes of serum HGF level, but not increased in the kidneys or lungs. HGF activator mRNA were detected only in the liver of both normal and CC14-treated monkeys, but not in the spleen, kidneys or lungs. The HGF activator mRNA level was increased in the monkey liver as early as 6 hours after CC14 administration, and then decreased and reached the minimum level at 24 hours. These findings indicate that the activity of HGF activator in the serum or tissue should be examined to clarify the mechanism of liver regeneration regulated by HGF.
P-167 HEPATOCYTE PROLIFERATION INDUCED IN RATS BY LEAD NITRATE IS SUPPRESSED BY SEVERAL TUMOR NECROSIS FACTOR-a INHIBITORS Y. Kubo ~, M. Yasunaga ~, K. Tanigawa ~, O. Tamura ~, S. Wasakf, S. Terai 1, M. Masuhara', [. Sakaida ~, T. Nakamura z, K. OkRa' 1The 1st Dept. of Int. Med., Yamaguchi Univ. School of Med., Yamagnchi, Japan 2Division of Biochemistry, Biomedical Research Center, Osaka Univ. school of Med., Osaka, Japan Lead nitrate induces liver cell proliferation in rats without accompanying liver cell necrosis. However, proliferative mechanism and effect on hepatocyte itself is still unknown. Therefore, our initial experiments were designated to determine whether lead nitrate modifies hepatocyte proliferation and alter the production of hepatocyte growth factor and tumor necrosis factor-a by examining both liver and blood level of these cytokines at various time periods. Finally, several tumor necrosis f a c t o r - a inhibitors were administered to rats. Hepatocyte proliferation occurred 24 hours and reached a peak 48 hours after a single intravenous injection of lead nitrate ( 1 0 0 m M / K g ) , and tumor necrosis f a c t o r - a mRNA expression in the liver was enhanced 1, 6 and 12 hours after the injection. However, no alteration was observed in the liver or blood level of hepatocyte growth factor. Pretreatment with dexamethasone (4.0 mg/Kg), E3330 (100 mg/Kg) adenosine (0.3 mM/Kg) and pentoxifylline (100mg/Kg) inhibited both tumor necrosis f a c t o r - a mRNA expression and hepatocyte proliferation 48 hours after the injection, These experimental results strongly support the hypothesis that tumor necrosis f a c t o r - a positively conduct and regulate hepatocyte proliferation induced in rats by direct mitogen, lead nitrate.
P-166
THE ROLEOF bFGF IN HEPATIC
REGENERATION AFTER ISCHEMIA REPERFUSION INJURY IN RATS S. Kawachi, M. Shimazu, G. Wakabayashi, N. Shirasugi, Y. Kumamoto, T. Karahashi, M. Yoshida, M. Kitajima Dept. of Surgery, Keio University School of Medicine, Tokyo,
Japan bFGF is the potent angiogenedc factor and plays an important role in remodeling of post ischemic neuronal damage and salvaging of infarcted myocardium by its angiogenic action. We examined synthesis and localilzation of bFGF to study the role of bFGF in hepatic regeneration after ischemia repeffusion injury. [Methods] Partial warm ischemia reperfusion was induced by clamping and de,clamping of afferent vessels of the median and left lateral lobes of the rat liver. Livers were removed at the indicated times after reperfusion and homogenized, bFGF levels were mesured in both of damaged and non damaged liver tissue homogenate by enzyme immunoassay. Using anti-bFGF monoclonal antibody, immunohistochemical staining was also performed in regenerating liver. [Results] On the 4th day after reperfusion, bFGF levels of damaged liver tissue were increased significantly as compared with those of non damaged and normal liver tissue. Histologically, intensive regenerating change was observed and immunohistochemical reaction was show only in damaged liver on the 4th day after reperfusion. [Conclusion] Those data suggested that bFGF was synthesized after ischemia reperfusion injury and might play an important role in hepatic regeneration.
P-168 ROLE OF HEPARIN-BINDING EGF-LIKE GROWTH FACTOR (HB-EGF) IN RAT LIVER REGENERATION :CELL SPECIFICITY OF HB-EGF GENE EXPRESSION AND INDUCTION OF HGF AND TGF-a GENE EXPRESSION BY HB-EGF S.Kiso 1, S.Kawata1, S.Tamura 1, N.Ito 1, H.Tsushima ~, O.Oshikawa1, S.Higashiyama 2, N.Taniguchi 2, Y. Matsuzawa1 1 2nd Dept. of nt Med., ~ Dept. of Biochem., Osaka Univ. Med. Sch., Osaka 565, Japan Heparin-binding EGF-like growth factor (HB-EGF) is anew member of the EGF family that was initially purified from conditioned medium of U-937 macrophage-like cells. Recently, we reported that HB-EGF has hepatotrophic effects in vitro, and that the level of HB-EGF mRNA in regenerating rat liver increased 1.5 h after 70% partial hepatectomy and reached a maximum (about 7-fold over normal) at 6 h. In this study we investigated that the cell specificity of HB-EGF ~ene expression expression of HB-EGF protein in regenerating rat hver, and induction of HGF and TGF-a gene express on by HB-EGF. MATERIALS AND METHODS (1)Male S-D rats were partially hepatectomized according to the method of Higgins and Anderson. (2)Hepatocyte and non-parenchymal liver cells (NPC) were isolated bKYuinsitu perfusion with collagenase. (3)Sinusoidal endothelial cells, pffer cells and lipocytes were isolated with a JE-6 elutdation rotor (Beckman). HB-EGF gene expression in these cells was investigated by RT-PCR. (4)HB-EGF protein expression in the liver of normal and hepatectomized rats was investigated by Western blot analysis. (5)HB-EGF(30ng/ml)was added to the separated hepatocytes and NPC in primary culture and HGF and TGF-c( gene expression in these cells were investigated by Northern blot analysis. RESULTS (1)RT-PCR showed HB-EGF gene expression predominantly in Kupffer cells and sinusoidal endothelial cells but not in lipocytes andhepatocytes. (2)HB-EGF protein expression increased about 2.8-fold over normal liver at 10 h after partial hepatectomy. (3)Marked HGF and TGF-(~ gene expression were induced by HB-EGF in NPC and in hepatocytes respectively. In the remmnant liver after partial hepatectomy, HB-EGF may participate in regulation of the expression of HGF and TGF-a gene. CONCLUSION HB-EGF may play an important role in liver regeneration after partial hepatectomy v/a a paracrine mechanism.
Posters/International Hepatoiogy Communications 3 SuppL (1995) $37-S169
P-165 EXPRESSION OF HEPATOCYTE GROWTH FACTOR (HGF) AND HGF ACTIVATOR IN CARBON TETRACHLORIDE-INDUCED ACUTE HEPATIC INJURY Y.Ishinoda,1 R.Matsushita,1 T.Etoh, l S.Hirono,1 K.KomuraJ T.Kitamura,1 K.Hayashi,1 T.Maruyama,] K.Miyazawa,2 N.Kitamura,2 H.Tsubouchfl 12nd Dept. of Internal Medicine, Miyazaki Medical College, Miyazaki, and 2Institute for Liver Research, Kansai Medical University, Osaka, Japan Recently, an HGF activator which processes HGF activation from the HGF precurssor (pro-HGF) to mature HGF has been purified. Herein, we examined the mRNA levels of HGF and HGF activator in various organs in normal and carbon tetrachloride (CC14)-treated Japanese monkeys by Northern blot analysis. Serum HGF levels of CCl4-treated monkeys markedly increased 12 hours after CC14 administration and reached a maximum at 24 hours. HGF protein and HGF mRNA levels were markedly increased only in the liver and spleen in parallel with the changes of serum HGF level, but not increased in the kidneys or lungs. HGF activator mRNA were detected only in the liver of both normal and CC14-treated monkeys, but not in the spleen, kidneys or lungs. The HGF activator mRNA level was increased in the monkey liver as early as 6 hours after CC14 administration, and then decreased and reached the minimum level at 24 hours. These findings indicate that the activity of HGF activator in the serum or tissue should be examined to clarify the mechanism of liver regeneration regulated by HGF.
P-167 HEPATOCYTE PROLIFERATION INDUCED IN RATS BY LEAD NITRATE IS SUPPRESSED BY SEVERAL TUMOR NECROSIS FACTOR-a INHIBITORS Y. Kubo ~, M. Yasunaga ~, K. Tanigawa ~, O. Tamura ~, S. Wasakf, S. Terai 1, M. Masuhara', [. Sakaida ~, T. Nakamura z, K. OkRa' 1The 1st Dept. of Int. Med., Yamaguchi Univ. School of Med., Yamagnchi, Japan 2Division of Biochemistry, Biomedical Research Center, Osaka Univ. school of Med., Osaka, Japan Lead nitrate induces liver cell proliferation in rats without accompanying liver cell necrosis. However, proliferative mechanism and effect on hepatocyte itself is still unknown. Therefore, our initial experiments were designated to determine whether lead nitrate modifies hepatocyte proliferation and alter the production of hepatocyte growth factor and tumor necrosis factor-a by examining both liver and blood level of these cytokines at various time periods. Finally, several tumor necrosis f a c t o r - a inhibitors were administered to rats. Hepatocyte proliferation occurred 24 hours and reached a peak 48 hours after a single intravenous injection of lead nitrate ( 1 0 0 m M / K g ) , and tumor necrosis f a c t o r - a mRNA expression in the liver was enhanced 1, 6 and 12 hours after the injection. However, no alteration was observed in the liver or blood level of hepatocyte growth factor. Pretreatment with dexamethasone (4.0 mg/Kg), E3330 (100 mg/Kg) adenosine (0.3 mM/Kg) and pentoxifylline (100mg/Kg) inhibited both tumor necrosis f a c t o r - a mRNA expression and hepatocyte proliferation 48 hours after the injection, These experimental results strongly support the hypothesis that tumor necrosis f a c t o r - a positively conduct and regulate hepatocyte proliferation induced in rats by direct mitogen, lead nitrate.
P-166
THE ROLEOF bFGF IN HEPATIC
REGENERATION AFTER ISCHEMIA REPERFUSION INJURY IN RATS S. Kawachi, M. Shimazu, G. Wakabayashi, N. Shirasugi, Y. Kumamoto, T. Karahashi, M. Yoshida, M. Kitajima Dept. of Surgery, Keio University School of Medicine, Tokyo,
Japan bFGF is the potent angiogenedc factor and plays an important role in remodeling of post ischemic neuronal damage and salvaging of infarcted myocardium by its angiogenic action. We examined synthesis and localilzation of bFGF to study the role of bFGF in hepatic regeneration after ischemia repeffusion injury. [Methods] Partial warm ischemia reperfusion was induced by clamping and de,clamping of afferent vessels of the median and left lateral lobes of the rat liver. Livers were removed at the indicated times after reperfusion and homogenized, bFGF levels were mesured in both of damaged and non damaged liver tissue homogenate by enzyme immunoassay. Using anti-bFGF monoclonal antibody, immunohistochemical staining was also performed in regenerating liver. [Results] On the 4th day after reperfusion, bFGF levels of damaged liver tissue were increased significantly as compared with those of non damaged and normal liver tissue. Histologically, intensive regenerating change was observed and immunohistochemical reaction was show only in damaged liver on the 4th day after reperfusion. [Conclusion] Those data suggested that bFGF was synthesized after ischemia reperfusion injury and might play an important role in hepatic regeneration.
P-168 ROLE OF HEPARIN-BINDING EGF-LIKE GROWTH FACTOR (HB-EGF) IN RAT LIVER REGENERATION :CELL SPECIFICITY OF HB-EGF GENE EXPRESSION AND INDUCTION OF HGF AND TGF-a GENE EXPRESSION BY HB-EGF S.Kiso 1, S.Kawata1, S.Tamura 1, N.Ito 1, H.Tsushima ~, O.Oshikawa1, S.Higashiyama 2, N.Taniguchi 2, Y. Matsuzawa1 1 2nd Dept. of nt Med., ~ Dept. of Biochem., Osaka Univ. Med. Sch., Osaka 565, Japan Heparin-binding EGF-like growth factor (HB-EGF) is anew member of the EGF family that was initially purified from conditioned medium of U-937 macrophage-like cells. Recently, we reported that HB-EGF has hepatotrophic effects in vitro, and that the level of HB-EGF mRNA in regenerating rat liver increased 1.5 h after 70% partial hepatectomy and reached a maximum (about 7-fold over normal) at 6 h. In this study we investigated that the cell specificity of HB-EGF ~ene expression expression of HB-EGF protein in regenerating rat hver, and induction of HGF and TGF-a gene express on by HB-EGF. MATERIALS AND METHODS (1)Male S-D rats were partially hepatectomized according to the method of Higgins and Anderson. (2)Hepatocyte and non-parenchymal liver cells (NPC) were isolated bKYuinsitu perfusion with collagenase. (3)Sinusoidal endothelial cells, pffer cells and lipocytes were isolated with a JE-6 elutdation rotor (Beckman). HB-EGF gene expression in these cells was investigated by RT-PCR. (4)HB-EGF protein expression in the liver of normal and hepatectomized rats was investigated by Western blot analysis. (5)HB-EGF(30ng/ml)was added to the separated hepatocytes and NPC in primary culture and HGF and TGF-c( gene expression in these cells were investigated by Northern blot analysis. RESULTS (1)RT-PCR showed HB-EGF gene expression predominantly in Kupffer cells and sinusoidal endothelial cells but not in lipocytes andhepatocytes. (2)HB-EGF protein expression increased about 2.8-fold over normal liver at 10 h after partial hepatectomy. (3)Marked HGF and TGF-(~ gene expression were induced by HB-EGF in NPC and in hepatocytes respectively. In the remmnant liver after partial hepatectomy, HB-EGF may participate in regulation of the expression of HGF and TGF-a gene. CONCLUSION HB-EGF may play an important role in liver regeneration after partial hepatectomy v/a a paracrine mechanism.