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Transforming Growth Factor α Levels in Liver and Blood Correlate Better than Hepatocyte Growth Factor with Hepatocyte Proliferation during Liver Regeneration

1998; Elsevier BV; Volume: 153; Issue: 3 Linguagem: Inglês

10.1016/s0002-9440(10)65637-4

ISSN

1525-2191

Autores

Tomoaki Tomiya, Itsuro Ogata, Kenji Fujiwara,

Tópico(s)

Organ Transplantation Techniques and Outcomes

Resumo

Transforming growth factor α (TGFα) and hepatocyte growth factor (HGF) are mitogens for hepatocytes in vitro andin vivo, produced by hepatocytes or nonparenchymal cells such as stellate cells in the liver. It is still uncertain whether TGFα and HGF are essential for liver regeneration. To assess the role of these growth factors in liver regeneration, their circulating and hepatic levels were studied in various rat models of liver regeneration. Hepatic and plasma HGF levels were increased with increased number of mitotic hepatocytes in rats after partial hepatectomy or carbon tetrachloride intoxication. However, hepatic HGF levels were decreased despite an increased number of mitotic hepatocytes and increased or unchanged plasma HGF levels in rats given phenobarbital and in rats after dimethylnitrosamine intoxication, which can induce hepatic necrosis after apoptosis of hepatic stellate cells. In contrast, hepatic and serum TGFα levels were increased in all of the models. In sham-operated rats with no increased number of mitotic hepatocytes, hepatic and circulating levels of HGF were increased, whereas those levels of TGFα were unchanged. The results indicate that TGFα levels in liver and blood more closely correlate with hepatocyte mitogenesis than HGF levels. Transforming growth factor α (TGFα) and hepatocyte growth factor (HGF) are mitogens for hepatocytes in vitro andin vivo, produced by hepatocytes or nonparenchymal cells such as stellate cells in the liver. It is still uncertain whether TGFα and HGF are essential for liver regeneration. To assess the role of these growth factors in liver regeneration, their circulating and hepatic levels were studied in various rat models of liver regeneration. Hepatic and plasma HGF levels were increased with increased number of mitotic hepatocytes in rats after partial hepatectomy or carbon tetrachloride intoxication. However, hepatic HGF levels were decreased despite an increased number of mitotic hepatocytes and increased or unchanged plasma HGF levels in rats given phenobarbital and in rats after dimethylnitrosamine intoxication, which can induce hepatic necrosis after apoptosis of hepatic stellate cells. In contrast, hepatic and serum TGFα levels were increased in all of the models. In sham-operated rats with no increased number of mitotic hepatocytes, hepatic and circulating levels of HGF were increased, whereas those levels of TGFα were unchanged. The results indicate that TGFα levels in liver and blood more closely correlate with hepatocyte mitogenesis than HGF levels. The liver has an ability to regenerate after loss of its volume, but its regulation mechanisms have not been known well. Many mitogens for hepatocytes have been found since hepatocytes in primary culture were utilized to detect and characterize contributing factors.1Fausto N Hepatic regeneration.in: Zakim D Boyer TD Hepatology. WB Saunders, Philadelphia1996: 32-57Google Scholar, 2Michalopoulos G Liver regeneration: molecular mechanisms of growth control.FASEB J. 1990; 4: 176-187Crossref PubMed Scopus (842) Google Scholar, 3Zarnegar R DeFrances MC Michalopoulos GK Hepatocyte growth factor: its role in hepatic growth and pathology.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter DA Shafritz DA Liver: Biology and Pathology. Raven Press, New York1994: 1047-1057Google Scholar Transforming growth factor α (TGFα) and hepatocyte growth factor (HGF) can stimulate hepatocyte proliferationin vivo as well as in vitro.4Jhappan C Stahle C Harkins RN Fausto N Smith GH Merlino GT TGFα overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas.Cell. 1990; 61: 1137-1146Abstract Full Text PDF PubMed Scopus (644) Google Scholar, 5Shiota G Wang TC Nakamura T Schmidt EV Hepatocyte growth factor in transgenic mice: effects on hepatocyte growth, liver regeneration and gene expression.Hepatology. 1994; 19: 962-972Crossref PubMed Scopus (153) Google Scholar, 6Ishiki Y Ohnishi H Muto Y Matsumoto K Nakamura T Direct evidence that hepatocyte growth factor is a hepatotropic factor for liver regeneration and has a potent antihepatitis effect in vivo.Hepatology. 1992; 16: 1227-1235PubMed Google Scholar, 7Fujiwara K Nagoshi S Ohno A Hirata K Ohta Y Mochida S Tomiya T Higashio K Kurokawa K Stimulation of liver growth by exogenous human hepatocyte growth factor in normal and partially hepatectomized rats.Hepatology. 1993; 18: 1443-1449Crossref PubMed Scopus (136) Google Scholar, 8Webber EM Godowski PJ Fausto N In vivo response of hepatocytes to growth factors requires an initial priming stimulus.Hepatology. 1994; 19: 489-497Crossref PubMed Scopus (204) Google Scholar, 9Liu ML Mars WM Zarnegar R Michalopoulos GK Collagenase pretreatment and mitogenic effects of hepatocyte growth factor and transforming growth factor-α in adult rat liver.Hepatology. 1994; 19: 1521-1527Crossref PubMed Scopus (111) Google Scholar TGFα exerts its action by binding to the TGFα/epidermal growth factor (EGF) receptor on the cell surface. In the liver, hepatocytes express both TGFα mRNA and TGFα/EGF receptor mRNA,10Todaro GJ Fryling C deLarco JE Transforming growth factors produced by certain human tumor cells: polypeptides that interact with epidermal growth factor receptors.Proc Natl Acad Sci USA. 1980; 77: 5258-5262Crossref PubMed Scopus (778) Google Scholar, 11Massague J Epidermal growth factor-like transforming growth factor. II. Interaction with epidermal growth factor receptors on human placenta membrane and A431 cells.J Biol Chem. 1983; 258: 13614-13620Abstract Full Text PDF PubMed Google Scholar, 12Derynck R Transforming growth factor α.Cell. 1988; 54: 593-595Abstract Full Text PDF PubMed Scopus (584) Google Scholar, 13Johnson AC Garfield SH Merlino GT Pastan I Expression of epidermal growth factor receptor proto-oncogene mRNA in regenerating rat liver.Biochem Biophys Res Commun. 1988; 150: 412-418Crossref PubMed Scopus (51) Google Scholar, 14Mead JE Fausto N Transforming growth factor α may be a physiological regulator of liver regeneration by means of an autocrine mechanism.Proc Natl Acad Sci USA. 1989; 86: 1558-1562Crossref PubMed Scopus (552) Google Scholar, 15Fisher DA Lakshmanan J Metabolism and effects of epidermal growth factor and related growth factors in mammals.Endocr Rev. 1990; 11: 418-442Crossref PubMed Scopus (353) Google Scholar, 16Fausto N Webber EM Liver regeneration.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter DA Shafritz DA The Liver: Biology and Pathology. Raven Press, New York1994: 1059-1084Google Scholar suggesting that TGFα can stimulate hepatocyte proliferation in an autocrine manner.1Fausto N Hepatic regeneration.in: Zakim D Boyer TD Hepatology. WB Saunders, Philadelphia1996: 32-57Google Scholar HGF is produced by nonparenchymal cells, principally by hepatic stellate cells, in the liver, and the c-met proto-oncogene product identified as its receptor is present in hepatocytes.17Kinoshita T Tashiro K Nakamura T Marked increase of HGF mRNA in non-parenchymal liver cells of rats treated with hepatotoxins.Biochem Biophys Res Commun. 1989; 165: 1229-1234Crossref PubMed Scopus (262) Google Scholar, 18Noji S Tashiro K Koyama E Nohno T Ohyama K Taniguchi S Nakamura T Expression of hepatocyte growth factor gene in endothelial and Kupffer cells of damaged rat livers, as revealed by in situ hybridization.Biochem Biophys Res Commun. 1990; 173: 42-47Crossref PubMed Scopus (293) Google Scholar, 19Zarnegar R DeFrances MC Oliver L Michalopoulos G Identification and partial characterization of receptor binding sites for HGF on rat hepatocytes.Biochem Biophys Res Commun. 1990; 173: 1179-1185Crossref PubMed Scopus (65) Google Scholar, 20Naldini L Vigna E Narsimhan RP Gaudino G Zarnegar R Michalopoulos GK Comoglio PM Hepatocyte growth factor (HGF) stimulates the tyrosine kinase activity of the receptor encoded by the proto-oncogene c-MET.Oncogene. 1991; 6: 501-504PubMed Google Scholar, 21Bottaro DP Rubin JS Faletto DL Chan AM-L Kmiecik TE Vande Woude GF Aaronson SA Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product.Science. 1991; 251: 802-804Crossref PubMed Scopus (2144) Google Scholar, 22Higuchi O Nakamura T Identification and change in the receptor for hepatocyte growth factor in rat liver after partial hepatectomy or induced hepatitis.Biochem Biophys Res Commun. 1991; 176: 599-607Crossref PubMed Scopus (131) Google Scholar, 23Schirmacher P Geerts A Pietrangelo A Dienes HP Rogler CE Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells.Hepatology. 1992; 15: 5-11Crossref PubMed Scopus (202) Google Scholar, 24Hu Z Evarts RP Fujio K Marsden ER Thorgeirsson SS Expression of hepatocyte growth factor and c-met genes during hepatic differentiation and liver development in the rat.Am J Pathol. 1993; 142: 1823-1830PubMed Google Scholar Thus, HGF is postulated to stimulate hepatocyte proliferation in a paracrine manner in the liver.3Zarnegar R DeFrances MC Michalopoulos GK Hepatocyte growth factor: its role in hepatic growth and pathology.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter DA Shafritz DA Liver: Biology and Pathology. Raven Press, New York1994: 1047-1057Google Scholar TGFα and HGF have been reported to increase in the liver in several experimental models of liver regeneration,13Johnson AC Garfield SH Merlino GT Pastan I Expression of epidermal growth factor receptor proto-oncogene mRNA in regenerating rat liver.Biochem Biophys Res Commun. 1988; 150: 412-418Crossref PubMed Scopus (51) Google Scholar, 25Webber EM FitzGerald MJ Brown PI Bartlett MH Fausto N Transforming growth factor-α expression during liver regeneration after partial hepatectomy and toxic injury, and potential interactions between transforming growth factor-α and hepatocyte growth factor.Hepatology. 1993; 18: 1422-1431Crossref PubMed Scopus (146) Google Scholar, 26Kinoshita T Hirao S Matsumoto K Nakamura T Possible endocrine control of liver regeneration after partial hepatectomy.Biochem Biophys Res Commun. 1991; 177: 330-335Crossref PubMed Scopus (153) Google Scholar, 27Zarnegar R DeFrances MC Kost DP Lindroos P Michalopoulos G Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy.Biochem Biophys Res Commun. 1991; 177: 559-565Crossref PubMed Scopus (210) Google Scholar and also in the circulation of patients after partial hepatectomy and patients with acute hepatitis.28Tomiya T Fujiwara K Serum levels of transforming growth factor-α in patients after partial hepatectomy as determined with an enzyme-linked immunosorbent assay.Hepatology. 1993; 18: 304-308Crossref PubMed Scopus (40) Google Scholar, 29Tomiya T Fujiwara K Liver Regeneration in fulminant hepatitis as evaluated by serum transforming growth factor α levels.Hepatology. 1996; 23: 253-257PubMed Google Scholar, 30Tomiya T Tani M Yamada S Hayashi S Umeda N Fujiwara K Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients.Gastroenterology. 1992; 103: 1621-1624Abstract PubMed Google Scholar, 31Tomiya T Nagoshi S Fujiwara K Significance of serum human hepatocyte growth factor levels in patients with hepatic failure.Hepatology. 1992; 15: 1-4Crossref PubMed Scopus (90) Google Scholar These findings suggest that TGFα and HGF can contribute to liver regeneration.3Zarnegar R DeFrances MC Michalopoulos GK Hepatocyte growth factor: its role in hepatic growth and pathology.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter DA Shafritz DA Liver: Biology and Pathology. Raven Press, New York1994: 1047-1057Google Scholar Recently, it was reported that there was no significant difference in hepatocyte proliferation after partial hepatectomy between TGFα knockout mice and wild-type mice.32Russell WE Kaufmann WK Sitaric S Luetteke NC Lee DC Liver regeneration and hepatocarcinogenesis in transforming growth factor-α-targeted mice.Mol Carcinog. 1996; 15: 183-189Crossref PubMed Scopus (98) Google Scholar Also, HGF has been shown to increase resulting from its active production in inflammatory cells as well as hepatic stellate cells in rats and human.18Noji S Tashiro K Koyama E Nohno T Ohyama K Taniguchi S Nakamura T Expression of hepatocyte growth factor gene in endothelial and Kupffer cells of damaged rat livers, as revealed by in situ hybridization.Biochem Biophys Res Commun. 1990; 173: 42-47Crossref PubMed Scopus (293) Google Scholar, 33Sakaguchi H Seki S Tsubouchi H Daikuhara Y Nittani T Kobayashi K Ultrastructural localization of human hepatocyte growth factor in human.Hepatology. 1994; 19: 1157-1163Crossref PubMed Scopus (53) Google Scholar Systemic and hepatic inflammation can occur even in the process of liver regeneration after resection.30Tomiya T Tani M Yamada S Hayashi S Umeda N Fujiwara K Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients.Gastroenterology. 1992; 103: 1621-1624Abstract PubMed Google Scholar, 34Mochida S Ogata I Hirata K Ohta Y Yamada S Fujiwara K Provocation of massive hepatic necrosis by endotoxin after partial hepatectomy in rats.Gastroenterology. 1990; 99: 771-777PubMed Google Scholar In fact, serum HGF levels in patients after partial hepatectomy are more closely related to the degree of systemic and hepatic inflammation than to the degree of liver regeneration.30Tomiya T Tani M Yamada S Hayashi S Umeda N Fujiwara K Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients.Gastroenterology. 1992; 103: 1621-1624Abstract PubMed Google Scholar Liver regeneration occurs generally after partial hepatectomy, acute hepatic injury, or small-for-size liver transplantation.1Fausto N Hepatic regeneration.in: Zakim D Boyer TD Hepatology. WB Saunders, Philadelphia1996: 32-57Google Scholar, 2Michalopoulos G Liver regeneration: molecular mechanisms of growth control.FASEB J. 1990; 4: 176-187Crossref PubMed Scopus (842) Google Scholar, 3Zarnegar R DeFrances MC Michalopoulos GK Hepatocyte growth factor: its role in hepatic growth and pathology.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter DA Shafritz DA Liver: Biology and Pathology. Raven Press, New York1994: 1047-1057Google Scholar, 35Kawasaki S Makuuchi M Ishizone S Matsunami H Terada M Kawarazaki H Liver regeneration in recipients and donors after transplantation.Lancet. 1992; 339: 580-581Abstract PubMed Scopus (184) Google Scholar The levels of essential factors for liver regeneration might increase in the liver or circulation, similarly reflecting the extent of liver regeneration after these events. The aim of this study is to determine whether TGFα and HGF levels in liver and blood correlate with hepatocyte proliferation. In different rat models of liver regeneration, we serially measured circulating levels as well as hepatic levels of both factors. Sep-Pak C18 minicolumns were obtained from Waters Chromatography Division (Millipore Corp., Milford, MA). Twenty percent neutral buffered formalin solution was purchased from Muto Pure Chemicals Ltd. (Tokyo, Japan) and phenylmethyl-sulfonyl fluoride from Sigma Chemical Co. (St. Louis, MO). HGF enzyme-linked immunosorbent assay (ELISA) kit was a product of the Institute of Immunology (Tokyo, Japan).36Yamada A Matsumoto K Iwanari H Sekiguchi K Kawata S Matsuzawa Y Nakamura T Rapid and sensitive enzyme-linked immunosorbent assay for measurement of HGF in rat and human tissues.Biomed Res. 1995; 16: 105-114Crossref Scopus (111) Google Scholar Other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan). Male Sprague-Dawley rats ages 5 to 6 weeks were obtained from Japan SLC (Shizuoka, Japan). They were housed in cages at 22 ± 2°C under a 12-hour light-dark cycle and fed a commercial diet and water ad libitum. All animal study protocols conformed to the guidelines of the Faculty of Medicine, University of Tokyo for humane care. The rats were subjected to either of the following: an operation of two-thirds resection of the liver under diethyl ether anesthesia, a single subcutaneous injection of 3.0 ml/kg body weight of carbon tetrachloride (CCl4) as a 20% solution in olive oil, a single intraperitoneal injection of 35 mg/kg body weight of dimethylnitrosamine (DMN) as a 3.5% solution in saline, or oral administration of phenobarbital sodium ad libitum at a concentration of 0.10% in the drinking water. In sham-operated rats, the abdomen was cut open under similar anesthesia, and the liver was briefly exposed outside the peritoneal cavity. Rats given water were used as controls for phenobarbital-treated rats. The rats were serially anesthetized with diethyl ether. Blood was collected through the inferior vena cava with an empty plastic syringe and a plastic syringe containing 3.1% sodium citrate solution (1:10 v/v) to prepare serum and plasma, respectively. The liver was perfused with 25 ml of saline at 37°C through the portal vein after near-total exsanguination, and excised. A portion of the liver was immersed in 20% neutral buffered formalin solution for histological examination. The remaining liver was frozen in liquid nitrogen and stored at −80°C until use for TGFα and HGF assays. Liver extracts were prepared by homogenizing 400 mg liver tissue in 1.8 ml of 0.1 mol/L phosphate-buffered saline (pH 7.2) using a Tenbrock tissue grinder as previously reported.37Brown PI Lam R Lakshmanan J Fischer DA Transforming growth factor alpha in developing rats.Am J Physiol. 1990; 259: E256-E260PubMed Google Scholar The supernatants were collected after centrifugation at 100,000 ×g for 60 minutes at 4°C. One milliliter of the supernatants or sera was applied to Sep-Pak C18minicolumns.38Inagaki H Katoh M Kurokawa-Ohsawa K Tanaka S A new sandwich enzyme-linked immunosorbent assay (ELISA) for transforming growth factor α (TGFα) based upon conformational modification by antibody binding.J Immunol Methods. 1990; 128: 27-37Crossref PubMed Scopus (19) Google Scholar, 39Katoh M Inagaki H Kurosawa-Ohsawa K Katsuura M Tanaka S Detection of transforming growth factor alpha in human urine and plasma.Biochem Biophys Res Commun. 1990; 167: 1065-1072Crossref PubMed Scopus (35) Google Scholar The columns were washed with 20 ml distilled water, and the trapped materials were eluted with 40% acetonitrile (3 ml). The column eluates were lyophilized, redissolved in 0.2 ml of 10 mmol/L phosphate-buffered saline (pH 7.2), and used for TGFα ELISA as previously reported.28Tomiya T Fujiwara K Serum levels of transforming growth factor-α in patients after partial hepatectomy as determined with an enzyme-linked immunosorbent assay.Hepatology. 1993; 18: 304-308Crossref PubMed Scopus (40) Google Scholar, 29Tomiya T Fujiwara K Liver Regeneration in fulminant hepatitis as evaluated by serum transforming growth factor α levels.Hepatology. 1996; 23: 253-257PubMed Google Scholar Liver extracts were prepared according to the protocol from the manufacturer of the HGF ELISA kit.36Yamada A Matsumoto K Iwanari H Sekiguchi K Kawata S Matsuzawa Y Nakamura T Rapid and sensitive enzyme-linked immunosorbent assay for measurement of HGF in rat and human tissues.Biomed Res. 1995; 16: 105-114Crossref Scopus (111) Google Scholar Briefly, liver tissues were homogenized in 4 vol/g of a buffer composed of 20 mmol/L Tris[hydroxymethyl]aminomethane-HCl (pH 7.5), 2 mol/L NaCl, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L ethylenediaminetetraacetic acid, and 0.1% polyoxyethylenesorbitan mono-oleate. The homogenates were centrifuged at 20,000 × g for 60 minutes at 4°C, and the resultant supernatants were collected. These supernatants and the plasma were applied to HGF ELISA.36Yamada A Matsumoto K Iwanari H Sekiguchi K Kawata S Matsuzawa Y Nakamura T Rapid and sensitive enzyme-linked immunosorbent assay for measurement of HGF in rat and human tissues.Biomed Res. 1995; 16: 105-114Crossref Scopus (111) Google Scholar Serum ALT levels were measured by modified Reitman-Framkel method using a commercial kit.40Reitman S Frankel S A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases.Am J Clin Pathol. 1957; 28: 56-63Crossref PubMed Scopus (8200) Google Scholar Liver specimens fixed in formalin were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The mitotic index of hepatocytes was determined by counting more than 3000 hepatocytes in each liver specimen.7Fujiwara K Nagoshi S Ohno A Hirata K Ohta Y Mochida S Tomiya T Higashio K Kurokawa K Stimulation of liver growth by exogenous human hepatocyte growth factor in normal and partially hepatectomized rats.Hepatology. 1993; 18: 1443-1449Crossref PubMed Scopus (136) Google Scholar The differences between unpaired two samples were defined as significant when P values by both Student'st-test and Mann-Whitney U test were less than 0.05. Spearman's correlation test was used for analysis of correlations. Mean (±SEM) hepatic and circulating levels of TGFα in five normal rats were 9.8 ± 1.2 ng/g liver and 5.3 ± 0.85 ng/ml, respectively, and those of HGF were 168 ± 17.2 ng/g liver and 0.14 ± 0.013 ng/ml, respectively. As shown in Figure 1, the mitotic index of hepatocytes was unchanged until 18 hours after partial hepatectomy, but increased to the maximal level at 24 hours. It decreased to the preoperative level at 120 hours, with a smaller peak at 72 hours. Hepatic and serum TGFα levels were significantly increased after 12 hours compared with the preoperative levels, reached the maximum at 24 hours, and thereafter changed similarly to the mitotic index of hepatocytes. Hepatic and plasma HGF levels were significantly increased at 6 hours compared with the preoperative levels, reached the maximum around 18 hours, and returned to the preoperative level at 48 hours. After sham operation, the mitotic index of hepatocytes and hepatic and serum TGFα levels showed no significant changes. However, hepatic and plasma HGF levels were significantly increased between 6 and 18 hours compared with the preoperative levels (Figure 2). As shown in Figure 3, serum ALT levels were increased with a peak on day 1 after intoxication with CCl4, and the mitotic index of hepatocytes and hepatic and serum TGFα levels peaked on day 3. Hepatic and plasma HGF levels changed similarly to serum ALT levels. In the rats on days 0.5, 1, and 2, plasma HGF levels were correlated with serum ALT levels as shown in Figure 4 (r = 0.81; P < 0.01).Figure 4Serum ALT levels and plasma HGF levels after CCl4 injection in rats. Open circles, closed circles, and triangles indicate values 0.5, 1, and 2 days after CCl4 injection, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Figure 5 demonstrates the results in rats given DMN. Serum ALT levels peaked on day 2 after intoxication, but the mitotic index of hepatocytes and hepatic and serum TGFα levels peaked on day 3. In contrast, hepatic HGF levels were significantly decreased compared with the basal level, but plasma HGF levels were unchanged. As shown in Figure 6, the mitotic index of hepatocytes was gradually increased and peaked on day 7, with liver weights increasing significantly after 12 hours compared with the controls. The mitotic index decreased thereafter and returned to the basal level on day 14. Hepatic and serum TGFα levels changed similarly to the mitotic index. Plasma HGF levels were increased, reached the maximum on day 1, and returned to the basal level on day 5. In contrast, hepatic HGF levels were significantly decreased from day 1 until day 14 compared with the basal level. Our results demonstrate that hepatic and circulating levels of TGFα were closely associated with liver regeneration. However, those levels of HGF were not necessarily related to it. We used four different rat models of liver regeneration. Partial hepatectomy induces compensatory hyperplasia of the remaining liver with minimal necrosis.1Fausto N Hepatic regeneration.in: Zakim D Boyer TD Hepatology. WB Saunders, Philadelphia1996: 32-57Google Scholar Intoxication with CCl4also induces compensatory hepatic hyperplasia after liver necrosis due to direct damage to hepatocytes.41Fujiwara K Ogata I Ohta Y Hirata K Oka Y Yamada S Sato Y Masaki N Oka H Intravascular coagulation in acute liver failure in rats and its treatment with antithrombin III.Gut. 1988; 29: 1103-1108Crossref PubMed Scopus (80) Google Scholar Similar hepatic hyperplasia after liver necrosis is also produced by intoxication with DMN, but the mechanism of development of liver necrosis is much different from that by CCl4 intoxication. In this model, liver necrosis occurs mainly as a result of microcirculatory disturbance due to sinusoidal fibrin deposition caused by endothelial cell destruction after apoptosis of hepatic stellate cells, which are the major HGF-producing cells in the liver.41Fujiwara K Ogata I Ohta Y Hirata K Oka Y Yamada S Sato Y Masaki N Oka H Intravascular coagulation in acute liver failure in rats and its treatment with antithrombin III.Gut. 1988; 29: 1103-1108Crossref PubMed Scopus (80) Google Scholar, 42Hirata K Ogata I Ohta Y Fujiwara K Hepatic sinusoidal cell destruction in the development of intravascular coagulation in acute liver failure of rats.J Pathol. 1989; 158: 157-165Crossref PubMed Scopus (80) Google Scholar Phenobarbital administration can induce augmentative hyperplasia of the liver without any damage to parenchymal and nonparenchymal cells in the liver.43Lindroos P Tsai WH Zarnegar R Michalopoulos GK Plasma levels of HGF in rats treated with tumor promoters.Carcinogenesis. 1992; 13: 139-141Crossref PubMed Scopus (34) Google Scholar Both hepatic and serum TGFα levels were increased in close association with the numbers of mitotic hepatocytes irrespective of the type of liver regeneration (Figure 1, Figure 3, Figure 5, Figure 6). Sham operation caused no changes in hepatic and serum TGFα levels (Figure 2). These results were in line with our previous observations in patients28Tomiya T Fujiwara K Serum levels of transforming growth factor-α in patients after partial hepatectomy as determined with an enzyme-linked immunosorbent assay.Hepatology. 1993; 18: 304-308Crossref PubMed Scopus (40) Google Scholar, 39Katoh M Inagaki H Kurosawa-Ohsawa K Katsuura M Tanaka S Detection of transforming growth factor alpha in human urine and plasma.Biochem Biophys Res Commun. 1990; 167: 1065-1072Crossref PubMed Scopus (35) Google Scholar and reports in rats showing increased hepatic TGFα mRNA levels after partial hepatectomy and CCl4intoxication.1Fausto N Hepatic regeneration.in: Zakim D Boyer TD Hepatology. WB Saunders, Philadelphia1996: 32-57Google Scholar, 14Mead JE Fausto N Transforming growth factor α may be a physiological regulator of liver regeneration by means of an autocrine mechanism.Proc Natl Acad Sci USA. 1989; 86: 1558-1562Crossref PubMed Scopus (552) Google Scholar, 25Webber EM FitzGerald MJ Brown PI Bartlett MH Fausto N Transforming growth factor-α expression during liver regeneration after partial hepatectomy and toxic injury, and potential interactions between transforming growth factor-α and hepatocyte growth factor.Hepatology. 1993; 18: 1422-1431Crossref PubMed Scopus (146) Google Scholar, 44Everts RP Nakatsukasa H Marsden ER Hu Z Thorgeirsson SS Expression of transforming growth factor-α in regenerating liver and during hepatic differentiation.Mol Carcinog. 1992; 5: 25-31Crossref PubMed Scopus (112) Google Scholar TGFα produced by hepatocytes in the liver may be an essential factor for liver regeneration. Recently, Russell et al found no difference in hepatocyte proliferation after partial hepatectomy between TGFα knockout mice and wild-type mice.32Russell WE Kaufmann WK Sitaric S Luetteke NC Lee DC Liver regeneration and hepatocarcinogenesis in transforming growth factor-α-targeted mice.Mol Carcinog. 1996; 15: 183-189Crossref PubMed Scopus (98) Google Scholar There are many ligands for the TGFα/EGF receptor, including EGF which is abundantly produced by salivary glands in mice.1Fausto N Hepatic regeneration.in: Zakim D Boyer TD Hepatology. WB Saunders, Philadelphia1996: 32-57Google Scholar, 15Fisher DA Lakshmanan J Metabolism and effects of epidermal growth factor and related growth factors in mammals.Endocr Rev. 1990; 11: 418-442Crossref PubMed Scopus (353) Google Scholar Loss of TGFα might be masked as a result of opportunistic compensation by one such ligand in TGFα knockout mice, as was discussed in the report.32Russell WE Kaufmann WK Sitaric S Luetteke NC Lee DC Liver regeneration and hepatocarcinogenesis in transforming growth factor-α-targeted mice.Mol Carcinog. 1996; 15: 183-189Crossref PubMed Scopus (98) Google Scholar Indeed, targeting of a gene can increase the expression of other functionally related proteins.45Zhang W Behringer RR Olson EN Inactivation of the myogenic bHLH gene MRF4 results in up-regulation of myogenin and rib anomalies.Genes Dev. 1995; 9: 1388-1399Crossref PubMed Scopus (250) Google Scholar Investigation with TGFα/EGF receptor knockout mice would be a way to clarify this controversy. Hepatic and plasma HGF levels were increased in rats after partial hepatectomy and CCl4 intoxication (Figure 1, Figure 3). In both models, the increase of hepatic HGF mRNA levels has also been reported.3Zarnegar R DeFrances MC Michalopoulos GK Hepatocyte growth factor: its role in hepatic growth and pathology.in: Arias IM Boyer JL Fausto N Jakoby WB Schachter DA Shafritz DA Liver: Biology and Pathology. Raven Press, New York1994: 1047-1057Google Scholar, 17Kinoshita T Tashiro K Nakamura T Marked increase of HGF mRNA in non-parenchymal liver cells of rats treated with hepatotoxins.Biochem Biophys Res Commun. 1989; 165: 1229-1234Crossref PubMed Scopus (262) Google Scholar, 26Kinoshita T Hirao S Matsumoto K Nakamura T Possible endocrine control of liver regeneration after partial hepatectomy.Biochem Biophys Res Commun. 1991; 177: 330-335Crossref PubMed Scopus (153) Google Scholar, 27Zarnegar R DeFrances MC Kost DP Lindroos P Michalopoulos G Expression of hepatocyte growth factor mRNA in regenerating rat liver after partial hepatectomy.Biochem Biophys Res Commun. 1991; 177: 559-565Crossref PubMed Scopus (210) Google Scholar, 46Tashiro K Hagiya M Nishizawa T Seki T Shimonishi M Shimizu S Nakamura T Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues.Proc Natl Acad Sci USA. 1990; 87: 3200-3204Crossref PubMed Scopus (386) Google Scholar, 47Okajima A Miyazawa K Kitamura N Primary structure of rat hepatocyte growth factor and induction of its mRNA during liver regeneration following hepatic injury.Eur J Biochem. 1990; 193: 375-381Crossref PubMed Scopus (113) Google Scholar However, such significant increases of hepatic and plasma HGF levels were also found after sham operation, with no increase in number of mitotic hepatocytes (Figure 2). Recently, it was reported that hepatic HGF mRNA levels were increased after sham operation, as well as after partial hepatectomy in rats.47Okajima A Miyazawa K Kitamura N Primary structure of rat hepatocyte growth factor and induction of its mRNA during liver regeneration following hepatic injury.Eur J Biochem. 1990; 193: 375-381Crossref PubMed Scopus (113) Google Scholar, 48Aldana PR Goerke ME Carr SC Tracy TF The expression of regenerative growth factors in chronic liver injury and repair.J Surg Res. 1994; 57: 711-717Abstract Full Text PDF PubMed Scopus (30) Google Scholar In addition, plasma HGF levels were positively correlated with serum ALT levels in rats treated with CCl4(Figure 4). These results are comparable with our previous finding in patients that serum HGF levels were increased in association with hepatic necrosis and systemic inflammation.30Tomiya T Tani M Yamada S Hayashi S Umeda N Fujiwara K Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients.Gastroenterology. 1992; 103: 1621-1624Abstract PubMed Google Scholar, 31Tomiya T Nagoshi S Fujiwara K Significance of serum human hepatocyte growth factor levels in patients with hepatic failure.Hepatology. 1992; 15: 1-4Crossref PubMed Scopus (90) Google Scholar It can be assumed that HGF production is stimulated in response to an inflammatory reaction after a surgical procedure or hepatic injury. This assumption may be supported by the recent report that preinflammatory cytokines such as interleukin-1 can regulate HGF production.49Matsumoto K Okazaki H Nakamura T Up-regulation of hepatocyte growth factor gene expression by interleukin-1 in human skin fibroblasts.Biochem Biophys Res Commun. 1992; 188: 235-243Crossref PubMed Scopus (172) Google Scholar Furthermore, the number of mitotic hepatocytes was increased despite decreased hepatic HGF levels after intoxication with DMN and during phenobarbital administration in rats (Figure 5, Figure 6). Considering that stellate cells are major HGF-producing cells in the liver,23Schirmacher P Geerts A Pietrangelo A Dienes HP Rogler CE Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells.Hepatology. 1992; 15: 5-11Crossref PubMed Scopus (202) Google Scholar such a decrease with unchanged plasma HGF levels in DMN-intoxicated rats is likely to result from apoptosis of stellate cells caused by DMN.41Fujiwara K Ogata I Ohta Y Hirata K Oka Y Yamada S Sato Y Masaki N Oka H Intravascular coagulation in acute liver failure in rats and its treatment with antithrombin III.Gut. 1988; 29: 1103-1108Crossref PubMed Scopus (80) Google Scholar, 42Hirata K Ogata I Ohta Y Fujiwara K Hepatic sinusoidal cell destruction in the development of intravascular coagulation in acute liver failure of rats.J Pathol. 1989; 158: 157-165Crossref PubMed Scopus (80) Google Scholar The decrease in hepatic HGF levels during phenobarbital administration was accompanied by transiently increased plasma HGF levels (Figure 6). These data are comparable with reported observations that plasma HGF levels were increased, even with no increase of hepatic HGF mRNA levels in the model of augmentative hepatic hyperplasia.43Lindroos P Tsai WH Zarnegar R Michalopoulos GK Plasma levels of HGF in rats treated with tumor promoters.Carcinogenesis. 1992; 13: 139-141Crossref PubMed Scopus (34) Google Scholar, 50Shinozuka H Kubo Y Katyal SL Coni P Ledda-Columbano GM Columbano A Nakamura T Roles of growth factors and tumor necrosis factor-α on liver cell proliferation induced in rats by lead nitrate.Lab Invest. 1994; 71: 35-41PubMed Google Scholar, 51Columbano A Shinozuka H Liver regeneration versus direct hyperplasia.FASEB J. 1993; 10: 118-128Google Scholar The reason for such an increase should be investigated, especially in relation to reduced uptake of HGF. There were some differences in the duration and the timing of the increase in TGFα and HGF levels in rats after partial hepatectomy between our results and reported observations.52Russell WE Dempsey PJ Sitaric S Peck AJ Coffey Jr, RJ Transforming growth factor-α (TGFα) concentrations increase in regenerating rat liver: evidence for a delayed accumulation of murine TGFα.Endocrinology. 1993; 133: 1731-1738Crossref PubMed Scopus (65) Google Scholar, 53Lindroos P Zarnegar R Michalopoulos GK Hepatocyte growth factor (hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration.Hepatology. 1991; 13: 743-749Crossref PubMed Scopus (352) Google Scholar The differences might arise from assay systems used. Russell et al reported that the increase of hepatic TGFα levels after partial hepatectomy in rats continued longer than in rats in the present study.52Russell WE Dempsey PJ Sitaric S Peck AJ Coffey Jr, RJ Transforming growth factor-α (TGFα) concentrations increase in regenerating rat liver: evidence for a delayed accumulation of murine TGFα.Endocrinology. 1993; 133: 1731-1738Crossref PubMed Scopus (65) Google Scholar The TGFα ELISA we used can measure exclusively mature TGFα,28Tomiya T Fujiwara K Serum levels of transforming growth factor-α in patients after partial hepatectomy as determined with an enzyme-linked immunosorbent assay.Hepatology. 1993; 18: 304-308Crossref PubMed Scopus (40) Google Scholar, 29Tomiya T Fujiwara K Liver Regeneration in fulminant hepatitis as evaluated by serum transforming growth factor α levels.Hepatology. 1996; 23: 253-257PubMed Google Scholar, 38Inagaki H Katoh M Kurokawa-Ohsawa K Tanaka S A new sandwich enzyme-linked immunosorbent assay (ELISA) for transforming growth factor α (TGFα) based upon conformational modification by antibody binding.J Immunol Methods. 1990; 128: 27-37Crossref PubMed Scopus (19) Google Scholar, 39Katoh M Inagaki H Kurosawa-Ohsawa K Katsuura M Tanaka S Detection of transforming growth factor alpha in human urine and plasma.Biochem Biophys Res Commun. 1990; 167: 1065-1072Crossref PubMed Scopus (35) Google Scholar whereas their radioimmunoassay seems to detect both the mature TGFα and its precursors. They found that mature TGFα was increased in regenerating liver but not in the liver of sham-operated rats by gel chromatography, suggesting that the increase of mature TGFα is related to liver regeneration. Lindroos et al showed two peaks of plasma HGF levels as early as 2 and 6 hours after operation, and this elevation continued for 72 hours,53Lindroos P Zarnegar R Michalopoulos GK Hepatocyte growth factor (hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration.Hepatology. 1991; 13: 743-749Crossref PubMed Scopus (352) Google Scholar whereas the levels in the present study reached the maximum around 18 hours and returned to the preoperative levels at 48 hours (Figure 1). In the determination of HGF, we used sandwich ELISA composed of a polyclonal antibody and a monoclonal antibody specific for the heavy chain of HGF.36Yamada A Matsumoto K Iwanari H Sekiguchi K Kawata S Matsuzawa Y Nakamura T Rapid and sensitive enzyme-linked immunosorbent assay for measurement of HGF in rat and human tissues.Biomed Res. 1995; 16: 105-114Crossref Scopus (111) Google Scholar In contrast, only one polyclonal antibody was utilized in their assay system. There are many forms of HGF.12Derynck R Transforming growth factor α.Cell. 1988; 54: 593-595Abstract Full Text PDF PubMed Scopus (584) Google Scholar, 39Katoh M Inagaki H Kurosawa-Ohsawa K Katsuura M Tanaka S Detection of transforming growth factor alpha in human urine and plasma.Biochem Biophys Res Commun. 1990; 167: 1065-1072Crossref PubMed Scopus (35) Google Scholar, 54Tang W Miyazawa K Kitamura N Hepatocyte growth factor remains as an inactive single chain after partial hepatectomy or unilateral nephrectomy.FEBS Lett. 1995; 362: 220-224Abstract Full Text PDF PubMed Scopus (16) Google Scholar Furthermore, HGF-like protein, a novel protein synthesized by hepatocytes, contains the same structural domains as HGF.55Bezerra JA Witte DP Aronow BJ Degen SJ Hepatocyte-specific expression of the mouse hepatocyte growth factor-like protein.Hepatology. 1993; 18: 394-398PubMed Google Scholar The ELISA we have used might detect a smaller number of HGF forms and HGF-like protein compared with other assays. From our study, both TGFα and HGF seemed not to initiate liver regeneration, because the timing of their increases in the liver and circulation occurred too late to act as initiation factors. Recently, tumor necrosis factor and interleukin-6 have been reported to initiate liver regeneration.56Yamada Y Kirillova I Peschon JJ Fausto N Initiation of liver growth by tumor necrosis factor: deficient liver regeneration in mice lacking type I tumor necrosis factor receptor.Proc Natl Acad Sci USA. 1997; 94: 1441-1446Crossref PubMed Scopus (851) Google Scholar, 57Cressman DE Greenbaum LE DeAngelis RA Ciliberto G Furth EE Poli V Taub R Liver failure and defective hepatocyte regeneration in interleukin-6 deficient mice.Science. 1996; 274: 1379-1383Crossref PubMed Scopus (1342) Google Scholar The relation of TGFα and HGF to tumor necrosis factor and interleukin-6 should be investigated in the future.

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