Increased technetium-99m-GSA uptake per hepatocyte in rats with administration of dimethylnitrosamine or hepatocyte growth factor

Technetium-99m-diethylenetriaminepentaacetic acid-galactosyl-human serum albumin (GSA) is a new scintigraphic agent that binds specifically to asialoglycoprotein receptors on hepatocytes, and can be used to evaluate hepatic function. Asialoglycoprotein receptor is a hepatocellular membrane receptor responsible for the endocytosis of asialoglycoproteins, and the function of this receptor is affected in various disease states. The aim of this study was to investigate GSA uptake per hepatocyte in the convalescent stage from hepatic damage. METHODS: We used rats with dimethylnitrosamine (DMN)-induced hepatic injury and rats with recombinant human hepatocyte growth factor (rhHGF) stimulation. Plasma clearance of GSA and the number of hepatocytes in whole liver were calculated. RESULTS: In the DMN-treated rats, the total number of hepatocytes and GSA plasma clearance were reduced significantly at 3 wk after the final administration of DMN. However, calculated GSA uptake per individual hepatocyte was significantly greater by 53.2% than in the normal controls. The area of hepatic nucleus was also significantly greater than in the normal controls. In the rhHGF-treated rats, an increase in the total number of hepatocytes was not demonstrated on the final day of rhHGF administration (Day 4). However, calculated GSA uptake per hepatocyte was significantly greater (59%) than in the controls. CONCLUSION: Augmented GSA uptake per hepatocyte during the convalescent stage after hepatic injury suggests a cellular compensation to the decreased number of hepatocyte. This mechanism may be caused by the secretion of some hepatotropic factors such as HGF.     

A high expression of heme oxygenase-1 in the liver of LEC rats at the stage of hepatoma: the possible implication of induction in uninvolved tissue

We have examined changes in the expression of heme oxygenase-1 (HO-1), an inducible isoform and HO-2, a constitutive isoform, in the liver of Long-Evans with a Cinnamon-like color (LEC) rat, a mutant strain which spontaneously develops acute hepatitis and hepatoma. HO-1 expression was highly enhanced in the LEC rat livers with jaundice, and then decreased slightly, but overall remained at a higher level than in the Long-Evans with Agouti color (LEA) control rats, as judged by Northern blotting analysis of the whole liver extract. The high expression of HO-1 in the LEC rat liver was, however, not due to the actual cancer lesion but, rather, due to the surrounding uninvolved tissues including hepatocytes. Immunohistochemical analysis also supported this conclusion. Among normal tissues, the expression of HO-1 but not HO-2 was high in only the spleen of both LEC and LEA rats. The high expression observed in the stage of acute hepatitis and hepatoma stages in the LEC rat is probably due to the oxidative stress caused by the accumulation of free copper and free iron levels which has been reported earlier by our group (Suzuki et al., Carcinogenesis, 1993, 14, 1881-1884 and Koizumi et al., Free Radical Research, in press) as well as by free heme levels. The inflammatory cytokines produced by the surrounding tissue at the hepatoma stage would also be expected to play a role in the induction mechanism. The physiological relevance of HO-1 induction might be an adaptive response to oxidative stress and vasodilatory effect of carbon monoxide on sinusoidal circulation.     

Stimulation of asialoglycoprotein uptake by recombinant human hepatocyte growth factor in normal and damaged rat liver

BACKGROUND: Hepatocyte growth factor (HGF) is a strong mitogen of hepatocytes. However, little is known about the effect of HGF on the asialoglycoprotein receptors (ASGPR) of hepatocytes. The aim of this study was to identify alterations in binding of ligand to ASGPR by recombinant human HGF (rhHGF) infusion. METHODS: RhHGF was administered to rats with either normal or dimethylnitrosamine (DMN)-damaged livers. Technetium-99m-diethylenetriaminepentaacetic acid-galactosyl-human serum albumin (GSA) blood clearance was used to measure ASGPR activity. RESULTS: In normal and damaged rats, liver weight, hepatocyte nuclear size, and number of hepatocytes (cells/mm2) were not altered by rhHGF, but GSA blood clearance after rhHGF infusion was significantly increased over the preinfusion rate. CONCLUSIONS: Independent of proliferation of hepatocytes, rhHGF stimulates a hepatocytic function of the receptor-mediated uptake of ASGP.     

pp60c-src activation in hepatocellular carcinoma of humans and LEC rats

For the related Src kinases, a close correlation exists between elevated tyrosine kinase activity and cell transformation. However, the involvement of pp60c-src in hepatocellular carcinoma (HCC) remains obscure. The aim of this study was to evaluate whether pp60c-src tyrosine kinase activity is elevated in HCC. We analyzed the kinase activity of pp60c-src in normal liver tissue, chronic hepatitis liver tissue, and tumorous and adjacent nontumorous portions of HCC tissue from patients and Long-Evans cinnamon (LEC) rats that are known to develop liver cancer spontaneously. The kinase activity of pp60c-src was rarely detected in the normal human liver tissue and chronic hepatitis liver tissue, but it was elevated in tumorous and nontumorous portions of HCC tissue. Furthermore, the kinase activity of pp60c-src was significantly elevated in tumorous tissues compared with nontumorous tissues. The kinase activity of pp60c-src was also higher in poorly differentiated HCC. In addition, the kinase activity of pp60c-src increased proportionately with the development of HCC of LEC rats. Our results suggest that activation of the protooncogene product pp60c-src may play an important role in the malignant transformation of hepatocytes in human and LEC rats, and that it may be closely related to the histopathological grading of human HCC.     

Abnormal accumulation of copper in LEC rat liver induces expression of p53 and nuclear matrix-bound p21(waf 1/cip 1)

The LEC rat is an inbred mutant strain which spontaneously develops liver injury and subsequent liver cancer. Liver injury in LEC rats has recently been shown to be closely related to abnormal copper accumulation in the liver. Previously, we reported that LEC rat hepatocytes lose their growth potential, probably allowing selective growth of preneoplastic cells. In this study, to elucidate the effects of copper accumulation on the growth activity of LEC rat hepatocytes, we examined the growth activity and the expression of p53 and p21(waf 1/cip 1) in the livers of LEC rats fed on either a control or a low-copper diet. Potential for cell proliferation of hepatocytes obtained from normal diet fed LEC rats was almost comparable to that of the cells from age-matched Sprague-Dawley (SD) rats. Northern blot analysis showed that the expression of p53 and p21(waf 1/cip 1) was significantly high in the livers of LEC rats fed a control diet, while the expression of p53 and p21(waf 1/cip 1) in the LEC rats fed a low-copper diet was as low as that of SD rat livers. Western blot analysis consistently showed that the amount of p21(waf 1/cip 1) bound to the nuclear matrix scaffold of the LEC rat liver was reduced by feeding a low-copper diet. These findings suggest that abnormal accumulation of copper induced the expression of p53 and p21(waf 1/cip 1), resulting in the inhibition of cell proliferation of LEC rat hepatocytes.     

Hepatocyte growth factor stimulates liver regeneration and elevates blood protein level in normal and partially hepatectomized rats

The effects of recombinant human hepatocyte growth factor (HGF) on liver growth and function of normal and partially hepatectomized rats have been examined. HGF was continuously administered into the jugular vein because it was rapidly eliminated from the plasma (t1/2 alpha; approximately 4.5 min) and degraded. In normal rats, the labeling index of hepatocytes was increased about 6 times by the administration of HGF. HGF also decreased the prothrombin time and increased the hepaplastin and serum albumin content. In 70%-hepatectomized rats, HGF stimulated liver regeneration and increased the level of blood proteins such as hepaplastin in a dose-dependent manner. The stimulation of serum protein level seemed to result from not only the increase of hepatic cell number but also the direct effect of HGF on the protein production in hepatocytes, because HGF rapidly enhanced the protein synthesis prior to the increase of cell number and increased the mRNA content of albumin in the liver in vivo. In addition, a combination of heparin with HGF further accelerated the effects of HGF described above, possibly due to the decrease of HGF clearance. These findings suggest that HGF accelerates both the hepatic regeneration and function in vivo, and that rhHGF is clinically expected to be a potent therapeutic agent in hepatectomy and liver injury.     

Interaction of c-myc with transforming growth factor alpha and hepatocyte growth factor in hepatocarcinogenesis

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein 1 promoter-human TGF-alpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing the interaction of nuclear oncogenes and growth factors in tumorigenesis. Coexpression of c-myc and TGF-alpha as transgenes in the mouse liver resulted in a tremendous acceleration of neoplastic development in this organ as compared to expression of either of these transgenes alone. The two distinct cellular reactions that occurred in the liver of the double transgenic mice prior to the appearance of liver tumors were dysplastic and apoptotic changes in the existing hepatocytes followed by emergence of multiple focal lesions composed of both hyperplastic and dysplastic cell populations. These observations suggest that the interaction of c-myc and TGF-alpha, during development of hepatic neoplasia contributes to the selection and expansion of the preneoplastic cell populations which consequently increases the probability of malignant conversion. These studies have now been extended to examine the interaction of hepatocyte growth factor (HGF) with c-myc during hepatocarcinogenesis in the transgenic mouse model. While sustained overexpression of c-myc in the liver leads to cancer, coexpression of HGF and c-myc in the liver delayed the appearance of preneoplastic lesions and prevented malignant conversion. Similarly, tumor promotion by phenobarbital was completely inhibited in the c-myc/HGF double transgenic mice whereas phenobarbital was an effective tumor promoter in the c-myc single transgenic mice. The results indicate that HGF may function as a tumor suppressor during early stages of liver carcinogenesis, and suggest the possibility of therapeutic application for this cytokine. Furthermore, we show for the first time that interaction of c-myc with HGF or TGF-alpha results in profoundly different outcomes of the neoplastic process in the liver.     

Sparse distribution of hepatocyte growth factor-producing cells inside hepatocellular foci in rats treated with hepatocarcinogens

The distribution of hepatocyte growth factor (HGF)-synthesizing cells in rat liver during development of glutathione S-transferase P form (GST-P)-positive nodules after diethylnitrosamine initiation followed by promotion with 2-acetylaminofluorene plus partial hepatectomy (PH) was investigated using in situ hybridization. HGF-producing cells were non-parenchymal in nature, and were suspected to be mainly of Kupffer type. They were mostly located outside GST-P-positive lesions, in the surrounding parenchyma. In the oval cell proliferation phase 1 week after PH, they increased and they were mainly localized around the portal triads. It is concluded that HGF is directly involved in an endogenous paracrine growth pathway controlling proliferation in oval cells and in normal, but not GST-P-positive, hepatocytes.     

Stimulation of asialoglycoprotein receptor activity after transcatheter arterial embolotherapy in clinical study and continuous infusion of human hepatocyte growth factor in rat

The concentration of hepatocyte growth factor (HGF) was increased immediately after transcatheter arterial embolization (TAE) for hepatocellular carcinoma (HCC). We measured the changes in serum HGF levels and those of the asialoglyco-protein receptors (ASGP) in hepatocytes using 99mTc-galactosyl human serum albumin (GSA) on 22 patients with HCC after TAE. HGF and Rmax were increased 33% +/- 32, 40% +/- 28, respectively. Effects of HGF administration were examined in normal rats. Liver weight, hepatocyte nuclear size, and number of hepatocytes (cells/mm2) were not altered by HGF, but GSA blood clearance per hepatocyte was significantly increased over the preinfusion rate. We conclude that increased HGF stimulates a hepatocytic function of the receptor-mediated uptake of ASGP.     

Reduced C-terminal Src kinase (Csk) activities in hepatocellular carcinoma

The proto-oncogene product pp60(c-src) is the cellular homologue of the Rous sarcoma transforming gene, and it is a non-receptor-linked and membrane-associated tyrosine kinase. There is a close correlation between elevated pp60(c-src) activity and cell transformation. We have recently reported that pp60(c-src) was activated in hepatocellular carcinoma (HCC) of human and Long-Evans cinnamon (LEC) rats. However, the mechanisms involved in this process remain unknown. C-terminal Src kinase (Csk) is a novel cytoplasmic protein tyrosine kinase that inactivates the members of the Src family protein tyrosine kinase in vitro. We investigated the role of Csk in hepatocarcinogenesis by analyzing the location, amount of Csk, and its kinase activity levels in nontumorous cirrhotic and tumorous sections of HCC of patients and an animal model of LEC rats. Csk tyrosine kinase activity was significantly reduced in tumorous tissues compared with nontumorous sections of patients as well as LEC rats. A single immunoreactive band at 50 kd was detected with Csk antibody in normal liver (NL), chronic hepatitis (CH), and nontumorous cirrhotic (NTC) segments of HCC of patients and LEC rats. In human tumorous tissues, Western blot revealed a 53-kd immunoreactive band, which was slightly larger than the usual 50-kd band of Csk. These results suggest that the reduced activity of tyrosine kinase of Csk may play an important role in the malignant transformation of hepatocytes in human and LEC rat, and the appearance of 53-kd Csk-related protein may be closely involved in the progression of cirrhosis to HCC in humans, and that 50-kd Csk may act as an antioncogene through the negative regulation of pp60(c-src) in the development of human HCC.     

Increased polyploidy, delayed mitosis and reduced protein phosphatase-1 activity associated with excess copper in the Long Evans Cinnamon rat

Until now, it is not known whether copper hepatotoxicity impairs mitosis. Enlarged hepatocytes with huge nuclei considered as polyploids are frequently observed in the Long Evans Cinnamon (LEC) rat which exhibits an abnormal accumulation of hepatic copper due to a defect in the gene homologous to human Wilson's disease gene responsible for intracellular copper delivery. This defect may lead to a abnormal mitotic progression in increased polyploidization and is associated with excessive hepatic copper. This study was designed to examine whether excess copper impairs mitotic progression and results in increased polyploidization using a model of LEC rat liver. Polyploidy was analyzed by flow cytometry. The rate of mitotic progression was investigated using the fraction of mitotic hepatocytes or a mitosis-specific phosphoprotein retained in regeneration. Nuclear protein phosphatase-1 (PP-1) activity essential to mitotic progression was measured. The effect of excess copper on incidence of polyploidy, the rate of mitotic progression and nuclear PP-1 activity was investigated using age- or copper overload-dependent changes in them in LEC rat, or genetic profile-dependent changes of them in backcrosses. LEC rat liver showed an increase of polyploidy, a delay of mitotic progression, and a reduction of nuclear PP-1 activity. These abnormal features concurred with increase of copper concentration accompanied by changes of age in LEC rats from 2 to 4 months of age, induced by dietary copper overload in LEC rat, or caused by single genetic defect in backcrosses. Excess copper impairs mitotic progression, resulting in increased polyploidization. Nuclear PP-1 activity is likely to be at least one of targets of copper hepatotoxicity leading to impairment of mitotic progression.     

Pustular psoriasis in childhood

The activity of tumor necrosis factor (TNF) in serum and in peripheral blood monocytes (PBMC) was determined in 8 patients with fulminating viral hepatitis (FVH), 10 patients with chronic active hepatitis (CAH) and 10 health controls. The activity was monitored in FVH patients before and after treatment with hepatocyte growth factor (HGF). It was found that TNF level was significantly higher in CAH patients than in controls (t = 3.56, P < 0.01) and also significantly higher in FVH than in CAH patients (t = 3.07, P < 0.01). The activity of TNF decreased gradually in FVH patients after HGF treatment and there was a positive correlation (r = 0.09, P < 0.01) between the activity of serum and PBMC TNF. It was found that in animal models of hepatic necrosis there was transient elevation of TNF activity with its peak occurring after 6 hours; the peak could be lowered with HGF treatment (t = 3.65, P < 0.05). The authors are of the opinion that TNF is an important mediator causing hepatic necrosis. The relationship between HGF and TNF was also discussed.     

Antiferromagnetic coupling in (111)-oriented Co/Pt superlattices

The LEC rat is an inbred mutant strain with spontaneous hepatitis isolated from Long-Evans rats. Since approximately 40% of LEC rats die of fulminant hepatitis, the rat serves an animal model for studying the pathogenesis and treatment of human fulminant hepatitis. The remaining 60% of LEC rats survive and develop chronic (prolonged) hepatitis and subsequently develop liver cancer. Therefore, the LEC rat serves an important animal model for studying the significance of chronic hepatitis in the development of human liver cancer, which often develops in association with chronic hepatitis. The LEC rat can also be used as an animal model of Wilson's disease, since recent studies have disclosed high copper accumulation in the liver and low ceruloplasmin concentration in the serum of this mutant rat.     

Immune response to HBsAg, HBcAg and e-antigen in patients with acute hepatitis and HBsAg carriers with and without liver diseases

Humoral and/or cell-mediated (CMI) immune responses to HBAg components, human and rabbit liver specific proteins (HLP and RLP) and tuberculin were tested in patients with acute virus B and non-B-hepatitis, asymptomatic HBsAg carriers and HBsAg positive chronic active hepatitis (CAH). Furthermore, the presence of HBsAg, HBcAg and/or "e"-antigen has been studied in patients with sera and/or liver tissue. Asymptomatic HBsAg carriers are characterized by a status of immunological tolerance against HBsAg. HBcAg in liver nuclei could not be detected. All sera were positive for anti-HBc, some had anti "e". - Patients with uneventful acute virus-B-hepatitis developed CMI against HBsAg 4-6 weeks and anti-HBs 4-6 months after onset of the disease. Acute virus hepatitis without detectable HBsAg are defined as non-B-hepatitis by negative humoral and cell-mediated immune reaction against HBsAg 1-12 months after onset of the disease. - Patients with type B chronic active hepatitis are characterized by inadequate CMI against HBsAg without immune elimination of virus and virusantigens. Acute and chronic type-B-hepatitis showed temporary or constant CMI against HLP. These findings suggest an alteration or a carrier function of membrane antigens of virus infected hepatocytes or an induction of new membrane antigens by a virus. The results indicate that recovery from type B-hepatitis is associated with the ability to elicit a specific immune response to HBsAg. Furthermore immune responses to virus, virus antigens and virusinfected hepatocytes seemed to be the pathogenic principle of virus induced acute and chronic liver diseases.     

Common proteins bind mRNAs encoding erythropoietin, tyrosine hydroxylase, and vascular endothelial growth factor

Lysophosphatidic acid (LPA) is a growth factor-like mediator for fibroblasts or smooth muscle cells produced and released by activated platelets. Platelet activation occurs with hepatic necrosis and subsequent liver regeneration and fibrosis. In the fibrosis, hepatic stellate cells proliferate with phenotypic transformation to myofibroblasts. Thus, effects of LPA on proliferation of hepatocytes and stellate cells were investigated. In cultured rat stellate cells, LPA increased DNA synthesis with enhanced MAP kinase activity. Pertussis toxin (PTX) attenuated this mitogenic action. In contrast, LPA decreased DNA synthesis by cultured rat hepatocytes induced by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) without affecting protein synthesis. Enhanced MAP kinase activity by HGF or EGF was not changed by LPA. This anti-mitogenic action was attenuated by PTX. TGFbeta level in the medium was less than the level effective for inhibiting the DNA synthesis in the presence of LPA. Our results suggest that LPA might affect proliferation of hepatocytes and stellate cells in liver diseases complicating platelet activation.     

Regulation of both erythroid and megakaryocytic differentiation of a human leukemia cell line, UT-7

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and various epithelial cells. Unexpectedly, it has been reported to inhibit the growth of hepatoma cells in vitro. To clarify this phenomenon, we examined the effects of recombinant baculovirus-expressed HGF on the growth of 6 human hepatoma cell lines. The growth of Hep3B and HepG2 cells was markedly stimulated to 1.8- and 1.7-fold, respectively, PLC/PRF/5 to 1.4-fold, and SK-Hep-1 to 1.2-fold in a dose-dependent manner under HGF concentrations below 20 ng/ml. Neither HuH-7 nor HCC36 were affected. None of these cells were inhibited. All these cells expressed c-Met, the membrane receptor for HGF, and their c-Met would be activated to be phosphorylated upon addition of HGF. They also contained the ERK2 subgroup of mitogen-activated protein kinases (MAPKs). When HGF was added, their ERK2 would also be phosphorylated. The extent of ERK2 phosphorylation was partially correlated to their growth response to HGF. In conclusion, HGF could stimulate the growth of certain human hepatoma cells, probably through activation of c-Met and MAPKs.     

Primary photosensitization related to ingestion of alfalfa silage by cattle

A herd of 650 Holstein cows was examined for skin disease. Approximately 400 of the lactating adults were affected, but heifers, calves, and nonlactating cows were clinically normal. The condition was characteristic of primary photosensitization. Milk production of the affected cows was normal. Affected cows did not appear to be ill, and none of the cows was icteric. Three of 7 cows had high serum gamma-glutamyltransferase activities, but in the other 4 cows, activity was within the reference range. Serum activities of other hepatic enzymes were within reference ranges in the 7 cows that were examined. Hepatic biopsy specimens from 3 cows were normal. Specimens from 4 other cows had changes that ranged from minimal to mild, chronic, lymphoplasmacytic periportal hepatitis to acute, random, necrotizing hepatitis. Development of photosensitivity was related to ingestion of alfalfa silage. Acetone extracts of the alfalfa silage, but not of other feedstuffs, were found to inhibit growth of Candida albicans under ultraviolet light. Cows experimentally fed a diet composed exclusively of the alfalfa silage developed skin lesions after 6 days, but did not have detectable serum concentrations of phylloerythrin.     

Can clamping of splenic vessels prevent abrupt increase of portal vein pressure and migration of transplanted hepatocytes to the liver after intrasplenic hepatocyte transplantation?

BACKGROUND/AIMS: It is well known that hepatocyte transplantation can retain some proper functions, significantly improve the survival rate of rats with different models of acute fulminant hepatic failure, correct some congenital genetic disorders, and improve liver function in cirrhosis. Portal hypertension and hepatic embolization have been described following intrasplenic hepatocyte transplantation. We evaluated the effect of temporary occlusion of splenic vessels on changes in portal vein pressure and on distribution of transplanted hepatocytes after hepatocyte transplantation into the spleen in normal rats. METHODOLOGY: Liver cirrhosis has been induced in rats by 1% dimethylnitrosamine (Sigma, St. Louis, Mo) dissolved in normal saline at the dose of 10 ml of DMN/Kg, i.p., 3 consecutive days a week for 4 weeks. Donor hepatocytes were harvested by in situ ethylenediaminetetraacetic acid (EDTA) perfusion. Changes in portal vein pressures were monitored by a pressure monitor and distribution of transplanted hepatocytes was assayed by measurement of radioactivity of 51Cr-labeled transplanted hepatocytes according to clamping or non-clamping during intrasplenic hepatocyte transplantation. RESULTS: The changes in portal pressure remained significantly high 10 min after hepatocyte transplantation in the nonocclusion groups compared to the occlusion groups. However, the changes in portal vein pressures in cirrhotic rats returned to normal faster than in normal rats after cell transplantation in the nonocclusion groups. The distribution of 51Cr-labeled transplanted hepatocytes into the spleen significantly diminished radioactivity of the liver at 10 min, 2 hours, and 24 hours in the occlusion groups compared to the nonocclusion groups. Also, duration of clamping time of splenic vessels did not influence the initial distribution of transplanted hepatocytes at the time of intrasplenic hepatocyte injection. CONCLUSIONS: These results suggested that temporary occlusion of splenic vessels should be routinely used during intrasplenic hepatocyte transplantation.     

Endothelin-A and -B antagonists protect myocardial and endothelial function after ischemia/reperfusion in a rat heart transplantation model

Excessive activity of the Fas system in the liver is an essential event and contributor to fulminant hepatic failure, whose prognosis is extremely poor with high mortality due to lack of effective therapy. Administration of agonistic anti-Fas antibody to mice rapidly led to massive liver apoptosis and fulminant hepatic failure. In contrast, administration of human recombinant hepatocyte growth factor (HGF) abrogated Fas-induced massive liver apoptosis and the lethal hepatic failure. Addition of anti-Fas antibody to hepatocytes in primary culture induced cell death, but Fas-mediated cell death was potently suppressed by HGF. HGF strongly induced Bcl-xL expression and subsequently blocked Fas-mediated signaling pathway upstream of CPP32 in the liver. These results implicate a potential therapeutic usage of HGF for treatment of fulminant hepatic failure.