epidermal-growth-factor and Liver-Diseases

Mesenchymal stem cells (MSCs) are a group of stem cells derived from the mesodermal mesenchyme. MSCs can be obtained from a variety of tissues, including bone marrow, umbilical cord tissue, umbilical cord blood, peripheral blood and adipose tissue. Under certain conditions, MSCs can differentiate into many cell types both in vitro and in vivo, including hepatocytes. To date, four main strategies have been developed to induce the transdifferentiation of MSCs into hepatocytes: addition of chemical compounds and cytokines, genetic modification, adjustment of the micro-environment and alteration of the physical parameters used for culturing MSCs. Although the phenomenon of transdifferentiation of MSCs into hepatocytes has been described, the detailed mechanism is far from clear. Generally, the mechanism is a cascade reaction whereby stimulating factors activate cellular signalling pathways, which in turn promote the production of transcription factors, leading to hepatic gene expression. Because MSCs can give rise to hepatocytes, they are promising to be used as a new treatment for liver dysfunction or as a bridge to liver transplantation. Numerous studies have confirmed the therapeutic effects of MSCs on hepatic fibrosis, cirrhosis and other liver diseases, which may be related to the differentiation of MSCs into functional hepatocytes. In addition to transdifferentiation into hepatocytes, when MSCs are used to treat liver disease, they may also inhibit hepatocellular apoptosis and secrete various bioactive molecules to promote liver regeneration. In this review, the capacity and molecular mechanism of MSC transdifferentiation, and the therapeutic effects of MSCs on liver diseases are thoroughly discussed.

Topics: Adipose Tissue; Bone Marrow Cells; Cell Differentiation; Cell Transdifferentiation; Cytokines; Epidermal Growth Factor; Fetal Blood; Fibroblast Growth Factors; Hepatocytes; Humans; Liver Diseases; Liver Regeneration; Liver Transplantation; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Signal Transduction

Apoptosis not only plays a key role in physiological demise of defunct hepatocytes, but is also associated with a plethora of acute and chronic liver diseases as well as with hepatotoxicity. The present paper focuses on the modelling of this mode of programmed cell death in primary hepatocyte cultures. Particular attention is paid to the activation of spontaneous apoptosis during the isolation of hepatocytes from the liver, its progressive manifestation upon the subsequent establishment of cell cultures and simultaneously to strategies to counteract this deleterious process. In addition, currently applied approaches to experimentally induce controlled apoptosis in this in vitro setting for mechanistic research purposes and thereby its detection using relevant biomarkers are reviewed.

Topics: Animals; Apoptosis; Biomarkers; Cell Culture Techniques; Cells, Cultured; Culture Media; Epidermal Growth Factor; Extracellular Matrix; Glucagon; Hepatocytes; Humans; Insulin; Liver; Liver Diseases

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Epidermal Growth Factor; fas Receptor; Heparin-binding EGF-like Growth Factor; Hepatitis C; Humans; Intercellular Signaling Peptides and Proteins; Liver Diseases; Mice; Signal Transduction

Topics: Adult; Aged; Endocrine System Diseases; Epidermal Growth Factor; Female; Humans; Kidney Diseases; Liver Diseases; Male; Middle Aged; Neoplasms; Radioimmunoassay; Radioligand Assay

The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p < 0.05). When cumulus cells were removed from cumulus-oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7 h of in vitro maturation (IVM) (p < 0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0 h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p < 0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.

Topics: Animals; Cattle; Cattle Diseases; Cell Communication; Cell Nucleus; Cells, Cultured; Cumulus Cells; Epidermal Growth Factor; Female; Gap Junctions; Liver Diseases; Meiosis; Oocytes; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Signal Transduction

We have previously reported that epiregulin is a growth factor that seems to act on liver progenitor cells (LPCs) during liver regeneration. However, the relationship between epiregulin and LPCs has remained unclear. The aim of the present study was to clarify the role of epiregulin during liver regeneration. The serum levels of epiregulin in patients with acute liver failure were examined. A liver injury model was developed using mice fed a diet containing 0.1% 3.5-diethoxycarbonyl-1.4-dihydrocollidine (DDC) to induce LPCs. We then evaluated the expression of epiregulin and LPCs in these mice. The proliferation of epithelial cell adhesion molecule + LPCs cultured with epiregulin was examined in vitro, and finally epiregulin was overexpressed in mouse liver. In patients with acute liver failure, serum epiregulin levels were elevated significantly. In DDC mice, LPCs emerged around the portal area. Epiregulin was also detected around the portal area during the course of DDC-induced liver injury and was partially coexpressed with Thy1. Serum epiregulin levels in DDC mice were also significantly elevated. Recombinant epiregulin augmented the proliferative capacity of the LPCs in a dose-dependent manner. In mice showing overexpression of epiregulin, the expression of PCNA on hepatocytes was increased significantly. Finally, LPCs emerged around the portal area after epiregulin gene delivery. We concluded that epiregulin promotes the proliferation of LPCs and DNA synthesis by hepatocytes and is upregulated in the serum of patients with liver injury. Furthermore, induction of epiregulin leads to the appearance of LPCs. Epiregulin would be a useful biomarker of liver regeneration.

Topics: Adult; Adult Stem Cells; Animals; Antigens, Neoplasm; Cell Adhesion Molecules; Cell Line; Cell Proliferation; DNA Replication; Dose-Response Relationship, Drug; Epidermal Growth Factor; Epiregulin; Epithelial Cell Adhesion Molecule; Female; Humans; Liver; Liver Diseases; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Middle Aged; Recombinant Proteins; Thy-1 Antigens; Time Factors

The hepatic peptide hormone hepcidin controls the duodenal absorption of iron, its storage, and its systemic distribution. Hepcidin production is often insufficient in chronic hepatitis C and alcoholic liver disease, leading to hyperabsorption of iron and its accumulation in the liver. Hepatocyte growth factor (HGF) and epidermal growth factor (EGF) mediate hepatic regeneration after liver injury. We examined the effect of these growth factors on hepcidin synthesis by hepatocytes. HGF and EGF treatment of primary mouse hepatocytes, as well as EGF administration in mice, suppressed hepcidin messenger RNA (mRNA) synthesis. The suppression of hepcidin by these growth factors was transcriptional, and was mediated by a direct effect of HGF and EGF on the bone morphogenetic protein (BMP) pathway regulating hepcidin synthesis. We further show that growth factors interfered with nuclear localization of activated sons of mothers against decapentaplegic (Smad) and increased the nuclear pool of the BMP transcriptional corepressor TG-interacting factor (TGIF). In a kinase screen with small-molecule kinase inhibitors, inhibitors in the PI3 kinase pathway and in the mitogen-activated ERK kinase/extracellular signal-regulated kinase (MEK/ERK) pathway prevented HGF suppression of hepcidin in primary mouse hepatocytes.. HGF and EGF suppress hepatic hepcidin synthesis, in part through PI3 kinase MEK/ERK kinase pathways which may be modulating the nuclear localization of BMP pathway transcriptional regulators including activated Smads1/5/8 and the corepressor TGIF. EGF, HGF, and possibly other growth factors that activate similar pathways may contribute to hepcidin suppression in chronic liver diseases, promote iron accumulation in the liver, and exacerbate the destructive disease processes.

Topics: Animals; Antimicrobial Cationic Peptides; Bone Morphogenetic Proteins; Cells, Cultured; Disease Models, Animal; Epidermal Growth Factor; Gene Expression Regulation; Hepatocyte Growth Factor; Hepatocytes; Hepcidins; Humans; Liver Diseases; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Random Allocation; RNA, Messenger; Sensitivity and Specificity; Signal Transduction; Transcriptional Activation; Transfection

Increased translocation of intestinal bacteria is a hallmark of chronic liver disease and contributes to hepatic inflammation and fibrosis. Here we tested the hypothesis that the intestinal microbiota and Toll-like receptors (TLRs) promote hepatocellular carcinoma (HCC), a long-term consequence of chronic liver injury, inflammation, and fibrosis. Hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota and TLR4 activation in non-bone-marrow-derived resident liver cells. TLR4 and the intestinal microbiota were not required for HCC initiation but for HCC promotion, mediating increased proliferation, expression of the hepatomitogen epiregulin, and prevention of apoptosis. Gut sterilization restricted to late stages of hepatocarcinogenesis reduced HCC, suggesting that the intestinal microbiota and TLR4 represent therapeutic targets for HCC prevention in advanced liver disease.

Topics: Animals; Apoptosis; Bacterial Translocation; Cell Proliferation; Epidermal Growth Factor; Epiregulin; Humans; Intestines; Liver Diseases; Liver Neoplasms, Experimental; Mice; Mice, Inbred C57BL; Toll-Like Receptor 4; Tumor Cells, Cultured

Hepatocyte cell death is a characteristic indication in the development of non-alcoholic steatohepatitis (NASH); however, the underlying mechanism is still unclear. In this study, we examined the potential mechanism(s) involved in the development of liver injury using a methionine-choline deficient (MCD) diet feeding NASH model. Male C57BL6/J mice were fed MCD and methionine-choline sufficient (MCS) diet for two weeks before being killed. Our results showed that MCD diet feeding resulted in fatty liver and liver injury, evidenced by increased hepatic triglyceride (TG), plasma alanine aminotransferases and hepatic thiobarbituric acid reactive substances levels in MCD-fed mice. Furthermore, we found that MCD diet feeding caused remarkable suppression of hepatic extracellular signal-regulated kinases (ERK) 1/2 activation and increased transforming growth factor (TGF)-beta1 levels in plasma and the liver tissue. In vitro investigations showed that intracellular MEK/ERK1/2 activation status played a critical role in the determination of sensitivity of hepatocytes to TGF-beta1-induced cell death. HepG2 cells, otherwise resistant to TGF-beta1 killing due to high level of ERK1/2 activation, was sensitized by U0126, a specific MEK/ERK1/2 inhibitor, to TGF-beta1 cytotoxicity. H4IIEC3 cells, which have lower level of constitutive ERK1/2 activity, are sensitive to TGF-beta1-induced cell death. Lastly, we demonstrated that administration of epidermal growth factor, a strong ERK1/2 activator, to MCD-fed mice attenuated liver injury without affecting hepatic TG accumulation. Our findings demonstrated that hepatic ERK1/2 inactivation aggravates TGF-beta1-induced hepatotoxicity, which may contribute, at least in part, to the initiation of liver injury in NASH.

Topics: Aldehydes; Animals; Cell Line; Choline Deficiency; Cysteine Proteinase Inhibitors; Enzyme Activation; Epidermal Growth Factor; Humans; Lipid Peroxidation; Liver Diseases; Male; Methionine; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Rats; Signal Transduction; Transforming Growth Factor beta1

Activation of the epidermal growth factor receptor (EGFR) plays an important role in liver regeneration and resistance to acute injury. However its chronic activation participates in the progression of liver disease, including fibrogenesis and malignant transformation. Hepatobiliary disease represents a constant feature in the clinically relevant Fechm1pas/Fechm1pas genetic model of erythropoietic protoporphyria (EPP). Similarly, chronic administration of griseofulvin to mice induces pathological changes similar to those found in patients with EPP-associated liver injury. We investigated the hepatic expression of the EGFR and its seven most relevant ligands in Fechm1pas/Fechm1pas mice bred in three different backgrounds, and in griseofulvin-induced protoporphyria. We observed that the expression of amphiregulin, betacellulin and epiregulin was significantly increased in young EPP mice when compared to aged-matched controls in all genetic backgrounds. The expression of these ligands was also tested in older (11 months) BALB/cJ EPP mice, and it was found to remain induced, while that of the EGFR was downregulated. Griseofulvin feeding also increased the expression of amphiregulin, betacellulin and epiregulin. Interestingly, protoporphyrin accumulation in cultured hepatic AML-12 cells readily elicited the expression of these three EGFR ligands. Our findings suggest that protoporphyrin could directly induce the hepatic expression of EGFR ligands, and that their chronic upregulation might participate in the pathogenesis of EPP-associated liver disease.

Topics: Amphiregulin; Animals; Betacellulin; Cell Line; EGF Family of Proteins; Epidermal Growth Factor; Epigen; Epiregulin; ErbB Receptors; Glycoproteins; Griseofulvin; Heparin-binding EGF-like Growth Factor; Intercellular Signaling Peptides and Proteins; Liver; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Protoporphyria, Erythropoietic; Protoporphyrins; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor alpha

Acute and intense psychological stressors induce cell damage in several organs, including the heart and the liver. Much less is known about social stress. In male mice, aggressive behavior is the most common social stressor. It is remarkable that upon fighting, submandibular salivary glands release a number of peptides into the bloodstream including epidermal growth factor (EGF). We showed previously that released EGF protects the heart from cell damage in this particular stressful situation. Here, we studied the effect of an aggressive encounter on the liver and whether EGF has a similar effect on this organ. An aggressive encounter in male mice caused inflammatory response and a transient increase in plasma alanine and aspartate transaminase activities. At 3 h, focal infiltration of neutrophils was observed in liver parenchyma. These cells accumulate on eosinophilic hepatocytes, which may correspond to dying cells. A few hours later, evidence of necrotic lesion was observed. Surgical excision of submandibular glands, sialoadenectomy, did not prevent the rise in plasma EGF concentration and did not affect the increase in plasma transaminase activities. Neither did the administration of tyrphostin AG-1478 (inhibitor of EGF receptor kinase) alter the increase in plasma alanine transaminase activity. However, it did enhance the rise in both aspartate transaminase and creatine kinase activity, suggesting heart damage. We conclude that an aggressive encounter causes mild liver damage and that released EGF does not protect this organ, in contrast to its effect on the heart.

Topics: Aggression; Animals; Cell Death; Corticosterone; Epidermal Growth Factor; ErbB Receptors; Hepatocytes; Interleukin-6; Kupffer Cells; Liver; Liver Diseases; Liver Glycogen; Male; Mice; Neutrophils; Shock, Septic; Stress, Psychological; Tyrphostins

It is unknown whether heparin-binding EGF-like growth factor (HB-EGF) can be a therapeutic agent, although previous studies suggested that HB-EGF might be a hepatotrophic factor. This study explores the potential of hepatic HB-EGF gene therapy in comparison with HGF.. Mice received an intraperitoneal injection of the agonistic anti-Fas antibody 72 h after an intravenous injection of either adenoviral vector (1x10(11) particles) expressing human HB-EGF (Ad.HB-EGF), human HGF (Ad.HGF) or no gene (Ad.dE1.3), and were sacrificed 24 or 36 h later to assess liver injury and regeneration.. Exogenous HB-EGF was predominantly localized on the membrane, suggesting the initial synthesis of proHB-EGF in hepatocytes. The control Ad.dE1.3-treated mice represented remarkable increases in serum ALT and AST levels and histopathologically severe liver injuries with numerous apoptosis, but a limited number of mitogenic hepatocytes. In contrast, the liver injuries and apoptotic changes were significantly inhibited, but the mitogenic hepatocytes remarkably increased, in both the Ad.HB-EGF- and Ad.HGF-treated mice. More mitogenic hepatocytes and milder injuries were observed in the Ad.HB-EGF-treated mice.. HB-EGF has more potent protective and mitogenic effects for hepatocytes than HGF, at least for the present conditions. In vivo hepatic HB-EGF gene transduction is therapeutic for Fas-induced liver injury.

Topics: Adenoviridae; Animals; Antibodies; Apoptosis; Cell Membrane; Epidermal Growth Factor; fas Receptor; Genetic Therapy; Heparin-binding EGF-like Growth Factor; Hepatocyte Growth Factor; Intercellular Signaling Peptides and Proteins; Liver; Liver Diseases; Liver Regeneration; Male; Mice; Mice, Inbred C57BL; Transduction, Genetic

To study the effect of IL-10 on the expression of growth factors--transforming growth factor-beta1 (TGF-beta1), epidermal growth factor (EGF), hepatocyte growth factor (HGF) and platelet-derived growth factor (PDGF) of hepatic stellate cells (HSCs) of hepatic fibrosis rat and the anti-fibrogenic role of exogenous IL-10.. Hepatic fibrosis was induced by CCl(4) administration intra-peritoneally. Sixty clean male Sprague-Dawley (SD) rats were randomly divided into three groups: normal control group (GN, 8 rats), hepatic fibrosis model group (GC, 28 rats) and IL-10 treated group (GI, 24 rats). At the beginning of the 7th and 11th wk, rats in each group were routinely perfused with pronase E and type IV collagenase through a portal vein catheter and the suspension obtained from the liver was spun by centrifugation with 11% Nycodenz density gradient to isolate HSCs. Histological examination was used to determine the degree of hepatic fibrosis. RT-PCR was employed to analyze mRNA expression from freshly isolated cells. Immunocytochemistry was performed to detect protein expression in primary cultured HSCs.. Rat hepatic fibrosis was developed with the increase of injection frequency of CCl(4), and HSCs were successfully isolated. At the 7th and 11th wk, TGF-beta1, EGF, and HGF mRNA in GC increased obviously compared with GN (P = 0.001/0.042, 0.001/0.001, 0.001/0.001) and GI (P = 0.001/0.007, 0.002/0.001, 0.001/0.001). For TGF-beta1, no difference was observed between GI and GN. For EGF, mRNA level in GI increased compared with GN during the 7th wk (P = 0.005) and 11th wk (P = 0.049). For HGF, mRNA level in GI decreased compared with GN at the 7th wk (P = 0.001) and 11th wk (P = 0.021). Between these two time points, TGF-beta1 expression at the 7th wk was higher than that of the 11th wk (P = 0.049), but for EGF, the former was lower than the latter (P = 0.022). As for PDGF mRNA, there was no significant difference between these groups, but difference seemed to exist in protein levels. Results by immunocytochemistry of TGF-beta1 and EGF were paralleled with the above findings.. The expression of TGF-beta1, EGF and HGF increased in HSC of hepatic fibrosis rat and decreased after treatment with IL-10. IL-10 plays an anti-fibrogenic role by suppressing growth factors expression.

Topics: Animals; Base Sequence; Carbon Tetrachloride Poisoning; DNA Primers; Epidermal Growth Factor; Gene Expression Regulation; Hepatocyte Growth Factor; Interleukin-10; Liver Diseases; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger; Transforming Growth Factor beta; Transforming Growth Factor beta1

We reported recently that heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family, is a new hepatotrophic factor for the regeneration of rat liver after partial hepatectomy. The current study examined changes in the amount of HB-EGF mRNA in liver injured by hepatotoxins. The level of HB-EGF mRNA was very low in normal rat liver, but increased markedly in the liver of rats injured by CCl4, showing two peaks, the first at 6 h and the second at 36 h. Western blot analysis showed that HB-EGF protein in the liver of CCl4-treated rats (at 6 h) was increased about 3.4-fold above normal. The level of HB-EGF mRNA also increased markedly in the liver of rats treated with D-galactosamine, showing a major peak at 18 h, and a smaller one at 6 h. These results indicate that HB-EGF may play a role in the regeneration of the liver following hepatotoxic injury.

Topics: Animals; Blotting, Western; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Epidermal Growth Factor; Galactosamine; Gene Expression; Heparin; Liver; Liver Diseases; Male; Rats; Rats, Sprague-Dawley; RNA, Messenger

This study was designed to evaluate the concentration and the regional distribution of TGF-alpha and EGF in normal and portal hypertensive human gastric mucosa. To this end we measured by RIA the gastric and duodenal concentration of TGF-alpha and EGF in subjects with chronic hepatitis, who had normal gastric endoscopic appearance, and in patients with liver cirrhosis with and without congestive gastropathy. Our results show that TGF-alpha concentration is significantly higher than EGF concentration in both the stomach and duodenum. No significant regional differences in the distribution of the two peptides were found. Moreover, the gastroduodenal tissue levels of TGF-alpha were comparable in subjects with and without hypertensive gastropathy. EGF gastric concentration was not altered in patients with congestive gastropathy. However, EGF duodenal tissue levels were significantly lower in patients with liver cirrhosis than in noncirrhotic subjects. We speculate that the higher level of TGF-alpha in the gastroduodenal mucosa may support the hypothesis that TGF-alpha and not EGF is the major physiological ligand for TGF-alpha/EGF receptor in the intact gut. Furthermore, the lower duodenal concentration of EGF in cirrhotics might partially explain the increased susceptibility of cirrhotic patients to duodenal ulcer.

Topics: Adult; Aged; Biopsy; Chronic Disease; Duodenum; Epidermal Growth Factor; Female; Gastric Mucosa; Humans; Hypertension, Portal; Intestinal Mucosa; Liver Diseases; Male; Middle Aged; Radioimmunoassay; Reference Values; Stomach Diseases; Transforming Growth Factor alpha

To characterize the receptor(s) for human hepatocyte growth factor (hHGF), a physiological hepatotrophic factor involved in liver regeneration following hepatic injury, recombinant hHGF (rhHGF) was radioiodinated. The labeled rhHGF retained its full biological activity on adult rat hepatocytes in primary culture. The specific binding of [125I]iodo-rhHGF to hepatocytes reached a plateau within 240 min at 4 degrees C. Scatchard plot analysis of the binding data suggested the presence of two classes of high affinity binding sites for [125I]iodo-rhHGF. One of the sites had a dissociation constant (Kd) of about 4.6 pM with 300 sites/cell and the other has a Kd of about 275 pM with 15,160 sites/cell. Unlabeled rhHGF displaced cell surface-bound [125I]iodo-rhHGF in a dose-dependent manner as did native hHGF purified from plasma of patients with fulminant hepatic failure. However, other growth factors to rat hepatocytes in primary culture such as insulin and human epidermal growth factor, and proteins which have high amino acid sequence-homology to hHGF such as plasminogen and prothrombin, did not compete with [125I]iodo-rhHGF in the binding, which suggests the binding was specific to hHGF. Covalent cross-linking experiment of [125I]iodo-rhHGF to cell surface receptor(s) on hepatocytes showed there were two macromolecular species with apparent molecular weights of 330,000 and 230,000. Unlabeled rhHGF and native hHGF competed for the binding of [125I]iodo-rhHGF to the two macromolecular species, but insulin, human epidermal growth factor, plasminogen, and prothrombin did not. Based upon our estimated molecular weight of rhHGF = 84,000, these results suggest that hHGF specifically binds to two polypeptides of 246,000 and 146,000 daltons which are likely to represent the hHGF receptors of primary cultured rat hepatocytes.

Topics: Animals; Autoradiography; Binding Sites; Cells, Cultured; Cross-Linking Reagents; DNA; Electrophoresis, Polyacrylamide Gel; Epidermal Growth Factor; Humans; Insulin; Liver; Liver Diseases; Plasminogen; Prothrombin; Proto-Oncogene Proteins c-met; Rats; Receptors, Cell Surface

One of the many hypotheses put forward to explain the mechanism by which phenobarbital (PB) promotes hepatocarcinogenesis is by differential mitoinhibition of surrounding hepatocytes while allowing the initiated hepatocytes to respond to growth stimuli and form foci and nodules. Given the similarity in structures between PB and orotic acid (OA), another rat liver tumor promoter, the present investigation was designed to determine (i) whether PB, like OA, exerts its mitoinhibitory effect at a site beyond the growth factor receptor and receptor mediated early events; and (ii) whether PB exerts a differential mitoinhibitory effect by selectively inhibiting the non-initiated hepatocytes but not the initiated hepatocytes in vitro. Our studies demonstrate that, like OA, PB also inhibits DNA synthesis in hepatocytes from normal rat liver in a dose dependent manner with 80-90% at a dose of 6 mM. One target site may lie beyond the growth factor receptor mediated early events because PB inhibited DNA synthesis in hepatocytes primed with the growth factor 24 h earlier. Interestingly, PB inhibited DNA synthesis not only in hepatocytes from non-nodular surrounding liver but also in hepatocytes from persistent hepatic nodules initiated with 1,2-dimethylhydrazine and promoted with OA. Therefore, our results suggest that although PB is a mitoinhibitor of DNA synthesis in hepatocytes, it does not appear to create as strong a differential mitoinhibition between non-nodular surrounding and initiated hepatocytes as is evident in the resistant hepatocyte and OA models. These results raise the question whether differential mitoinhibition is the major contributing factor in the PB mediated rat liver tumor promotion.

Topics: Animals; Carcinogens; Cells, Cultured; DNA Replication; Epidermal Growth Factor; ErbB Receptors; Liver; Liver Diseases; Male; Orotic Acid; Phenobarbital; Rats; Rats, Inbred F344; Transforming Growth Factor alpha

We carried out studies on the expression of liver-specific genes during regeneration of the liver and searched for changes in the expression of oncogenes and housekeeping genes. Albumin and ornithine transcarbamylase genes were the liver-specific genes examined by Northern blot analysis, using total RNAs isolated from residual livers of Sprague-Dawley rats subjected to a 68% partial hepatectomy. The mRNA levels of both genes began to decrease 8 hr after hepatectomy, both reaching the lowest levels at 24 hr, and then recovered to some extent at 48 hr. In contrast, these levels in the housekeeping and growth-related genes were augmented during this period. This would suggest that there is a selective expression of growth-related and housekeeping genes, in preference to liver-specific genes during liver regeneration. The expression of these genes in the regenerating liver was simulated in primary cultured hepatocytes during the dedifferentiation processes. It would appear that the first step in regeneration of the residual liver is dedifferentiation, in which the depression of liver-specific genes may be linked to liver dysfunction following hepatectomy.

Topics: Animals; Cells, Cultured; Epidermal Growth Factor; Gene Expression; Hepatectomy; Liver; Liver Diseases; Liver Regeneration; Ornithine Carbamoyltransferase; Postoperative Complications; Postoperative Period; Rats; Rats, Inbred Strains; Serum Albumin; Transcription, Genetic

In certain etiological groups of patients with fulminant hepatic failure, poor survival may be due to lack of liver regeneration. In vitro experiments have shown that fulminant hepatic failure serum is cytotoxic to rabbit hepatocytes and inhibits DNA synthesis on short-term incubation with isolated regenerating rat hepatocytes. When fulminant hepatic failure serum is injected into partially hepatectomized rats at the time of maximal DNA synthesis, [3H]thymidine incorporation into hepatic DNA is reduced significantly. The effect is greater with sera obtained from patients with fulminant hepatic failure due to non-A, non-B hepatitis or an adverse drug reaction and is associated with a less than 10,000-dalton fraction. No stimulation of DNA synthesis is observed with injection of the greater than 10,000-dalton serum fraction into normal rats. In preliminary experiments, no increase in epidermal growth factor production has been found in liver failure. Overall, the substances present in fulminant hepatic failure serum appear to be inhibitory rather than stimulatory for liver cell regeneration.

Topics: Animals; Cells, Cultured; DNA; Epidermal Growth Factor; Hepatectomy; Humans; Liver; Liver Diseases; Liver Regeneration; Rabbits; Rats

We have recently purified human hepatocyte growth factor (hHGF), a heterodimer with molecular weight of about 83,000, from plasma of patients with fulminant hepatic failure (Gohda, E. et al., J. Clin. Invest. 81, 414-419, 1988). Biological and immunological properties of hHGF were examined. Out of the well-known growth factors tested, only epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) stimulated DNA synthesis of adult rat hepatocytes in primary culture. hHGF enhanced the DNA synthesis at less than one-tenth of the molar concentrations of EGF and TGF-alpha. Half-maximal stimulations by hHGF, EGF and TGF-alpha were observed at 30, 400 and 900 pM, respectively. Maximal stimulation by TGF-alpha, however, was greater than those caused by hHGF and EGF. The effect of hHGF was additive with the maximal effects of EGF and TGF-alpha. Anti-hHGF antiserum was prepared in a rabbit by injecting with purified hHGF. This antiserum recognized nonreduced hHGF, but not reduced hHGF. The antiserum for hHGF did not inhibit growth-promoting activity of EGF, that was neutralized by incubation with anti-EGF antiserum. The activity of hHGF was completely inhibited by anti-hHGF antiserum, but not by anti-EGF antiserum. hHGF did not show any cross-reactivity to anti-EGF antiserum as measured by enzyme immunoassay for EGF. Thus, biological and immunological properties of hHGF are different from those of EGF and TGF-alpha.

Topics: Animals; Antigens; Cells, Cultured; DNA; Epidermal Growth Factor; Growth Substances; Hepatocyte Growth Factor; Humans; Immunoblotting; Immunoenzyme Techniques; Liver; Liver Diseases; Male; Molecular Weight; Rats; Rats, Inbred Strains; Transforming Growth Factors

Hepatic transport of epidermal growth factor (EGF) was studied in D-galactosamine-intoxicated rats by the multiple-indicator dilution (MID) method. The extraction ratio of 125I-labeled EGF in the intoxicated rats, obtained from a model-independent analysis of the dilution curves, decreased to 45% of the control values. A distributed two-compartment model was fitted to the dilution data by nonlinear least-squares regression, and the kinetic parameters, kon.PT (product of on-rate constant and receptor density), koff (off-rate constant) and ks (sequestration rate constant) were determined. The values of kon.PT and ks in the intoxicated rats decreased to approximately one-half and one-third of those in the control rats respectively. Similar decreases in the kon.PT and ks values in the intoxicated rats were also observed for the transport of 125I-labeled insulin, a positive control, into the liver. The 125I-labeled EGF binding experiment at equilibrium using liver homogenates revealed that the intoxication reduced the receptor density (PT) to one-third of the control values, whereas the equilibrium dissociation constant (kd) did not change significantly. The activities of Na+,K+-ATPase, cytochrome P-450 and glutathione S-transferase decreased in the intoxicated rats to 70-80% of the control values. The number of nuclei per unit area of tissue slices was also reduced to 70% of the control. Thus, the extent to which the enzyme activities and the number of nuclei decreased in the intoxicated liver was smaller than that of the number of EGF receptors. It is concluded that the reduction of EGF receptors cannot be explained by the "intact hepatocyte hypothesis" but rather by the functional change of hepatocytes induced by the administration of D-galactosamine.

Topics: Animals; Chemical and Drug Induced Liver Injury; Epidermal Growth Factor; ErbB Receptors; Galactosamine; Indicator Dilution Techniques; Liver; Liver Diseases; Male; Rats; Rats, Inbred Strains

Topics: Animals; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; DNA; Epidermal Growth Factor; Glucagon; Insulin; Liver; Liver Diseases; Liver Regeneration; Male; Necrosis; Rats; Rats, Inbred Strains

Topics: Adult; Aged; Bile; Biliary Tract Diseases; Epidermal Growth Factor; Female; Humans; Liver Diseases; Male; Middle Aged

The partial purification and characterization of a hepatocyte proliferation stimulatory factor (HPSF) isolated from the liver of D-galactosamine-treated rats are described. The HPSF was a heat-labile, acid-stable and trypsin-sensitive protein. The partially purified HPSF stimulated DNA synthesis and increased the labeling index of parenchymal hepatocytes at 5 micrograms/ml and maximally at 50 micrograms/ml. The effect of HPSF in stimulating DNA synthesis was synergistic with that of insulin plus epidermal growth factor (EGF). The HPSF was scarcely detected in normal rat liver. The results obtained indicate that this HPSF is distinct from insulin, multiplication-stimulating activity (MSA), EGF and other hepatocyte growth factors previously reported, and suggest a plausible role for HPSF in the regeneration of liver tissue following hepatotoxic damage.

Topics: Animals; Chemical and Drug Induced Liver Injury; Chemical Precipitation; Chromatography; Dialysis; DNA; Epidermal Growth Factor; Galactosamine; Growth Substances; Hot Temperature; Hydrogen-Ion Concentration; Insulin; Liver; Liver Diseases; Male; Rats; Rats, Inbred Strains; Trypsin

The binding of epidermal growth factor, asialoorosomucoid, and apoprotein E-rich lipoproteins to isolated hepatocytes was investigated at various time intervals during the step-by-step development of liver cancer in rats. The degree of binding of the three ligands showed a progressive reduction in early persistent and late persistent putative preneoplastic hepatocyte nodules. This was further decreased in hepatocytes isolated from unequivocal hepatocellular carcinomas. Regenerating liver hepatocytes bound lesser amounts of epidermal growth factor and asialoorosomucoid than did hepatocytes from control resting liver but increased amounts of apoprotein E-rich lipoproteins. The progressive decrease in ligand binding during the precancerous phase of hepatocarcinogenesis, the nodule-to-cancer sequence, may render nodules less responsive to the influences of their external environments.

Topics: Animals; Apolipoproteins E; Asialoglycoproteins; Chemical and Drug Induced Liver Injury; Epidermal Growth Factor; Ligands; Lipoproteins; Liver; Liver Diseases; Liver Neoplasms, Experimental; Liver Regeneration; Male; Orosomucoid; Precancerous Conditions; Rats; Rats, Inbred F344; Receptors, Cell Surface

Plasma from patients with fulminant hepatic failure obtained during plasma exchange therapy, like their serum, demonstrated marked stimulatory activity on DNA synthesis in cultured rat hepatocytes. Heat treatment at 56 degrees C for 30 min did not affect this activity of the plasma, but reduced that of the serum. This growth-promoting activity was confirmed by showing that the patients' serum and plasma increased the labeling index with [3H]thymidine and the total number of nuclei in hepatocyte cultures. The activity of pooled active fractions obtained by gel filtration of the heated plasma was lost completely on heat treatment at 80 degrees C for 10 min or on treatment with trypsin or chymotrypsin, which suggests that it was due to a protein. The human hepatocyte growth factor was purified about 600-fold from heated plasma of a patient by ammonium sulfate precipitation and chromatographies on Affi-Gel Blue and hydroxylapatite. The maximum effect of this partially purified factor on DNA synthesis in cultured hepatocytes was greater than that of epidermal growth factor. The molecular weight of the hepatocyte growth factor was about 85,000 as determined by SDS-PAGE.

Topics: Animals; Cells, Cultured; DNA; Epidermal Growth Factor; Growth Substances; Hot Temperature; Humans; Insulin; Liver; Liver Diseases; Molecular Weight; Rats