Sustained induction of hepatocyte growth factor by sensitization with freeze-thawed hepatic tissue

Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, and has therapeutic potential for fibrotic/cirrhotic liver. Therefore, the induction of HGF in vivo is considered to be useful in the treatment of liver dysfunction caused by cirrhosis, chronic hepatitis, or extensive surgical resection. In this study, we examined the sustained induction of HGF by inoculation of freeze-thawed hepatic tissue (FTHT). Serum from rats inoculated with FTHT increased [³H]thymidine incorporation i.e., increased DNA synthesis, in primary cultured rat hepatocytes. The DNA synthesis was significantly promoted by the addition of the FTHT-sensitized serum, while this DNA synthesis was inhibited by neutralizing anti-rat HGF antibody. The concentration of HGF in the FTHT-sensitized serum was increased by day 3 after the inoculation. The time of HGF induction was dependent on the inoculated volume of FTHT, but peaks of HGF concentration were found on day 5 with different volumes of FTHT. Injurins, inducers of HGF, were also induced in the FTHT-sensitized rats, with their peak levels on day 3. The FTHT inoculated tissue showed inflammatory cell infiltration, which was gradually absorbed, and had completely disappeared by day 14 after the inoculation. Although mild inflammatory cell infiltration was observed in non-freeze-thawed inoculated hepatic tissue (NFHT) a tight capsule formed around the NFHT, and was scarcely phagocytized on day 14. These results suggest that FTHT inoculation induces HGF sustainedly through the increased synthesis of injurins, and that freeze-thawed tissue, which is easily phagocytized, is important for the sustained induction of HGF.     

Recombinant Human Interleukin-6 Induces Hepatocyte Growth Factor Production in Cancer Patients

Background: Experiments in animals demonstrate an important role for interleukin-6 (IL-6) in liver regeneration. It is suggested that IL-6 initiates hepatocyte growth factor (HGF) synthesis. Methods: The aim of the study was to examine the effect of exogenously administered recombinant human IL-6 (rhIL6), in doses of 0.5, 1.0, 2.5, 5, 10 and 20 µg/kg/day, on HGF serum levels in humans. Serum HGF levels were measured on days 1, 2, 3, 8 and 15 and were correlated with serum amyloid A (SAA) and C-reactive protein (CRP). Results: Median HGF levels increased to 124% at day 3 (P < 0.05) and 157% (P < 0.05) at day 8 as compared to 100% levels at day 1. An IL-6 dose-dependent increase in HGF was found at day 8 (R = 0.53, P < 0.02). The percentual change in serum HGF level at day 8 correlated with IL-6 serum levels at day 1 R = 0.59, P < 0.01). HGF levels did not correlate with CRP and SAA. Conclusion: In humans, rhIL-6 administration resulted in an increase in serum HGF levels.     

Free 67Ga enters into the hepatocytes of carbon tetrachloride-treated rats

Gallium-67 (⁶⁷Ga) has been used as tumor or inflammation-imaging agent in nuclear medicine for decades. Although many hypotheses concerning the mechanism of uptake of ⁶⁷Ga into tumors and inflammation have been proposed, consensus has not been reached. If the mechanism of ⁶⁷Ga uptake is clarified, we can improve the sensitivity of diagnostic imaging with ⁶⁷Ga. We attempted to clarify the mechanism of ⁶⁷Ga uptake by the liver of carbon tetrachloride (CCl₄)-treated rats. First, we investigated whether or not transferrin (Tf) is involved in ⁶⁷Ga uptake by the liver tissue of CCl₄-treated rats. It is well known that Fe³⁺ can inhibit the binding of ⁶⁷Ga to Tf. The administration of FeCl₃ 5 min before the injection of ⁶⁷Ga slightly enhanced the uptake of ⁶⁷Ga by the liver tissue of CCl₄-treated rats. The entering of ⁶⁷Ga into hepatocytes of CCl₄-treated rats was similar to the uptake by the liver tissue. In addition, the administration of FeCl₃ slightly increased the entering of ⁶⁷Ga to hepatocytes. These results suggest that free ⁶⁷Ga enters into hepatocytes from the liver of CCl₄-treated rats.     

Enhancement of retrovirus-mediated gene transfer to rat liver in vivo by infusion of hepatocyte growth factor and triiodothyronine

BACKGROUND/AIMS: Gene transfer using recombinant Moloney murine leukemia viruses (rMoMuLV) requires mitosis of the target cell. Previously, we and others have used partial hepatectomy for induction of hepatocellular proliferation for gene transfer to the liver in vivo by exsanguineous perfusion with rMo-MuLV. We hypothesized that induction of hepatocellular proliferation by combined administration of two hepatocellular mitogens, hepatocyte growth factor (HGF) and triiodothyronine (T3), should permit rMo-MuLV-mediated gene transfer into liver without invasive approaches. METHODS: HGF (1 mg/kg) was perfused continuously into the portal vein of Wistar male rats and T3 (2 mg/kg) was injected subcutaneously. Twenty-four hours after injecting HGF and T3, the state of proliferation of hepatocytes was estimated from the incorporation of 5'-bromo-2'-deoxy-uridine (BrdU). The amphotropic retroviral receptor (Ram-1) expression of liver was evaluated at different time points after injecting HGF and T3 by means of Northern blotting using Ram-1 cDNA probe. In order to evaluate the role of hormone treatment on gene transfer, the liver was perfused exsanguineously with rMoMuLV 24 h after injection with hormones. RESULTS: Rats treated with a combination of HGF and T3 expressed BrdU and beta-galactosidase in 8.3% and 0.7% of hepatocytes, respectively. On the other hand, there was near absence of gene transfer in untreated rats perfused with rMoMuLV Twenty-four hours after the initial manipulation, abundant expression of Ram-1 mRNA was observed in rat hepatocytes treated with HGF plus T3. CONCLUSIONS: Stimulation of hepatocellular mitosis and upregulation of Ram-1 expression by HGF and T3 augment retrovirus-mediated gene transfer into hepatocytes.     

Triiodothyronine and interleukin‐6 (IL‐6) induce expression of HGF in an immortalized rat hepatic stellate cell line

Abstract: Background/Aims: Despite its being considered a primary mitogen for hepatocytes, triiodothyronine (T3) has no effect on the proliferation of hepatocytes in vitro, and in our studies, induces significant in vivo hepatocyte proliferation only during liver injury. We hypothesized that T3 may affect hepatocytes proliferation indirectly, by inducing other cells in the liver to secrete hepatic mitogens. Methods: In vivo studies: Lipopolysaccharide, T3 and a combination of the two were injected into rats, and hepatocyte proliferation was determined by PCNA staining and mitotic index. In vitro studies: a rat hepatic stellate cell line (HSC‐6T) was cultured with T3, IL‐6 and a combination of the two, and we assessed the effect of these cytokine/hormone combinations on the cell proliferation and on secretion of IL‐6 and HGF, measured by ELISA. Expression of thyroid hormone receptors was assessed by RT‐PCR. Results: In vivo: T3, together with lipopolysaccharide, enhances PCNA staining and the mitotic index of hepatocytes in the treated rats. In vitro: the hepatic stellate cell line expresses thyroid hormone receptor α1, but not β1. Proliferation of stellate cells is not affected by T3, with or without IL‐6. T3 has no effect on secreted levels of IL‐6 in the stellate cell line. Hepatic stellate cells cultured with T3 and IL‐6 show significantly increased amounts of secreted HGF after 48 h in culture. Conclusion: T3 may induce hepatocyte proliferation in vivo during injury by turning on expression of HGF in stellate cells and acting together with IL‐6.     

Recombinant human interleukin-6 induces hepatocyte growth factor production in cancer patients.

BACKGROUND: Experiments in animals demonstrate an important role for interleukin-6 (IL-6) in liver regeneration. It is suggested that IL-6 initiates hepatocyte growth factor (HGF) synthesis. METHODS: The aim of the study was to examine the effect of exogenously administered recombinant human IL-6 (rhIL-6), in doses of 0.5, 1.0, 2.5, 5, 10 and 20 micrograms/kg/day, on HGF serum levels in humans. Serum HGF levels were measured on days 1, 2, 3, 8 and 15 and were correlated with serum amyloid A (SAA) and C-reactive protein (CRP). RESULTS: Median HGF levels increased to 124% at day 3 (P < 0.05) and 157% (P < 0.05) at day 8 as compared to 100% levels at day 1. An IL-6 dose-dependent increase in HGF was found at day 8 (R = 0.53, P < 0.02). The percentual change in serum HGF level at day 8 correlated with IL-6 serum levels at day 1 R = 0.59, P < 0.01). HGF levels did not correlate with CRP and SAA. CONCLUSION: In humans, rhIL-6 administration resulted in an increase in serum HGF levels.     

Protein and glycoprotein synthesis and secretion by the diabetic liver

Summary Protein and glycoprotein synthesis and secretion by isolated perfused livers and isolated hepatocytes from control and streptozotocin diabetic rats have been investigated. ³H-Leucine and ¹⁴Cglucosamine incorporation were used as markers for protein and glycoprotein synthesis and secretion. Total protein secretion was reduced by 50% in the perfusate (p <0.001) and by 36% in hepatocytes (p <0.05), but glycoprotein secretion was unchanged in both preparations from diabetic animals. These differences were not due to changes in the available pool sizes of the different labels. On liver fractionation, all membrane components from the liver of diabetic animals had lowered ³H-leucine:¹⁴Cglucosamine ratios in relation to the control animals. This was caused by enhanced glucosamine incorporation in relation to that of leucine. It is suggested that whereas protein synthesis is decreased in acutely diabetic rats, the production of glycoproteins is normal and occurs by the same pathway as in control animals.     

The role of a protease inhibitor against hepatectomy

BACKGROUND/AIMS: Nafamostat Mesilate (NM) is a synthetic serine protease inhibitor that is capable of inhibiting the various coagulation factors. To determine whether NM may also be useful in attenuating operative invasiveness, we investigated the effects of perioperative administration of NM on postoperative serum levels of proinflammatory cytokine IL-6 and hepatocyte growth factor (HGF). METHODOLOGY: Thirty patients undergoing hepatectomy with hepatocellular carcinoma, biliary carcinoma and metastatic colorectal cancer were enrolled in this study. These patients were separated into two groups; high invasive group (resected liver volume: 1000cm3 <) and less invasive group (resected liver volume: 1000cm3 >). The high invasive group of 11 patients received perioperative administration of NM (Group NM), while the less invasive group of 19 patients did not (Group C). Serum levels of IL-6, HGF and soluble IL-6 receptor (sIL-6R) were simultaneously measured on preoperative and postoperative day ('day 0', 'day 7'). RESULTS: Serum IL-6 levels on day 0 were significantly elevated and returned to preoperative levels on day 7 in both groups, and the serum IL-6 level in Group NM on day 0 was significantly lower than that in Group C on day 0. Serum HGF levels on day 0 and day 7 were significantly higher in Group NM than those in Group C. Compared with healthy control subjects, the higher serum level of HGF on the preoperative day in all patients was attributable to tumor-burden. The sIL-6R levels on day 0 decreased in both groups, and their levels in Group NM were significantly lower than those in Group C, suggesting that increased synthesis of IL-6/sIL-6R complex which could accelerate liver regeneration. CONCLUSIONS: These results suggested that perioperative administration of NM may attenuate surgical stress by decreasing production of proinflammatory cytokine IL-6, and may accelerate liver regeneration through stimulation with the IL-6/sIL-6R complex and possible involvement of increased production of HGF.     

Serum HGF and TGF‐β1 levels after right portal vein embolization

Aim: The changes in the serum hepatocyte growth factor (HGF) and transforming growth factor (TGF)‐beta1 levels after portal vein embolization (PVE), and their clinical significance, remain unclear and we aimed to assess their relationship. Methods: The serum HGF and TGF‐beta1 levels were prospectively measured in 22 patients before and 1, 3, 5, 7, and 14 day after right PVE. Computed tomographic volumetry was performed before and at a mean of 26 ± 4 days after right PVE. Results: Three to four weeks after right PVE, the volume of embolized lobe significantly decreased from 704 ± 157 cm³ before PVE to 539 ± 168 cm³ after PVE (P < 0.001). In contrast, the volume of nonembolized lobe significantly increased from 426 ± 142 cm³ to 560 ± 165 cm³ (P < 0.001). The serum HGF level significantly increased on day 3 after PVE compared with the pretreatment level (P = 0.005), while the serum TGF‐beta1 level significantly decreased and reached its lowest value on day 3 (P = 0.002). Using Pearson's correlation analysis, we found that the serum HGF and TGF‐beta1 levels on day 14 negatively associated with the large hypertrophic response in the nonembolized lobe (HGF: r = ?0.490, P = 0.021; TGF‐beta1: r = ?0.473, P = 0.026). Conclusions: PVE induced an increase in the serum HGF level and reduced the serum TGF‐beta1 level. Measurement of serum HGF and TGF‐beta1 levels on day 14 after right PVE may be useful for assessment of the future liver hypertrophy in nonembolized lobe after right PVE.     

Effect of age on RNA synthesis by rat hepatocytes.

The incorporation of (³H) orotic acid into RNA by hepatocytes from rats of various ages was studied. A 37% decrease in the incorporation of (³H) orotic acid into RNA was observed by hepatocytes isolated from 30-month-old rats compared to 12-month-old rats. There was no age-related difference in the incorporation of (³H) orotic acid into acid-insoluble material by hepatocytes. The incorporation of (³H) orotic acid into UTP by hepatocytes was determined by high pressure liquid chromatography. The (³H) orotic acid was rapidly converted to UTP by hepatocytes, and the specific activity of the UTP reached a steady state after 10 min of incubation. There was no significant difference in the specific activity of the UTP in hepatocytes isolated from rats ranging in age from 6 to 25 months. Therefore, the age-related decrease in the incorporation of (³H) orotic acid into RNA by hepatocytes is not due to an age-related difference in the uptake or conversion of (³H) orotic acid into UTP.     

Antirheumatic drugs ands macrophage function: Effects on tumour cell crowth in vitro

Summary The ability of the antirheumatic drugs D-penicillamine, chloroquine and levamisole to modify macrophage-mediated inhibition of tumour cell growth in vitro was investigated. Increasing numbers of rat peritoneal macrophages were cocultured with a fixed number of ascites hepatoma AH-13 rat tumour cells. Tumour cell growth was assessed as the uptake of³H-thymidine (³H-TdR) by AH-13 cells at the end of a 24h period of coculture with macrophages treated in vitro or in vivo with the various drugs. In vitro, preincubation of macrophages with D-penicillamine or chloroquine (50 – 250 g/ml) increased tumour cell³H-TdR incorporation, compared to cultures with untreated macrophages. Macrophages from rats treated with D-penicillamine or chloroquine (50 mg/kg/day orally) for 4 days similarly increased tumour cell³H-TdR incorporation, compared to cultures with macrophages from untreated rats. These effects persisted for at least 3 to 4 weeks of treatment. Preincubation with levamisole (10 – 100 g/ml) in vitro had no effect on macrophage-mediated inhibition of tumour cells, whereas increased tumour cell³H-TdR incorporation was observed in cultures with macrophages from rats treated with levamisole (5 mg/kg/day orally) in vivo. Macrophages from rats with experimentally induced chronic inflammation, i.e. adjuvant arthritis, were found to increase tumour cell³H-TdR incorporation, compared to macrophages from nonarthritic rats. This trend was further enhanced by treatment with D-penicillamine, chloroquine or levamisole.     

Insulin binding and action in isolated rat hepatocytes: Evidence for spare receptors

An in vitro assay for insulin action on hepatocytes is described. The ¹²⁵I-insulin binding and the effects of insulin on net ¹⁴C-glucose incorporation into glycogen were studied in suspensions of isolated hepatocytes from fed, 250 gram adult rats. Insulin doubled the basal value (mean ± SEM) of 9.0 ± 1.0 nmoles glucose/10⁶ cells/hr with a one-half maximal concentration of 3 ng/ml (75 μU/ml) and a maximum effect between 10 and 20 ng/ml (250 μU/ml). Insulin binding was half-maximal at 40 ng/ml and maximal between 100 and 300 ng/ml. Thus, maximal stimulation occurred at approximately 35% of maximum binding implying that hepatocytes have spare receptors for insulin action on net incorporation of ¹⁴C-glucose into glycogen. This assay was then used to investigate the time course of activation of insulin action. Isolated hepatocytes were preincubated at 37°C in the presence or absence of 40 ng/ml of insulin for 2, 15, or 30 min, washed, and then tested for action in fresh insulin-free media containing ¹⁴C-glucose. No activation was seen after 2 min, a partial activation after 15 min and maximum activation was seen only after a 30 min preincubation. Therefore, insulin activation of glucose incorporation into glycogen in liver is a time-dependent phenomenon that is reversible by early dissociation.     

Regulation of ovarian follicle differentiation in gonadotrophin-stimulated rats

The aim of the present study was to investigate the regulation of the in vitro DNA synthesis of ovarian cells recovered from prepubertal rats 48 h after administration of pregnant mare’s serum gonadotrophin alone (granulosa cells) or followed by human chorionic gonadotrophin (luteal cells). Isolated granulosa cells were cultured in serum-free medium, different stimuli added for periods of 48 h, and³H-thymidine incorporation was measured. Both follicle-stimulating hormone (FSH) and luteinizing hormone (LH) inhibited³H-thymidine incorporation by cultured granulosa cells in a dose-dependent manner (FSH: 10, 100, 200 ng/mL-26, 41, 49% inhibition, respectively; LH: 0.1, 1, 10 ng/mL=11, 37, 75% inhibition, respectively). On the other hand, estradiol was found to stimulate³H-thymidine incorporation in granulosa cells (Estradiol: 5, 50, 500 ng/mL=17, 37, 76% stimulation, respectively). In luteal cells, the rate of basal³H-thymidine incorporation was very low (granulosa cells: 2560±310; luteal cells: 661±92 cpm/100,000 cells) and not modified by any stimulus. To determine the possible production of an inhibitory growth factor by the early corpus luteum,³H-thymidine incorporation by granulosa cells was assessed in the presence of 10% conditioned media (CM) recovered from luteal cell cultures. A marked inhibition both in basal and estradiol-stimulated³H-thymidine incorporation was observed (74 and 76% of inhibition, respectively). Results suggest that an inhibitory growth factor produced by luteal cells after luteinizing gonadotrophin stimulus could be involved in the differentiation of growing follicles to corpus luteum.     

Sensitization by interleukin-6 of rat hepatocytes to tumor necrosis factor α-induced apoptosis

Background/Aims: Tumor necrosis factor (TNF) elicits hepatocyte apoptosis in toxic liver injury and is also central in hepatocyte proliferation after partial hepatectomy. In both circumstances interleukin (IL)-6 levels are also elevated. In mouse liver IL-6 attenuated Fas receptor-mediated apoptosis indicating its interference with pro-apoptotic signal chains. It was, therefore, the aim to examine the modulation by IL-6 of TNFα-induced apoptosis in rat hepatocytes.Methods: Primary rat hepatocytes were treated with IL-6 prior to induction of apoptosis with TNFα/actinomycin D or anti-Fas antibody M-20. Apoptosis was detected by determination of caspase-3 activation and bisbenzimide staining of condensed nuclei. Expression of TNFα receptors was analyzed by semi-quantitative polymerase chain reaction and ligand binding studies with [¹²⁵I]-TNFα.Results: IL-6 treatment doubled TNFα/actinomycin D-induced caspase-3 activity and significantly enhanced chromatin condensation. By contrast IL-6 inhibited Fas-induced increase in caspase-3 activity by 45% and significantly reduced chromatin condensation. IL-6 increased the mRNA level of TNF-R1 1.35-fold and augmented cell surface binding of [¹²⁵I]-TNFα 3-fold. The latter and TNFα-mediated caspase activation was attenuated by prostaglandin E₂.Conclusions: IL-6 – in contrast to its anti-apoptotic modulation of the Fas-induced pathway – exerted a pro-apoptotic effect on the TNFα/actinomycin D-induced apoptosis by increasing the number of TNF-R on hepatocytes.     

Effect of estradiol on the synthesis of DNA in the female rat liver

The effect of estradiol on DNA synthesis in hepatocytes was investigated for a study of the functional importance and mechanism of action of sex steroids in the liver. After administration of 17, beta-estradiol dipropionate at a dose of 20 micrograms per 100 g of body mass to ovariectomized albino female rats for 11 days the kinetics of incorporation of 3H-thymidine triphosphate in liver cell nuclei and chromatin was determined. A stimulating effect of the hormone on the tracer incorporation in the nuclei and chromatin was established. A similar tendency was noted in immature female rats. The tracer incorporation depends on the cytosol presence in the system. The rate of incorporation has been shown to increase under the influence of the liver cytosol of intact rats. In can be assumed that estradiol is involved in the regulation of liver DNA synthesis raising matrix activity and modulating cytosol factor activity.     

Treatment of cirrhotic rats with epidermal growth factor and insulin accelerates liver DNA synthesis after partial hepatectomy

Prevention of postoperative hepatic failure is important after hepatic resection. In patients with cirrhosis, impaired liver function and regenerative capacity after major hepatic resection are associated with increased morbidity and mortality. In this study, a combination of epidermal growth factor (EGF) and insulin were used as hepatotrophic factors in an attempt to stimulate DNA synthesis after 70% hepatectomy (HTX). Regenerative capacity was evaluated in normal and cirrhotic rat liver by measuring DNA synthesis in vivo. Micronodular liver cirrhosis was established by the simultaneous oral administration of CCl₄ and phenobarbital. Epidermal growth factor plus insulin was injected subcutaneously immediately after and 12 h after HTX or sham operation was performed. Rats were killed 24 h after the operation and liver regeneration was estimated by [³H]-thymidine incorporation into DNA as well as an autoradiographic nuclear labelling index. Hepatectomy increased [³H]-thymidine incorporation significantly in both normal and cirrhotic rats. In cirrhotic rats, [³H]-thymidine incorporation after HTX was significantly lower than in normal rats and administration of a combination of EGF and insulin after HTX enhanced [³H]-thymidine incorporation. In conclusion, DNA synthesis 24 h after HTX is decreased in cirrhotic rats compared with normal rats and EGF supplementation with insulin accelerates DNA synthesis in hepatectomized cirrhotic rats. The data suggest that administration of combinations of exogenous hepatotrophic factors may play a useful role in the treatment of cirrhotic patients undergoing major hepatic resection.     

Alpha1 acid glycoprotein in streptozotocin diabetic rats. Some aspects of sugar moiety structure and metabolism

Summary The pure alpha₁ acid glycoprotein (AGP) preparation from streptozotocin diabetic rat sera showed diminished sialic acid content and lower ratios of galactose to mannose and galactose to fucose.In vivo incorporation of¹⁴C-leucine and³H-galactose into AGP of control and diabetic animals was studied 30, 60 and 120 min after administration of the radioisotopic precursors. The changed¹⁴C/³H ratio in AGP of diabetic rats suggested disturbed glycosylation of AGP in this disease. Similar activity of UDP-galactose 4-epimerase in control and diabetic rat liver was found suggesting that this enzyme has no influence on the decreased level of serum protein bound galactose.