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.     

Evidence for a functional hepatocyte growth factor receptor in human mesangial cells

In a previous study, mu-opioid receptor binding was decreased by chronic treatment of rats with a mu-opioid receptor-selective agonist [CH3Phe3, D-Pro4]morphiceptin (PL-017) [Tao, P.L., Lee, H.Y., Chang, L.R., Loh, H.H., 1990. Decrease in mu-opioid receptor binding capacity in rat brain after chronic PL-017 treatment. Brain Res. 526, 270-275]. However, there was a lack of correlation between the time course of receptor down-regulation and the loss of pharmacological effects of the drug. In the current study, we used immunohistochemistry to reinvestigate this issue. Male Sprague-Dawley rats were chronically treated with PL-017 i.c.v. for 1, 3 or 5 days, using an escalating dosage paradigm (0.75-6.0 microg), which resulted in a 1.4 to 32-fold increase in the AD50. Rat brains were removed, frozen, coronally sectioned (14 microm) and processed for mu-, delta- or kappa-opioid receptor immunohistochemistry by the avidin-biotin complex (ABC) method. Significant decreases in OP3 immunodensity were found in many brain regions which are enriched with OP3 after chronic treatment of PL-017. Time-dependent decreases in OP3 were detected and reached a plateau around 3 days of PL-017 treatment. No significant change in OP1 or OP2 immunodensity after chronic treatment with PL-017 was found. Our conclusion is that chronic treatment with PL-017 of rats selectively down-regulates mu-opioid receptors in the brain. This may be an important mechanism for PL-017 tolerance.     

Recombinant human hepatocyte growth factor facilitates biliary transport after hepatocyte transplantation in Eisai hyperbilirubinemic rats

Hepatocyte transplantation may offer an attractive treatment for inborn errors of liver metabolism. However, factor(s) are required as stimuli to induce proliferation of the limited number of hepatocytes transplanted. The Eisai hyperbilirubinemic rat (EHBR) is a Sprague-Dawley (SD) mutant rat with conjugated hyperbilirubinemia. EHBRs have impaired canalicular excretory transport of organic anions, bile acid glucuronide, and sulfate. Recombinant human hepatocyte growth factor (rhHGF) (100 microg/kg) was injected intravenously at 2-hr intervals for 10 hr, immediately and 35 days following the intraportal injection of 1 x 10(7) wild-type SD rat hepatocytes. Serum bilirubin concentrations decreased significantly within 35 days and were maintained at significantly reduced levels for 120 days following transplantation. Biliary excretion was demonstrated by the biliary transport of indocyanine green and sulfobromophthalein sodium into the bile. These results indicate that hepatic transport of bile acid conjugates in EHBRs can be restored by hepatocyte transplantation combined with repeated administration of exogenous rhHGF, in conjunction with functioning of the recipient's excretory biliary system.     

Coexpression of hepatocyte growth factor and receptor (Met) in human breast carcinoma

Expression of hepatocyte growth factor (HGF) and HGF receptor (HGFR, product of the met proto-oncogene) mRNA were examined by nonisotopic in situ hybridization in a spectrum of benign and malignant human breast tissues. mRNA for both HGFR and HGF was detected in benign ductal epithelium. Epithelial expression of HGF mRNA was particularly intense in regions of ductal epithelial hyperplasia. Positive expression of HGF (but not HGFR) mRNA was also found in adipocytes, endothelial cells, and to varying degrees in stromal fibroblasts. In 12 of 12 cases of ductal carcinoma in situ and infiltrating ductal carcinoma, carcinoma cells showed a heterogeneous pattern of expression for both HGFR and HGF mRNA. In infiltrating ductal carcinomas, intense expression of HGFR mRNA was not restricted to ductular structures but as also seen in non-duct-forming carcinoma cells. The same zones of the tumors (most commonly at the advancing margins) that expressed strongly HGFR mRNA often were also strongly positive for HGF mRNA, suggesting a possible autocrine effect. The expression pattern of HGFR protein in 25 cases including the same series of tissues used for in situ hybridization analysis was similar to that of HGFR mRNA, as determined by an immunoperoxidase technique. The finding that HGFR is expressed by both benign and malignant epithelium, and its not restricted to duct-forming structures, suggests that, although the potential for HGF/HGFR binding is maintained in malignancy, the response to ligand binding at the level of the receptor or the cellular response to receptor activation may change at some point during progression.     

The expression of cMET/hepatocyte growth factor receptor in colorectal cancer

In this study, we estimated the expression of c-MET/Hepatocyte Growth Factor receptor in colorectal cancers by immunohistochemistry. In 118 patients, c-MET wee expressed in 65 patients (55%). About the clinicopathological findings of metastasis, the proportion of c-MET-positive in the patients with liver metastasis, 78% (18/23), was significantly higher than that without liver metastasis, 49% (47/95), but there was no significant difference about lymph node metastasis and peritoneal dissemination. About the pathological findings of primary lesion, the proportion of c-MET-positive in the patients with infiltration into lymphatic vessels, 63% (48/76), was significantly higher than that without infiltration, 40% (17/42), but there was no significant difference about infiltration into veins. The proportion of c-MET-positive increased as the tumor stage proceeded from t1 to t4 and as the histopathological stage proceeded from I to IV. These results suggest that c-MET may play an important role in the growth and scattering of colorectal cancer cells.     

Stimulation by histamine of TPA-dependent hepatocyte growth factor production in promyelocytic leukemia HL-60 cells

Hepatocyte growth factor (HGF) is most likely a physiological hepatotrophic factor that triggers regeneration of the injured liver. Histamine may also be important in the pathophysiology of the injured liver. Previously we showed that histamine production was increased in liver macrophages of mice injected with CCI4, a well-known hepatotoxin. Therefore, it is likely that the biological actions of histamine in repairing processes of the injured liver are mediated by HGF. This study was aimed at examining the effects of histamine on production of HGF using, as a model, the human promyelocytic leukemia cells, HL-60. 12-o-Tetradecanoylphorbol-13-acetate (TPA) markedly stimulated HGF production and release from the cells; the maximal amount of HGF was released at a concentration of 3 ng/ml of TPA. Histamine significantly stimulated the TPA-induced HGF production and release in these cells, depending on incubation time and its dose. These actions of histamine were abrogated by a H2 receptor antagonist, ranitidine.     

Overexpression and activation of hepatocyte growth factor/scatter factor in human non-small-cell lung carcinomas

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates the invasive growth of epithelial cells via the c-MET oncogene-encoded receptor. In normal lung, both the receptor and the ligand are detected, and the latter is known to be a mitogenic and a motogenic factor for both cultured bronchial epithelial cells and non-small-cell carcinoma lines. Here, ligand and receptor expression was examined in 42 samples of primary human non-small-cell lung carcinoma of different histotype. Each carcinoma sample was compared with adjacent normal lung tissue. The Met/HGF receptor was found to be 2 to 10-fold increased in 25% of carcinoma samples (P = 0.0113). The ligand, HGF/SF, was found to be 10 to 100-fold overexpressed in carcinoma samples (P < 0.0001). Notably, while HGF/SF was occasionally detectable and found exclusively as a single-chain inactive precursor in normal tissues, it was constantly in the biologically-active heterodimeric form in carcinomas. Immunohistochemical staining showed homogeneous expression of both the receptor and the ligand in carcinoma samples, whereas staining was barely detectable in their normal counterparts. These data show that HGF/SF is overexpressed and consistently activated in non-small-cell lung carcinomas and may contribute to the invasive growth of lung cancer.     

Levels of vascular endothelial growth factor, hepatocyte growth factor/scatter factor and basic fibroblast growth factor in human gliomas and their relation to angiogenesis

Angiogenesis is a possible target in the treatment of human gliomas. To evaluate the role of 3 growth factors, vascular endothelial growth factor (VEGF), hepatocyte growth factor/scatter factor (HGF/SF) and basic fibroblast growth factor (bFGF), in the angiogenic cascade, we determined their levels in extracts of 71 gliomas by enzyme-linked immunosorbent assay (ELISA). The levels of bFGF were only marginally different between gliomas of World Health Organization (WHO) grade II (low grade) and grades III and IV (high grade). In contrast, the mean concentrations of VEGF were 11-fold higher in high-grade tumors and those of HGF/SF 7-fold, respectively. Both were highly significantly correlated with microvessel density (p < 0.001) as determined by immunostaining for factor VIII-related antigen. In addition, VEGF and HGF/SF appeared to be independent predictive parameters for glioma microvessel density as determined by multiple regression analysis. We measured the capacity of all 3 factors to induce endothelial tube formation in a collagen gel. In this assay, bFGF was found to be an essential cofactor with which VEGF as well as HGF/SF were able to synergize independently. According to the concentrations of angiogenic factors, extracts from high-grade tumors were significantly more potent in the tube formation assay than the low-grade extracts (p = 0.02). Adding neutralizing antibodies to bFGF, VEGF and HGF/SF together with the extracts, tube formation was inhibited by up to 98%, 62% and 54%, respectively. Our findings suggest that bFGF is an essential cofactor for angiogenesis in gliomas, but in itself is insufficient as it is present already in the sparsely vascularized low-grade tumors. Upon induction of angiogenesis in high-grade tumors, bFGF may synergize with rising levels of not only VEGF but possibly also with HGF/SF, which appears here to be an independent angiogenic factor.     

Activation of epidermal growth factor receptor in the early phase after renal ischemia-reperfusion in rat

This article describes 2 white male patients with probable splenic crisis caused by sickle cell trait after travel from sea level to a moderately high altitude. Both did well with return to lower altitude and conservative treatment. Although rare, this entity should be considered in patients presenting to the emergency department with left-sided chest or abdominal pain after travel to moderate or high altitude.     

Serum hepatocyte growth factor activity and hepatocyte proliferation in Long-Evans with a cinnamon-like coat color rats with hepatic lesions

We classified hepatic lesions spontaneously developed by Long-Evans with a cinnamon-like coat color (LEC) rats into the following four stages: Normal liver, acute hepatitis, chronic hepatitis, and hepatoma, by biochemical tests of the sera, and anatomical and histopathological examination of the livers. Hepatocyte growth factor (HGF) activity in the sera of LEC rats which developed acute hepatitis, chronic hepatitis, and hepatoma was higher than that of normal LEC rats. In particular, HGF activity in the sera of the LEC rats with acute hepatitis was about 70-fold that of normal LEC rats. However, primary cultured hepatocytes of LEC rats with hepatic lesions were hardly proliferated by stimulation with EGF and insulin in vitro or with increased HGF in vivo. These results suggest that the hepatocytes of LEC rats with hepatic lesions disorder the signal transduction of growth factors.     

Analysis of transforming growth factor (TGF)-alpha/epidermal growth factor receptor, hepatocyte growth Factor/c-met,TGF-beta receptor type II, and p53 expression in human hepatocellular carcinomas

There is high current interest in developing synthetic routes to oligosaccharides involved in glycoconjugates. Significant attention has been focused on the application of glycosidase-catalyzed transglycosylation for practical synthesis of oligosaccharides. The enzymatic synthesis has become more practical by the use of several glycosidases available in sufficient quantities. This review describes convenient syntheses of di- and trisaccharide units, which are related to molecular recognition, by using regioselective transgalactosylation, trans-N-acetylglucosaminylation, transfucosylation, and transmannosylation. The regioselectivity could be controlled to some extent by using the following techniques: (1) varying enzymes, (2) organic co-solvent system, (3) the configuration of the existing glycosidic linkage of the acceptor and (4) inclusion complex of acceptor glycoside with cyclodextrin. Furthermore, glycopolymers carrying a series of disaccharides containing beta-D-galactosyl residues were synthesized and used as a model in oligosaccharide-lectin interaction analysis. These water-soluble glycopolymers were shown to be useful as probes of carbohydrate recognition.     

Platelet-derived growth factor receptor expression after neural grafting in a rat model of Parkinson's disease

Platelet-derived growth factor (PDGF) has trophic effect on dopaminergic neurons in vitro. We have previously shown dynamic changes in the expression of PDGF in embryonic mesencephalic grafts and surrounding host striatal tissue following intracerebral transplantation in a rat model of Parkinson's disease. In this study the expression of the PDGF receptors was examined in the same model using immunohistochemistry. Most ventral mesencephalic (VM) cells from E13-E15 rat embryos possessed both PDGF alpha- and beta-receptors before implantation. Double immunofluorescence staining revealed that about 10% of the cells also expressed tyrosine hydroxylase (TH). The PDGF alpha-receptor was detectable in the graft up to 1 wk after transplantation but had disappeared at 3 wk. In the host tissue, scattered glial cells were positive for the alpha-receptor but the expression was unchanged following transplantation. The beta-receptor expression almost completely disappeared from the grafted tissue by 4 h following transplantation, and only a few cells of the host striatum showed immunoreactivity. However, after 3 wk beta-receptor positive cells were again detectable in the graft. These cells appeared to be endothelial cells as identified by an antibody against von Willebrand's factor. Our data suggest that PDGF might act locally on embryonic dopaminergic cells in an autocrine or juxtacrine manner before and shortly after transplantation, and on surrounding glial cells in a paracrine manner after transplantation. Furthermore, PDGF-BB might influence neovascularization in the graft.     

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.     

Ameloblast-lineage cells of rat tooth germs proliferate and scatter in response to hepatocyte growth factor in culture

Hepatocyte growth factor (HGF) is considered to be one of the mediators of epithelial-mesenchymal interactions during early organogenesis and to be involved in the development of murine molars. In this study, the immunohistochemical localization of HGF and of its receptor, c-Met, revealed that HGF was distributed in the proliferating mesenchymal cells in the dental papillae and that c-Met was continuously expressed in the epithelial cells during the development of rat incisors. These observations confirmed the involvement of HGF in the development of rat incisors, as demonstrated previously in molars. We then used a primary culture of ameloblast-lineage cells, prepared from mandibular incisors of young rats, to examine the direct effects of HGF on the growth and differentiation of ameloblasts. We found that HGF at 2-20 ng/ml induced a marked increase in the number of ameloblast-lineage cells and in the scattering of such cells. Our results suggest that HGF promotes the proliferation and scattering of ameloblast-lineage cells simultaneously.     

Controlling insulin-like growth factor activity and the modulation of insulin-like growth factor binding protein and receptor binding

The insulin-like growth factors (IGF) and insulin perform seemingly unique roles by causing the same metabolic effect: cellular hypertrophy. Although overlapping, there are different consequences to cellular hypertrophy induced by IGF and that induced by insulin. The IGF enhance the cell hypertrophy that is requisite for cell survival, hyperplasia, and differentiation, and insulin enhances cell hypertrophy primarily as a means to increase nutrient stores. The effects of IGF and insulin are controlled by the segregation of their receptors between different cell types. A model is discussed that describes the need for three hormones (IGF-I, IGF-II, and insulin) to control nutrient partitioning. Insulin receptor localization, as well as an episodic mode of secretion, evolved to perform the short-term action of clearing excess nutrients from the circulation. In contrast, a complex and interactive set of factors ensure that maximal IGF activity occurs only when conditions are optimal for growth. A relatively invariant rate of secretion and the IGF binding proteins serve to maintain a large mutable pool of IGF. This pool exists to ensure a constant supply of IGF to maintain the basal metabolic rate and to ensure that, once a cell begins to proliferate or differentiate, adequate exposure is available to complete the process even after severe short-term physiological insults. The IGF concentrations only change in response to prolonged differences in protein and energy availabilities, environmental and body temperatures, and external stress. Also, evidence is now emerging that describes a discrete role for trace nutrients in the regulation of IGF activity. In this latter regard, zinc has the notable role of targeting IGF binding proteins to the cell surface. New data are presented showing that zinc also changes the affinity of the type 1 IGF receptor and cell-associated IGF binding proteins to optimize IGF activity.     

Growth hormone receptor activity is stimulated by insulin-like growth factor binding protein 5 in rat osteosarcoma cells

Osteoblast-like UMR-106.01 rat osteosarcoma cells express high affinity growth hormone (GH) receptors (GHRs). Because osteoblasts secrete insulin-like growth factor binding protein-5 (IGFBP-5), we evaluated whether it also modulates GH binding and GHR expression in UMR cells. Human recombinant intact IGFBP-5 stimulated 125I-hGH binding in a dose-dependent manner (dose range 300-3000 ng/ml), inducing an increase to 193.6 +/- 2.1% of control binding at 3000 ng/ml (P < 0.001). Carboxy-truncated IGFBP-5 also stimulated GH binding but with less potency (125 +/- 2.7% of control at 3000 ng/ml, P < 0.01). GHRs identified by chemical crosslinking of 125I-hGH to cell monolayers increased after treatment with IGFBP-5 and decreased in response to insulin-like growth factor-I (IGF-I). GHR mRNA levels, as quantitated by a solution hybridization RNAse protection assay, increased up to 3 to 7-fold in a time-dependent manner by intact IGFBP-5 but not by carboxy-truncated IGFBP-5. An antiserum to IGFBP-5 reduced basal GH binding to 56.7 +/- 4.3% of control value at a concentration of 0.5% (P < 0.001), showing that IGFBP-5 produced by the cells is a strong regulator of GH binding. IGFBP-5 antiserum also decreased GH binding to 85.9 +/- 0.9% of IGFBP-5 stimulated value (P < 0.001), showing the specificity of IGFBP-5 stimulation. To determine whether the GHR upregulation was physiologically significant, cell proliferation was evaluated after coincubation of IGFBP-5 with low, non-stimulatory concentrations of GH. IGFBP-5 (1000 ng/ml) induced cell proliferation to 116.2 +/- 3.2% of control levels, and coincubation with hGH at 10 ng/ml induced an increase to 133.3 +/- 0.1% of control levels. We conclude that exogenous and endogenous IGFBP-5 upregulate GHR mRNA levels and GH binding and this interaction potentiates GH-stimulated mitogenesis in osteoblastic cells.