Immediate increase in circulating hepatocyte growth factor/scatter factor by heparin

Circulating levels of hepatocyte growth factor (HGF)/scatter factor have been recently found to be increased in the early phase of myocardial infarction, and it has been hypothesized that HGF plays a role in angiogenesis and collateral vessel growth. Heparin has also been shown to enhance angiogenesis and to improve collateral blood flow. This study was designed to study the effect of heparin on the release of HGF. In an experimental study, heparin was given to rats intravenously and plasma was collected for measurements of HGF by enzyme-linked immunosorbent assay. A dose-dependent increase in circulating HGF was measured with peak levels occurring 10 min after injection of 300 units/kg of heparin (15.4+/-2.0 ng/ml after v 0. 17+/-0.14 ng/ml before injection,P<0.0001). In a subsequent clinical study, 12 patients received 3000 units of heparin during cardiac catheterization. Circulating HGF increased steeply within 3 min of the injection. Comparable changes in plasma concentrations were measured in samples obtained from femoral vein (8.7+/-3.5 after v 0. 33+/-0.07 before injection P<0.05) or artery (10.5+/-3.2 ng/mlv 0. 27+/-0.05 P<0.01), pulmonary artery (9.1+/-2.0 ng/mlv 0.36+/-0.06 ng/ml,P=0.07 ) or right atrium (8.5+/-1.6 ng/mlv 0.42+/-0.11,P<0.01). This study suggests that heparin-induced effects such as the promotion of angiogenesis may be at least partly due to the release of HGF.     

Strain-dependent embryonic lethality in mice lacking the retinoblastoma-related p130 gene

The retinoblastoma-related p130 protein is a member of a conserved family, consisting of Rb, p107 and p130, which are believed to play important roles in cell-cycle control and cellular differentiation. We have generated a null mutation in p130 by gene targeting and crossed the null allele into Balb/cJ and C57BL/6J strains of mice. In an enriched Balb/cJ genetic background, p130(-/-) embryos displayed arrested growth and died between embryonic days 11 and 13. Histological analysis revealed varying degrees of disorganization in neural and dermamyotomal structures. Immunohistochemistry with antibody reactive with Islet-1 indicated markedly reduced numbers of neurons in the spinal cord and dorsal root ganglia. Immunohistochemistry with antibody reactive with desmin indicated a similar reduction in the number of differentiated myocytes in the myotome. The myocardium of mutant embryos was abnormally thin and resembled an earlier staged two-chambered heart consisting of the bulbus cordis and the ventricular chamber. TUNEL analysis indicated the presence of extensive apoptosis in various tissues including the neural tube, the brain, the dermomyotome, but not the heart. Immunohistochemistry with antibody reactive with PCNA revealed increased cellular proliferation in the neural tube and the brain, and decreased proliferation in the heart. The placentas of p130(-/-) embryos did not display elevated apoptosis and were indistinguishable from wild type suggesting that the phenotype was not due to placental failure. Following a single cross with the C57BL/6 mice, p130(-/-) animals were derived that were viable and fertile. These results indicate that p130 in a Balb/cJ genetic background plays an essential role that is required for normal development. Moreover, our experiments establish that second-site modifier genes exist that have an epistatic relationship with p130.     

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.     

Wild-type but not mutant p53 activates the hepatocyte growth factor/scatter factor promoter

p53 transactivates the expression of a variety of genes by binding to specific DNA sequences within the promoter. We have investigated the ability of wild-type p53 and a non-DNA binding p53 mutant to activate the hepatocyte growth factor/scatter factor (HGF/SF) promoter using chloramphenicol acetyltransferase reporter constructs. We also used deletion sequences of the HGF/SF promoter to identify which regions, if any, were responsible for p53 binding. Our results show that wild-type but not mutant p53 activates the HGF/SF promoter when using -3000 and -755 bp upstream of the HGF/SF gene. This activation is lost when promoter sequences covering -365 and -239 bp are used. Analysis of the DNA sequence between -365 and -755 bp shows one putative p53 half-site with 80% homology to the consensus sequence and another half-site 3 bases downstream of this with 100% homology to the consensus sequence. In contrast to previously identified p53 binding DNA sequences, the downstream half-site is inverted. We propose that the HGF/SF promoter can be activated by wild-type p53 in vivo and that this could be as a result of a novel form of sequence-specific DNA binding.     

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.     

Circular ruffle formation and closure lead to macropinocytosis in hepatocyte growth factor/scatter factor-treated cells

Treatment with hepatocyte growth factor/scatter factor (HGF/SF) rapidly induced the formation of conspicuous circular ruffles on the apical surfaces of two kidney cell lines, MDCK and PtK2. The ruffles were found to contain significant amounts of F-actin and myosin as judged by immunofluorescence microscopy. Time-lapse photomicroscopy demonstrated that the ruffles constrict, closing over, and were followed by the formation of phase bright structures. That these structures were macropinocytotic vesicles was confirmed using fluorescein isothiocyanate (FITC)-dextran as a marker for fluid uptake. It is hypothesized that the constriction of the ruffles followed by membrane fusion causes the vesicles to form. Treatment with suramin blocked both circular ruffle formation and scattering, suggesting that ligand binding was the causal agent for ruffle formation. The drugs amiloride and SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) also completely inhibited ruffle formation, suggesting that ion transport was an early consequence of HGF/SF binding and that these transport effects had a major role in the cytoskeletal changes leading to circular ruffle formation. The appearance of macropinocytotic vesicles was also blocked by amiloride treatment. Surprisingly though, subsequent scattering was not blocked by amiloride treatment, although suramin and SITS both entirely inhibited scattering.     

Insights into the structure of hepatocyte growth factor/scatter factor (HGF/SF) and implications for receptor activation

The modular structure of HGF/SF offers a reductionist or 'divide and rule' approach to the analysis of structure and function. Domain deletion experiments have established that the N domain, kringle 1 and kringle 2 are essential for HGF/SF activity and that truncated variants containing the N domain and kringle 1 (NK1) or kringles 1 and 2 (NK2) can exhibit partial agonistic or antagonistic activity depending on target cells. Comparative modelling has been used to predict the 3D structures of the six domains of HGF/SF. More recently, NMR methods have shown that the N domain has a novel fold, the charge distribution of which suggests a heparin binding site. Crystals of NK1 indicate the relationship of this domain to the kringle 1, offering further insights into the mechanism of domain interactions and receptor activation.     

Autocrine hepatocyte growth factor/scatter factor-Met signaling induces transformation and the invasive/metastastic phenotype in C127 cells

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic effector of cells expressing the Met tyrosine kinase receptor. C127 is a non-tumorigenic mouse cell line which expresses negligible levels of HGF/SF and Met proteins. In the present report we have generated C127 cells which overexpress HGF/SF and/or Met proteins, and have analysed the effect of HGF/SF-Met signaling in these cells. We show that this signaling pathway stimulates the growth and invasiveness of C127 cells in vitro and that cells overexpressing both HGF/SF and Met proteins (but neither alone) are phenotypically transformed and highly tumorigenic and metastatic in vivo. Our data unequivocally demonstrates the autocrine dependency of HGF/SF-Met-induced transformation and metastasis in this system and supports the theory that the inappropriate expression of HGF/SF and Met proteins could play a role in the development and spread of human tumors. In addition, this system may be useful for identifying metastasis-associated genes that are activated by HGF/SF-Met signaling.     

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.     

Hepatocyte growth factor/scatter factor stimulates the Ras-guanine nucleotide exchanger

Hepatocyte growth factor/scatter factor (HGF/SF) induces mitogenesis and cell dissociation upon binding to the protein-tyrosine kinase receptor encoded by the MET proto-oncogene (p190MET). The signal transduction pathways downstream from the receptor activation are largely unknown. We show that HGF/SF activates Ras protein. HGF/SF stimulation of metabolically labeled A549 cells raised the amount of Ras-bound radiolabeled guanine nucleotides by over 5-fold. Furthermore, following HGF/SF stimulation of these cells, 50% of Ras was in the GTP-bound active state. The uptake by Ras of radiolabeled GTP was also increased by 5-fold following HGF/SF stimulation in digitonin-permeabilized A549 cells. Moreover, HGF/SF treatment of A549 cells leads to stimulation of the cytosolic Ras-guanine nucleotide exchange activity, measured as accelerated release of [3H]GDP from purified recombinant Ras protein in vitro, in a dose- and time-dependent manner. Likewise, treatment with the protein-tyrosine kinase inhibitor 3-(1',4'-dihydroxytetralyl)methylene-2-oxindole of GTL-16 cells (featuring a p190MET receptor constitutively active) significantly decreased the cytosolic Ras-guanine nucleotide exchange activity. These data demonstrate that HGF/SF activates Ras protein by shifting the equilibrium toward the GTP-bound state and increases the uptake of guanine nucleotides by Ras, through mechanism(s) including the activation of a Ras-guanine nucleotide exchanger.     

Hepatocyte growth factor/scatter factor stimulates chemotaxis and growth of malignant mesothelioma cells through c-met receptor

AIMS: To assess the ability of clinical characteristics, admission ECG and continuous ST segment monitoring in determining long-term prognosis in unstable angina. METHODS: Two hundred and twelve patients with unstable angina (mean age 59 years), presenting within 24 h of an acute episode of angina were recruited at three hospitals and treated with standardized medical therapy. All patients kept chest pain charts and underwent ST segment monitoring for 48 h. The occurrence of death, myocardial infarction, and need for revascularization was assessed over a median follow-up of 2.6 years. RESULTS: The risk of death of myocardial infarction was greatest in the first 6-8 weeks after admission. Admission ECG ST depression and the presence of transient ischaemia predicted increased risk of subsequent death or myocardial infarction, whereas a normal ECG predicted a good prognosis. In 14 patients, ST segment monitoring provided the only evidence of recurrent ischaemia, and 72% of this group suffered an adverse event. Transient ischaemia and a history of hypertension were the most powerful independent predictors of death or myocardial infarction. CONCLUSIONS: Adverse events in unstable angina occur early after admission and can be predicted by clinical and ECG characteristics, and by the presence of transient ischaemia during ST segment monitoring. Risk stratification by these simple assessments can identify patients with unstable angina at high risk.     

Abnormal astrocyte development and neuronal death in mice lacking the epidermal growth factor receptor

Stimulation of the epidermal growth factor receptor (EGF-R) produces numerous effects on central nervous system (CNS) cells in vitro including neuronal survival and differentiation, astrocyte proliferation and the proliferation of multipotent progenitors. However, the in vivo role of EGF-R is less well understood. In the present study, we demonstrate that EGF-R null mice generated on a 129Sv/J Swiss Black background undergo focal but massive degeneration the olfactory bulb, piriform cortex, neocortex, and thalamus between postnatal days 5 and 8 which is due, at least in part, to apoptosis. Some of the neuronal populations that degenerate do not normally express EGF-R, indicating an indirect mechanism of neuronal death. There were also delays in GFAP expression within the glia limitans and within structures outside the germinal zones in early postnatal ages. At or just prior to the onset of the degeneration, however, there was an increase in GFAP expression in these areas. The brains of EGF-R (-/-) animals were smaller but cytoarchitecturally normal at birth and neuronal populations appeared to be intact, including striatal GABAergic and midbrain dopaminergic neurons which have previously been shown to express EGF-R. Multipotent progenitors and astrocytes derived from EGF-R (-/-) mice were capable of proliferating in response to FGF-2. These data demonstrate that EGF-R expression is critical for the maintenance of large portions of the postnatal mouse forebrain as well as the normal development of astrocytes.     

Targeted disruption of the ubiquitous CNC-bZIP transcription factor, Nrf-1, results in anemia and embryonic lethality in mice

The CNC-basic leucine zipper (CNC-bZIP) family is a subfamily of bZIP proteins identified from independent searches for factors that bind the AP-1-like cis-elements in the beta-globin locus control region. Three members, p45-Nf-e2, Nrf-1 and Nrf-2 have been identified in mammals. Expression of p45-Nf-e2 is largely restricted to hematopoietic cells while Nrf-1 and Nrf-2 are expressed in a wide range of tissues. To determine the function of Nrf-1, targeted disruption of the Nrf-1 gene was carried out. Homozygous Nrf-1 mutant mice are anemic due to a non-cell autonomous defect in definitive erythropoiesis and die in utero.     

Hepatocyte growth factor/scatter factor overexpression induces growth, abnormal development, and tumor formation in transgenic mouse livers

To investigate the in vivo role of hepatocyte growth factor/scatter factor (HGF/SF) in liver function, we generated transgenic mice using a mouse HGF/SF cDNA under the control of the mouse metallothionein gene promoter and 5'/3' flanking sequences. In adult HGF/SF transgenic mice, liver weight as a percentage of total body weight was at least twice that of wild-type mice. Comparison of transgenic and control liver morphology revealed dramatic heterogeneity in the size and appearance of hepatocytes as a distinctive feature of HGF/SF overexpression. Transgenic livers exhibited a significant increase in the number of small hepatocytes with a 2N DNA content, accounting for the observed increase in liver mass. The DNA labeling index of hepatocytes increased 11-fold at 4 weeks of age, when liver enlargement first became apparent, and was still elevated about 5-fold in adult HGF/SF transgenic mice. Moreover, hepatocytes isolated by perfusion of transgenic livers doubled every 2 days in culture, whereas little or no growth was observed with isolated control hepatocytes. The mechanistic basis of hepatocyte proliferation was elucidated as the chronic activation of the c-met proto-oncogene product. Met and substrates such as phosphatidylinositol 3-kinase, Src homology and collagen-like, pp60c-src, focal adhesion kinase p125FAK, and paxillin were associated with tyrosine-phosphorylated complexes in a hepatocyte cell line established from the transgenic liver. This proliferative stimulus triggered the formation of hepatocellular adenomas and/or carcinomas in most transgenic mice > or = 1.5 years of age. Finally, the rate of transgenic mouse liver regeneration was increased 3-fold over control livers following partial hepatectomy.     

Membrane type 1-matrix metalloproteinase is involved in the formation of hepatocyte growth factor/scatter factor-induced branching tubules in madin-darby canine kidney epithelial cells

The cyclin-dependent kinase inhibitor p27 is a negative regulator of the cell cycle and a potential tumor suppressor gene. Because we had previously demonstrated that loss of p27 protein is associated with aggressive behavior in colorectal adenocarcinomas, we used immunohistochemistry and in situ hybridization to evaluate the potential role of alterations in p27 expression in primary and metastatic colorectal adenocarcinomas. Parallel immunostaining was performed for Ki-67 and p53. We evaluated 13 cases of metachronous and 23 cases of synchronous primary and metastatic colorectal tumor pairs. In the synchronous subgroup (Stage IV tumors), 57% of the primary tumor and metastases pairs did not express p27 protein and the remainder were low expressors. In the metachronous subgroup, 54% of the primary tumors were low expressors and the remainder high expressors of p27 protein. There was a significant reduction in the expression of p27 in the metachronous metastases (mean positive cells: 14.5%) when compared to the corresponding primary tumors (mean positive cells: 41.8%), P = 0.0023. All the primary and metastatic tumors in the metachronous subgroup showed high levels of p27 mRNA expression. There was no association between loss of p27 and either Ki-67 count or p53 expression. Because p27 is known to be up-regulated when epithelial cells are grown in suspension, the down-regulation of p27 in circulating tumor cells may confer the ability to grow in an environment of altered extracellular matrix or intercellular adhesion properties, two situations which may facilitate metastases.     

Hemodialysis stimulates hepatocyte growth factor release

Studies were performed in 26 patients on regular dialysis treatment with cuprophane (CU), polymethylmetacrilate (PMMA) or cuprammonium (CAM) dialyzers. Controls were six patients with chronic renal failure but not on regular dialysis treatment (CRF) and six healthy subjects (N). Blood was collected at the start (T0), and at 15 (T15) and 240 (T240) minutes of dialysis to measure the serum hepatocyte growth factor (HGF) concentration and to study HGF production by peripheral blood mononuclear cells (PBMC) in vitro. The form of HGF (that is, inactive/monomeric, active/dimeric) present in the serum was analyzed by immunoblotting. In addition, the ability of serum to stimulate proliferation of tubular cells (HK-2) and HGF release by PBMC and fibroblasts (MRC-5) was investigated. At T0, serum HGF levels were identical to that of the controls. In patients treated with CU, serum HGF rose from 0.24 ng/ml at T0 to 7.44 ng/ml at T15, and remained high at T240. PBMC collected at T15 and T240 released significantly more HGF in vitro than those collected at T0. Serum at T15 stimulated proliferation of HK-2 cells and the release of HGF by PBMC and MRC-5 cells. The PMMA and CAM dialyzers had similar effects as the CU. These results indicate that dialysis induces a striking rise in serum HGF and a prompt circulation of factor(s) stimulating HGF release. Dialysis-activated PBMC release HGF.     

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.     

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.