Increased hepatocyte growth factor level in cerebrospinal fluid in Alzheimer's disease.

BACKGROUND & OBJECTIVE: Hepatocyte growth factor (HGF), also known as the scatter factor, is a potent mitogen for mature hepatocytes, and also has multifunctional effects on some cells in various organs. Recently, we have found expression and localization of HGF in white matter astrocytes in human brain tissues. Furthermore, immunohistochemistry using anti-HGF antibody revealed more intense immunolabeling in Alzheimer's disease (AD) than control brains. The aim of the study is to investigate the level of HGF in cerebrospinal fluid (CSF) from patients with AD. MATERIAL AND METHODS: We examined the level of HGF in CSF from 34 AD and 15 age-matched disease control patients by highly sensitive enzyme-linked immunoabsorbent assay (ELISA) system. RESULTS: Consistent with the immunohistochemical data, a significantly higher concentration of HGF in AD CSF was found as compared with controls. A significant correlation was also seen between CSF HGF levels and white matter high-signal foci determined on brain magnetic resonance imaging (MRI) in AD patients. CONCLUSION: These results indicate that CSF HGF levels correspond with the white matter damage in AD brain.

Hepatocyte growth factor in gingival crevicular fluid and the distribution of hepatocyte growth factor-activator in gingival tissue from adult periodontitis.

Hepatocyte growth factor (HGF), also known as scatter factor, is a broad-spectrum and multifunctional cytokine required for the development, growth and regeneration of various organs and tissues. The expression of HGF in human gingival fibroblasts is induced by inflammatory cytokines such as interleukin 1. Thus, although it is possible that content of HGF in gingival crevicular fluid (GCF) in periodontitis is increased, this has not so far been reported because the volume of GCF is too small to determine HGF by the available enzyme-linked immunosorbent assay (ELISA). A recently developed, highly sensitive ELISA for HGF, with a detection limit of 1 pg/ml sample, has now enabled HGF to be measured in GCF.The mean HGF content in GCF from sites with clinically healthy gingiva, defined by the absence of overt signs of gingival inflammation and a probing depth (PD) <3 mm, was 1.7 ng/ml, and that of periodontitis, defined by obvious alveolar bone loss detected by radiographic examination and a PD> or =3 mm, was 3.23 ng/ml. Although treating the periodontitis did not significantly decrease the HGF concentration despite significantly improved clinical scores such as PD and Gingival Index, the total amount of HGF in GCF did decrease significantly after treatment. HGF was expressed by gingival fibroblasts and inflammatory cells as determined by in situ hybridization. HGF-activator (HGFA), which converts inactive pro-HGF to active mature HGF, was detected in gingival epithelial cells by immunostaining. The expression of HGFA was also confirmed in gingival tissue by reverse transcription-polymerase chain reaction (RT-PCR). These findings indicate that HGF is synthesized and activated in gingiva that is clinically healthy or associated with periodontitis.

Hepatocyte growth factor in cerebrospinal fluid in neurologic disease.

OBJECTIVE: To investigate hepatocyte growth factor (HGF) concentration in cerebrospinal fluid (CSF) in neurologic disease. MATERIALS AND METHODS: We determined CSF concentration of HGF with human-HGF-specific enzyme-linked immunosorbent assays (ELISA) in 121 patients: Alzheimer's disease (AD) (33), amyotrophic lateral sclerosis (ALS) (10), Parkinson's disease (PD) (5), progressive supranuclear palsy (PSP) (3), spinocerebellar degeneration (7), acute disseminating encephalomyelitis (ADEM) (6), human T-lymphotropic virus-1 (HTLV-1)-associated myelopathy (HAM) (6), multiple sclerosis (MS) (7), aseptic meningitis (AM) (12), and peripheral neuropathy and myopathy as control diseases (32). RESULTS: HGF concentrations in CSF were significantly higher with diseases of the central nervous system (CNS) than control diseases and were slightly higher with AD than other neurodegenerative diseases. Values were highest with ADEM but decreased during corticosteroid treatment. We found no relationship between HGF in CSF and CSF cells or protein, immunoglobulin index, or Q albumin. CONCLUSION: It is suggested that high concentrations of HGF in CSF may be partially related to CNS pathology, especially to demyelinating disease.

HGF/SF induces mesothelial cell migration and proliferation by autocrine and paracrine pathways.

Mesothelial repair differs from that of other epithelial-like surfaces as healing does not occur solely by centripetal in-growth of cells as a sheet from the wound margins. Mesothelial cells lose their cell-cell junctions, divide, and adopt a fibroblast-like morphology while scattering across and covering the wound surface. These features are consistent with a cellular response to hepatocyte growth factor/scatter factor (HGF/SF). In this study, we examined the ability of mesothelial cells to secrete HGF/SF and investigated its possible role as an autocrine regulator of mesothelial cell motility and proliferation. We found that human primary mesothelial cells expressed HGF/SF mRNA and secreted active HGF/SF into conditioned medium as determined by ELISA and in a scattering bioassay. These cells also expressed the HGF/SF receptor, Met, as shown by RT-PCR and by Western blot analysis and immunofluorescence. Incubation of mesothelial cells with neutralizing antibodies to HGF/SF decreased cell migration to 25% of controls, whereas addition of HGF/SF disrupted cell-cell junctions and induced scattering and enhanced mesothelial cell migration. Furthermore, HGF/SF showed a small but significant mitogenic effect on all mesothelial cell lines examined. In conclusion, HGF/SF is produced by mesothelial cells and induces both motility and proliferation of these cells. These data are consistent with HGF/SF playing an autocrine role in mesothelial healing.

Circulating hepatocyte growth factor as a marker of thrombus formation in unstable angina pectoris.

A new enzyme-linked immunosorbent assay can detect 10 pg/ml of human hepatocyte growth factor (HGF). Circulating HGF was significantly higher in patients with unstable angina (296+/-184 pg/ml, mean+/-SD, n=36) than in healthy volunteers (201+/-64 pg/ml, n=250, p<0.0001). Individual concentrations exceeded the mean control value +2 SD (329 pg/ml) in 12 of the 36 (33%) patients with unstable angina. The present study indicates that this new, sensitive HGF assay can successfully detect thrombosis in patients with unstable angina.

Development of highly sensitive enzyme-linked immunosorbent assays for hepatocyte growth factor/scatter factor (HGF/SF): determination of HGF/SF in serum and urine from normal human subjects.

A sandwich enzyme-linked immunosorbent assay (ELISA) using rabbit anti-hepatocyte growth factor (HGF) IgG for human HGF, also known as the scatter factor, has previously been developed for determining increases in serum HGF levels in various liver diseases. The sensitivity limit of the ELISA is, however, approximately 0.2 ng/ml sample, and HGF concentrations in about 50% of normal subjects are not accurately measurable by this method, because the mean level of HGF in normal serum is close to the sensitivity limit. In the present study, chicken Fab' from egg yolk anti-HGF immunoglobulin Y and rabbit Fab' from rabbit anti-HGF IgG were conjugated with beta-D-galactosidase. With these conjugates as the second antibodies, we developed two sandwich ELISAs for human HGF and found that the sensitivities were about 20 pg/ml with the former conjugate and 2 pg/ml with the latter. The HGF concentration in sera from 138 normal subjects determined by the ELISA with the rabbit conjugate was 244+/-65 (SD) pg/ml serum, and it correlated very well with the number of leukocytes. Moreover, the ELISA with the rabbit conjugate permitted the determination of HGF levels in urine from normal subjects without first concentrating the sample. The determination of HGF in various biological fluids other than blood and urine by these ELISAs may aid the diagnosis and prognosis of various diseases.

Fetuin in the developing neocortex of the rat: distribution and origin.

Immunocytochemical distribution of the fetal protein fetuin in the neocortex of developing rat brain and the presence of its mRNA, as detected by using reverse transcriptase-polymerase chain reaction analysis, was studied in fetuses at embryonic day 15 (E15) through E22, in neonates at postnatal day 0 (P0) through P20, and in adults. Quantitative estimates of fetuin in cerebrospinal fluid (CSF) and plasma were obtained over the same period. Exogenous (bovine) fetuin injected intraperitoneally into fetal and postnatal rats was used to study the uptake of fetuin into CSF and brain and its distribution compared with endogenous fetuin; bovine albumin was used as a control. Fetuin was identified immunocytochemically in the cortical plate and subplate cells of the developing neocortex. In the rat fetus, fetuin first was apparent at E17, mainly in cell processes, but a few subplate cells also were positive. By E18, there was strong staining in subplate neurons and in inner cells of the cortical plate. At E21, these inner cells of the cortical plate were beginning to differentiate into layer VI neurons, many of which were positive for fetuin. By P0-P1, more layer VI neurons and some layer V neurons had become positive for fetuin. Fetuin immunoreactivity generally was weaker at P1, and, by P2-P3, it had disappeared from all of the layers of the developing neocortex. Bovine fetuin (but not albumin), probably taken up through CSF over the neocortical dorsal surface, had a cytoplasmic distribution; endogenous rat fetuin was both cytoplasmic and membrane bound. Thus, much of this fetuin can be accounted for by uptake, although the presence of fetuin mRNA indicates that in situ synthesis may also contribute.

Prostaglandin E2 predominantly induces production of hepatocyte growth factor/scatter factor in human dental pulp in acute inflammation.

Hepatocyte growth factor (HGF), which is also known as the scatter factor, is a broad-spectrum and multifunctional cytokine, mediates epithelial-mesenchyme interaction, and is shown to be involved in the development and regeneration of various tissues, including tooth. Here, we report that HGF was present in adult human dental pulps, and its levels increased during acute inflammation of the tissue. Levels of HGF mRNA in dental pulps also increased with inflammation, as determined by reverse-transcription/polymerase chain-reaction. The production of HGF in fibroblasts from dental pulps in culture was dose-dependently stimulated by inflammatory cytokines such as interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha, and by prostaglandin (PG) E2, as determined by an enzyme-linked immunosorbent assay. We also showed that indomethacin did not affect the increase in HGF production by the cells with IL-1alpha, TNF-alpha, and PGE2. The levels of HGF mRNA in the cells were simultaneously increased by these stimulants, as determined by Northern blotting. Since the production of PGs is known to increase at the beginning of inflammation, PGE2 may be involved in the regeneration of dental pulps by the induction of HGF expression after inflammation.

Enhancement of hepatocyte growth factor (scatter factor) production by human gingival fibroblasts in culture stimulated with Porphyromonas gingivalis fimbriae.

Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a motogen, mitogen and morphogen produced by mesenchymal cells that mainly acts on epithelial cells and is involved in osteoclast stimulation. This study examined the possible enhanced production of HGF/SF by human gingival fibroblasts upon stimulation with killed cells of Porphyromonas gingivalis strain 381 and its representative bioactive cellular components, fimbriae and lipopolysaccharide (LPS). P. gingivalis whole cells enhanced the production of HGF/SF detected by ELISA in culture supernates of the fibroblasts. Fimbriae prepared from P. gingivalis exhibited powerful HGF/SF-inducing activity in a concentration-dependent manner, with peak activity observed at 100-200 microg/ml. The fimbriae-induced HGF/SF mRNA expression by the cells was also detected by reverse transcription-PCR. P. gingivalis LPS exhibited weak HGF/SF-inducing activity. The study also examined the HGF/SF-inducing activity of seven synthetic peptides corresponding to the segments of P. gingivalis fimbrial subunit protein. The peptides of residues 282-301 and 302-321, which exhibited antagonistic effects against P. gingivalis fimbriae-binding to human gingival fibroblasts in a previous study, showed weak activity, whereas other non-antagonistic peptides showed no significant activity. These findings indicated that P. gingivalis fimbriae enhanced production of HGF/SF by human gingival fibroblasts, whereas synthetic peptide segments of fimbrial subunit protein were not sufficient to exert the activity.

Involvement of a cis-acting element in the suppression of carbamoyl phosphate synthetase I gene expression in the liver of carnitine-deficient mice.

The expression of carbamoyl phosphate synthetase I (CPS) gene is suppressed in the liver of carnitine-deficient juvenile visceral steatosis (JVS) mice at weaning and under starvation at adult age. To clarify the suppression mechanism, we produced CPSL transgenic JVS mice carrying a transgene composed of the chloramphenicol acetyltransferase (CAT) gene with the upstream region (-12 kb to +138) of the rat CPS gene and CPSE transgenic JVS mice carrying a transgene composed of the luciferase gene with minimal promoter (299 bp from -161 to +138) and enhancer (469 bp around -6.3 kb) fragments of the rat gene. The expression of the CAT gene as well as the endogenous CPS was suppressed in CPSL transgenic JVS mice, but luciferase gene expression was not suppressed in CPSE transgenic JVS mice. We isolated the 5'-upstream region of the mouse CPS gene and identified an activator protein-1 (AP-1) site downstream of the minimum enhancer region of both rat and mouse CPS genes. In conjunction with the 313-bp mouse promoter region, the 714-bp mouse enhancer fragment conferred a cell-type-dependent hormone responsiveness. In rat primary cultured hepatocytes, the addition of oleic acid suppressed reporter gene expression induced by dexamethasone in the construct containing the enhancer fragment of 714 bp with the AP-1 site, but not in its AP-1 site mutants or in 519 bp without the AP-1 site. These results strongly suggest that direct protein-protein interaction between AP-1 and glucocorticoid receptor is not involved in the suppression of the CPS gene in JVS mice and that the AP-1 element is the cis-element which is responsible for the suppression.

Effects of rat fetuin on stimulation of bone resorption in the presence of parathyroid hormone.

Rat fetuin, which is the rat counterpart of human alpha 2-HS glycoprotein and bovine fetuin, is only detectable in calcified tissues such as bone matrices and dentin, and bone cells such as osteoblasts and osteocytes immunohistochemically. The effect of this protein on bone resorption was examined to study its physiological role in bone metabolism. Rat fetuin increased bone resorption in the presence of low concentrations of parathyroid hormone (PTH), but it had no activity on bone resorption without PTH. The increase in bone resorption by PTH and PTH plus rat fetuin was inhibited by the addition of chymostatin, an inhibitor for cathepsin L. Moreover, we found that when type I collagen from rat was preincubated with rat fetuin, the digestion of rat type I collagen by cathepsin L was increased. These findings suggest that rat fetuin present in bone matrix is important in bone resorption.

Involvement of oxidative stress in tumor cytotoxic activity of hepatocyte growth factor/scatter factor.

In this study, we show that N-acetylcysteine (NAC), a precursor of glutathione and an intracellular free radical scavenger, almost completely prevented hepatocyte growth factor (HGF)-suppressed growth of Sarcoma 180 and Meth A cells, and HGF-induced apoptosis, assessed by DNA fragmentation, and increase in caspase-3 activity, in Sarcoma 180 cells. The reduced form of glutathione also prevented HGF-suppressed growth of the cells as effective as NAC. Ascorbic acid partially prevented the effect of HGF, but other antioxidants such as superoxide dismutase, catalase, and vitamin E, and the free radical spin traps N-t-butyl-alpha-phenylnitrone and 3,3,5, 5-tetramethyl-1-pyrroline-1-oxide did not have protective effects. HGF caused morphological changes of the cells, many cells showing condensation and rounding, and enhanced the generation of intracellular reactive oxygen species (ROS) as judged by flow cytometric analysis using 2',7'-dichlorofluorescein diacetate. NAC completely prevented both HGF-induced morphological changes and the enhancement of ROS generation in the cells. However, NAC did not prevent the HGF-induced scattering of Madin-Darby canine kidney cells. To our knowledge, this is the first report that HGF stimulates the production of ROS, and our results suggest the involvement of oxidative stress in the mechanism by which HGF induces growth suppression of tumor cells.

Expression of hepatocyte growth factor/scatter factor and c-Met in human dental papilla and fibroblasts from dental papilla.

Hepatocyte growth factor/scatter factor (HGF/SF), a broad-spectrum and multifunctional cytokine, is essential for the development of tissues including tooth. Here it was found that the HGF/SF content of human dental papillae obtained from 8 to 16-year-old individuals decreased significantly with age. Cultured fibroblasts prepared from the dental papillae of individuals of different ages produced HGF/SF at almost the same rate, but the sensitivities of the cells to interleukin-1alpha and tumour necrosis factor-alpha for the production of HGF/SF increased with age. Generally, mesenchymal cells such as fibroblasts produce HGF/SF but do not express c-Met, a receptor for HGF/SF, yet fibroblasts in dental papilla and cultured fibroblasts prepared from dental papilla did express c-Met, as determined by immunohistochemistry, in situ hybridization and reverse transcription-polymerase chain reaction. Recombinant human [125I]iodo-HGF/SF specifically bound to cell-surface macromolecules with a mol. wt of 146,000, which is the same as that of the beta-subunit of c-Met. The physiological role of c-Met on fibroblasts in dental papilla is unknown, but the addition of 2 ng of HGF/SF per ml to the culture medium significantly stimulated DNA synthesis in the cells, as determined by pulse labelling with [3H]thymidine. Exogenous HGF/SF also stimulated secretion by the cells of vascular endothelial growth factor, a cytokine that induces blood vessel-formation. These results suggest that HGF/SF may be involved in tooth development via autocrine mechanisms.

Changes with age of the rat fetuin concentration in serum and its mRNA expression.

Rat fetuin, a counterpart of human alpha2-HS glycoprotein and bovine fetuin, shows strong intermolecular binding and association with other serum proteins. Therefore, to measure its concentration in rat serum, we pretreated serum samples with 1% SDS plus 5% (ca. 0.7 M) 2-mercaptoethanol at 100 degrees C for 3 min, and then subjected them to SDS-PAGE under reducing conditions followed by Western blotting. We found that the fetuin concentrations in normal rat serum determined by Western blotting were 2.5-4.5 mg/ml. These concentrations were three orders of magnitude higher than the previously reported concentrations. We also tried to measure the fetuin concentration in rat serum by means of an enzyme-linked immunosorbent assay after treatment of the samples with 0.1% sodium dodecyl sulfate (SDS) plus 10 mM 2-mercaptoethylamine at 100 degrees C for 3 min, but it gave a value of about 1/4 of that on Western blotting. Rat fetuin is expressed mainly in the liver, with a peak 2-4 weeks after birth, as determined by Northern blot analysis. The fetuin mRNA level in the liver changes almost in parallel with its serum concentration. The tibia also expresses fetuin, but much less than the liver.

Expression of HGF/SF in mesothelioma cell lines and its effects on cell motility, proliferation and morphology.

The expression of hepatocyte growth factor/scatter factor (HGF/SF) was studied in 12 mesothelioma cell lines characterized by either an epithelioid or a fibroblast-like phenotype. Conditioned media from these lines were analysed by bioassay and ELISA, and HGF/SF was detected in three cell lines, all with a fibroblast-like or mixed morphology. None of eight epithelioid cell lines expressed the factor. Thus, for these cell lines, the ability to secrete HGF/SF correlated with the cell phenotype. Following on from these observations, two cell lines, BR and BT, with a fibroblast-like and an epithelioid phenotype, respectively, were further investigated. Both cell lines expressed the Met receptor but only BR secreted HGF/SF. Both cell lines responded to exogenous HGF/SF treatment by a change of morphology but in different ways: BR became more elongated and bipolar, while BT formed more spread-out cell colonies. HGF/SF acted as a paracrine effector on the epithelioid BT cells and stimulated both cell-spreading and proliferation. Interestingly, BT cells spread but did not scatter in response to exogenous HGF/SF. In contrast BR cells showed only some stimulation of cell motility with HGF/SF and no increase in cell proliferation was observed. Because HGF/SF was previously found in the pleural effusion fluids of patients with malignant mesothelioma and in paraffin-embedded tumour tissues, it is concluded that HGF/SF may well stimulate the growth and spread of malignant mesothelioma in vivo by paracrine and/or autocrine mechanisms.

Hepatocyte growth factor/scatter factor activates the apoptosis signaling pathway by increasing caspase-3 activity in sarcoma 180 cells.

Hepatocyte growth factor, which is now known to be the same protein as scatter factor, induced oligonucleosomal fragmentation of nuclear DNA of Sarcoma 180 cells and increased the activity of caspase-3, a key component in control of the apoptotic cell death pathway to about 2.6 times that in control cells on 48 hr incubation, but did not increase the activity of caspase-1. Both HGF-induced DNA fragmentation and caspase-3 activity were completely inhibited by co-incubation with an inhibitor of caspase-3, Ac-DEVD-H. In contrast, HGF did not affect the expression of the apoptosis suppressors Bcl-2 and Bcl-x. These results indicate that HGF activates the apoptosis signaling pathway by increasing caspase-3 activity in Sarcoma 180 cells.

Effect of phosphorylated rat fetuin on the growth of hepatocytes in primary culture in the presence of human hepatocyte-growth factor. Evidence that phosphorylated fetuin is a natural modulator of hepatocyte-growth factor.

Rat fetuin, a counterpart of human alpha 2-HS glycoprotein and bovine fetuin, that is synthesized and secreted by hepatocytes is mostly phosphorylated, though rat fetuin isolated from bone matrix does not contain phosphorus. A rat 63-kDa phosphorylated N-glycoprotein (pp63) is the phosphorylated form of rat fetuin and pp63 has been shown to inhibit insulin-receptor tyrosine kinase activity. Therefore, we examined the effect of phosphorylated rat fetuin (phosphofetuin) on DNA synthesis in rat hepatocytes in culture in the presence of human hepatocyte-growth factor (HGF), since the human receptor of HGF, c-Met, is known to contain a tyrosine-kinase domain in its intracellular domain. We found that phosphofetuin from conditioned medium of rat-hepatocyte cultures dose-dependently decreased HGF-stimulated DNA synthesis in hepatocytes, whereas addition of non-phosphorylated rat fetuin had no effect. Addition of anti-(rat fetuin) Ig to the culture medium increased HGF-stimulated DNA synthesis by hepatocytes. Immunoprecipitation and cross-linking experiments showed that phosphofetuin bound to human HGF. We found that phosphofetuin interfered with binding of HGF to its specific receptor(s). These observations suggest that phosphofetuin synthesized by hepatocytes may be a natural modulator of HGF as a chalone, and that regulation of expression of phosphofetuin by growth factors and cytokines may be involved in liver regeneration under inflammatory conditions, such as in hepatitis.

Levels of human hepatocyte growth factor (hHGF) in peritoneal fluid after partial hepatectomy.

BACKGROUND/AIMS: Human hepatocyte growth factor (hHGF), now known to be identical with scatter factor and tumor cytotoxic factor, is thought to be involved in the regeneration of the liver in humans. MATERIAL AND METHODS: We measured the levels of immunoreactive hHGF levels in peritoneal fluid before and after partial hepatectomy in 37 patients, 10 with and 27 without cirrhosis. The presence of hHGF was confirmed in human peritoneal fluid with rat hepatocytes in primary culture and Western blot. RESULTS: Levels of hHGF increased significantly in peritoneal fluid after surgery, with its total concentration being correlated with the weight of resected liver 168 hours postoperatively. CONCLUSION: Findings suggest that the hHGF levels in peritoneal fluid may indicate the extent of hepatic damage following partial hepatectomy and other liver diseases.

Liver hepatocyte growth factor does not always correlate with hepatocellular proliferation in human liver lesions: its specific receptor c-met does.

Increased levels of expression of hepatocyte growth factor (HGF) and its specific receptor c-met have been shown in the liver of several benign and malignant pathologies, both in experimental models and humans. We investigated by immunohistochemistry the presence of both HGF and c-met protoocogene product (c-met pp) in 20 hepatocellular carcinomas (HCCs), 5 focal nodular hyperplasias (FNHs), 4 cases of fulminant hepatitis (FH), and 1 case of regenerated liver. The c-met protooncogene product was expressed in all cases with marked overexpression in the HCCs and in ductular metaplasia. HGF was detected in the Ito cells of all cases and in neoplastic hepatocytes of 9 of 20 HCCs (45%). The proliferative index of each lesion was evaluated by means of the polyclonal antibody anti-cyclin A. When the level of expression of HGF and c-met protooncogene product with the percentage of cyclin A+ nuclei were compared, the closest relationship was between c-met protooncogene product and cyclin A+ nuclei were compared, the closest relationship was between c-met protooncogene product and cyclin A. In 11 of 20 HCCs (55%), there wa no correlation between HGF positivity and cyclin A. This seems to suggest that, independently of the levels of native liver HGF, c-met protooncogene product is the most active modulator of liver cell proliferation.