Doses Of Hepatocyte Growth Factor Research Articles

Quantitation of ligand is critical for ligand-dependent MET signalling activation and determines MET-targeted therapeutic response in gastric cancer.

Despite the promising preclinical antitumor activity of MET-targeting therapies, most clinical trials have failed. We introduced a new concept of quantitation of stroma-induced hepatocyte growth factor (HGF) to assess the actual MET signalling activity in gastric cancer (GC). We treated serially diluted HGF and conditioned media (CM) from cancer-associated fibroblasts (CAFs) on low MET-expressing cancer cells and investigated the phenotypical and signalling changes. Stromal proportion and MET expression in GC samples were assessed, and gene set enrichment analysis (GSEA) from the public database was performed. The antitumor effect of anti-MET treatment was examined, especially when cancer cells were activated in a ligand-dependent manner. Relatively high doses of HGF or high-concentrated CM fully activated MET signalling cascades and promoted cell proliferation/invasion. High stromal proportion denoted worse patient survival in MET-positive GCs than in MET-negative ones. GSEA showed that the gene sets regarding proliferation, migration, and CAF as well as MET pathway signature were enriched in simultaneously MET- and HGF-positive samples. Sufficient ligand-dependent MET signalling activation increased the sensitivity to crizotinib. We conclude that patients whose tumours have a high stromal proportion and at least low MET expression may benefit more from MET-targeted therapies.

Disproportionately high levels of HGF induce the degradation of the c-met receptor through the proteasomal degradation pathway

Hepatocyte growth factor (HGF) receptor is a member of the receptor tyrosine kinases (RTKs) and has been reported to perform diverse functions in various cell types during both the developmental and adult stages. Among different roles, HGF is best known for its angiogenic effects of inducing the migration of endothelial cells. Because angiogenesis is one of the prerequisite steps for tumor metastasis, HGF-dependent cell migration has to be tightly controlled. However, the underlying mechanisms regulating the optimum level of HGF/c-met signaling have been poorly understood. In this study, we tested whether the migration of endothelial cells is regulated by a negative feedback mechanism under disproportionately large amounts of HGF. Data from endothelial cell migration assays showed that HGF activity increased as its concentration increased, but declined beyond a certain point. Under limiting conditions, amounts of phosphorylated Erk and Akt surged, reaching a plateau in which the enhanced level was more or less maintained. The c-met receptor was degraded when unnecessarily large amounts of HGF were present. Under these conditions, HGF could no longer activate downstream signaling pathways even if cells were re-treated with optimal amounts of HGF. Excessive doses of HGF increased the phosphorylation of tyrosine residue 1003 involved in the ubiquitination of c-met, and phosphorylated c-met was diverted toward the proteasomal degradation pathway. Taken together, HGF/c-met signaling is tightly regulated by a negative feedback loop through an ubiquitin-proteasomal degradation pathway.

Towards an in vitro fibrogenesis model of human vocal fold scarring

BackgroundVocal fold (VF) scarring remains a therapeutic dilemma and challenge in modern laryngology. To facilitate corresponding research, we aimed to establish an in vitro fibrogenesis model employing human VF fibroblasts (hVFF) and the principles of macromolecular crowding (MMC).MethodsFibrogenesis was promoted by addition of transforming growth factor-β1 to standard medium and medium containing inert macromolecules (MMC). Hepatocyte growth factor (HGF) and Botox type A were tested for their antifibrotic properties in various doses. Experiments were analyzed with respect to the biosynthesis of collagen, fibronectin, and α-smooth muscle actin using immunofluorescence, silver stain and western blot.ResultsMMC led to favourable enhanced deposition of collagen and other extracellular matrix components, reflecting fibrotic conditions. Low doses of HGF were able to dampen profibrotic effects. This could not be observed for higher HGF concentrations. Botox type A did not show any effects.ConclusionBased on the principles of MMC we could successfully establish a laryngeal fibrogenesis model employing hVFF. Our finding of dose-dependent HGF effects is important before going into clinical trials in humans and has never been shown before. Our model provides a novel option to screen various potential antifibrotic compounds under standardized conditions in a short time.

Antifibrotic Effects of Hepatocyte Growth Factor on Endothelial-to-Mesenchymal Transition via Transforming Growth Factor-Beta1 (TGF-β1)/Smad and Akt/mTOR/P70S6K Signaling Pathways.

BackgroundThe related mechanisms involved in allograft interstitial fibrosis and chronic allograft dysfunction (CAD), following renal transplant, remain largely unknown. Here, we explored the role of hepatocyte growth factor (HGF) treatment on the endothelial-to-mesenchymal transition (EndMT) as a new way to target and prevent kidney fibrosis and improve outcomes for renal transplant recipients.Method/MaterialWe extracted proteins and mRNAs from human umbilical vein endothelial cells (HUVECs) and human renal glomerular endothelial cells (HRGECs) treated with transforming growth factor-beta1 (TGF-β1) and/or varying doses of HGF, and assessed the effect of HGF on the EndMT using western blotting, qRT-PCR, and ELISA assays. We utilized cell motility and migration assays to evaluate cell movement, and applied western blotting to assess the mechanism by which TGF-β1 induced the EndMT.ResultsHGF significantly attenuated the development of TGF-β1-induced EndMT in a concentration-dependent way, and weakened the abilities of motility and migration of both HUVECs and HRGECs. Moreover, our results reveal that the antifibrotic effect of HGF on the EndMT was associated with the TGF-β/Smad and Akt/mTOR/p70S6K signaling pathways.ConclusionsOur study suggests that HGF treatment significantly attenuates the development of EndMT induced by TGF-β1 via the TGFβ/Smad and Akt/mTOR/P70S6K signaling, which provides novel insights into the prevention and treatment of interstitial fibrosis and CAD following renal transplant.

HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway

BackgroundThe hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration.MethodsWe evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting.ResultsWe demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin.ConclusionsWe demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.

Hepatocyte growth factor protects human mesangial cells against apoptosis induced by lead damage.

Lead is a kind of nephrotoxic metal which frequently threats human health. Hepatocyte growth factor (HGF) is a multifunctional growth factor that protects cell apoptosis. In this study, human mesangial cells (HMCs) were treated with a single HGF dose of 20 and 40 μl/ml in order to investigate the effect of HGF on proliferation and apoptosis ability of HMCs induced by lead acetate. In HGF-treated group, HMCs were incubated with HGF (20, 40 μl/ml) half an hour prior to lead inducing. After lead-induced damage 48 h, the proliferation of HMCs was measured by MTT assay, and the apoptosis was assessed by flow cytometry. RT-PCR was used to detect the expression of P53, Bcl-2, Bax, and caspase-3 mRNA. The expression of Bax protein was measured by Western blot analysis. The results showed that HGF inhibits proliferation of HMCs induced lead acetate in a dose-dependent manner (P < 0.05). HGF significantly promoted the proliferation of HMCs, and flow cytometry revealed that HGF can inhibit apoptosis of HMCs. RT-PCR and Western blot showed that P53, Bax, and caspase-3 expression decreased, while Bcl-2 expression increased. HGF may afford a protection to HMCs against lead-induced damage.

Dose Variation of Hepatocyte Growth Factor and its Effects on an Animal Model of TPN-Induced Liver Injury

Total parenteral nutrition (TPN) induced liver failure is the leading indication for transplantation in children. Our previous research demonstrated the benefit of a specific intravenous dose of hepatocyte growth factor (HGF) in the amelioration of TPN-induced liver injury. This study was designed to ascertain the optimum concentration of HGF in an animal model of TPN-induced liver injury. Twenty adult female Sprague-Dawley rats underwent 70% small bowel resection and placement of venous catheters connected to subcutaneous osmotic minipumps. Four groups (n=5 each) based on the contents of the osmotic pump were utilized as follows: group 1 (control): saline; group 2: HGF 75 mcg/kg/d; group 3: HGF 150 mcg/kg/d; and group 4: HGF 250 mcg/kg/d. Each rat received 14d of TPN without enteral nutrition. After sacrifice, the liver was harvested. Hepatic inflammation was evaluated using antibodies for TNF-α and IL-6. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) technique. All concentrations induced statistically significantly less IL-6 and TNF- α expression compared to the control animals. Increased efficacy was demonstrated with increasing dose concentration up to 150 mcg/kg/d but not 250 mcg/kg/d. Apoptotic activity was decreased statistically significantly for all dose concentrations compared with the controls, as well as to increases in dose concentration. Increasing concentrations of HGF were directly correlated with increased modulation of inflammatory response and apoptotic index in this animal model for TPN-induced liver injury, up to 150mcg/kg/d. Further increases were significant with respect to apoptotic index only. Further investigations are warranted to determine if HGF may be useful to minimize TPN-induced liver injury in children.

Recombinant kringle 5 from plasminogen antagonises hepatocyte growth factor-mediated signalling

The blood protein plasminogen is proteolytically cleaved to produce angiostatin and kringle 5 (K5), both of which are known angiogenesis inhibitors. A common structural element between K5, angiostatin and other endogenous angiogenesis inhibitors is the presence of the kringle protein-interacting domain. Another kringle domain-containing protein, hepatocyte growth factor (HGF), promotes angiogenesis by binding to and stimulating the tyrosine kinase receptor Met. HGF binding to Met is dependent on the kringle domains of HGF. Because both K5 and HGF contain kringle motifs and because these proteins have opposite effects on angiogenesis, we hypothesised that K5 can antagonise HGF-mediated signalling in a Met-dependent manner. We determined that K5 binding to H1299 cells is competed by HGF suggesting that these two proteins bind to the same protein. Purified K5 immunoprecipitates with Met and this interaction is abolished by increasing doses of HGF. Using proliferation, phosphorylation of Met and Akt as markers of HGF activity, we determined that K5 inhibits HGF-mediated signalling. Taken together, these data support a model by which K5 binds to Met and functions as a competitive antagonist of HGF signalling and presents a novel mechanism of action of K5.

Possible implication of satellite cells in regenerative motoneuritogenesis: HGF upregulates neural chemorepellent Sema3A during myogenic differentiation

Regenerative coordination and remodeling of the intramuscular motoneuron network and neuromuscular connections are critical for restoring skeletal muscle function and physiological properties. The regulatory mechanisms of such coordination remain unclear, although both attractive and repulsive axon guidance molecules may be involved in the signaling pathway. Here we show that expression of a neural secreted chemorepellent semaphorin 3A (Sema3A) is remarkably upregulated in satellite cells of resident myogenic stem cells that are positioned beneath the basal lamina of mature muscle fibers, when treated with hepatocyte growth factor (HGF), established as an essential cue in muscle fiber growth and regeneration. When satellite cells were treated with HGF in primary cultures of cells or muscle fibers, Sema3A message and protein were upregulated as revealed by reverse transcription-polymerase chain reaction and immunochemical studies. Other growth factors had no inductive effect except for a slight effect of epidermal growth factor treatment. Sema3A upregulation was HGF dose dependent with a maximum (about 7- to 8-fold units relative to the control) at 10-25 ng/ml and occurred exclusively at the early-differentiation stage, as characterized by the level of myogenin expression and proliferation (bromodeoxyuridine incorporation) of the cells. Neutralizing antibody to the HGF-specific receptor, c-met, did not abolish the HGF response, indicating that c-met may not mediate the Sema3A expression signaling. Finally, in vivo Sema3A was upregulated in the differentiation phase of satellite cells isolated from muscle regenerating following crush injury. Overall, the data highlight a heretofore unexplored and active role for satellite cells as a key source of Sema3A expression triggered by HGF, hence suggesting that regenerative activity toward motor innervation may importantly reside in satellite cells and could be a crucial contributor during postnatal myogenesis.

Hepatocyte growth factor induces anoikis resistance by up-regulation of cyclooxygenase-2 expression in uterine endometrial cancer cells

Cyclooxygenase-2 (COX-2) has been implicated in the promotion of carcinogenesis. Although the role of COX-2 in endometrial cancer remains unclear, recent experiments suggest that COX-2 antagonizes cell apoptosis, increases the invasiveness of malignant cells, and promotes angiogenesis. Hepatocyte growth factor (HGF) is a mesenchymal-derived cytokine and the interaction between HGF and its tyrosine kinase receptor, c-Met proto-oncogene, is associated with tumor progression and metastasis. To investigate the molecular mechanism of HGF-induced anoikis resistance, we analyzed the signal transduction and COX-2 expression in endometrial cancer cells. Here, we show i) the expression of COX-2 protein significantly increased in a dose-dependent manner after HGF stimulation in endometrial cancer cell lines (HEC-IB and RL95-2), reaching 200-270% stimulation at the highest doses of HGF tested (40 ng/ml); ii) flow cytometry and TUNEL analyses revealed that HGF significantly inhibited anoikis of RL95-2 cells; iii) phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), but not mitogen-activated protein kinase/ERK kinase (MEK) inhibitor (PD98059), specifically blocked HGF-mediated anoikis resistance in RL95-2 cells; and iv) COX-2 inhibitor, Meloxicam, abrogated HGF-mediated anoikis resistance. Our data suggest that HGF induces anoikis resistance in endometrial cancer cells possibly through PI3K/Akt pathway-dependent up-regulation of COX-2 expression.

Suppression of the production of extracellular matrix and α-smooth muscle actin induced by transforming growth factor-β1 in fibroblasts of the flexor tendon sheath by hepatocyte growth factor

We examined the effectiveness of hepatocyte growth factor (HGF) in blocking production of transforming growth factor (TGF)-beta1-induced collagen I, fibronectin, and alpha-smooth muscle actin (alpha-SMA) in the flexor tendon sheath of rabbits in vitro. Fibroblasts were obtained from the sheaths. Cell culture was supplemented with TGF-beta1 5 ng/ml and increasing doses of HGF (10-40 ng/ml). The production of collagen I and fibronectin in supernatants culture were examined using an enzyme-linked immunosorbent assay (ELISA). alpha-SMA expression was assessed by western blot. TGF-beta1 stimulated production of collagen I, fibronectin, and alpha-SMA greatly, while HGF significantly (p<0.05) reduced production of all components induced by TGF-beta1 in a dose-dependent manner. This suggests that HGF effectively antagonises the action of TGF-beta1 in cultured fibroblasts from flexor tendon sheaths. The results provide a cellular and molecular basis for HGF acting as a therapeutic agent for adhesions in flexor tendons.

Promotion of Fas‐mediated apoptosis in Type II cells by high doses of hepatocyte growth factor bypasses the mitochondrial requirement

The death receptor pathway is coupled to the mitochondria apoptosis pathway. However, mitochondrial participation, which is stimulated by Bid but suppressed by Bcl-2/Bcl-x(L), is required in certain cells (Type II), but not in others (Type I). While these differences were originally characterized in the lymphoid cell lines, the typical Type II cells are represented by hepatocytes in vivo. The molecular mechanisms that distinguish Type II from Type I cells and the regulation are not fully understood. Fas can be sequestered by the HGF receptor c-Met and high doses of HGF can promote cell death by freeing Fas from c-Met complex. We thus reasoned that treatment of the Type II cells with high doses of HGF could enhance Fas-mediated apoptosis and spare the mitochondria amplification. Indeed, such treatment led to increased apoptosis in Type II lymphoid cells, which could not be blocked by Bcl-x(L). Moreover, significant hepatocyte apoptosis was induced by this scheme in the absence of Bid with increased dissociation of Fas from c-Met. These findings indicate that high doses of HGF could be used to promote apoptosis in Type II cells bypassing the requirement for mitochondria activation.

HGF‐mediated control of IL‐6 production in primary rat hepatocyte cultures

Hepatocyte Growth Factor (HGF) is an omnipotent growth factor that targets various kinds of cells. As an extracellular signal, HGF is able to trigger many downstream signaling pathways through its receptor, MET. One such pathway involves the transcription factor, NF-kappaB. We report here that two different concentrations of HGF (20ng/ml, growth-promoting; 500 ng/ml, growth-suppressing) in serum-free rat hepatocyte cultures generate different responses of both NF-kappaB and its regulated gene product, interleukin-6 (IL-6). The low dose of HGF leads to translocation of the NF-kappaB complex (p50/p65) to the hepatocellular nucleus whereas; the high dose has the opposite effect, blocking the complex in the cytoplasm. Transcription of the well-known downstream target of macrophage NF-kappaB, IL-6, mimicked these results. Surprisingly, immuno-staining showed that the transcriptional results were reflected at the protein level in hepatocytes, i.e. the IL-6 staining was hepatocellular and changed with the concentration of HGF. These findings emphasize the importance of HGF concentration effects and reveal what may be a previously undisclosed biological role for IL-6 in hepatocyte cultures.

247. Gene Therapy with a Retroviral Vector Expressing Canine β-Glucuronidase to Juvenile (7 Week Old ) Dogs Improves the Biochemical Manifestations of MPS VII

Top of pageAbstract Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease due to deficiency of |[beta]|-glucuronidase (GUSB). Manifestations include bone and joint disease, heart disease, and neurological dysfunction. The MPS VII dog has a missense mutation in GUSB (R166H) that results in clinical manifestations that resemble those in humans. We have previously demonstrated that neonatal gene therapy with 3|[times]|109 TU/kg of a retroviral vector (RV) expressing canine GUSB from the human |[alpha]|1-antitrypsin promoter (hAAT-cGUSB-WPRE) resulted in transduction of hepatocytes and 195+/-36 U/ml of GUSB in serum. This resulted in a marked improvement in cardiac, bone and joint, and other manifestations. However, most patients with MPS VII are not identified at birth, and it will be necessary to determine the effect of transfer into older animals. Seven week-old MPS VII dogs were injected IV with 1|[times]|1010 transducing units (TU)/kg of hAAT-cGUSB-WPRE. Some received a cumulative dose of hepatocyte growth factor (HGF) over 24 hours of 2.5 mg/kg, then were injected with RV at 24, 48, 72, and 96 hours after the first dose of HGF (HGF/RV). Others received RV once a day for 4 days without preceding HGF (RV alone). Dogs that received HGF/RV or RV alone at 7 weeks had transduced hepatocytes and achieved stable expression of GUSB in serum at 185+/-54 U/ml or 51+/-5 U/ml for up to 1 year, respectively, although differences were not statistically significant. The serum GUSB achieved per TU/kg given was 28% and 8%, respectively, of that achieved after neonatal gene transfer, which likely reflects lower levels of hepatocyte replication in juveniles. Some animals were sacrificed at 6 months after gene transfer, and organs analyzed for biochemical correction of disease. The results in both juvenile transfer groups were similar and were pooled for statistical analyses. MPS VII results in elevation of the secondary lysosomal enzyme |[beta]|-hexosaminodase (|[beta]|-hex) and glycosaminoglycan (GAG) levels, and normalization of these occurs with successful therapies. In liver, spleen, jejunum, and lung, GUSB activity was 1% to 15% of normal levels and there was a marked reduction in |[beta]|-hex and GAG levels. In thymus, pancreas, kidney, and muscle, GUSB activity was 0.25% to 1% of normal and there was a marked or partial reduction in |[beta]|-hex and GAG levels. However, brain GUSB activity was only 0.2% of normal and there was little reduction in |[beta]|-hex (GAG was not evaluable as untreated MPS VII dogs do not have elevated brain GAG levels). Despite these biochemical improvements, the mobility of the dogs was only slightly improved and all were unable to walk by 12 months or earlier. We conclude that gene therapy into juvenile dogs results in expression similar to that observed after neonatal gene transfer, and marked biochemical improvements in somatic organs. This is the first demonstration of using juvenile gene therapy to improve disease in a large animal model for LSD. However, there was little biochemical improvement in brain, and the bone disease remained severe.

Hepatocyte growth factor treatment ameliorates diarrhea and bowel inflammation in a rat model of inflammatory bowel disease

Background/purpose Transfection of the HLA-B27 gene into normal Fischer rats induces phenotypic changes similar to inflammatory bowel disease (IBD). This study investigated the benefits of 2 doses of hepatocyte growth factor (HGF) on the manifestations of IBD in this rat model. Methods Fischer rats and HLA-B27 rats were divided into 4 groups: Fischer rats treated with saline, HLA-B27 rats treated with saline, HGF at 150 μg/kg/d, and HGF at 300 μg/kg/d. HGF or saline was infused for 14 days via an osmotic pump attached to a catheter in the internal jugular vein. After treatment, rats were evaluated for diarrhea and reduction in gross and microscopic bowel inflammation. Statistics were determined using analysis of variance (ANOVA). A P value ≤ .05 was considered significant. Results Administration of HGF at 150 μg/kg/d decreased diarrhea by 40%, gross inflammation by 41%, and microscopic inflammation by 72% ( P ≤ .05). At 300 μg/kg/d HGF decreased diarrhea by 46%, gross inflammation by 45%, and microscopic inflammation by 54% (P ≤ .05). Conclusions HGF administration reduces the clinical manifestations of IBD in this rat model. Similar effects were seen at both doses of HGF administration, implying that there is a plateau above which further increases in HGF levels provides no added benefit. HGF administration may be clinically useful in the management of IBD.

A trial on regeneration therapy of rat liver cirrhosis by controlled release of hepatocyte growth factor

This paper is an investigation of therapeutic trial on the liver cirrhosis by the controlled release of hepatocyte growth factor (HGF). Biodegradable microspheres were prepared from gelatin for the controlled release of HGF. Rats with liver cirrhosis were prepared by the intraperitoneal injection of thioacetamide at a dose of 0.2 g/kg every other day for 10 weeks. The rats received single intraperitoneal injection of gelatin microspheres incorporating 2 or 0.4 mg of HGF, 2 or 0.4 mg of free HGF, and HGF-free, empty gelatin microspheres 3 weeks after the last thioacetamide injection. Histological observation of the rat liver revealed that injection of gelatin microspheres incorporating HGF effectively allowed to recovery from the liver fibrosis, inducing liver regeneration. When the histological score, the area of fibrous linkage, and the hydroxyproline content in the liver were evaluated, all values were significantly smaller than those of the free HGF and empty gelatin microspheres injection, irrespective of the HGF dose. It is concluded that the gelatin microspheres incorporating HGF are a promising therapeutic method of inducing successful liver regeneration by fibrosis digestion in rats with cirrhosis.

Localization and functional role of hepatocyte growth factor (HGF) and its receptor c-met in the rat developing cerebral cortex

Development of the cerebral cortex is a series of precisely timed proliferative, migratory, and maturational processes. Hepatocyte growth factor (HGF) is a pleiotrophic cytokine, which plays important roles in the organogenesis and regeneration of various tissues, both during development and in the adult, due to its mitogenic, motogenic and morphogenic activities. In the present study, we examined expression and functional roles of HGF and c-Met during development of the rat cerebral cortex. Quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) revealed that expression levels of c- met and HGF mRNAs were increased in the cerebral cortex during late embryonic development and peaked at E18. Immunohistochemical analyses revealed that c-Met-immunoreactivity (IR) was localized to the preplate (PP), with weaker-IR in neuroepithelial layer (NE) at embryonic day 14 (E14). At E16, c-Met-IR was present in the cortical plate (CP) and the intermediate zone (IZ), with a weak presence in the ventricular zone (VZ). On the other hand, HGF-IR was present in NE and VZ at E14 and E16, respectively. HGF-IR appeared in cortical plate tissue from E16 onward. Double labeling immunofluorescent cytochemical studies revealed that c-Met-IR was localized both in TuJ-1-IR- and non-TuJ-1-IR-cells, purified from E18 cerebral cortex in vitro, suggesting the presence of c-Met-IR in postmitotic neurons as well as in neuroepithelial cells. c-Met-IR was strong in cell bodies and neurites shortly after in vitro culture, while at 7DIV c-Met-IR decreased in neurites and was evident in growth cones. HGF dose dependently supported neuronal survival in vitro under serum-deprived conditions. In a transwell culture chamber, HGF increased neuronal migration, and co-incubation with functional blocking antibody against HGF abrogated this motogenic effect of HGF. These lines of evidence suggest that HGF is involved in the development and maintenance of cortical neurons during differentiation, motogenesis, neuritogenesis and neuronal survival.

Hepatocyte Growth Factor Stimulates Fetal Gastric Epithelial Cell Growthin Vitro

Background.The growth and development of the fetal gastrointestinal tract is likely mediated, in part, by peptide growth factors. We compared the mitogenic effects of graded doses of hepatocyte growth factor (HGF) to epidermal growth factor (EGF), transforming growth factor-α (TGF-α), and insulin-like growth factor-1 (IGF-1) on fetal rabbit gastric epithelial cells.Materials and Methods.Fetal rabbit gastric epithelial cells were purified by mechanical dissociation and selected culture and grown in short-term (24 h) and long-term (12 days) culture. Stimulation of fetal gastric epithelial cell growth in response to individual peptide growth factors was measured by [3H]thymidine incorporation and cell counting.Results.In short-term culture, HGF stimulated [3H]thymidine incorporation in a dose-dependent manner from a threshold at 10 pM to a maximum at 100 pM. For EGF and TGF-α, maximal stimulation occurred at 100 pM. For HGF, maximal [3H]thymidine incorporation was 3.6 ± 0.7 times basal. For EGF and TGF-α, maximal [3H]thymidine incorporation was 4.3 ± 0.4, and 3.6 ± 0.4 times basal, respectively. For IGF-1, maximal [3H]thymidine incorporation was only 70% of the maximal effect observed for the other growth factors tested. Rabbit amniotic fluid increased [3H]thymidine uptake in a dose-dependent manner. In long-term culture, purification to greater than 90% epithelial cells was attained after 12 days treatment. For HGF, EGF, TGF-α, and 20% rabbit amniotic fluid, significant increases in cell number above control (P< 0.05) were observed at 1 nM concentrations. None of these individual factors, however, increased cell growth as significantly as that of 10% fetal bovine serum.Conclusions.Our results suggest that: (1) HGF stimulates [3H]thymidine uptake and cell proliferation in fetal rabbit gastric epithelial cellsin vitro,and (2) HGF's mitogenic effect on fetal rabbit gastric epithelial cell growth is comparable to that observed for EGF and TGF-α, but superior to the effect observed for IGF-1.

Intestinal fibroblasts regulate intestinal epithelial cell proliferation via hepatocyte growth factor.

Although the presence of subepithelial intestinal fibroblasts has been well recognized, the effects of fibroblasts on intestinal epithelial cell (IEC) growth are incompletely understood. In vitro studies were undertaken to evaluate the effects of fibroblasts on the proliferation of model IEC lines. IECs (Caco-2, T84, and IEC-6) were grown alone or in the presence of human intestinal (CCD-18), lung (CCD-37), or skin explant-derived fibroblasts. Cocultures were carried out directly on irradiated fibroblasts or by Transwell coculture technique with fibroblasts and epithelial cells separated by a porous filter. Cell proliferation was assessed by [3H]thymidine incorporation and cell counts. Hepatocyte growth factor (HGF) and c-met transcript expression in IECs and fibroblasts was examined by RT-PCR and Northern blotting; protein expression was evaluated by immunoblotting. Intestinal as well as lung and skin fibroblasts substantially stimulated proliferation of Caco-2, T84, and IEC-6 cells in both direct and Transwell cocultures. In addition, fibroblast-conditioned medium stimulated IEC proliferation, suggesting a paracrine mechanism. Anti-human HGF-neutralizing antibodies blocked the growth-promoting effects in both fibroblasts and fibroblast-conditioned medium. Recombinant human HGF dose dependently promoted IEC proliferation. HGF mRNA and protein expression was restricted to fibroblasts. High levels of c-met expression were found in Caco-2 and T84 cells; in contrast, expression in fibroblasts was weak. In summary, fibroblasts stimulate IEC proliferation through a paracrine mechanism mediated predominantly by HGF.

Human placental extract stimulates liver regeneration in rats.

The effect of human placental extract (HPE) on liver regeneration in rats was investigated. After intravenous administration of HPE to a-naphthylisothiocyanate (ANIT)-intoxicated rats, the labeling index in hepatocytes was significantly increased to a level 16.5 times higher than that of the control. A 1/500 dilution of HPE directly stimulated DNA synthesis of the hepatocytes in primary culture. HPE heated at 121 degrees C did not stimulate the labeling index in vivo or hepatocyte DNA synthesis in primary culture, suggesting that HPE contains heat-unstable but potent mitogens for hepatocytes. HPE contains hepatocyte growth factor (HGF), but the mitogenic effect of HPE cannot be explained by the effect exerted by HGF alone, since both the labeling index in vivo and hepatocellular DNA synthesis in vitro stimulated by HPE were much higher than those stimulated by HGF alone when the applied doses of HGF were set to be almost the same level between each case. When HPE was fractionated on a heparin-sepharose column, the mitogenic effect of HPE was found to be located mainly in the heparin-bound fraction. Hepatocyte DNA synthesis induced by this fraction was enhanced cooperatively by the heparin-unbound fraction, suggesting that there are some modulators in the heparin-unbound fraction which enhance the proliferative activity of the heparin-bound fraction by a synergetic mechanism. Both HPE and heated HPE completely recovered the biochemical marker activity for liver function (glutamic-pyruvic transaminase, GPT; alkaline phosphatase, ALP; lactate dehydrogenase, LAP; gamma-glutamyltransferase, gamma-GTP activities and the bilirubin concentration) almost to the control level in the serum of ANIT-intoxicated rats, indicating that HPE also contains a heat-stable fraction which repairs liver function.