Recombinant Human Hepatocyte Growth Factor Research Articles

Suppression of Tacrolimus on the expression of MMP-9 in rhHGF-stimulated ECV304 cell line

To investigate the stimulating effect of recombinant human hepatocyte growth factor (rhHGF) on the expression of matrix metalloproteinase-9 (MMP-9) in ECV304 cells and the suppression effect of FK506. The growth curve of ECV304 cells was drawn and the valid concentration of rhHGF and FK506 were determined. The expression levels of MMP-9 were analyzed by flow cytometry (FCM). (1) The optimal growth concentration of ECV304 cell was 1.0E+4/ml and logarithm growth time was 3-5 days. The valid concentration of rhHGF was 8 ng/ml and IC50 was 8.27 ng/ml. The valid concentration of FK506 was 50 ng/ml and IC50 was 51.63 ng/ml. (2) The expression level of MMP-9 was 39.74% in controls. Cell livability was 1.388169 +/-0.102033 (P<0.01) when 8 ng/ml rhHGF was added to culture medium, and the expression level of MMP-9 was 40.32%. Cell livability was 0.398764 +/-0.092476 (P<0.01) when 50 ng/ml FK506 was added to culture medium, and the expression level of MMP-9 was 14.61%. Cell livability was 0.767203 +/-0.02639 (P<0.01) when 8 ng/ml rhHGF was added 15 minutes after 50 ng/ml FK506 was added to culture medium, and the expression level of MMP-9 was 35.08%. rhHGF can stimulate the proliferation of ECV304 cells and increase the protein expression level of MMP-9. FK506 can suppress the proliferation of ECV304 cells and decrease the expression level of MMP-9. FK506 can inhibit the proliferation of ECV304 cells induced by rhHGF and decrease MMP-9 protein level.

Myocardial Protective Effect of Human Recombinant Hepatocyte Growth Factor for Prolonged Heart Graft Preservation in Rats

In heart transplantation, myocardial apoptosis during hypothermic storage contributes to graft dysfunction. On the other hand, hepatocyte growth factor (HGF) has been reported to be an antiapoptotic factor in the heart. Therefore, we assessed whether the administration of recombinant human HGF (rh-HGF) prevents apoptosis in the prolonged preserved myocardium, resulting in an improvement in the cardiac function of the graft. Isolated rat hearts were subjected to 4 hr (group A), 6 hr (group B), and 8 hr (group C: without rh-HGF vs. group D: with 100 microg of rh-HGF) of hypothermic storage followed by 60 min of normothermic reperfusion (n=5 in each group). Compared with non-HGF-treated hearts (group C), HGF-treated hearts (group D) showed a significantly higher recovery rate of left ventricular developed pressure (38+/-5% vs. 58+/-6%, P<0.01) and maximum dp/dt (53+/-7% vs. 74+/-4%, P<0.01) and a lower rate of TUNEL-positive cardiomyocytes (7.8+/-6.0% vs. 25.3+/-8.9%, P<0.05) after 60 min of reperfusion. Western blot analysis revealed that c-Met/HGF receptor expression was stronger in the HGF-treated myocardium than in the non-HGF-treated myocardium after 8 hr of storage and was associated with a weaker expression of caspase-3 and a stronger expression of Bcl-xL after 60 min of reperfusion. The administration of rh-HGF before storage improved cardiac function after prolonged myocardial preservation by preventing apoptosis through the c-Met/HGF receptor. Thus, the addition of rh-HGF in the storage solution may be a promising strategy for prolonged heart graft preservation.

Effect of hepatocyte growth factor on induction of cytokine-induced neutrophil chemoattractant in rat hepatocytes

Background Restrictions on graft size are a serious obstacle to the expansion of indications for adult recipients in living donor liver transplantation. Hepatocyte growth factor (HGF) has a crucial role in regeneration following hepatic injury. Rat cytokine-induced neutrophil chemoattractant (CINC), a member of the interleukin-8 superfamily in humans, has been implicated in chronic liver diseases or development of liver ischemia-reperfusion injury. Studies were performed to examine whether HGF influences the induction of CINC in hepatocytes. Methods Primary cultures of rat hepatocytes were treated with or without recombinant human (rh) HGF. The release of CINC into the culture medium and levels of CINC mRNA were measured using an enzyme-linked immunosorbent assay and Northern blot analysis. Transcription of nuclear factor (NF)-kappa B was detected by electrophoretic mobility shift assays. Results rhHGF increased the release of CINC in the medium dose- and time-dependently, showing a maximal effect at 100 ng/mL. Genistein (100 μmol/L) blocked the release of CINC stimulated by rhHGF. Levels of CINC mRNA were also increased, reaching a maximum at 8 hours after addition of rhHGF. Electrophoretic mobility shift assays revealed rhHGF activated transcription factor, NF-kappa B. Conclusion These results suggest that HGF stimulates the induction of CINC gene expression through activation of NF-kappa B. CINC may be involved in the function of HGF during liver regeneration.

HEPATOCYTE GROWTH FACTOR IMPROVES SURVIVAL OF STEATOTIC LIVER TRANSPLANTATION IN RATS

P71 Background/Aims; Transplantation of liver with severe steatosis may result in primary nonfunction (PNF) of the graft. As described previously, human recombinant hepatocyte growth factor (hrHGF) protected fatty grafts against cold preservation injury in rats. In the present study, we examined the effects of hrHGF on survival and graft function in rat fatty liver transplantation model. Methods; Severe steatosis was induced by a 15-day choline-deficient diet in male Lewis rats. The fatty graft was orthotopically transplanted in normal male Lewis rat after 12 hr cold preservation in UW solution. In the HGF group, donor rats were pretreated with a single bolus of systemic injection of hrHGF (10μg/rat), and 30μg of hrHGF was also added to the perfusion/preservation solution. Animal survival, graft function and morphological changes were compared with those of the untreated group. Liver cell apoptosis was detected by TUNEL stain and liver regeneration was evaluated by BrdU stain at 2 hrs after reperfusion of the liver graft. Results; One week survival was significantly improved in the HGF group compared with the untreated group (83% vs. 33%, p<0.01, n=18/group). AST, ALT and LDH levels in the rinse effluent at the end of cold preservation and serum ALT and LDH at 2 hrs after reperfusion were significantly lower in the HGF group. Although there was no significant difference in hyaluronic acid (HA) level in the rinse effluent at the end of preservation between the two groups, exogenous HA uptake rate (HAUR) at 2 hrs after reperfusion was improved in the HGF group (0.62±0.05 vs. 0.79±0.11, p<0.05). hrHGF pretreatment also significantly reduced the sinusoidal endothelial cell (SEC) apoptotic index (1.65±0.60% vs. 4.90±1.25%, p<0.01) and increased BrdU incorporation index (2.07±0.62% vs. 0.06±0.04%, p<0.01) at 2 hrs after reperfusion. More severe sinusoidal SEC damage was observed by transmission electron microscope in the untreated group than that in HGF group. Conclusions; hrHGF prevented the death of PNF in steatotic liver transplantation by attenuating preservation–reperfusion injury and facilitating recovery of the graft, probably through its antiapoptotic properties and acceleration of liver regeneration at early stage after transplantation.

Hepatocyte growth factor attenuates cerebral ischemia-induced learning dysfunction

Hepatocyte growth factor (HGF) acts as an organotropic factor for regeneration and protection in various organs and has the ability to attenuate cerebral ischemia-induced cell death. However, the effect of HGF on learning and memory function after a cerebral ischemic event is unknown. We demonstrate here that administration of human recombinant HGF (hrHGF) into the ventricle reduced the prolongation of the escape latency in the acquisition and retention tests in the water maze task on days 12–28 after microsphere embolism-induced cerebral ischemia. In addition, disruption of the blood–brain barrier at the early stage after microsphere embolism, which was determined by FITC–albumin leakage, was markedly reduced by treatment with hrHGF. We demonstrated that this effect of hrHGF on the blood–brain barrier was associated with protection against the apoptotic death of the cerebral endothelial cells at the early stage after the ischemia. These results suggest that hrHGF can prevent the learning and memory dysfunction soon after sustained cerebral ischemia by protecting against injury to the endothelial cells. The use of HGF may be a potent strategy for the treatment of cerebrovascular diseases, including cerebral infarct and vascular dementia.

No influence of tumor growth by intramuscular injection of hepatocyte growth factor plasmid DNA: safety evaluation of therapeutic angiogenesis gene therapy in mice

Recently, a novel therapeutic treatment for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As intramuscular injection of naked human hepatocyte growth factor (HGF) plasmid DNA induced therapeutic angiogenesis in several animal test subjects, we have started a clinical trial to treat peripheral arterial disease. However, one might assume that over-expression of angiogenic growth factors could enhance tumor growth. To resolve this issue, we examined the over-expression of HGF in tumor bearing mice. Tumors on their backs were prepared with an intradermal inoculation of A431, human epidermoid cancer cells expressing c-Met. These mice were intramuscularly injected with human HGF plasmid or control plasmid into the femoral muscle. Human HGF concentration was increased only in the femoral muscle, but not in blood. Although recombinant HGF stimulated the growth of A431 cells in vitro, temporally and locally HGF elevation in hindlimb had no effect on tumor growth in mice.

Lack of association between the hepatocyte growth factor receptor, c-met, and the anti-apoptotic action of bag-1 in endothelial cells.

Bag-1 is a novel multifunctional protein. It was identified based on its ability to bind the anti-apoptotic protein, bcl-2, and also reported to interact with the heat shock protein 70 kDa (Hsp70). Thus, bag-1 may modulate apoptosis and the chaperone activity. More interestingly, bag-1 can bind to several growth factor receptors or steroid hormone receptors and regulate their function and signaling. The receptor of hepatocyte growth factor (HGF), c-met, associated with bag-1 in a study measuring immunoprecipitation in endothelial cells, we decided to investigate the contribution of bag-1 to the anti-apoptotic action of HGF. Endogenous expression of bag-1 in endothelial cells was confirmed mainly in the cytosol fraction. The treatment of human recombinant HGF (rHGF) increased tyrosine kinase and ERK phosphorylation, whereas over-expression of bag-1 had no effect on this phosphorylation. In DNA synthesis as assessed by thymidine incorporation, over-expression of bag-1 also did not induce any additional increase. In contrast, in an assay of cell death as assessed by caspase activity and lactate dehydrogenase release, over-expression of bag-1 alone attenuated serum-free and tumor necrosis factor-alpha-induced cell death in endothelial cells. No synergistic effect was observed between bag-1 and rHGF. To further study the association of HGF and bag-1, we examined the effect of a deletion mutant of the bag-1 C-terminal region (CTR), because bag-1 CTR is necessary to bind to c-met. Unexpectedly, over-expression of bag-1 CTR also attenuated the endothelial cell death, similar to rHGF. Taken together, these results indicate that over-expression of bag-1 has an anti-apoptotic effect on endothelial cells independent of HGF signaling.

Derivation and characterization of a Wilms' tumour cell line, WiT 49.

Wilms' tumour is a pediatric neoplasm exhibiting histologic features of developing kidney. Although the majority of Wilms' tumour patients are treated effectively, approximately 15% develop metastases and of these, 30% succumb to their disease. The biologic factors governing Wilms' tumour metastasis are largely unknown. Attempts at deriving representative Wilms' tumour cell lines, which could facilitate functional studies, have only been partially successful thus far. We now report on derivation and characterization of a Wilms' tumour cell line, WiT 49, from a first-generation xenograft of a human Wilms' tumour lung metastasis. WiT 49 recapitulates the phenotype of the parent tumours (primary and lung metastasis) and expresses normal WT1, overexpresses IGFII and carries a frequently identified p53 mutation. We recently reported overexpression of hepatocyte growth factor(HGF) and its receptor met in a series of Wilms' tumours with higher levels in homotypic metastatic cases. We therefore examined WiT 49 for expression of HGF/met and for met signaling targets associated with cell adhesion and cytoplasmic mediators of transcription using Western blot, co-immunoprecipitation, immunofluorescence labeling and zymography. Our results show co-expression of HGF and met protein, absence of E-cadherin, high levels of beta-catenin co-immunolocalized to met at the cell membrane and moderate levels of gamma-catenin and ezrin protein expression. After cell fractionation, beta-catenin was detected in the cytoplasm and nuclei of WiT 49 with relatively higher levels in the cytoplasm as compared to nuclei. Examination of MMP expression in WiT 49 showed constitutive activation of MMP 9 and latent MMP 2 supporting possible beta-catenin-mediated transcriptional activation. The WiT 49 cell line responded to recombinant human HGF by an increase in the expression of the met receptor, recruitment of the Gab-1 adapter protein to met and release of bound beta-catenin from met. Our studies therefore establish WiT 49 as a representative Wilms' tumour cell line derived from a lung metastasis that co-expresses HGF/met and shows absence of the cadherin-catenin complex supporting a role for these factors in regulation of the invasive and metastatic phenotype in Wilms' tumour.

Hepatocyte growth factor facilitates colonic mucosal repair in experimental ulcerative colitis in rats.

Hepatocyte growth factor (HGF) modulates intestinal epithelial cell proliferation and migration, serving as a critical regulator of intestinal wound healing. In this study, we examined the effect of administration of recombinant human HGF on colonic mucosal damage in vivo. Acute colitis was induced in rats by feeding with 5% dextran sulfate sodium (DSS) for 7 days, and colitis was subsequently maintained by feeding with 1% DSS. On the 5th day of DSS administration, osmotic pumps releasing recombinant human HGF (200 microg/day) were implanted into the peritoneum of the rats. Continuous intraperitoneal delivery of HGF led to both increased serum human HGF levels and c-Met tyrosine phosphorylation within the colonic mucosa. Compared with mock-treated rats, those administered human HGF showed a reduction in colitis-associated weight loss, large intestinal shortening, and improved colonic erosions. Enhanced epithelial regeneration and cellular proliferation were observed in rats treated with recombinant human HGF. The weights of the liver, kidneys, and spleen were not affected by HGF administration. These results indicate that HGF administration accelerates colonic mucosal repair in rats with DSS-induced colitis and suggest that recombinant human HGF may be a useful therapeutic tool to facilitate intestinal wound healing in patients with ulcerative colitis.

Involvement of hepatocyte growth factor in branching morphogenesis of murine salivary gland.

We investigated the involvement of hepatocyte growth factor (HGF) in salivary gland (SG) branching morphogenesis. The mouse submandibular gland (SMG) starts to develop at embryonic day 11.5-12 (E11.5-E12), and branching morphogenesis occurs in the area between the mandibular bone and tongue between E14 and E16.5. Real-time reverse transcriptase-polymerase chain reaction showed that the expression of the c-met/HGF receptor gene in SMG increased and peaked between E14 and E16.5, concomitant with epithelial branching, and high levels of HGF mRNA were detected in the surrounding mesenchyme at E14-E15.5. Although strong expression of the HGF and c-met transcripts was observed in the tongue muscles, this expression was limited at E13.5-E14.5. Serum-free organ cultures were established, in which SG rudiments that contained SMG and sublingual gland (SLG) primordia (explant 1) and SMG/SLG rudiments with peripheral tissue that included part of the tongue muscle (explant 2) were isolated from E13.5 or E14 embryos. Mesenchyme-free SMG epithelium was obtained by the removal of mesenchymal tissue from explant 1. In the explant 1 and 2 organ cultures, SMG/SLG rudiments showed growth and branching morphogenesis, while mesenchyme-free epithelium failed to grow. When E13.5 or E14 mesenchyme-free epithelium and a recombinant human HGF (rh-HGF) -soaked bead were placed on Matrigel, the epithelium migrated toward the bead and formed branches, while the E13 epithelium failed to branch. The exogenous application of rh-HGF and anti-HGF antibody to the SMG/SLG rudiment cultures resulted in stimulation and inhibition, respectively, of branching morphogenesis. However, the response of E13.5 SMG to rh-HGF was very weak, while the branching of E14 SMG was enhanced strongly by rh-HGF. The branching morphogenesis of SMG was also inhibited by the addition of either antisense HGF or c-met oligodeoxynucleotides to the cultures. The development of SMG in explant 2, which was significantly better than in explant 1, was comparable to that seen in vivo. Moreover, the expression of both HGF and c-Met in the SMG of explant 2 was higher than in the SMG of explant 1. These findings provide the first demonstration that the branching morphogenesis of SMG is regulated by interactions with the surrounding mesenchyme-derived HGF and c-met expression in SMG, which occur concomitant with epithelial branching. The present data also suggest that the HGF that is released transiently from tongue muscles may contribute to the rapid development of SMG at the branching stage.

Heparan sulfates and coxsackievirus-adenovirus receptor: each one mediates coxsackievirus B3 PD infection.

Amino acid exchanges in the virus capsid protein VP1 allow the coxsackievirus B3 variant PD (CVB3 PD) to replicate in decay accelerating factor (DAF)-negative and coxsackievirus-adenovirus receptor (CAR)-negative cells. This suggests that molecules other than DAF and CAR are involved in attachment of this CVB3 variant to cell surfaces. The observation that productive infection associated with cytopathic effect occurred in Chinese hamster ovary (CHO-K1) cells, whereas heparinase-treated CHO-K1 cells, glucosaminoglycan-negative pgsA-745, heparan sulfate (HS)-negative pgsD-677, and pgsE-606 cells with significantly reduced N-sulfate expression resist CVB3 PD infection, indicates a critical role of highly sulfated HS. 2-O-sulfate-lacking pgsF-17 cells represented the cell line with minimum HS modifications susceptible for CVB3 PD. Inhibition of virus replication in CHO-K1 cells by polycationic compounds, pentosan polysulfate, lung heparin, and several intestinal but not kidney HS supported the hypothesis that CVB3 PD uses specific modified HS for entry. In addition, recombinant human hepatocyte growth factor blocked CVB3 PD infection. However, CAR also mediates CVB3 PD infection, because this CVB3 variant replicates in HS-lacking but CAR-bearing Raji cells, infection could be prevented by pretreatment of cells with CAR antibody, and HS-negative pgsD-677 cells transfected with CAR became susceptible for CVB3 PD. These results demonstrate that the amino acid substitutions in the viral capsid protein VP1 enable CVB3 PD to use specific modified HS as an entry receptor in addition to CAR.

Hepatocyte Growth Factor-Stimulating Endothelial Cell Growth and Accelerating Glomerular Capillary Repair in Experimental Progressive Glomerulonephritis

Hepatocyte growth factor (HGF) is one of the major growth factors that stimulate the growth and the migration of vascular endothelial cells. In this study, we examined the beneficial effects of HGF for glomerular repair in an experimental progressive glomerulonephritis (GN) model prepared by injecting both anti-Thy-1.1 antibody (day 0) and habu-snake venom (day 1) in rats. The rats received continuous intraperitoneal administration of recombinant human HGF (80 µg/100 g/day) or vehicle control at an early stage (day 2 to day 9), after severe glomerular injury. The vehicle-infused control rats initially showed severe mesangiolysis with large ballooning (day 2), followed by the prominent proliferation of mesangial cells with minimal capillary regeneration (day 5 to week 2), and global sclerosis with chronic renal failure (week 4 to week 8). Although mesangiolysis with large ballooning and mesangial cell proliferation were also observed in the HGF-infused rats, glomerular capillary regeneration with marked endothelial cell proliferation occurred during HGF administration from day 2 to day 9. Subsequently, the glomerulus was repaired with the development of the capillary network and the reduction of mesangial hypercellularity from week 2 to week 4, and almost all of the glomeruli showed a normal structure by week 8. The HGF-treated rats showed significantly better renal functions (Cr: 0.3 ± 0.1 vs. 3.5 ± 1.1 mg/dl in control, p < 0.001), less proteinuria (21.2 ± 8.0 mg/day vs. 421.4 ± 45.1 mg/day in control, p < 0.001) and less glomerular sclerosis at week 8 than the vehicle-infused rats. We conclude that HGF accelerated glomerular repair through the growth of capillary endothelial cells and capillary regeneration in experimental progressive GN. Administering HGF is a logical and efficient strategy for treating progressive GN with severe capillary destruction.

The impairment of hepatocytes and sinusoidal endothelial cells during cold preservation in rat fatty liver induced by alcohol and the beneficial effect of hepatocyte growth factor

Abstract A fatty liver resulting from alcohol intake is often unattractive for grafting. In this study, we investigated the impairment of hepatocytes and sinusoidal endothelial cells (SECs) during cold preservation of alcohol-induced fatty liver and examined the efficacy of human recombinant hepatocyte growth factor (hrHGF). Rats were fed an alcohol diet. We performed histological examinations of the hepatocytes and observed the ultrastructural alteration of the SECs. Additionally, we measured hepatic transminase and peroxidative lipids for hepatocellular injury and the hyaluronic acid uptake rate (HUR) to determine SEC injury. We added hrHGF to University of Wisconsin (UW) solution to assess the protective effect of the agent. Numerous fatty deposits were observed in ethanol-induced fatty livers. These grew with the duration of cold storage. Hepatic transminases of the effluents increased during cold preservation in the livers of alcohol-treated rats. Additionally, peroxidative lipids in the effluents increased during cold preservation in the livers of alcohol-treated rats, whereas they were undetectable in non-alcohol-treated rat livers. The sinusoidal endothelium had severely deteriorated in the livers of alcohol-treated rats. Further, the HUR decreased with ethanol treatment and/or cold preservation. The addition of hrHGF suppressed the increase of hepatic transaminase in the effluent of cold-preserved alcohol-treated livers. Peroxidative lipids in the same effluents were undetectable. In fatty livers, both hepatocytes and SECs Received: severe damage during cold preservation. Furthermore, we demonstrated that hepatocellular injury was significantly inhibited by hrHGF.

The impairment of hepatocytes and sinusoidal endothelial cells during cold preservation in rat fatty liver induced by alcohol and the beneficial effect of hepatocyte growth factor.

A fatty liver resulting from alcohol intake is often unattractive for grafting. In this study, we investigated the impairment of hepatocytes and sinusoidal endothelial cells (SECs) during cold preservation of alcohol-induced fatty liver and examined the efficacy of human recombinant hepatocyte growth factor (hrHGF). Rats were fed an alcohol diet. We performed histological examinations of the hepatocytes and observed the ultrastructural alteration of the SECs. Additionally, we measured hepatic transaminase and peroxidative lipids for hepatocellular injury and the hyaluronic acid uptake rate (HUR) to determine SEC injury. We added hrHGF to University of Wisconsin (UW) solution to assess the protective effect of the agent. Numerous fatty deposits were observed in ethanol-induced fatty livers. These grew with the duration of cold storage. Hepatic transaminases of the effluents increased during cold preservation in the livers of alcohol-treated rats. Additionally, peroxidative lipids in the effluents increased during cold preservation in the livers of alcohol-treated rats, whereas they were undetectable in non-alcohol-treated rat livers. The sinusoidal endothelium had severely deteriorated in the livers of alcohol-treated rats. Further, the HUR decreased with ethanol treatment and/or cold preservation. The addition of hrHGF suppressed the increase of hepatic transaminase in the effluent of cold-preserved alcohol-treated livers. Peroxidative lipids in the same effluents were undetectable. In fatty livers, both hepatocytes and SECs received severe damage during cold preservation. Furthermore, we demonstrated that hepatocellular injury was significantly inhibited by hrHGF.

Decreased microsomal triglyceride transfer protein activity contributes to initiation of alcoholic liver steatosis in rats

Background/Aims : To elucidate the role of microsomal triglyceride transfer protein (MTP) in the pathogenesis of alcoholic fatty liver, the effects of ethanol on MTP activity and gene expression were investigated. Methods and results : Male Sprague–Dawley rats fed an ethanol-containing liquid diet for 37 days, respectively, showed 2.9- and 4.9-fold increases in hepatic cholesterol and triglyceride content in comparison with rats fed an isocaloric ethanol-free diet ( P <0.01). Furthermore, a significant decrease in MTP activity and mRNA expression (by 27 and 58%, respectively) was observed after ethanol administration. Intravenous injection of human recombinant hepatocyte growth factor (hrHGF) on each of the last 7 days markedly suppressed ethanol-induced lipid accumulation in the liver. This inhibition of fatty change by hrHGF was accompanied by recovery of MTP activity and gene expression. No inhibitory effect of hrHGF on ethanol-induced acyl-CoA synthetase activation was observed. Experiments using human hepatoma-derived HepG2 cells indicated a direct positive effect of hrHGF on MTP gene expression as well as apolipoprotein B secretion. Conclusions : These results suggest that reduced MTP activity is crucial to development of alcoholic fatty liver, while promotion of MTP activity by HGF might serve as a therapeutic measure against alcoholic liver steatosis.

Effect of Hepatocyte Growth Factor on Lung Injury and Development of Clinical Application

Hepatocyte growth factor (HGF) is a humoral mediator of epithelial-mesenchymal interactions, acting on a variety of epithelial cells. In our previous studies, higher HGF levels were detected in the BAL fluid of patients with pulmonary fibrosis, 1 Sakai T Satoh K Matsushina K et al. Hepatocyte growth factor in bronchoalveolar lavage fluids and cells in patients with inflammatory chest diseases of the lower respiratory tract: detection by RIA and in situ hybridization. Am J Respir Cell Mol Biol. 1997; 16: 388-397 Crossref PubMed Scopus (52) Google Scholar and HGF could prevent the progression of bleomycin-induced lung fibrosis in mice using human recombinant HGF. 2 Yaekashiwa M Nakayama S Ohnuma K et al. Simultaneous or delayed administration of hepatocyte growth factor equally represses the fibrotic changes in murine lung injury induced by bleomycin: a morphologic study. Am J Respir Crit Care Med. 1997; 156: 1937-1944 Crossref PubMed Scopus (198) Google Scholar To step up the clinical application of HGF for respiratory diseases, we examined the adenoviral-mediated HGF treatment. While the intratracheal administration of adenoviral vector expressing rat HGF (AdCAG.HGF) yielded high transient HGF expression (312.5 ng/g lung tissue, day 3), adenovirus caused neutrophil alveolitis. In spite of indirect intraperitoneal administration of AdCAG.HGF (6 × 108 pfu) to C57BL/6 mice with bleomycin-induced lung injury, reduced hydroxyproline content (128 ± 5%; n = 5, day 28) resulted in AdCAG.HGF-treated mice, compared with mice treated with adenovirus without complementary DNA insertion (177 ± 9%, p < 0.005). Furthermore, the alveolar apoptotic cells in bleomycin-induced lung injury determined by deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling stain was also reduced by the intraperitoneal administration of AdCAG.HGF (35 ± 4% vs 13 ± 1%; p < 0.05). These results indicate the potential therapeutic effect of HGF for lung injury and fibrosis, and thus await the development of the clinical administration.

Hepatocyte growth factor reduces the infarct volume after transient focal cerebral ischemia in rats

Hepatocyte growth factor (HGF) was originally discovered as a powerful mitogen for hepatocytes. HGF also has been reported to function as a neurotrophic factor as well as an angiogenetic factor. The present study examined the neuroprotective effect of HGF against transient focal cerebral ischemia in rats, in which an anti-apoptotic and an angiogenetic effect of HGF was assumed to contribute to the reduction of the infarct volume. The intraventricular administration of human recombinant HGF prevented neuronal death after 120 min of occlusion in the right middle cerebral artery and the bilateral common carotid arteries. HGF significantly reduced the infarct volume in a dose-dependent manner. In a separate series of experiments, we next histopathologically investigated both the anti-apoptotic effect on neurons and the angiogenetic effect of HGF. A large number of TUNEL positive neurons were observed in the inner boundary of the infarct area in both the control and the vehicle group whereas only a few TUNEL positive neurons were observed in the corresponding area in the HGF group. In the HGF group, Bcl-2 protein was obviously represented in surviving neurons subjected to ischemia. The number of the vascular lamina in HGF group were significantly higher than those in the vehicle group. These data suggest that HGF appears to have an ability to prevent apoptotic neuronal cell death while also possessing an angiogenetic effect in the central nervous system which was affected with transient focal cerebral ischemia.

Hepatocyte growth factor suppresses interstitial fibrosis in a mouse model of obstructive nephropathy

As tubulointerstitial fibrosis (TIF) reflects the prognosis of patients with various chronic renal diseases, the pathogenesis of TIF has to be clarified. Transforming growth factor-beta (TGF-beta) is a key mediator for renal fibrosis. We reported that hepatocyte growth factor (HGF) prevents renal fibrosis in nephrotic mice. However, the function of HGF in chronic renal failure, except for nephrotic syndrome, remains to be determined. Using mice subjected to unilateral ureter-ligated obstruction (UUO), we investigated the roles of HGF in TIF, as induced by obstructive nephropathy. Pathophysiological changes in the kidney after UUO treatment were analyzed focusing on expressions of renal HGF and TGF-beta, TIF, tubular proliferation, and apoptosis. Neutralizing antibody against rodent HGF, or recombinant human HGF (rhHGF), was administrated to the UUO mice, and pathophysiological changes after neutralization or supplements of HGF were analyzed. In this UUO model, TIF with tubular apoptosis became evident, and it was accompanied by a decrease in renal HGF expression and an increase in renal TGF-beta expression. Neutralization of endogenous HGF accelerated the progression of TIF, accompanied by increases in TGF-beta expression and tubular apoptosis as well as by decreases in tubular proliferation. In contrast, rhHGF attenuated TIF progression, and there were decreases in TGF-beta expression and tubular apoptosis, and an increase in tubular proliferation. Endogenous as well as exogenous HGF attenuated the progression of the fibrosis caused by obstructive nephropathy in these mice. Thus, local reduction in HGF levels may account for TIF in chronic renal diseases.

C-MET Expression in Myofibroblasts: Role in Autocrine Activation and Prognostic Significance in Lung Adenocarcinoma

Hepatocyte growth factor (HGF) plays important roles in tumor development and progression. It is currently thought that the main action of HGF is of a paracrine nature: HGF produced by mesenchymal cells acts on epithelial cells that express its receptor c-MET. In this investigation, we explored the significance of c-MET expression in myofibroblasts, both in culture and in patients with lung adenocarcinoma. We first showed that human myofibroblasts derived from primary lung cancer expressed c-MET mRNA and protein by reverse transcription-polymerase chain reaction and Western blot analysis. Proliferation of myofibroblasts was stimulated in a dose-dependent manner by exogenously added recombinant human HGF whereas it was inhibited in a dose-dependent manner by neutralizing antibody to HGF. The addition of HGF in the culture medium stimulated tyrosine phosphorylation of c-MET. The c-MET protein was immunohistochemically detected in myofibroblasts in the invasive area of lung adenocarcinoma. Finally, the prognostic significance of c-MET expression in stromal myofibroblasts was explored in patients with small-sized lung adenocarcinomas. c-MET-positive myofibroblasts were observed in 69 of 131 cases (53%). A significant relationship between myofibroblast c-MET expression and shortened patient survival was observed in a whole cohort of patients including all pathological stages (two-sided P: = 0.0089 by log-rank test) and in patients with stage IA disease (two-sided P: = 0.0019 by log-rank test). These data suggest that the HGF/c-MET system constitutes an autocrine activation loop in cancer-stromal myofibroblasts. This autocrine system may play a role in invasion and metastasis of lung adenocarcinoma.

Hepatocyte growth factor attenuates collagen accumulation in a murine model of pulmonary fibrosis.

We investigated the in vivo effects of recombinant human hepatocyte growth factor (HGF) on epithelial cell proliferation in normal mouse lung and on the repair process that follows bleomycin-induced lung injury. Intratracheal administration of 100 micrograms of rhHGF to C57BL/6 mice led to proliferation of bronchial and alveolar epithelial cells as indicated by an increased number of cells staining for proliferating cell nuclear antigen (PCNA). The effect of HGF on the lung repair process was examined by administration of 100 micrograms of rhHGF on Day 3 and Day 6 after intratracheal injection of bleomycin to mice. We found that HGF significantly attenuated collagen accumulation induced by bleomycin as determined by quantitation of hydroxyproline content and by scoring of the extent of fibrosis. To explore the potential mechanisms involved in the beneficial effects of HGF, we performed in vitro studies with A549 pulmonary epithelial cells and found that HGF enhanced cell surface plasmin generation, expression of u-PA activity, and cell migration. In summary, HGF has potent in vivo and in vitro effects on epithelial cells, which suggests it may have a role in the therapy of pulmonary fibrosis.