Mouse Hepatocyte Growth Factor Research Articles

Hepatocyte growth factor upregulates MMP1 and MMP10 expression and resolves corneal fibrosis

Different therapeutic modalities, including steroids, have been used to treat corneal scarring. However, the ability of steroids to reduce corneal scarring is limited and associated with numerous side effects. Our previous studies have demonstrated that topical hepatocyte growth factor (HGF) after corneal injury suppresses the development of stromal scars. Here, we investigated whether HGF can re-establish corneal clarity and normalize tissue structure in corneas with pre-existing scars. Corneal scarring was induced by mechanically removing the corneal epithelium and the anterior third of the stroma using a hand-held Algerbrush II in C57BL/6 mice. Substantial scar tissue formed by day 10 post-injury, at which time the epithelium was debrided and treated with 0.1% recombinant mouse HGF, 0.1% dexamethasone (steroid) or 0.1% control protein thrice a day for 10 days. Corneal clarity was significantly restored in the HGF treatment group, compared to both the steroid and control protein treatment groups. Moreover, HGF treatment downregulated the expression of αSMA and upregulated the expression of extracellular matrix-remodeling matrix metalloproteinases 1 and 10 (MMP1 and MMP10), suggesting HGF upregulates tissue remodeling molecule MMP1 and 10 to promote tissue restoration. These findings offer novel insights into the mechanisms by which HGF re-establishes corneal clarity, and promotes epithelial regeneration in corneas with pre-existing stromal scarring.

Intercellular Interactions Mediated by HGF And TGF-Β Promote the 3D Spherical and Xenograft Growth of Liver Cancer Cells.

Recently, the importance of the interactions between liver cancer cells and fibroblasts has been increasingly recognized; however, many details remain to be explored. In this work, we first studied their intercellular interactions using conditioned medium from mouse embryonic fibroblasts (MEFs), then through a previously established coculture model. Culturing in a conditioned medium from MEFs could significantly increase the growth, migration, and invasion of liver cancer cells. The coculture model further demonstrated that a positive feedback loop was formed between transforming growth factor-β (TGF-β) from HepG2 cells and mHGF (mouse hepatocyte growth factor) from MEFs during coculture. In this feedback loop, c-Met expression in HepG2 cells was significantly increased, and its downstream signaling pathways, such as Src/FAK, PI3K/AKT, and RAF/MEK/ERK, were activated. Moreover, the proportion of activated MEFs was also increased. More importantly, the growth-promoting effects caused by the interaction of these two cell types were validated in vitro by a 3D spheroid growth assay and in vivo by a xenograft mouse model. Collectively, these findings provide valuable insights into the interactions between fibroblasts and liver cancer cells, which may have therapeutic implications for the treatment of liver cancer.

Effect of hepatocyte growth factor on mice with hypoxic pulmonary arterial hypertension: a preliminary study.

To study the association between hepatocyte growth factor (HGF) and treatment response in mice with hypoxic pulmonary arterial hypertension (HPAH) and the possibility of HGF as a new targeted drug for HPAH. After successful modeling, the HPAH model mice were randomly divided into two groups: HPAH group and HGF treatment group (tail vein injection of recombinant mouse HGF 1 mg/kg), with 10 mice in each group. Ten normal mice were used as the control group. After 5 weeks, echocardiography was used to measure tricuspid peak velocity, right ventricular systolic pressure, right ventricular hypertrophy index, and right ventricular/body weight ratio; the Griess method was used to measure the content of nitric oxide in serum; ELISA was used to measure the serum level of endothelin-1; transmission electron microscopy was used to observe changes in the ultrastructure of pulmonary artery. Compared with the HGF treatment and normal control groups, the HPAH group had significantly higher tricuspid peak velocity, right ventricular systolic pressure, right ventricular hypertrophy index, and right ventricular/body weight ratio (P<0.05). The transmission electron microscopy showed that the HPAH group had massive destruction of vascular endothelial cells and disordered arrangement of the elastic membrane of arteriolar intima with rupture and loss. The structure of vascular endothelial cells was almost complete and the structure of arterial intima elastic membrane was almost normal in the HGF treatment group. Compared with the normal control and HGF treatment groups, the HPAH group had significantly higher serum levels of nitric oxide and endothelin-1 (P<0.05). Increasing serum HGF level can alleviate the impact of HPAH on the cardiovascular system of mice, possibly by repairing endothelial cell injury, improving vascular remodeling, and restoring the normal vasomotor function of pulmonary vessels.

A Potential Role of Adhesion Molecules on Lung Metastasis Enhanced by Local Inflammation.

Local and systemic inflammations are associated with negative long-term outcomes; however, their precise mechanism of action remains unclear. We previously demonstrated that hepatocyte growth factor (HGF)/c-Met signaling contributed to the enhancement of liver metastasis associated with peritonitis model. The aim of this study is to investigate the effect of local inflammation on the development of lung metastasis. NL-17 cells were injected into BALB/c mice via the tail vein to produce a high potential model for lung metastasis. After injection of NL-17 cells, lipopolysaccharide (LPS) and live Pseudomonas aeruginosa, and phosphate-buffered saline were administered intratracheally to induce acute lung injury (ALI) and pneumonia, respectively. In both ALI and pneumonia mice, lung metastasis was significantly promoted compared to control mice. Concentrations of Interleukin-6, tumor necrosis factor-α, and HGF in the bronchoalveolar lavage fluid were significantly higher in ALI and pneumonia mice than in control mice. Neither administration of recombinant mouse HGF nor c-Met knockdown in NL-17 cells influenced the magnitude of lung metastasis. Yet stimulation with LPS increased the expression of α2 integrin, vascular cell-adhesion protein-1, and intercellular adhesion molecule-1 (ICAM-1) in the lung. Invasive activity of NL-17 cells was significantly up-regulated by LPS, but was suppressed by anti-ICAM-1 antibody. While LPS-stimulated NL-17 cells showed significantly promoted lung metastasis, E-selectin expression in the lungs of mice with ALI or pneumonia was significantly enhanced compared with control mice. Up-regulation of adhesion molecules, but not HGF/c-Met signaling, may contribute to the lung metastasis enhanced by local infection/inflammation.

Efficient Selection of Antibodies Reactive to Homologous Epitopes on Human and Mouse Hepatocyte Growth Factors by Next-Generation Sequencing-Based Analysis of the B Cell Repertoire.

YYB-101 is a humanized rabbit anti-human hepatocyte growth factor (HGF)-neutralizing antibody currently in clinical trial. To test the effect of HGF neutralization with antibody on anti-cancer T cell immunity, we generated surrogate antibodies that are reactive to the mouse homologue of the epitope targeted by YYB-101. First, we immunized a chicken with human HGF and monitored changes in the B cell repertoire by next-generation sequencing (NGS). We then extracted the VH gene repertoire from the NGS data, clustered it into components by sequence homology, and classified the components by the change in the number of unique VH sequences and the frequencies of the VH sequences within each component following immunization. Those changes should accompany the preferential proliferation and somatic hypermutation or gene conversion of B cells encoding HGF-reactive antibodies. One component showed significant increases in the number and frequencies of unique VH sequences and harbored genes encoding antibodies that were reactive to human HGF and competitive with YYB-101 for HGF binding. Some of the antibodies also reacted to mouse HGF. The selected VH sequences shared 98.3% identity and 98.9% amino acid similarity. It is therefore likely that the antibodies encoded by them all react to the epitope targeted by YYB-101.

Liver Macrophage Depletion Ameliorates The Effect of Mesenchymal Stem Cell Transplantation in a Murine Model of Injured Liver

Mesenchymal stem cells (MSCs) therapy show different levels of effectiveness in the context of different types of liver damage, suggesting that the microenvironment of the injured liver is a key determinant for effective stem cell therapy. The objective was to assess the modulatory effect of hepatic stem cell niche components on the transplanted MSCs during liver injury induced by carbon tetrachloride (CCl4). Superparamagnetic iron oxide (SPIO)-labeled human MSCs were injected intravenously into mice treated with CCl4 and subjected to hepatic macrophage-depletion. Liver tissues were collected at different intervals post transplantation for subsequent histopathological, morphometric, immunohistochemical, gene expression and ultrastructural studies. The homing of the transplanted MSCs was evidenced by tracing them within the niche by iron staining and immunohistochemical studies. MSCs differentiated into hepatocyte-like cells and intimal smooth muscle cells as evidenced by their expression of human albumin and α-smooth muscle actin with a concomitant increase in the level of mouse hepatocyte growth factor. A post transplantation reduction in the liver fibro-inflammatory reaction was found and was promoted by liver macrophages depletion. Thus, it could be concluded from the present study that prior manipulation of the microenvironment is required to improve the outcome of the transplanted cells.

MTOR/STAT-3 pathway mediates mesenchymal stem cell-secreted hepatocyte growth factor protective effects against lipopolysaccharide-induced vascular endothelial barrier dysfunction and apoptosis.

Mesenchymal stem cells (MSCs) protect the endothelial barrier complex and survival, implicated in the pathogenesis of acute lung injury (ALI) via paracrine hepatocyte growth factor (HGF). However, the mechanism of HGF in endothelial regulation remains unclear. Here, we introduced a coculture protocol of pulmonary microvascular endothelial cells (PMVECs) and overexpression of the HGF gene of MSCs (MSC-HGF). Immunofluorescence and endothelial permeability analysis revealed that MSC-HGF protected endothelial tight junction protein occludin expression and attenuated cellular permeability as well as endothelial apoptosis. To investigate the novel mechanism mammalian TOR (mTOR)/ signal transducer and activator of transcription 3 (STAT-3) signaling in HGF protective effects against endothelial barrier and apoptosis, we used recombinant mouse HGF in endothelial cells. In addition, we used mTOR inhibitor rapamycin to inhibit the mTOR pathway. Our study demonstrated that rapamycin decreased the protective effects of HGF on the endothelium by decreasing tight junction protein occludin expression and cell proliferation, and raising lipopolysaccharide (LPS)-induced endothelial permeability, endothelial cell injury factors ET-1 and vWF. Similarly, the protective effects of HGF on reducing endothelial barrier and apoptosis were weakened when PMVECs were treated with the STAT-3 inhibitor S3I-201. Moreover, mTOR/STAT-3 were activated by HGF demonstrated as raising mTOR (Ser2448) and STAT3 (Ser727) phosphorylation proteins, leading to endothelial barrier improvement and survival. Reversely, rapamycin or S3I-201 inhibited mTOR/STAT-3 activation. Taken together, our findings highlight that the activation of the mTOR/STAT-3 pathway provides novel mechanistic insights into MSC-secreted HGF protection against LPS-induced vascular endothelial permeability dysfunction and apoptosis, which contributes to decreasing microvascular loss and lung injury.

Abstract 3923: Insensitivity to c-Met inhibition at physiologic HGF concentrations in a prostate carcinoma model

Abstract C-Met is a receptor tyrosine kinase critical for embryogenesis and liver repair. In tumors, including breast, prostate, glioblastoma, and thyroid cancer, Met protein levels are often elevated and associated with disease progression and metastasis. The c-Met pathway is activated by the endogenous ligand Hepatocyte Growth Factor (HGF). HGF is produced by mesenchymal cells and binds to the c-Met protein, leading to phosphorylation and upregulation of a variety of downstream signaling pathways that result in increased tumor cell migration, proliferation, survival, and induction of angiogenesis. C-Met is associated with disease progression and metastasis. Consequently, there has been significant interest in the development of c-Met inhibitors as anti-cancer therapeutics. The efficacy of such agents usually initially are determined in vitro utilizing HGF concentrations of 25-50 ng/mL. However, HGF serum levels in humans typically range from 0.4-0.8 ng/mL. In our studies, we examined the prostate carcinoma cell line DU145, and BMS-777607. Migration and phosphorylation of c-Met increased in a dose-dependent manner with HGF treatment. Additionally, c-Met inhibition with BMS-777607 reduced migration and invasion when in the presence of 25-50 ng/mL HGF. However, at physiologically relevant HGF concentrations the drug had no effect on migration or invasion of this cell line. In vivo models of c-Met inhibition have been limited mainly to mice genetically engineered to express HGF, on the basis that mouse HGF does not stimulate human c-Met. However, in our studies, mouse HGF did stimulate c-Met in the DU145 tumor cell line. Subsequent in vivo evaluation revealed treatment with BMS-777607 had no reduction on DU145 tumor cell-induced angiogenesis in cells pretreated with the drug, nor in mice receiving oral administration of the drug. An experimental metastasis model also resulted in no significant reduction in metastasis formation, as assessed by a tail vein injection model. To characterize HGF concentration in vivo, an ELISA was used to determine an average concentration of 7.7 ng/g of DU145 tumor tissue. Further evaluations of phospho-Met are underway utilizing immunofluorescence of FFPE samples. Preliminary results indicate low levels of phospho-Met in DU145 tumor sections that can be induced by treating tumor-bearing mice with exogenous HGF. Our results suggest that in vitro evaluation of c-Met inhibitors utilizing non-physiological concentrations of HGF may produce an abnormally high phospho-Met that can subsequently be reduced by treatment with c-Met inhibition. In vitro studies performed at high HGF concentrations may not accurately predict the in vivo drug efficacy. Further characterization of HGF concentrations within the tumor is necessary to properly conduct in vitro evaluation of c-Met inhibitors. Citation Format: Veronica S. Hughes, Mumtaz Rojiani, Dietmar Siemann. Insensitivity to c-Met inhibition at physiologic HGF concentrations in a prostate carcinoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3923.

Abstract B157: Effect of HGF concentration on c-Met phosphorylation and inhibition in paracrine-activated tumor cells

Abstract c-Met is a receptor tyrosine kinase critical for embryogenesis and liver repair. In tumors, including breast, prostate, glioblastoma, and thyroid cancer, Met protein levels are often elevated and associated with disease progression and metastasis. The c-Met pathway is activated by the endogenous ligand hepatocyte growth factor (HGF). HGF is produced by mesenchymal cells and binds to the c-Met protein, leading to phosphorylation and upregulation of a variety of downstream signaling pathways that result in increased tumor cell migration, proliferation, survival, and induction of angiogenesis. Consequently, there has been significant interest in the development of c-Met inhibitors as anticancer therapeutics. The efficacy of such agents usually initially are determined in vitro utilizing HGF concentrations of 25-50 ng/mL. However, HGF serum levels in humans typically range from 0.4-0.8 ng/mL. To examine the impact of exogenous HGF levels on c-Met inhibitor activity, human prostate (PC-3, DU145) and lung (A549) cancer cell lines were utilized in this study. These cell lines have been previously characterized as paracrine-activated: cell lines that express the c-Met receptor, but not HGF. In the presence of 25-50 ng/mL HGF, these cell lines demonstrated a reduction in migration, invasion, and phosphorylation of Met after treatment with the small-molecule c-Met inhibitor BMS-777607. However, such antitumor efficacy was absent when these paracrine-activated cell lines were treated with BMS-777607 in the presence of physiologically normal concentrations of HGF (0.4-0.8 ng/mL). A human HGF-expressing mouse model has been developed for xenograft studies of human c-Met inhibition, on the basis that mouse HGF does not activate human c-Met. However, we found that mouse HGF was able to induce phosphorylation of Met in the human DU145 cell line. In vivo, DU145 xenografts in NSG mice treated with BMS-777607 showed no reduction in c-Met phosphorylation unless the mice were administered exogenous HGF during tumor growth. Taken together, these findings indicate that the utilization of nonphysiologic concentrations of HGF in tissue culture evaluations of c-Met targeting agents may result in an overestimation of agent activity not likely to be observed in the tumor microenvironment. Citation Format: Veronica S. Hughes, Dietmar W. Siemann. Effect of HGF concentration on c-Met phosphorylation and inhibition in paracrine-activated tumor cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B157.

Stroma-derived HGF drives metabolic adaptation of colorectal cancer to angiogenesis inhibitors.

The role of paracrine Hepatocyte Growth Factor (HGF) in the resistance to angiogenesis inhibitors (AIs) is hidden in xenograft models because mouse HGF fails to fully activate human MET. To uncover it, we compared the efficacy of AIs in wild-type and human HGF knock-in SCID mice bearing orthotopic human colorectal tumors. Species-specific HGF/MET signaling dramatically impaired the response to anti-angiogenic agents and boosted metastatic dissemination. In cell-based assays mimicking the consequences of anti-angiogenic therapy, colorectal cancer cells were completely resistant to hypoxia but extremely sensitive to nutrient deprivation. Starvation-induced apoptosis could be prevented by HGF, which promoted GLUT1-mediated glucose uptake, sustained glycolysis and activated autophagy. Pharmacological inhibition of GLUT1 in the presence of glucose killed tumor cells as effectively as glucose deprivation, and this effect was antagonized by HGF. Concomitant targeting of GLUT1 and HGF potently suppressed growth and dissemination of AI-resistant human tumors in human HGF knock-in SCID mice without exacerbating tumor hypoxia. These data suggest that stroma-derived HGF protects CRC cells against glucose starvation-induced apoptosis, promoting resistance to both AIs and anti-glycolytic agents. Combined inhibition of glucose metabolism and HGF/MET signaling (‘anti-METabolic therapy’) may represent a more effective CRC treatment compared to utterly blocking tumor blood supply.

Regulation of hepatocyte growth factor in mice with pneumonia by peptidases and trans-alveolar flux.

Hepatocyte growth factor (HGF) promotes lung epithelial repair after injury. Because prior studies established that human neutrophil proteases inactivate HGF in vitro, we predicted that HGF levels decrease in lungs infiltrated with neutrophils and that injury is less severe in lungs lacking HGF-inactivating proteases. After establishing that mouse neutrophil elastase cleaves mouse HGF in vitro, we tested our predictions in vivo by examining lung pathology and HGF in mice infected with Mycoplasma pulmonis, which causes neutrophilic tracheobronchitis and pneumonia. Unexpectedly, pneumonia severity was similar in wild type and dipeptidylpeptidase I-deficient (Dppi-/-) mice lacking neutrophil serine protease activity. To assess how this finding related to our prediction that Dppi-activated proteases regulate HGF levels, we measured HGF in serum, bronchoalveolar lavage fluid, and lung tissue from Dppi+/+ and Dppi-/- mice. Contrary to prediction, HGF levels were higher in lavage fluid from infected mice. However, serum and tissue concentrations were not different in infected and uninfected mice, and HGF lung transcript levels did not change. Increased HGF correlated with increased albumin in lavage fluid from infected mice, and immunostaining failed to detect increased lung tissue expression of HGF in infected mice. These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid. However, levels of intact HGF from infected mice, normalized for albumin concentration, were two-fold higher in Dppi-/- versus Dppi+/+ lavage fluid, suggesting regulation by Dppi-activated proteases. Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues. These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair.

GW24-e1374 Hyperhomocysteinemia Impair Angiogenesis via HGF Inhibition

ObjectivesAbundant epidemiological and clinical studies have revealed the close relationship between hyperhomocysteinemia (HHcy) and the increased frequency of CVD. During the last decade, we and others have demonstrated that HHcy...

Abstract 4085: Experimental metastasis by the prostate adenocarcinoma-derived cell line PC3M is driven by partial activation of the human Met pathway.

Abstract Interspecies cross-reactivity is an important consideration when interpreting the results of tissue xenograft studies. For many paracrine signaling pathways, murine growth factors may not activate their orthologous human receptors in tumor xenografts, thereby failing to recapitulate a potentially relevant human condition in the mouse model. Unrecognized or poorly defined species-specific differences of this type can seriously hinder basic research progress as well as the development of therapeutic or diagnostic agents. For example, the contributions of both tumor- and stromal-cell derived vascular endothelial growth factor (VEGF)-A to the vascularization of human tumor xenografts in immunodeficient mice prevented the direct comparison of anti-VEGF antibodies with different abilities to block host VEGF, and led to the production of mice expressing humanized VEGF with biological activity comparable to both human and mouse VEGF-A. Hepatocyte growth factor (HGF) and its receptor, Met, are also highly sought targets in cancer drug and diagnostic development. HGF/Met signaling drives tumor cell growth and motility, tumor invasiveness and metastasis. However, the failure of murine HGF to stimulate the growth of human tumor xenografts has restricted preclinical drug studies to a handful of human tumor cell lines that possess autocrine pathway activation or other more rare Met activation mechanisms, and ultimately forced the costly development of human HGF transgenic and knock-in mice so that preclinical studies could begin to encompass the known breadth of the pathway's involvement across human cancers. We report here that despite the failure of murine HGF to drive human tumor xenograft growth, mouse HGF potently stimulates human Met autophosphorylation, increased cell motility and matrix invasion, but not proliferation. As a result, the growth of xenografted PC3M human prostate adenocarcinoma cells as primary tumors in mice was not inhibited by a selective HGF antagonist, but spontaneous tumor metastasis was completely blocked. These results improve our understanding of the cross-reactivity between human and murine HGF/Met signaling pathways and suggest that the preclinical evaluation of agents for their ability to block HGF-driven tumor metastasis may incorporate cell lines expressing human Met but not HGF, i.e. a wide variety of human carcinoma-derived cell lines, without the need for using mice engineered to express human HGF. Citation Format: Fabiola Cecchi, Chih-Jian Lih, Young H. Lee, William D. Walsh, Michael P. Williams, Donald P. Bottaro. Experimental metastasis by the prostate adenocarcinoma-derived cell line PC3M is driven by partial activation of the human Met pathway. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4085. doi:10.1158/1538-7445.AM2013-4085

Human platelets inhibit liver fibrosis in severe combined immunodeficiency mice

To investigate the role of human platelets in liver fibrosis. Severe combined immunodeficiency (SCID) mice were administered CCl4 and either phosphate-buffered saline (PBS group) or human platelet transfusions (hPLT group). Concentrations of hepatocyte growth factor (HGF), matrix metallopeptidases (MMP)-9, and transforming growth factor-β (TGF-β) in the liver tissue were compared between the PBS and the hPLT groups by enzyme-linked immunosorbent assay (ELISA) and Western blotting. The effects of a human platelet transfusion on liver fibrosis included the fibrotic area, hydroxyproline content, and α-smooth muscle actin (α-SMA) expression, which were evaluated by picrosirius red staining, ELISA, and immunohistochemical staining using an anti-mouse α-SMA antibody, respectively. Phosphorylations of mesenchymal-epithelial transition factor (Met) and SMAD3, downstream signals of HGF and TGF-β, were compared between the two groups by Western blotting and were quantified using densitometry. Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling. Furthermore, the accumulation of human platelets in the liver 2 h after platelet transfusion was compared between normal and fibrotic livers by immunohistochemical staining using an anti-human CD41 antibody. The fibrotic area and hydroxyproline content in the liver were both significantly lower in the hPLT group when compared to the PBS group (fibrotic area, 1.7% ± 0.6% vs 2.5% ± 0.6%, P = 0.03; hydroxyproline content, 121 ± 26 ng/g liver vs 156 ± 47 ng/g liver, P = 0.04). There was less α-smooth muscle actin staining in the hPLT group than in the PBS group (0.5% ± 0.1% vs 0.8% ± 0.3%, P = 0.02). Hepatic expression levels of mouse HGF and MMP-9 were significantly higher in the hPLT group than in the PBS group (HGF, 109 ± 13 ng/g liver vs 88 ± 22 ng/g liver, P = 0.03; MMP-9, 113% ± 7%/GAPDH vs 92% ± 11%/GAPDH, P = 0.04). In contrast, the concentration of mouse TGF-β in the liver tissue was significantly lower in the hPLT group than in the PBS group (22 ± 5 ng/g liver vs 39 ± 6 ng/g liver, P = 0.02). Phosphorylation of Met was more prevalent in the hPLT group than in the PBS group (37% ± 4%/GAPDH vs 20% ± 8%/GAPDH, P = 0.03). Phosphorylation of SMAD3 was weaker in the hPLT group than in the PBS group (60% ± 12%/GAPDH vs 84% ± 12%/GAPDH, P = 0.1), although this difference was not significant. Furthermore, a lower rate of hepatocyte apoptosis was observed in the hPLT group than in the PBS group (5.9% ± 1.7% vs 2.9% ± 2.1%, P = 0.02). Significant human platelet accumulation was observed in the fibrotic liver tissues, whereas few platelets accumulated in the normal liver. Human platelets inhibit liver fibrosis in SCID mice. Increased concentration of HGF in the liver suppresses hepatic stellate cell activation, induces MMPs, and inhibits hepatocyte apoptosis.

Dissociation of c‐Met phosphotyrosine sites in human cells in response to mouse hepatocyte growth factor but not human hepatocyte growth factor: the possible roles of different amino acids in different species

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c-Met. We previously demonstrated that HGF α-chain hairpin-loop, K1 domain and β-chain are required for c-Met signalling. The sequential phosphorylation of tyrosine residues, from c-Met kinase domain to multidocking regions, is required for HGF-signalling transduction. Herein, we provide evidence that the disconcerted activation of c-Met tyrosine regions fails to induce biological functions. When human cells were incubated with 'mouse HGF', kinase domain activation (i.e. phospho-Tyr-1230/34/35) became evident, but the multidocking site (i.e. Tyr-1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α-chain, or of β-chain, to human c-Met was lower than that of human HGF, as evidenced by HGF-chimera assay. Notably, only four amino acid positions in HGF α-chain hairpin-loop and K1 domain and six positions in β-chain differed between human HGF and mouse HGF. The human-specific amino acids (such as Gln-95 in hairpin-loop, Arg-134 in K1 domain and Cys-561 in β-chain) may be important for accurate c-Met assembly and signalling transduction.

Hepatocyte growth factor has a role in the amelioration of diabetic vascular complications via autophagic clearance of advanced glycation end products: Dispo85E, an HGF inducer, as a potential botanical drug

The aim of this study was to evaluate the effect and elucidate the potential mechanism of the extract of rhizomes from Dioscorea alata L. cv. Phyto, Dispo85E, on accelerating the elimination of advanced glycation end products (AGEs) in vitro and in vivo. Primary mouse nonparenchymal cells (NPCs) were used to evaluate the drug effect on AGEs clearance and autophagic-lysosomal activity. In an animal study, we used AGEs-induced diabetic mice to evaluate the drug effect on AGEs-induced vascular complications. Our results indicated that Dispo85E enhanced the endocytosis and degradation activity of AGEs in hepatic NPCs. Furthermore, the hepatocyte growth factor (HGF) expression level was positively correlated with the clearance capacity of the AGEs in NPCs after Dispo85E treatment. In addition, the effects of Dispo85E on the degradation and uptake capability of 14C-AGEs were abolished in the presence of an anti-HGF neutralizing antibody. We further demonstrated that recombinant mouse HGF could enhance the endocytosis and autophagic clearance of AGEs in NPCs. The in vivo data indicated that Dispo85E increased hepatic HGF messenger RNA expression levels and decreased serum AGEs level in diabetic mice. Moreover, the function of retina and kidneys was improved by Dispo85E treatment in AGEs-induced diabetic mice. These results suggest that HGF may have an important role in the elimination of AGEs. This study suggests that Dispo85E is a botanical drug with a novel mechanism that enhances the clearance of AGEs through HGF-induced autophagic-lysosomal pathway and is a candidate drug for the treatment of diabetic vascular complications.

Abstract 621: High-dose gefitinib overcomes the resistance to gefitinib related to hepatocyte growth factor

Abstract Molecular targeted agents are being a mainstay in non-small cell lung cancer. Dramatic responses to the epidermal growth factor receptor (EGFR) -tyrosine kinase inhibitor (TKI) are observed in advanced non-small cell lung cancer (NSCLC), especially with EGFR mutation. Different from cytotoxic agents, it remains unclear whether EGFR-TKI exerts its anti-tumor effect dose-dependently. Gefitinib could not show survival benefit in previously treated unselected patients with NSCLC in a randomized phase III trial, whereas erlotinib did. The difference is considered resulting from the fact that erlotinib was dosed at its maximum-tolerated dose (MTD), while gefitinib was dosed at about one third of its MTD. To investigate the dose-dependency of gefitinib in the cells harboring mutated EGFR, we compared two doses of gefitinib (15 or 50 mg/kg) using PC-9 (adenocarcinoma of lung) xenografts. Both doses of gefitinib reduced tumors. However, the tumors regrew in the low-dose group after several weeks of treatment, whereas did not in the high dose group. This result indicates that gefitinib might have the dose-dependency in the tumors harboring mutated EGFR. We resected tumors at 10 weeks after gefitinib treatment. The removed tumors did not acquired T790M mutation and MET amplification. Interestingly, the cells (PC-9/RX) that were derived from the xenograft tumors treated with low-dose gefitinib remained the sensitivity to gefitinib. Thus, we investigated hepatocyte growth factor (HGF) as the environmental factor. Tumor tissues treated with gefitinib were positively stained with anti-mouse HGF. Mouse HGF mRNA and protein expression in the tumor tissues treated with gefitinib were significantly high when compared to those treated with vehicle alone, and similar results were obtained by ELISA. We confirmed that mouse HGF induced resistance to gefitinib in an ex vivo experiment. To elucidate the role of HGF in vivo, PC-9 xenografts were treated with gefitinib in combination with rabbit anti-HGF antibody. The combination of gefitinib with anti-HGF antibody inhibited tumor growth partially compared with the low-dose gefitinib alone. Taken together, HGF might induce resistance to low-dose of gefitinib treatment in an in vivo xenograft model before the tumor cells themselves acquired the resistant genetic changes. In conclusion, higher-dose of gefitinib might prevent resistance to gefitinib related to HGF and produce longer response duration. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 621.

Estradiol modulation of hepatocyte growth factor by stromal fibroblasts in the female reproductive tract

Primary human uterine, cervical, and ectocervical stromal fibroblasts constitutively secrete hepatocyte growth factor (HGF), yet only uterine stromal fibroblasts increase HGF secretion in response to estradiol. Estradiol-induced HGF secretion by uterine stromal fibroblasts may have a significant effect on uterine cancer and endometriosis.

Hepatocyte Growth Factor Enhances Engraftment and Function of Nonhuman Primate Islets

OBJECTIVE—Adenoviral delivery of hepatocyte growth factor (HGF) to rodent islets improves islet graft survival and function, markedly reducing the number of islets required to achieve glucose control. Here, we asked whether these prior observations in rodent models extend to nonhuman primate (NHP) islets.RESEARCH DESIGN AND METHODS—NHP islets were transduced with murine (Ad.mHGF) or human (Ad.hHGF) adenoviral HGF (Ad.HGF) at low multiplicity of infection and studied in vitro. To study the function of Ad.HGF-transduced NHP islets in vivo, a renal subcapsular marginal mass islet transplant model was developed in streptozotocin-induced diabetic NOD-SCID mice.RESULTS—Baseline glucose values were 454.7 ± 11.3 mg/dl (n = 7). Transplant of 500 NHP islet equivalents (IE) had only a marginal effect on blood glucose (369.1 ± 9.7 mg/dl, n = 5). In striking contrast, 500 NHP IE transduced with Ad.mHGF promptly and continuously corrected blood glucose (142.0 ± 6.2 mg/dl, n = 7) for the 6-week duration of the experiment. Unilateral nephrectomy resulted in an immediate return of glucose to baseline diabetic levels. Interestingly, adenoviral DNA, as well as mouse HGF (mHGF) mRNA derived from the adenovirus, were present for 42 days posttransplantation. Surprisingly, transplant of 500 IE with Ad.hHGF, as compared with Ad.mHGF, resulted in only marginal correction of blood glucose, suggesting that human HGF is less efficient than mHGF in this system.CONCLUSIONS—These studies demonstrate that mHGF markedly improves islet transplant outcomes in the highest preclinical species examined to date. HGF has promise as an agent that can improve islet mass and function in transplant models and likely in other models of types 1 and 2 diabetes.

Hepatocyte Growth Factor Enhances Engraftment and Function of Nonhuman Primate Islets

OBJECTIVE—Adenoviral delivery of hepatocyte growth factor (HGF) to rodent islets improves islet graft survival and function, markedly reducing the number of islets required to achieve glucose control. Here, we asked whether these prior observations in rodent models extend to nonhuman primate (NHP) islets. RESEARCH DESIGN AND METHODS—NHP islets were transduced with murine (Ad.mHGF) or human (Ad.hHGF) adenoviral HGF (Ad.HGF) at low multiplicity of infection and studied in vitro. To study the function of Ad.HGF-transduced NHP islets in vivo, a renal subcapsular marginal mass islet transplant model was developed in streptozotocin-induced diabetic NOD-SCID mice. RESULTS—Baseline glucose values were 454.7 ± 11.3 mg/dl (n = 7). Transplant of 500 NHP islet equivalents (IE) had only a marginal effect on blood glucose (369.1 ± 9.7 mg/dl, n = 5). In striking contrast, 500 NHP IE transduced with Ad.mHGF promptly and continuously corrected blood glucose (142.0 ± 6.2 mg/dl, n = 7) for the 6-week duration of the experiment. Unilateral nephrectomy resulted in an immediate return of glucose to baseline diabetic levels. Interestingly, adenoviral DNA, as well as mouse HGF (mHGF) mRNA derived from the adenovirus, were present for 42 days posttransplantation. Surprisingly, transplant of 500 IE with Ad.hHGF, as compared with Ad.mHGF, resulted in only marginal correction of blood glucose, suggesting that human HGF is less efficient than mHGF in this system. CONCLUSIONS—These studies demonstrate that mHGF markedly improves islet transplant outcomes in the highest preclinical species examined to date. HGF has promise as an agent that can improve islet mass and function in transplant models and likely in other models of types 1 and 2 diabetes.