anti-HGF Antibody Research Articles

Amniotic fluid-borne hepatocyte growth factor protects rat pups against experimental necrotizing enterocolitis

Fetal swallowing of amniotic fluid, which contains numerous cytokines and growth factors, plays a key role in gut mucosal development. Preterm birth interrupts this exposure to amniotic fluid-borne growth factors, possibly contributing to the increased risk of necrotizing enterocolitis (NEC) in premature infants. We hypothesized that supplementation of formula feeds with amniotic fluid can provide amniotic fluid-borne growth factors and prevent experimental NEC in rat pups. We compared NEC-like injury in rat pups fed with infant formula vs. formula supplemented either with 30% amniotic fluid or recombinant hepatocyte growth factor (HGF). Cytokines/growth factors in amniotic fluid were measured by immunoassays. Amniotic fluid and HGF effects on enterocyte migration, proliferation, and survival were measured in cultured IEC6 intestinal epithelial cells. Finally, we used an antibody array to investigate receptor tyrosine kinase (RTK) activation and immunoblots to measure phosphoinositide 3-kinase (PI3K) signaling. Amniotic fluid supplementation in oral feeds protected rat pups against NEC-like injury. HGF was the most abundant growth factor in rat amniotic fluid in our panel of analytes. Amniotic fluid increased cell migration, proliferation, and cell survival in vitro. These effects were reproduced by HGF and blocked by anti-HGF antibody or a PI3K inhibitor. HGF transactivated several RTKs in IEC6 cells, indicating that its effects extended to multiple signaling pathways. Finally, similar to amniotic fluid, recombinant HGF also reduced the frequency and severity of NEC-like injury in rat pups. Amniotic fluid supplementation protects rat pups against experimental NEC, which is mediated, at least in part, by HGF.

Targeting Receptor Tyrosine Kinase MET in Cancer: Small Molecule Inhibitors and Clinical Progress

The HGF/MET signaling pathway is critical in mediating a wide range of normal physiological functions including embryological development, wound healing, and tissue regeneration. Aberrant activation of the pathway has frequently been found in human cancers via protein overexpression, mutation, gene amplification, and also paracrine or autocrine up-regulation. In addition, the activation of HGF/MET signaling confers resistance to the effects of cancer treatments. Therefore, inhibition of the HGF/MET signaling pathway has great potential for therapeutic intervention in cancer. Currently, there are three approaches toward modulating HGF/MET signaling in human clinical studies of cancer: anti-HGF monoclonal antibodies, MET monoclonal antibodies, and small molecule MET inhibitors. Preliminary clinical benefit from inhibition of HGF or MET has been reported. This Perspective will provide an overview of the HGF/MET signaling pathway in cancer and then will review the development of small molecule MET inhibitors and their progress in clinical applications.

Inhibition of Tumor Growth in a Mouse Xenograft Model by the Humanized Anti-HGF Monoclonal Antibody YYB-101 Produced in a Large-Scale CHO Cell Culture

The humanized anti-hepatocyte growth factor (HGF) monoclonal antibody (mAb) YYB-101 is a promising therapeutic candidate for treating various cancers. In this study, we developed a bioprocess for large-scale production of YYB-101 and evaluated its therapeutic potential for tumor treatment using a xenograft mouse model. By screening diverse chemically defined basal media formulations and by assessing the effects of various feed supplements and feeding schedules on cell growth and antibody production, we established an optimal medium and feeding method to produce 757 mg/l of YYB-101 in flask cultures, representing a 7.5-fold increase in titer compared with that obtained under non-optimized conditions. The optimal dissolved oxygen concentration for antibody production was 70% pO2. A pH shift from 7.2 to 7.0, rather than controlled pH of either 7.0 or 7.2, resulted in productivity improvement in 5 L and 200 L bioreactors, yielding 737 and 830 mg/ml of YYB-101, respectively. The YYB-101 mAb highly purified by affinity chromatography using a Protein A column and two-step ion exchange chromatography effectively neutralized HGF in a cell-based assay and showed potent tumor suppression activity in a mouse xenograft model established with human glioblastoma cells.

Quantitative Phosphoproteomics Analysis Reveals Broad Regulatory Role of Heparan Sulfate on Endothelial Signaling

Heparan sulfate (HS) is a linear, abundant, highly sulfated polysaccharide that expresses in the vasculature. Recent genetic studies documented that HS critically modulates various endothelial cell functions. However, elucidation of the underlying molecular mechanism has been challenging because of the presence of a large number of HS-binding ligands found in the examined experimental conditions. In this report, we used quantitative phosphoproteomics to examine the global HS-dependent signaling by comparing wild type and HS-deficient endothelial cells that were cultured in a serum-containing medium. A total of 7222 phosphopeptides, corresponding to 1179 proteins, were identified. Functional correlation analysis identified 25 HS-dependent functional networks, and the top five are related to cell morphology, cellular assembly and organization, cellular function and maintenance, cell-to-cell communication, inflammatory response and disorder, cell growth and proliferation, cell movement, and cellular survival and death. This is consistent with cell function studies showing that HS deficiency altered endothelial cell growth and mobility. Mining for the underlying molecular mechanisms further revealed that HS modulates signaling pathways critically related to cell adhesion, migration, and coagulation, including ILK, integrin, actin cytoskeleton organization, tight junction and thrombin signaling. Intriguingly, this analysis unexpectedly determined that the top HS-dependent signaling is the IGF-1 signaling pathway, which has not been known to be modulated by HS. In-depth analysis of growth factor signaling identified 22 HS-dependent growth factor/cytokine/growth hormone signaling pathways, including those both previously known, such as HGF and VEGF, and those unknown, such as IGF-1, erythropoietin, angiopoietin/Tie, IL-17A and growth hormones. Twelve of the identified 22 growth factor/cytokine/growth hormone signaling pathways, including IGF-1 and angiopoietin/Tie signaling, were alternatively confirmed in phospho-receptor tyrosine kinase array analysis. In summary, our SILAC-based quantitative phosphoproteomic analysis confirmed previous findings and also uncovered novel HS-dependent functional networks and signaling, revealing a much broader regulatory role of HS on endothelial signaling.

Hepatocyte Growth Factor Protects Against Isoniazid/Rifampicin-Induced Oxidative Liver Damage

The worldwide increment of multidrug- and extensively drug-resistant tuberculosis has emphasized the importance of looking for new options in therapeutics. Long-time usage or higher doses of isoniazid and rifampicin have been considered for the treatment of multidrug-resistant tuberculosis; however, the risk of liver failure is proportionally increased. Hepatocyte growth factor (HGF) is a multitask growth factor that stimulates both antiapoptotic and antioxidant responses that counteract the toxic effects of drug metabolism in the liver. The present work was focused to address the antioxidant and antiapoptotic effects of HGF on isoniazid- and rifampicin-induced hepatotoxicity. BALB/c mice were subjected to rifampicin (150mg/kg, intragavage [ig]) plus isoniazid (75mg/kg, ig) for 7 days. Increments in alanine aminotransferase activity, steatosis, apoptosis, and oxidative stress markers were found in animals. Recombinant HGF (iv) prevented all the harmful effects by increasing the activation of Erk1/2 and PKCδ signaling pathways and glutathione (GSH) synthesis. Furthermore, inhibition of endogenous HGF with anti-HGF antibody (iv) enhanced the isoniazid- and rifampicin-induced oxidative stress damage and decreased the GSH content, aggravating liver damage. In conclusion, HGF demonstrated to be a good protective factor against antituberculosis drug-induced hepatotoxicity and could be considered a good adjuvant factor for the use of high doses of or the reintroduction of these antituberculosis drugs.

Adipose mesenchymal stem cells protect chondrocytes from degeneration associated with osteoarthritis

Our work aimed at evaluating the role of adipose stem cells (ASC) on chondrocytes from osteoarthritic (OA) patients and identifying the mediators involved. We used primary chondrocytes, ASCs from different sources and bone marrow mesenchymal stromal cells (MSC) from OA donors. ASCs or MSCs were co-cultured with chondrocytes in a minimal medium and using cell culture inserts. Under these conditions, ASCs did not affect the proliferation of chondrocytes but significantly decreased camptothecin-induced apoptosis. Both MSCs and ASCs from different sources allowed chondrocytes in the cocultures maintaining a stable expression of markers specific for a mature phenotype, while expression of hypertrophic and fibrotic markers was decreased. A number of factors known to regulate the chondrocyte phenotype (IL-1β, IL-1RA, TNF-α) and matrix remodeling (TIMP-1 and -2, MMP-1 and -9, TSP-1) were not affected. However, a significant decrease of TGF-β1 secretion by chondrocytes and induction of HGF secretion by ASCs was observed. Addition of a neutralizing anti-HGF antibody reversed the anti-fibrotic effect of ASCs whereas hypertrophic markers were not modulated. In summary, ASCs are an interesting source of stem cells for efficiently reducing hypertrophy and dedifferentiation of chondrocytes, at least partly via the secretion of HGF. This supports the interest of using these cells in therapies for osteo-articular diseases.

754 Tumorigenesis Promoted by Colibactin-Producing Escherichia coli Explained by the Dark Side of Cellular Senescence

Background & Aims: Microbiota has been shown to play a critical role in colorectal cancer (CRC). Most of CRC mucosa-associated Escherichia coli harbor the pks genomic island (pks+ E. coli) responsible for the synthesis of colibactin, a genotoxic compound able to promote colon tumorigenesis in a genetically susceptible mouse model. Our aim was to investigate the mechanisms underlying the tumorigenic activity of E. coli. Methods: Human intestinal epithelial cells HCT116 were infected with pks+ or pksE. coli. Cellular senescence and proliferation were assessed by β-galactosidase activity and MTT assay, respectively. Effect of growth factors secreted from infected cells on the proliferation of uninfected cells was assessed using specific neutralizing antibodies and inhibitors in vitro and in a xenograft mouse model. Human biopsies isolated from CRC tumors colonized with pks+ or pksE. coli were used to validate the experimental data. Results: A transient infection of HCT16 cells with pks+ E. coli increased tumor growth in the xenograft mouse model compared to pksE. coli infection, and the conditioned media derived from pks+ E. coli-infected cells stimulated the growth of uninfected cells. A pretreatment of the conditioned media with an anti-HGF antibody and inhibition of HGF receptor in the xenograft mouse model abolished the pro-proliferative effect of pks+ E. coli, indicating the involvement of the growth factor HGF. HGF was produced by pks+ E. coli-induced senescent cells, which is driven by posttranslational modification of p53 involving small ubiquitin-like modifier (SUMO) peptides. The pks+ E. coli-infected cells displayed a modified pattern of SUMO-conjugated proteins and decreased expression of the SUMO peptidase SENP1, a key regulator of SUMOylation. Over-expression of SENP1 in HCT116 cells abolished the deregulated SUMOylation, cellular senescence, HGF production and the pro-proliferative effect induced by pks+ E. coli infection. Finally, the human CRC tumors colonized by pks+ E. coli expressed a higher level of HGF and decreased SENP1 expression compared to those colonized by pksE. coli, supporting the pathological relevance of the in vitro data. Conclusions: Our study elucidates a new paradigm for the mechanism underlying the carcinogenesis mediated by colibactin-producing bacteria. We propose that those bacteria modulate tumor microenvironment by favoring the emergence of senescent cells, which in turn promote tumor growth by the secretion of growth factors, notably HGF.

757 Impact of Metabolic Syndrome on Incident or Recurrent Advanced Adenoma After Colonoscopy

Background & Aims: Microbiota has been shown to play a critical role in colorectal cancer (CRC). Most of CRC mucosa-associated Escherichia coli harbor the pks genomic island (pks+ E. coli) responsible for the synthesis of colibactin, a genotoxic compound able to promote colon tumorigenesis in a genetically susceptible mouse model. Our aim was to investigate the mechanisms underlying the tumorigenic activity of E. coli. Methods: Human intestinal epithelial cells HCT116 were infected with pks+ or pksE. coli. Cellular senescence and proliferation were assessed by β-galactosidase activity and MTT assay, respectively. Effect of growth factors secreted from infected cells on the proliferation of uninfected cells was assessed using specific neutralizing antibodies and inhibitors in vitro and in a xenograft mouse model. Human biopsies isolated from CRC tumors colonized with pks+ or pksE. coli were used to validate the experimental data. Results: A transient infection of HCT16 cells with pks+ E. coli increased tumor growth in the xenograft mouse model compared to pksE. coli infection, and the conditioned media derived from pks+ E. coli-infected cells stimulated the growth of uninfected cells. A pretreatment of the conditioned media with an anti-HGF antibody and inhibition of HGF receptor in the xenograft mouse model abolished the pro-proliferative effect of pks+ E. coli, indicating the involvement of the growth factor HGF. HGF was produced by pks+ E. coli-induced senescent cells, which is driven by posttranslational modification of p53 involving small ubiquitin-like modifier (SUMO) peptides. The pks+ E. coli-infected cells displayed a modified pattern of SUMO-conjugated proteins and decreased expression of the SUMO peptidase SENP1, a key regulator of SUMOylation. Over-expression of SENP1 in HCT116 cells abolished the deregulated SUMOylation, cellular senescence, HGF production and the pro-proliferative effect induced by pks+ E. coli infection. Finally, the human CRC tumors colonized by pks+ E. coli expressed a higher level of HGF and decreased SENP1 expression compared to those colonized by pksE. coli, supporting the pathological relevance of the in vitro data. Conclusions: Our study elucidates a new paradigm for the mechanism underlying the carcinogenesis mediated by colibactin-producing bacteria. We propose that those bacteria modulate tumor microenvironment by favoring the emergence of senescent cells, which in turn promote tumor growth by the secretion of growth factors, notably HGF.

755 Obesity Accelerates Helicobacter felis-Induced Gastric Carcinogenesis by Enhancing TH17 Responses and Immature Myeloid Cell Trafficking

Background & Aims: Microbiota has been shown to play a critical role in colorectal cancer (CRC). Most of CRC mucosa-associated Escherichia coli harbor the pks genomic island (pks+ E. coli) responsible for the synthesis of colibactin, a genotoxic compound able to promote colon tumorigenesis in a genetically susceptible mouse model. Our aim was to investigate the mechanisms underlying the tumorigenic activity of E. coli. Methods: Human intestinal epithelial cells HCT116 were infected with pks+ or pksE. coli. Cellular senescence and proliferation were assessed by β-galactosidase activity and MTT assay, respectively. Effect of growth factors secreted from infected cells on the proliferation of uninfected cells was assessed using specific neutralizing antibodies and inhibitors in vitro and in a xenograft mouse model. Human biopsies isolated from CRC tumors colonized with pks+ or pksE. coli were used to validate the experimental data. Results: A transient infection of HCT16 cells with pks+ E. coli increased tumor growth in the xenograft mouse model compared to pksE. coli infection, and the conditioned media derived from pks+ E. coli-infected cells stimulated the growth of uninfected cells. A pretreatment of the conditioned media with an anti-HGF antibody and inhibition of HGF receptor in the xenograft mouse model abolished the pro-proliferative effect of pks+ E. coli, indicating the involvement of the growth factor HGF. HGF was produced by pks+ E. coli-induced senescent cells, which is driven by posttranslational modification of p53 involving small ubiquitin-like modifier (SUMO) peptides. The pks+ E. coli-infected cells displayed a modified pattern of SUMO-conjugated proteins and decreased expression of the SUMO peptidase SENP1, a key regulator of SUMOylation. Over-expression of SENP1 in HCT116 cells abolished the deregulated SUMOylation, cellular senescence, HGF production and the pro-proliferative effect induced by pks+ E. coli infection. Finally, the human CRC tumors colonized by pks+ E. coli expressed a higher level of HGF and decreased SENP1 expression compared to those colonized by pksE. coli, supporting the pathological relevance of the in vitro data. Conclusions: Our study elucidates a new paradigm for the mechanism underlying the carcinogenesis mediated by colibactin-producing bacteria. We propose that those bacteria modulate tumor microenvironment by favoring the emergence of senescent cells, which in turn promote tumor growth by the secretion of growth factors, notably HGF.

Autocrine HGF/c‐MET signaling in prostate cancer stem‐like cells/cancer‐initiating cells. Prostate adenocarcinoma tissue was stained by anti‐SOX2 and anti‐HGF antibodies. HGF is expressed in prostate adenocarcinoma tissue

Cancer ScienceVolume 104, Issue 4 p. April cover-April cover Cover ImageOpen Access Autocrine HGF/c-MET signaling in prostate cancer stem-like cells/cancer-initiating cells. Prostate adenocarcinoma tissue was stained by anti-SOX2 and anti-HGF antibodies. HGF is expressed in prostate adenocarcinoma tissue Sachiyo Nishida, Sachiyo NishidaSearch for more papers by this authorYoshihiko Hirohashi, Yoshihiko HirohashiSearch for more papers by this authorToshihiko Torigoe, Toshihiko TorigoeSearch for more papers by this authorRyuta Inoue, Ryuta InoueSearch for more papers by this authorHiroshi Kitamura, Hiroshi KitamuraSearch for more papers by this authorToshiaki Tanaka, Toshiaki TanakaSearch for more papers by this authorAkari Takahashi, Akari TakahashiSearch for more papers by this authorHiroko Asanuma, Hiroko AsanumaSearch for more papers by this authorNaoya Masumori, Naoya MasumoriSearch for more papers by this authorTaiji Tsukamoto, Taiji TsukamotoSearch for more papers by this authorNoriyuki Sato, Noriyuki SatoSearch for more papers by this author Sachiyo Nishida, Sachiyo NishidaSearch for more papers by this authorYoshihiko Hirohashi, Yoshihiko HirohashiSearch for more papers by this authorToshihiko Torigoe, Toshihiko TorigoeSearch for more papers by this authorRyuta Inoue, Ryuta InoueSearch for more papers by this authorHiroshi Kitamura, Hiroshi KitamuraSearch for more papers by this authorToshiaki Tanaka, Toshiaki TanakaSearch for more papers by this authorAkari Takahashi, Akari TakahashiSearch for more papers by this authorHiroko Asanuma, Hiroko AsanumaSearch for more papers by this authorNaoya Masumori, Naoya MasumoriSearch for more papers by this authorTaiji Tsukamoto, Taiji TsukamotoSearch for more papers by this authorNoriyuki Sato, Noriyuki SatoSearch for more papers by this author First published: 22 March 2013 https://doi.org/10.1111/cas.12113AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume104, Issue4April 2013Pages April cover-April cover RelatedInformation

Prostate cancer stem‐like cells/cancer‐initiating cells have an autocrine system of hepatocyte growth factor

Prostate cancer cells include a small population of cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) that have roles in initiation and progression of the cancer. Recently, we isolated prostate CSCs/CICs as aldehyde dehydrogenase 1-highh (ALDH1(high) ) cells using the ALDEFLUOR assay; however, the molecular mechanisms of prostate CSCs/CICs are still elusive. Prostate CSCs/CICs were isolated as ALDH1(high) cells using the ALDEFLUOR assay, and the gene expression profiles were analyzed using a cDNA microarray and RT-PCR. We found that prostate CSCs/CICs expressed higher levels of growth factors including hepatocyte growth factor (HGF). Hepatocyte growth factor protein expression was confirmed by enzyme linked immunosorbent assay and Western blotting. On the other hand, c-MET HGF receptor was expressed in both CSCs/CICs and non-CSCs/CICs at similar levels. Hepatocyte growth factor and the supernatant of myofibloblasts derived from the prostate augmented prostasphere formation in vitro, and prostasphere formation was inhibited by an anti-HGF antibody. Furthermore, c-MET gene knockdown by siRNA inhibited the prostasphere-forming ability in vitro and tumor-initiating ability in vivo. Taken together, the results indicate that HGF secreted by prostate CSCs/CICs and prostate myofibroblasts has a role in the maintenance of prostate CSCs/CICs in an autocrine and paracrine fashion.

Breast cancer stroma influences the migratory behaviour of epithelial malignant cells: the role of secreted hepatocyte growth factor

In breast tumor, interactions occurring between malignant cells and stromal microenvironment heavily influence various aspects of cancer biology. We recently reported that this cross-talk affects structural and functional features correlated with the invasive phenotype of breast cancer cells by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts, particularly those isolated from breast tumor stroma (cancer-associated fibroblasts, CAFs). In the present study, we aimed to elucidate some aspects of the epithelium-stroma interaction, related to two linked CAF-triggered effects we previously described: a) increase in cancer cell plasma membrane fluidity; b) increase in speed and directness of cancer cell migration. Thus, we tested the effect of fibroblast/breast cancer cell co-culturing on the expression of Stearoyl-CoA desaturase 1 (SCD1), the main enzyme regulating the fatty acid membrane composition whose up-regulation has been reported in different cancer cells, generally leading to loosely packed membranes. Western blot analysis revealed a dramatic CAF-promoted increase in SCD1 expression in both MCF-7 and MDA-MB-231 cells. Quantitative real-time PCR demonstrated similar variations in the SCD1 transcript levels. With the aim of clarifying the possible role of soluble factors in the CAF-induced increase in speed and directness of cancer cell migration, we performed co-culture assays in presence of a neutralizing antibody against hepatocyte growth factor (HGF) which is released by CAFs and notoriously affects cancer cell motility. We demonstrated that speed and directness increases were strongly reduced or completely abolished by the addition of the anti-HGF neutralizing antibody to the culture medium. These results provide new insights in understanding the role of CAFs, the main host stromal component of breast cancer, in promoting the tumor cell invasive attitude and may help in defining further promising therapeutic targets.

A methachromatic-based experimental model for identification of bowel as the focus of an acute inflammation

Diarrhea is the most common symptom of acute inflammation in gastrointestinal tract and the patients are isolated in order to inhibit transmission and to conduct investigations. Yet there is no standard test to distinguish gastrointestinal infection from more generalized diseases at admittance which might cause delay in therapy. Hepatocyte growth factor (HGF) is produced upon injury by mesenchymal cells. On the contrary to chronic inflammation, HGF produced in the course of acute inflammation is biologically active and shows binding affinity to heparan sulphate proteoglycan (HSPG) and dextran sulphate (DS). Based on this phenomenon, an agarose gel containing DS was prepared and immobilized on loops to investigate the feces samples for the presence or absence of growth factors such as HGF with affinity to DS. The study is conducted as a clinical evaluation of an experimental model to distinguish acute infectious gastroenteritis from other causes of diarrhea. 656 fecal samples gathered consequently from patients seeking for bowel disturbances and healthy were tested by the test and the medical reports were investigated. Upon interaction with DS, methylene blue changes color to pink. This phenomenon was inhibited by HGF and converted by addition of anti-HGF antibodies to the samples. The test distinguished acute infectious gastroenteritis with high sensitivity and specificity (96% and 92% respectively) from other causes of diarrhea. We introduce a metachromatic experimental model that might distinguish acute inflammation in alimentary tract from other causes of diarrhea. This model might be used in developing rapid diagnostic tests.

HGF regulates VEGF expression via the c-Met receptor downstream pathways, PI3K/Akt, MAPK and STAT3, in CT26 murine cells

In the present study, we assessed the involvement of hepatocyte growth factor (HGF)/c-Met signalling with vascular endothelial cell growth factor (VEGF) and hypoxia inducible factor (HIF)-1α expression in the downstream pathways phosphatidylinositol 3-kinase (PI3K)/Akt, mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) in CT26 cells, to determine the mechanisms of the potent anti-angiogenic effect of NK4. We established genetically modified CT26 cells to produce NK4 (CT26-NK4). VEGF expression in subcutaneous CT26 tumours in vivo and in culture supernatants in vitro was determined by ELISA. HIF-1α expression in nuclear extracts was evaluated by western blot analysis. VEGF and HIF-1α mRNA levels were examined by real-time reverse transcription-polymerase chain reaction (RT-PCR). The DNA binding activity of HIF-1α was evaluated using an HIF-1α transcription factor assay kit. Our results demonstrated that VEGF expression was reduced in homografts of CT26-NK4 cells, compared to those of the control cells. In vitro, VEGF expression, which was induced by HGF, was inhibited by anti-HGF antibody, NK4 and by kinase inhibitors (PI3K, LY294002; MAPK, PD98059; and STAT3, Stattic). HGF‑induced HIF‑1α transcriptional activity was also inhibited by the kinase inhibitors. Real-time RT-PCR demonstrated that HGF‑induced HIF‑1α mRNA expression was not inhibited by LY294002 and PD98059, but was inhibited by Stattic. These data suggest that the PI3K/Akt, MAPK and STAT3 pathways, downstream of HGF/c‑Met signalling, are involved in the regulation of VEGF expression in CT26 cells. HGF/c‑Met signalling may be a promising target for anti-angiogenic strategies.

Beneficial effect of a synthetic prostacyclin agonist, ONO-1301, in rat autoimmune myocarditis model

Injury to the heart can result in cardiomyocyte hypertrophy, fibrosis, and cell death. Myocarditis sometimes progresses to dilated cardiomyopathy. We previously reported that ONO-1301, a synthetic prostacyclin agonist with thromboxane-synthase inhibitory activity, promotes production of hepatocyte growth factor (HGF) from various cell types and ameliorates ischemia-induced left ventricle dysfunction in the mouse, rat and pig. Here, we investigated the therapeutic efficacy of ONO-1301 in a rat model of myosin-induced experimental autoimmune myocarditis, in which the heart transits from an acute inflammatory phase to a chronic dilated cardiomyopathy phase. Four weeks after myosin injection to Lewis rats, ONO-1301 (6mg/kg/day) was orally administered for 4 weeks (ONO-1301 group). Hemodynamic parameters and plasma brain natriuretic peptide (BNP) level were significantly improved by ONO-1301. Histological analysis revealed that capillary density in the myocardium was significantly increased by ONO-1301. ONO-1301 increased circulating endothelial progenitor cells (EPC) as determined by FACS analysis. These beneficial effects of ONO-1301 were partially abrogated by a neutralizing anti-HGF antibody (8mg/kg/dose). These findings indicate beneficial effects of ONO-1301 in a rat experimental autoimmune myocarditis model.

Role of Hepatocyte Growth Factor in Promoting the Growth of Human Corneal Endothelial Cells Stimulated byl-Ascorbic Acid 2-Phosphate

To investigate the mechanisms by which L-ascorbic acid 2-phosphate (Asc-2P) increases the proliferation of human corneal endothelial cells (HCECs). Growth of cultured HCECs was examined in the presence of various antioxidants, including Asc-2P, retinyl acetate (vitamin A), reduced glutathione, oxidized glutathione, carnosine, and sodium alpha-tocopherol phosphate (a water-soluble vitamin E derivative). Synthesis of type I, III, and IV collagen by HCECs cultured with or without Asc-2P was evaluated by measuring cell lysates and conditioned medium with Western blotting, immunocytochemistry, or enzyme-linked immunosorbent assay (ELISA). The gene expression profiles of HCECs cultured with or without Asc-2P were compared by microarray analysis to determine critical proliferative factors, and the proliferative response of these cells to selected factors was tested. Among the antioxidants tested, only Asc-2P promoted the growth of HCECs. Asc-2P did not promote deposition of type I, III, or IV collagen. Microarray analysis revealed that several cytokines were potently upregulated by Asc-2P, but among them, only hepatocyte growth factor (HGF) stimulated HCEC growth. ELISA revealed the upregulation of HGF protein production by Asc-2P, while the stimulatory effect of Asc-2P was abolished by an anti-HGF neutralizing antibody or PHA-665752 (a specific inhibitor of the HGF receptor, c-Met). Asc-2P increases the proliferation of cultured HCECs through upregulation of HGF production via an HGF/c-Met autocrine loop.

Improvement of metabolic syndrome by irbesartan via the PPARγ/HGF pathway in apolipoprotein E knockout mice

Irbesartan, a partial agonist of peroxisome proliferators activated receptor-γ (PPARγ), has been reported to improve insulin resistance and lipid profile in patients with diabetes mellitus or metabolic syndrome (MS). However, the down effectors of PPARγ have yet to be elucidated. Thus, in this study, we focused on the role of the hepatocyte growth factor (HGF) in the anti-metabolic effects of irbesartan, using apolipoprotein E (ApoE) knockout (KO) mice. ApoE KO mice placed on a high-fat diet (HFD) for 12 weeks were divided into four groups: i) the control (HFD only), ii) the HFD + irbesartan (5 mg/kg/day), iii) the HFD + irbesartan + GW9662, a PPARγ antagonist (0.5 mg/kg/day) and iv) the HFD + irbesartan + anti-HGF neutralizing antibody (200 μg/week). The liver and epididymal adipose tissues were evaluated histologically. Serum adiponectin and HGF levels were also measured by ELISA. Fatty liver (as detected by oil-red O staining) and macrophage infiltration were markedly reduced by irbesartan. Irbesartan treatment also reduced macrophage infiltration into epididymal adipose tissue and hypertrophy of adipocytes. However, these effects of irbesartan were attenuated by GW9662 as well as by anti-HGF neutralizing antibody. Serum and hepatic HGF levels were also markedly increased by irbesartan, whereas GW9662 decreased the HGF level. In conclusion, irbesartan, an angiotensin (Ang) receptor blocker (ARB) and partial agonist of PPARγ (metabosartan), demonstrated a reduction in fatty liver and chronic inflammation, such as macrophage infiltration, beyond its blood pressure-lowering effect. These favorable characteristics of irbesartan might be due to local HGF activation through its partial PPARγ agonistic action, in addition to Ang II blockade. Upregulation of local HGF by irbesartan might provide a novel advantage in a strategy for the prevention and treatment of cardiovascular diseases (CVDs).

Autologous cell therapy for cisplatin-induced acute kidney injury by using non-expanded adipose tissue-derived cells

Recent studies have demonstrated that cultured mesenchymal stromal cells derived from adipose tissue are useful for regenerative cell therapy. The stromal vascular fraction (SVF) can be obtained readily without culturing and may be clinically applicable. We investigated the therapeutic effects of SVF and used it in the treatment of acute kidney injury (AKI). Liposuction aspirates were obtained from healthy donors who had provided written informed consent. We harvested the SVF and determined the growth factor secretion and anti-apoptotic ability with conditioned medium. To investigate the effect of SVF on AKI, cisplatin was injected into rats and SVF was administrated into the subcupsula of the kidney. Both human and rat SVF cells secreted vascular endothelial growth factor-A (VEGF) and hepatocyte growth factor (HGF). Human SVF-conditioned media had an anti-apoptotic effect, which was inhibited by anti-HGF antibody (Ab) but not by anti-VEGF Ab. In vivo, SVF significantly ameliorated renal function, attenuated tubular damage and increased the cortical blood flow speed. In the SVF-treated group, VEGF levels in the cortex and HGF levels in both the cortex and medulla, especially tubules in the medulla, were significantly higher. Immunostaining revealed that SVF cells expressing VEGF and HGF and remained in the subcapsule on day 14. The present study demonstrates that a subcapsular injection of non-expanded SVF cells ameliorates rat AKI, and that the mechanism probably involves secretion of renoprotective molecules. Administration of human SVF may be clinically applicable and useful as a novel autologous cell therapy against kidney diseases.

Inhibition of c-Met activation sensitizes osteosarcoma cells to cisplatin via suppression of the PI3K–Akt signaling

Osteosarcoma is a common malignant bone tumor. Cisplatin (CDDP) achieves a high response rate in osteosarcoma. However, osteosarcoma usually exhibits cisplatin resistance. Many members of receptor tyrosine kinases (RTKs)1Abbreviations used: RTKs, receptor tyrosine kinases; HGFR/c-Met, Hepatocyte growth factor receptor; rh-HGF, recombinant human HGF; EGFR, epidermal growth factor receptor; PDGFR, platelet-derived growth factor receptor; HGF, hepatocyte growth factor; HRP, horseradish peroxidase.1 have been demonstrated to be overexpressed and constitutively activated in various tumors including osteosarcoma, resulting in malignant progression and insensitivity to chemotherapy. Hepatocyte growth factor receptor (HGFR/c-Met) also appears overexpressed and activated in osteosarcoma cells. Nevertheless, which role of c-Met activation in cisplatin efficacy against osteosarcoma cells remains still elusive. This study found that inhibition of c-Met activity by PHA-665752 or blockade of the interaction of autocrined HGF with c-Met with neutralizing anti-HGF antibody promoted cisplatin efficacy in osteosarcoma cells, while addition of recombinant human HGF (rh-HGF) counteracts cisplatin cytotoxicity. Specifically, we demonstrated that inhibition of c-Met activity led to suppression of the PI3K–Akt pathway, thus enhancing cisplatin chemosensitivity. Our study clearly suggests that inhibition of c-Met activity can effectively sensitize osteosarcoma cells to cisplatin via suppression of the PI3K–Akt signaling.

Identification of Basophils as a Major Source of Hepatocyte Growth Factor in Chronic Myeloid Leukemia: A Novel Mechanism of BCR-ABL1-Independent Disease Progression

Chronic myeloid leukemia (CML) is a hematopoietic neoplasm characterized by the Philadelphia chromosome and the related BCR-ABL1 oncoprotein. Acceleration of CML is usually accompanied by basophilia. Several proangiogenic molecules have been implicated in disease acceleration, including the hepatocyte growth factor (HGF). However, little is known so far about the cellular distribution and function of HGF in CML. We here report that HGF is expressed abundantly in purified CML basophils and in the basophil-committed CML line KU812, whereas all other cell types examined expressed only trace amounts of HGF or no HGF. Interleukin 3, a major regulator of human basophils, was found to promote HGF expression in CML basophils. By contrast, BCR-ABL1 failed to induce HGF synthesis in CML cells, and imatinib failed to inhibit expression of HGF in these cells. Recombinant HGF as well as basophil-derived HGF induced endothelial cell migration in a scratch wound assay, and these effects of HGF were reverted by an anti-HGF antibody as well as by pharmacologic c-Met inhibitors. In addition, anti-HGF and c-Met inhibitors were found to suppress the spontaneous growth of KU812 cells, suggesting autocrine growth regulation. Together, HGF is a BCR-ABL1-independent angiogenic and autocrine growth regulator in CML. Basophils are a unique source of HGF in these patients and may play a more active role in disease-associated angiogenesis and disease progression than has so far been assumed. Our data also suggest that HGF and c-Met are potential therapeutic targets in CML.