Showing papers on "Hepatocyte growth factor published in 2001"

Anti-apoptotic signaling by hepatocyte growth factor/Met via the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways

Abstract: Hepatocyte growth factor (HGF) is a ligand of the receptor tyrosine kinase encoded by the c-Met protooncogene. HGF/Met signaling has multifunctional effects on various cell types. We sought to determine the role of HGF/Met in apoptosis and identify signal transducers involved in this process. In experiments with human SK-LMS-1 leiomyosarcoma cells, we show that the Akt kinase is activated by HGF in a time- and dose-dependent manner by phosphatidylinositol 3-kinase (PI3-kinase). Akt is also activated by active tumorigenic forms of Met, i.e., ligand-independent Tpr-Met, a truncated and constitutively dimerized form of Met, and a mutationally activated version of Met corresponding to that found in human hereditary papillary renal carcinoma. In NIH 3T3 cells transfected with wild-type Met, HGF inhibits apoptosis induced by serum starvation and UV irradiation. HGF-induced survival correlates with Akt activity and is inhibited by the specific PI3-kinase inhibitor LY294002, indicating that HGF inhibits cell death through the PI3-kinase/Akt signal transduction pathway. Furthermore, transiently transfected Tpr-Met activates Akt (both Akt1 and Akt2) and protects cells from apoptosis. Mitogen-activated protein kinase (MAPK) also is activated by HGF and rescues cells from apoptosis, although the cytoprotective effect is less marked than for PI3-kinase/Akt. Blocking MAPK with the specific MAPK kinase inhibitor PD098059 impairs the ability of HGF to promote cell survival. Similar results were obtained with NIH 3T3 cells expressing the fusion protein Trk-Met and stimulated with nerve growth factor, the Trk ligand. These results demonstrate that HGF/Met is capable of protecting cells from apoptosis by using both PI3-kinase/Akt and, to a lesser extent, MAPK pathways.

The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells

Abstract: The mammary gland develops its adult form by a process referred to as branching morphogenesis. Many factors have been reported to affect this process. We have used cultured primary mammary epithelial organoids and mammary epithelial cell lines in three-dimensional collagen gels to elucidate which growth factors, matrix metalloproteinases (MMPs) and mammary morphogens interact in branching morphogenesis. Branching stimulated by stromal fibroblasts, epidermal growth factor, fibroblast growth factor 7, fibroblast growth factor 2 and hepatocyte growth factor was strongly reduced by inhibitors of MMPs, indicating the requirement of MMPs for three-dimensional growth involved in morphogenesis. Recombinant stromelysin 1/MMP3 alone was sufficient to drive branching in the absence of growth factors in the organoids. Plasmin also stimulated branching; however, plasmin-dependent branching was abolished by both inhibitors of plasmin and MMPs, suggesting that plasmin activates MMPs. To differentiate between signals for proliferation and morphogenesis, we used a cloned mammary epithelial cell line that lacks epimorphin, an essential mammary morphogen. Both epimorphin and MMPs were required for morphogenesis, but neither was required for epithelial cell proliferation. These results provide direct evidence for a crucial role of MMPs in branching in mammary epithelium and suggest that, in addition to epimorphin, MMP activity is a minimum requirement for branching morphogenesis in the mammary gland.

Hepatocyte growth factor enhances vascular endothelial growth factor-induced angiogenesis in vitro and in vivo

Abstract: Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis in both physiological and pathological processes. Hepatocyte growth factor (HGF) is a mesenchyme-derived mitogen that also stimulates cell migration, and branching and/or tubular morphogenesis of epithelial and endothelial cells. In the present study, we tested the hypothesis that simultaneous administration of HGF and VEGF would synergistically promote new blood vessel formation. HGF acted in concert with VEGF to promote human endothelial cell survival and tubulogenesis in 3-D type I collagen gels, a response that did not occur with either growth factor alone. The synergistic effects of VEGF and HGF on endothelial survival correlated with greatly augmented mRNA levels for the anti-apoptotic genes Bcl-2 and A1. Co-culture experiments with human neonatal dermal fibroblasts and human umbilical vein endothelial cells demonstrated that neonatal dermal fibroblasts, in combination with VEGF, stimulated human umbilical vein endothelial cells tubulogenesis through the paracrine secretion of HGF. Finally, in vivo experiments demonstrated that the combination of HGF and VEGF increased neovascularization in the rat corneal assay greater than either growth factor alone. We suggest that combination therapy using HGF and VEGF co-administration may provide a more effective strategy to achieve therapeutic angiogenesis.

Hepatocyte Growth Factor/Scatter Factor-induced Activation of MEK and PI3K Signal Pathways Contributes to Expression of Proangiogenic Cytokines Interleukin-8 and Vascular Endothelial Growth Factor in Head and Neck Squamous Cell Carcinoma

Abstract: The proangiogenic activity of hepatocyte growth factor (HGF)/scatter factor has been closely associated with its ability to stimulate endothelial cell chemotaxis, migration, proliferation, and capillary formation. However, the potential of HGF as a paracrine factor in regulating the expression of angiogenesis factors by tumor cells is not widely appreciated. We observed that increased HGF was correlated with higher levels of angiogenesis factors interleukin (IL)-8 and vascular endothelial growth factor (VEGF) in serum of patients with head and neck squamous cell carcinoma (HNSCC) as compared with that in normal volunteers and hypothesized that HGF may regulate angiogenesis factor production by tumor cells through the activation of its receptor c-Met, which is expressed by HNSCC cells. To test this hypothesis, we examined the effect of HGF treatment on IL-8 and VEGF expression by a panel of primary keratinocytes and HNSCC lines. HGF induced a significant dose-dependent increase in IL-8 and/or VEGF cytokine production in eight HNSCC lines tested, which is not observed in normal keratinocytes. In addition, HGF increased mRNA expression of IL-8 in 3 of 6 and VEGF in 5 of 6 HNSCC lines. The increase in induction of these factors by HGF corresponded to an increase in phosphorylation of c-Met in HNSCC. HGF-induced phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) pathway substrate p42/p44 erk and phosphatidylinositol 3′-kinase (PI3K) pathway substrate Akt provided evidence for downstream activation of MEK and PI3K pathways in HNSCC. Inhibitors of MEK (U0126) and PI3K (LY294002) blocked p42/p44 erk and Akt, respectively, and partially blocked HGF-induced production of IL-8 and VEGF, whereas the combination of U0126 and LY294002 completely inhibited expression of IL-8 and VEGF by UMSCC-11A. Our results demonstrate that HGF can promote expression of angiogenesis factors in tumor cells through both MEK- and PI3K-dependent pathways. Understanding HGF/Met paracrine regulatory mechanisms between tumor and host cells may provide critical information for targeting of therapies against angiogenesis.

Glypican-1 is overexpressed in human breast cancer and modulates the mitogenic effects of multiple heparin-binding growth factors in breast cancer cells.

Abstract: Glypicans are a family of glycosylphosphatidylinositol-anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. Here we show that glypican-1 is strongly expressed in human breast cancers, whereas expression of glypican-1 is low in normal breast tissues. In contrast, the expression of glypican-3 and -4 is only slightly increased in breast cancers by comparison with normal breast tissues, and glypican-2 and -5 are below the level of detection by Northern blotting in both normal and cancer samples. Treatment of MDA-MB-231 and MDA-MB-468 breast cancer cells with phosphoinositide-specific phospholipase-C abrogated the mitogenic response to two heparin-binding growth factors, heparin-binding epidermal growth factor-like growth factor and fibroblast growth factor 2. Stable transfection of these cells with a glypican-1 antisense construct markedly decreased glypican-1 protein levels and the mitogenic response to the same heparin-binding growth factors, as well as that to heregulin alpha, heregulin beta, and hepatocyte growth factor. Syndecan-1 was also expressed at high levels in both breast cancer tissues and breast cancer cells when compared with normal breast tissues. There was a good correlation between glypican-1 and syndecan-1 expression in the tumors. However, clones expressing the glypican-1 antisense construct did not exhibit decreased syndecan-1 levels, indicating that loss of responsiveness to heparin-binding growth factors in these clones was not due to altered syndecan-1 expression. Furthermore, 8 of 10 tumors with stage 2 or 3 disease exhibited high levels of glypican-1 by Northern blot analysis. In contrast, low levels of glypican-1 mRNA were evident in 1 of 10 tumors with stage 2 or 3 disease and in 9 of 10 tumors with stage 1 disease. Taken together, these data suggest that glypican-1 may play a pivotal role in the ability of breast cancer cells to exhibit a mitogenic response to multiple heparin-binding growth factors and may contribute to disease progression in this malignancy.

Neutralizing monoclonal antibodies to hepatocyte growth factor/scatter factor (HGF/SF) display antitumor activity in animal models.

Abstract: The hepatocyte growth factor (HGF/SF) receptor, Met, regulates mitogenesis, motility, and morphogenesis in a cell type-dependent fashion. Activation of Met via autocrine, paracrine, or mutational mechanisms can lead to tumorigenesis and metastasis and numerous studies have linked inappropriate expression of this ligand-receptor pair to most types of human solid tumors. To prepare mAbs to human HGF/SF, mice were immunized with native and denatured preparations of the ligand. Recloned mAbs were tested in vitro for blocking activity against scattering and branching morphogenesis. Our results show that no single mAb was capable of neutralizing the in vitro activity of HGF/SF, and that the ligand possesses a minimum of three epitopes that must be blocked to prevent Met tyrosine kinase activation. In vivo, the neutralizing mAb combination inhibited s.c. growth in athymic nu/nu mice of tumors dependent on an autocrine Met-HGF/SF loop. Importantly, growth of human glioblastoma multiforme xenografts expressing Met and HGF/SF were markedly reduced in the presence of HGF/SF-neutralizing mAbs. These results suggest interrupting autocrine and/or paracrine Met-HGF/SF signaling in tumors dependent on this pathway is a possible intervention strategy.

Glioma cell invasion: Regulation of metalloproteinase activity by TGF-β

Abstract: Matrix metalloproteinases (MMPs) are a family of extracellular endopeptidases that selectively degrade components of the extracellular matrix MMPs are implicated in tumor cell invasion because they mediate the breakdown of the basal membrane In addition, they seem to be important for the creation and maintenance of a microenvironment that facilitates tumor cell survival Among the essential characteristics of human malignant gliomas are infiltrative growth, angiogenesis and suppression of antitumor immune surveillance Transforming growth factor-beta (TGF-β) is intimately involved in the regulation of these processes We have previously demonstrated that TGF-β promotes the migration of LN-18 and LN-229 glioma cells via a process that may involve the upregulation of αVβ3 integrin expression Furthermore, we have defined a novel pathway for hepatocyte growth factor (HGF)-induced glioma cell migration and invasion which requires the induction of TGF-β2 expression Here, we demonstrate that TGF-β2 induces MMP-2 expression and suppresses tissue inhibitor of metalloproteinases (TIMP)-2 expression and that concentration-dependently promotes the invasion of U87MG and LN-229 glioma cells in a matrigel invasion assay Similarly, ectopic expression of the anti-apoptotic BCL-xL protein leads to enhanced matrigel invasion by LN-18 and LN-229 glioma cells We outline the possible interrelations of TGF-β, proteins of the BCL-2 family, integrins and metalloprotease activity By virtue of its promotion of glioma invasion and its growth regulatory and immunomodulatory properties, TGF-β continues to be one of the most promising targets for the experimental therapy of human malignant glioma

Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat and rabbit hindlimb ischemia models: preclinical study for treatment of peripheral arterial disease.

Abstract: Hepatocyte growth factor (HGF) exclusively stimulates the growth of endothelial cells without replication of vascular smooth muscle cells, and acts as a survival factor against endothelial cell death. Recently, a novel therapeutic strategy for ischemic diseases using angiogenic growth factors to expedite and/or augment collateral artery development has been proposed. We have previously reported that intra-arterial administration of recombinant HGF induced angiogenesis in a rabbit hindlimb ischemia model. In this study, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease rather than recombinant therapy, due to its disadvantages. Initially, we examined the transfection of 'naked' human HGF plasmid into a rat hindlimb ischemia model. Intramuscular injection of human HGF plasmid resulted in a significant increase in blood flow as assessed by laser Doppler imaging, accompanied by the detection of human HGF protein. A significant increase in capillary density was found in rats transfected with human HGF as compared with control vector, in a dose-dependent manner (P < 0.01). Importantly, at 5 weeks after transfection, the degree of angiogenesis induced by transfection of HGF plasmid was significantly greater than that caused by a single injection of recombinant HGF. As an approach to human gene therapy, we also employed a rabbit hindlimb ischemia model as a preclinical study. Naked HGF plasmid was intramuscularly injected in the ischemic hindlimb of rabbits, to evaluate its angiogenic activity. Intramuscular injection of HGF plasmid once on day 10 after surgery produced significant augmentation of collateral vessel development on day 30 in the ischemia model, as assessed by angiography (P < 0.01). Serial angiograms revealed progressive linear extension of collateral arteries from the origin stem artery to the distal point of the reconstituted parent vessel in HGF-transfected animals. In addition, a significant increase in blood flow was assessed by a Doppler flow wire and the ratio in blood pressure of the ischemic limb to the normal limb was observed in rabbits transfected with HGF plasmid as compared with rabbits transfected with control vector (P < 0.01). Overall, intramuscular injection of naked human HGF plasmid induced therapeutic angiogenesis in rat and rabbit ischemic hindlimb models, as potential therapy for peripheral arterial disease.

Downregulation of E-cadherin and Desmoglein 1 by autocrine hepatocyte growth factor during melanoma development.

Abstract: During melanoma development, transformed cells evade keratinocyte-mediated control by downregulating cell adhesion molecules. This study investigated the regulation of cell adhesion by hepatocyte growth factor (HGF) in melanoma. Melanocytes and two melanoma lines, WM164 and WM35, expressed normal level E-cadherin and Desmoglein 1, whereas most melanomas (18 out of 20) expressed no E-cadherin and significantly reduced Desmoglein 1. Overexpression of dominant negative E-cadherin and Desmoglein in melanocytes demonstrated that both molecules contribute to adhesion between melanocytes and keratinocytes. In contrast to melanocytes, most melanomas expressed HGF. All melanocytic cells expressed the HGF receptor c-Met, and autocrine HGF caused constitutive activation of c-Met, MAPK and PI3K. When autocrine activation was induced with HGF-expressing adenovirus, E-cadherin and Desmoglein 1 were decreased in melanocytes, WM164 and WM35. MAPK inhibitor PD98059 and PI3K inhibitor wortmannin partially blocked the downregulation, suggesting that both pathways are involved in this process. c-Met was coimmunoprecipitated with E-cadherin, Desmoglein 1 and Plakoglobin, suggesting that they form a complex (es) that acts to regulate intercellular adhesion. Together, the results indicate that autocrine HGF decouples melanomas from keratinocytes by downregulating E-cadherin and Desmoglein 1, therefore frees melanoma cells from the control by keratinocytes and allows dissemination of the tumor mass.

Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (hif-1) activity in hepg2 hepatoma cells

Abstract: Hepatocyte growth factor (HGF), a multifunctional cytokine of mesenchymal origin, activates the DNA binding of hypoxia inducible factor-1 (HIF-1) in the HepG2 cell line: the activated complex contained the inducible alpha subunit. An increased expression of HIF-1alpha (mRNA and nuclear protein levels) was observed. To investigate the molecular basis of the HIF-1 response under this non-hypoxic condition, we evaluated first the expression of putative target genes. We found a time-dependent increase in steady-state mRNA levels of heme oxygenase and urokinase plasminogen activator at 4 h, followed by that of urokinase receptor at 10 h. The enhanced expression of these genes might confer the invasive phenotype, since HGF is a proliferative and scatter factor. Second, we examined some aspects of HIF-1 activity regulation in HGF-treated cells with the following findings: (i) the activation of HIF-1 DNA binding was prevented by proteasome blockade, probably because stabilization of the cytosolic alpha-subunit protein level is not sufficient to generate a functional form: also under these conditions nuclear protein level of HIF-1alpha did not increase; (ii) N-acetylcysteine, a free radical scavenger, strongly decreased HIF-1 activation suggesting a role of reactive oxygen species in this process; (iii) the thiol reducing agent dithiothreitol was ineffective. Third, consistent with these data, N-acetylcysteine reduced the stimulatory effect of HGF on stress kinase activities, while p42/44 mitogen activated kinase (MAPK) was unmodified, suggesting an involvement of c-Jun-N-terminal kinase (JNK) and p38 MAPK in HIF-1 activation. Finally, LY 294002 induced the blockade of phosphatidylinositol 3-kinase (PI3K), one of the principal transducers of HGF/Met receptor signalling, prevented the enhancement of HIF-1 DNA binding and JNK activity, but the inhibition of p42/44 MAPK phosphorylation with PD 98059 was ineffective. In conclusion, we suggest that HGF triggers a signal transduction cascade involving PI3K and ultimately activates HIF-1.

Expression of the Invasion Factor Laminin γ2 in Colorectal Carcinomas Is Regulated by β-Catenin

Abstract: The migration-inducing γ2 chain of laminin-5, one of the best known invasion markers, is strongly overexpressed in disseminating and infiltrating tumor cells at the invasive front of colorectal carcinomas. The same tumor cells show nuclear accumulation of the oncoprotein β-catenin, which together with T-cell factor-DNA-binding proteins, functions as transcriptional activator of genes involved in tumor progression. Here we show that β-catenin activates the human laminin-5 γ2 gene through two T-cell factor-binding elements in a synergistic manner together with hepatocyte growth factor and conclude that laminin-5 γ2 is another important target gene of nuclear β-catenin during tumor progression.

Skeletal muscle cell activation by low-energy laser irradiation: a role for the MAPK/ERK pathway.

Abstract: Low-energy laser irradiation (LELI) has been shown to promote skeletal muscle regeneration in vivo and to activate skeletal muscle satellite cells, enhance their proliferation and inhibit differentiation in vitro. In the present study, LELI, as well as the addition of serum to serum-starved myoblasts, restored their proliferation, whereas myogenic differentiation remained low. LELI induced mitogen-activated protein kinase/extracellular signal-regulated protein kinase (MAPK/ERK) phosphorylation with no effect on its expression in serum-starved myoblasts. Moreover, a specific MAPK kinase inhibitor (PD098059) inhibited the LELI- and 10% serummediated ERK1/2 activation. However, LELI did not affect Jun N-terminal kinase (JNK) or p38 MAPK phosphorylation or protein expression. Whereas a 3-sec irradiation induced ERK1/2 phosphorylation, a 12-sec irradiation reduced it, again with no effect on JNK or p38. Moreover, LELI had distinct effects on receptor phosphorylation: it caused phosphorylation of the hepatocyte growth factor (HGF) receptor, previously shown to activate the MAPK/ERK pathway, whereas no effect was observed on tumor suppressor necrosis alpha (TNF-alpha) receptor which activates the p38 and JNK pathways. Therefore, by specifically activating MAPK/ERK, but not JNK and p38 MAPK enzymes, probably by specific receptor phosphorylation, LELI induces the activation and proliferation of quiescent satellite cells and delays their differentiation.

Matrilysin Mediates Extracellular Cleavage of E-Cadherin from Prostate Cancer Cells: A Key Mechanism in Hepatocyte Growth Factor/Scatter Factor-induced Cell-Cell Dissociation and in Vitro Invasion

Abstract: PURPOSE: The current study examined the effects of hepatocyte growth factor/scatter factor (HGF/SF) on cell-cell dissociation, invasion, and its association with the mediated release of matrix metalloproteinase-7 (Matrilysin) on the extracellular cleavage of E-cadherin in prostate cancer cells. EXPERIMENTAL DESIGN: The effects of HGF/SF on cell-cell dissociation, in vitro invasion, and on the expression of E-cadherin at both protein and mRNA levels were assessed in cells whose expression of Matrilysin was altered by treatment with antisense oligonucleotide. RESULTS: Incubation with HGF/SF mediated the release of active Matrilysin (M(r) 19,000), resulting in extracellular cleavage of E-cadherin from prostate cancer cells. This resultant soluble M(r) 80,000 fragment of E-cadherin was subsequently recognized upon immunoprobing with an anti-E-cadherin antibody. Both recombinant human Matrilysin (rh-Matrilysin) and/or HGF/SF increased the level of soluble E-cadherin and decreased the level of full-length (M(r) 120,000) E-cadherin as detected by Western blotting. The effects of rh-Matrilysin and HGF/SF were inhibited by an antisense oligonucleotide specifically directed toward human Matrilysin. In addition, stimulation with either rh-Matrilysin or HGF/SF resulted in disruption to the E-cadherin/beta-catenin complex, as shown by a significant increase (P < 0.05) in both cell scattering and invasion index. CONCLUSIONS: Treatment with HGF/SF induced Matrilysin-mediated cleavage to the extracellular domain of E-cadherin, resulting in its dissociation from the cadherin/catenin complex. This provides a new mechanism in HGF/SF-induced cell scattering, resulting in a switch to a more invasive phenotype in LNCapFGC cells, as demonstrated by in vitro invasion.

Skin antigens in the steady state are trafficked to regional lymph nodes by transforming growth factor-β1-dependent cells

Abstract: Antigen capturing in the skin and antigen trafficking into regional lymph nodes (LN) initiate immune responses. In this study, employing melanin granule (MG) as an easily traceable antigen in two mouse strains that carried steel factor or hepatocyte growth factor transgenes and had melanocytosis in the epidermis or in the dermis respectively, we investigated the mechanism of antigen trafficking from the skin. MG captured in the epidermis or dermis accumulated in the regional LN, but not other tissues. Only in alymphoplastic mice did MG-laden cells pass through the lymphatics and reached many tissues. Since inflammatory regions were not observed in the skin of either type of transgenic mouse, our developmental system enables us to investigate constitutive capturing and trafficking of insoluble antigens in the steady state. Both dendritic cells and macrophages were laden with MG in the regional LN. To determine which cells traffic antigens to the LN, we prepared double mutants that carried the transgenes and lacked transforming growth factor (TGF)-β1, since mice lacking TGF-β1 are reported to be deficient of Langerhans cells. Few MG were observed in the regional LN of these double-mutant mice. We also showed that signaling via macrophage colony stimulating factor receptor or Flt3/Flk2 is not essential for development of the cells for this antigen trafficking. These results indicate that antigens in the epidermis and dermis in the steady state are trafficked into regional LN only by TGF-β1-dependent cells, which may be a dendritic cell lineage.

Renal fibrosis: an update.

Abstract: Tubulointerstitial fibrosis invariably accompanies the course of chronic renal failure towards end-stage renal disease. Tubular epithelial cells, the predominant cell type in the tubulointerstitium, are increasingly being recognized for playing a dominant role as mediators of renal fibrogenesis. Tubular epithelial cells become activated either by the glomerular ultrafiltrate from their apical side or by mononuclear cells from their basolateral side. They initiate the scarring process by secreting chemokines, which in return attract mononuclear cells as well as growth factors that stimulate interstitial fibroblasts. In later phases of renal fibrogenesis, cellular changes of tubular epithelial cells contribute to the chronic impairment of renal function. Whereas tubular epithelial cells react by proliferation or hypertrophy to initial stimuli, they may undergo apoptosis or transdifferentiate into fibroblasts, and thus contribute to tubular atrophy in later stages of progressive renal disease. Resident interstitial fibroblasts are also important in renal fibrogenesis, and recent research has demonstrated that these cells are much more heterogeneous than expected. Cytokines such as fibroblast growth factor type 2 and epithelial growth factor have been shown to be pro-fibrogenic, whereas hepatocyte growth factor and bone morphogenic protein type 7 may inhibit fibrogenesis. Despite recent progress, further research is mandatory for a better understanding and the development of novel therapeutic approaches.

Therapeutic angiogenesis induced by human hepatocyte growth factor gene in rat diabetic hind limb ischemia model: molecular mechanisms of delayed angiogenesis in diabetes.

Abstract: Background Because no study has documented the angiogenic properties of hepatocyte growth factor (HGF) in a diabetes model, we examined the feasibility of gene therapy using HGF to treat peripheral arterial disease in diabetes. Methods and Results Because intramuscular injection of luciferase plasmid by the hemagglutinating virus of Japan (HVJ)–liposome method had much higher efficiency than injection of naked plasmid, we used the HVJ-liposome method to transfect the human HGF gene into the rat diabetic hindlimb model. As expected, transfection of human HGF vector resulted in a significant increase in blood flow as assessed by laser Doppler imaging and capillary density, even in the diabetes model, accompanied by the detection of human HGF protein. Interestingly, the degree of natural recovery of blood flow was significantly greater in nondiabetic rats than in diabetic rats. Thus, in an in vitro culture system, we further studied the molecular mechanisms of how diabetes delayed angiogenesis. Importantly, high–d-glucose treatment of endothelial cells resulted in a significant decrease in matrix metalloproteinase (MMP)-1 protein and ets-1 expression in human aortic endothelial cells. Similarly, high d-glucose significantly decreased mRNA and protein of HGF in endothelial cells. Downregulation of MMP-1 and ets-1 by high d-glucose might be due to a significant decrease in HGF, because HGF stimulated MMP-1 production and activated ets-1. Conclusions Overall, intramuscular injection of human HGF plasmid induced therapeutic angiogenesis in a rat diabetic ischemic hindlimb model as a potential therapy for peripheral arterial disease. The delay of angiogenesis in diabetes might be due to downregulation of MMP-1 and ets-1 through a decrease in HGF by high d-glucose. Received June 22, 2001; revision received August 22, 2001; accepted August 22, 2001.

Inhibition of growth, invasion, and metastasis of human pancreatic carcinoma cells by NK4 in an orthotopic mouse model.

Abstract: Hepatocyte growth factor (HGF) is involved in malignant behavior of cancers as a mediator in tumor-stromal interactions through enhancing tumor invasion and metastasis. We found recently that NK4, a four-kringle fragment of HGF, functions as both an HGF-antagonist and an angiogenesis inhibitor. We have now determined whether blockade of the HGF-c-Met/HGF receptor pathway and tumor angiogenesis by administration of recombinant NK4 would inhibit growth, invasion, and metastasis of human pancreatic carcinoma implanted into the pancreas of nude mice. When treatment with NK4 or anti-HGF neutralizing antibody was initiated from the third day after orthotopic injection of SUIT-2 human pancreatic cancer cells, both NK4 and anti-HGF antibody suppressed the conversion of orthotopic pancreatic tumors from carcinoma in situ to aberrantly invading cancers during days 3-14. On the other hand, when the treatment was begun on day 10, a time when cancer cells were already invading surrounding tissues, NK4 but not anti-HGF antibody inhibited tumor growth, peritoneal dissemination, and ascites accumulation at 4 weeks after the inoculation. Antitumor effects of NK4 correlated with decreased microvessel density in pancreatic tumors thereby indicating that the antiangiogenic activity of NK4 may have mainly contributed to its antitumor effects. Moreover, although NK4-treatment was initiated from the end stage (day 24 after tumor inoculation), NK4 prolonged survival time of mice, and the suppression of peritoneal dissemination, ascites accumulation, and invasion of metastasized cancer cells into the peritoneal wall were remarkable. We propose that simultaneous targeting of both tumor angiogenesis and the HGF-mediated invasion-metastasis may prove to be a new approach to treating patients with pancreatic cancer.

Transgenic Overexpression of Hepatocyte Growth Factor in the β-Cell Markedly Improves Islet Function and Islet Transplant Outcomes in Mice

Abstract: Recent advances in human islet transplantation have highlighted the need for expanding the pool of beta-cells available for transplantation. We have developed three transgenic models in which growth factors (hepatocyte growth factor [HGF], placental lactogen, or parathyroid hormone-related protein) have been targeted to the beta-cell using rat insulin promoter (RIP). Each displays an increase in islet size and islet number, and each displays insulin-mediated hypoglycemia. Of these three models, the RIP-HGF mouse displays the least impressive phenotype under basal conditions. In this study, we show that this mild basal phenotype is misleading and that RIP-HGF mice have a unique and salutary phenotype. Compared with normal islets, RIP-HGF islets contain more insulin per beta-cell (50 +/- 5 vs. 78 +/- 9 ng/islet equivalent [IE] in normal vs. RIP-HGF islets, P < 0.025), secrete more insulin in response to glucose in vivo (0.66 +/- 0.06 vs. 0.91 +/- 0.10 ng/ml in normal vs. RIP-HGF mice, P < 0.05) and in vitro (at 22.2 mmol/l glucose: 640 +/- 120.1 vs. 1,615 +/- 196.9 pg. microg protein(-1). 30 min(-1) in normal vs. RIP-HGF islets, P < 0.01), have two- to threefold higher GLUT2 and glucokinase steady-state mRNA levels, take up and metabolize glucose more effectively, and most importantly, function at least twice as effectively after transplantation. These findings indicate that HGF has surprisingly positive effects on beta-cell mitogenesis, glucose sensing, beta-cell markers of differentiation, and transplant survival. It appears to have a unique and unanticipated effective profile as an islet mass- and function-enhancing agent in vivo.

Mitogenic and Antiapoptotic Actions of Hepatocyte Growth Factor Through ERK, STAT3, and Akt in Endothelial Cells

Abstract: Hepatocyte growth factor (HGF), a member of the angiogenic growth factors, may play a pivotal role in the regulation of endothelial cells, inasmuch as HGF shows mitogenic and antiapoptotic actions ...

Differential regulation of sphingosine-1-phosphate- and VEGF-induced endothelial cell chemotaxis. Involvement of G(ialpha2)-linked Rho kinase activity.

Abstract: We compared stimulus-coupling pathways involved in bovine pulmonary artery (PA) and lung microvascular endothelial cell migration evoked by sphingosine-1-phosphate (S1P), a potent bioactive lipid released from activated platelets, and by vascular endothelial growth factor (VEGF), a well-recognized angiogenic factor. S1P-induced endothelial cell migration was maximum at 1 μ M ( ∼ 8-fold increase with PA endothelium) and surpassed the maximal response evoked by either VEGF (10 ng/ml) ( ∼ 2.5-fold increase) or hepatocyte growth factor (HGF) ( ∼ 2.5-fold increase). Migration induced by S1P, but not by VEGF, was significantly inhibited by treatment with antisense oligonucleotides directed to Edg-1 and Edg-3 (endothelial differentiation gene) S1P receptors and by G protein modification. These strategies included pretreatment with pertussis toxin, or transfection with mini-genes encoding a βγ subunit inhibitory peptide of the β -adrenergic receptor kinase, or an 11-amino-acid peptide that inhibits G1 α 2 signali...