Platelet-rich plasma (PRP) containing hepatocyte growth factor (HGF) and other growth factors are widely used in orthopaedic/sports medicine to repair injured tendons. While PRP treatment is reported to decrease pain in patients with tendon injury, the mechanism of this effect is not clear. Tendon pain is often associated with tendon inflammation, and HGF is known to protect tissues from inflammatory damages. Therefore, we hypothesized that HGF in PRP causes the anti-inflammatory effects. To test this hypothesis, we performed in vitro experiments on rabbit tendon cells and in vivo experiments on a mouse Achilles tendon injury model. We found that addition of PRP or HGF decreased gene expression of COX-1, COX-2, and mPGES-1, induced by the treatment of tendon cells in vitro with IL-1β. Further, the treatment of tendon cell cultures with HGF antibodies reduced the suppressive effects of PRP or HGF on IL-1β-induced COX-1, COX-2, and mPGES-1 gene expressions. Treatment with PRP or HGF almost completely blocked the cellular production of PGE2 and the expression of COX proteins. Finally, injection of PRP or HGF into wounded mouse Achilles tendons in vivo decreased PGE2 production in the tendinous tissues. Injection of platelet-poor plasma (PPP) however, did not reduce PGE2 levels in the wounded tendons, but the injection of HGF antibody inhibited the effects of PRP and HGF. Further, injection of PRP or HGF also decreased COX-1 and COX-2 proteins. These results indicate that PRP exerts anti-inflammatory effects on injured tendons through HGF. This study provides basic scientific evidence to support the use of PRP to treat injured tendons because PRP can reduce inflammation and thereby reduce the associated pain caused by high levels of PGE2.

Cross-communication between the Met receptor tyrosine kinase and the epidermal growth factor receptor (EGFR) has been proposed to involve direct association of both receptors and EGFR kinase-dependent phosphorylation. Here, we demonstrate that in human hepatocellular and pancreatic carcinoma cells the Met receptor becomes tyrosine phosphorylated not only upon EGF stimulation but also in response to G protein-coupled receptor (GPCR) agonists. Whereas specific inhibition of the EGFR kinase activity blocked EGF- but not GPCR agonist-induced Met receptor transactivation, it was abrogated in the presence of a reducing agent or treatment of cells with a NADPH oxidase inhibitor. Both GPCR ligands and EGF are further shown to increase the level of reactive oxygen species within the cell. Interestingly, stimulation of the Met receptor by either GPCR agonists, EGF or its cognate ligand HGF, resulted in release of Met-associated beta-catenin and in its Met-dependent translocation into the nucleus, as analyzed by small interfering RNA-mediated knockdown of the Met receptor. Our results provide a new molecular explanation for cell surface receptor cross-talk involving the Met receptor and thereby link the wide diversity of GPCRs and the EGFR to the oncogenic potential of Met signaling in human carcinoma cells.

OBJECTIVE

Hepatocyte growth factor (HGF), a potent mitogen for hepatocytes, binds to heparan sulfate. Because immunoreactive HGF can be detected in the interstitial extracellular matrix (ECM), where little heparan sulfate is found, the aim of this study was to investigate binding of HGF to several collagens and noncollagenous ECM proteins in vitro.

METHODS

125I-labeled HGF was incubated with collagens I-VI, single collagen chains and their cyanogen bromide peptides, with fibronectin, fibrinogen, and laminin that were either immobilized on polystyrene or blotted to nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biological activity of collagen-bound HGF was investigated in cell culture.

RESULTS

HGF displayed binding of moderate affinity (Kd approximately 10(-9) mol/L) to immobilized collagen types I, III, IV, V, and VI. Binding of HGF to all collagens could be inhibited by single chains of either collagens I, III, or VI. Fragmentation with cyanogen bromide indicated unique collagenous peptides mediating the interaction. Collagen-bound HGF induced primary hepatocyte proliferation and MDCK cell scattering in a dose-dependent manner.

CONCLUSIONS

Interstitial collagens I, III, V, and VI serve as abundant, low-affinity binding sites for HGF in the ECM. This interaction is mediated by unique collagenous peptides, opening the potential to modulate HGF availability and activity by collagen-derived peptide analogues.

Hepatocyte growth factor (HGF) and its high affinity receptor, the tyrosine kinase Met, play a key role in embryo development and tumor invasion. Both HGF and Met are established targets for cancer therapy. However, the mechanism of their interaction is complex and remains elusive. HGF is secreted as a monomeric precursor (pro-HGF) that binds to but does not activate Met. Mature HGF is a alpha/beta heterodimer containing a high affinity Met-binding site in the alpha-chain (HGF-alpha) and a low affinity Met-binding site in the beta-chain (HGF-beta). The extracellular portion of Met contains a semaphorin (Sema) domain, a cysteine-rich hinge (plexin-semaphorin-integrin), and four immunoglobulin-like domains (immunoglobulin-like regions in plexins and transcription factors (IPT) 1-4). HGF-beta binds to Sema through a low affinity contact. The domain of Met responsible for high affinity binding to HGF-alpha has not been identified yet. Here we show that this long sought after binding site lies in the immunoglobulin-like region of Met and more precisely in IPT 3 and 4. We also show that IPT 3 and 4 are sufficient to transmit the signal for kinase activation to the cytoplasm, although the lack of Sema makes the receptor equally sensitive to mature HGF and pro-HGF. Finally, we provide evidence that soluble Met-derived proteins containing either the low affinity or high affinity HGF-binding site antagonize HGF-induced invasive growth both in vitro and in xenografts. These data suggest that the immunoglobulin-like region of Met cooperates with the Sema domain in binding to HGF and in controlling Met kinase activity. Although the IPT-HGF-alpha interaction provides binding strength, the Sema-HGF-beta contact confers selective sensitivity to the active form of the ligand.

Hepatocyte growth factor/scatter factor (HGF/SF) has a cofactor requirement for heparan sulfate (HS) and dermatan sulfate (DS) in the optimal activation of its signaling receptor MET. However, these two glycosaminoglycans (GAGs) have different sugar backbones and sulfation patterns, with only the presence of iduronate in common. The structural basis for GAG recognition and activation is thus very unclear. We have clarified this by testing a wide array of natural and modified GAGs for both protein binding and activation. Comparisons between Ascidia nigra (2,6-O-sulfated) and mammalian (mainly 4-O-sulfated) DS species, as well as between a panel of specifically desulfated heparins, revealed that no specific sulfate isomer, in either GAG, is vital for interaction and activity. Moreover, different GAGs of similar sulfate density had comparable properties, although affinity and potency notably increase with increasing sulfate density. The weaker interaction with CS-E, compared with DS, shows that GlcA-containing polymers can bind, if highly sulfated, but emphasizes the importance of the flexible IdoA ring. Our data indicate that the preferred binding sites in DS in vivo will be comprised of disulfated, IdoA(2S)-containing motifs. In HS, clustering of N-/2-O-/6-O-sulfation in S-domains will lead to strong reactivity, although binding can also be mediated by the transition zones where sulfates are mainly at the N- and 6-O- positions. GAG recognition of HGF/SF thus appears to be primarily driven by electrostatic interactions and exhibits an interesting interplay between requirements for iduronate and sulfate density that may reflect in part a preference for particular sugar chain conformations.

OBJECTIVE

To explore the expression of stem cell genes in breast cancer and the relationship between stem cell gene expression and clinical and pathological characteristics and prognosis of breast cancer.

BACKGROUND

By now, stem cell differentiation-related genes and the relationship between the genes and clinic-pathological characteristics and prognosis of breast cancer are still unclear.

METHODS

CD44+/CD24- tumor cells were selected by Flow cytometry. The differential expression of genes between CD44+/CD24- tumor cells and non-CD44+/CD24- tumor cells were detected by RT(2) Profiler™ PCR Array. The expression of stem cell gene Octamer-4 (Oct-4) was analyzed by immunohistochemistry staining and the relationship between Oct-4 and clinicopathological parameters of breast cancer was determined.

RESULTS

Seven different genes including stem cell differentiation-related factors (CD44, Oct-4, and nestin), cell cycle regulators (APC and CDC2), and growth factors (HGF and TGF) were detected as significantly differently expressed between CD44+/CD24- tumor cells and non-CD44+/CD24- tumor cells. Oct-4 protein expressed significantly higher in cancerous tissues than adjacent-tumor tissues (P = 0.001). Moreover, we observed that the expression of Oct-4 protein was related to histological type, lymph node status and molecular type of breast cancer (P = 0.001, 0.006, and 0.001, respectively). After survival analysis, the cases with highly expressed Oct-4 protein attained a significantly poorer postoperative disease-specific survival than those with none/low expressed Oct-4 protein (P = 0.001). In the Cox regression test, tumor size, histological type, disease stage, lymph node metastasis, Her-2 and Oct-4 were detected as the independent prognostic factors (P = 0.031, 0.012, 0.001, 0.002, 0.030, and 0.003, respectively).

CONCLUSIONS

Oct-4 was highly expressed in CD44+/CD24- tumor cells, and may be a potential biomarker for the initiation, progression, and differentiation of breast cancer.

Hepatocyte growth factor (HGF), a potent mitogen for mature hepatocytes, possesses mitogenic and morphogenic activities for renal epithelial cells. To examine the renotropic function of HGF, we investigated the expression of HGF mRNA and HGF activity in the rat kidney after acute renal failure. When acute renal failure was induced by ischemia or by HgCl2 administration, a DNA synthesis occurred predominantly in the renal tubular cells located in the outer medulla with a peak at 48 h after the treatments. In both renal injuries, HGF mRNA in the kidney increased markedly, reaching a maximum 6 to 12 h after the treatments. HGF activity in the kidney also increased to three- to fourfold higher level than the normal level at 12 h after ischemic treatment or HgCl2 administration. In situ hybridization and immunohistochemical analysis indicated that both HGF mRNA and HGF protein were expressed in renal interstitial cells, presumably endothelial cells and macrophages, but not in tubular epithelial cells. In addition, HGF activity in the plasma of rats with renal ischemia or HgCl2 administration rapidly increased, reaching a maximum at 6 h after the treatment. One week after these injuries, HGF mRNA and HGF activity reverted to normal levels, and renal tubular cell regeneration ceased. Moreover, intravenous injection of human recombinant HGF into mice with acute renal failure caused by HgCl2 administration stimulated DNA synthesis of renal tubular cells in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Two major types of glycosaminoglycan (GAG) polysaccharides, heparan sulfate and chondroitin sulfate, are polymerized and modified by enzymes that are encoded by more than 40 genes in animal cells. Because of the expression repertoire of the GAG assembly and modification enzymes, each heparan sulfate and chondroitin sulfate chain has a sulfation pattern, chain length, and fine structure that is potentially unique to each animal cell. GAGs interact with hundreds of proteins. Such interactions protect growth factors, chemokines, and cytokines against proteolysis. GAGs catalyze protease (such as thrombin) inhibition by serpins. GAGs regulate multiple signaling pathways including, but not limited to, fibroblast growth factor (FGF)/FGFR, hepatocyte growth factor (HGF)/c-Met, glial cell line-derived neurotrophic factor (GDNF)/c-Ret/GFRalpha1, vascular endothelial growth factor (VEGF)/VEGFR, platelet derived growth factor (PDGF)/PDGFR, BAFF/TACI, Indian hedgehog, Wnt, and BMP signaling pathways,where genetic studies have revealed an absolute requirement for GAGs in these pathways. Most importantly, protein/GAG aggregates induce thrombin generation and immune system upregulation by activating the contact system. Abnormal protein/GAG aggregates are associated with a variety of devastating human diseases including, but not limited to, Alzheimer's, diabetes, prion or transmissible spongiform encephalopathies, Lupus, heparin-induced thrombocytopenia/thrombosis, and different kinds of cancers. Therefore, GAGs are essential components of modern molecular biology and human physiology. Understanding GAG structure and function at molecular level with regard to development and health represents a unique opportunity in combating different kinds of human diseases.

Mechanical ventilation at high tidal volumes compromises the blood-gas barrier and increases lung vascular permeability, which may lead to ventilator-induced lung injury and pulmonary edema. Using pulmonary endothelial cell (ECs) exposed to physiologically [5% cyclic stretch (CS)] and pathologically (18% CS) relevant magnitudes of CS, we evaluated the potential protective effects of hepatocyte growth factor (HGF) on EC barrier dysfunction induced by CS and vascular endothelial growth factor (VEGF). In static culture, HGF enhanced EC barrier function in a Rac-dependent manner and attenuated VEGF-induced EC permeability and paracellular gap formation. The protective effects of HGF were associated with the suppression of Rho-dependent signaling triggered by VEGF. Five percent CS promoted HGF-induced enhancement of the cortical F-actin rim and activation of Rac-dependent signaling, suggesting synergistic barrier-protective effects of physiological CS and HGF. In contrast, 18% CS further enhanced VEGF-induced EC permeability, activation of Rho signaling, and formation of actin stress fibers and paracellular gaps. These effects were attenuated by HGF pretreatment. EC preconditioning at 5% CS before HGF and VEGF further promoted EC barrier maintenance. Our data suggest synergistic effects of HGF and physiological CS in the Rac-mediated mechanisms of EC barrier protection. In turn, HGF reduced the barrier-disruptive effects of VEGF and pathological CS via downregulation of the Rho pathway. These results support the importance of HGF-VEGF balance in control of acute lung injury/acute respiratory distress syndrome severity via small GTPase-dependent regulation of lung endothelial permeability.

Previous studies from our laboratory have demonstrated that hepatocytes can transdifferentiate into biliary epithelium (BE) both in vivo and in vitro; however, the mechanisms are unclear. The current study was designed to investigate the mechanisms of hepatocyte transdifferentiation in vitro. Rat hepatocytes were cultured in roller bottles to obtain hepatocyte organoid cultures, which were stimulated with various growth factors (GFs) including hepatocyte growth factor (HGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), stem cell factor (SCF), macrophage-stimulating protein (MSP), fibroblast growth factor-a (FGF-a), fibroblast growth factor-b (FGF-b), and fibroblast growth factor-8b (FGF-8b). Only the cultures treated with HGF, EGF, and their combination exhibited formation of hepatocyte-derived biliary epithelium (BE) despite the presence and activation of all the pertinent cognate membrane receptors of the rest of the GFs. Microarray analysis of the organoid cultures identified specific up-regulation of approximately 500 target genes induced by HGF and EGF, including members of the extracellular matrix (ECM) protein family, Wnt/beta-catenin pathway, transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) pathway, and CXC (cysteine-any amino acid-cysteine) chemokines. To investigate the downstream signaling involved in hepatocyte to biliary epithelial cell (BEC) transdifferentiation, we investigated expression and activities of mitogen-activated protein (MAP) kinases [extracellular signal-regulated kinase (ERK)1/2, p38, and c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK)] as well as serine/threonine kinase AKT. The analysis indicated that AKT phosphorylation was particularly increased in cultures treated with HGF, EGF, and their combination. Whereas phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 completely inhibited biliary epithelium formation, AKT inhibitor could only moderately reduce formation of BE in the organoid cultures treated with HGF+EGF. Most of the HGF+EGF target genes were altered by LY294002.

CONCLUSIONS

Taken together, these data indicate that hepatocyte to BE transdifferentiation is regulated by HGF and EGF receptors and that PI3 kinase-mediated signaling independent of AKT is a crucial component of the transdifferentiation process.

RON is a member of the receptor tyrosine kinase gene family that includes the MET oncogene, whose germline mutations have been causally related to human tumorigenesis. In vitro, RON and MET receptors cross-talk, synergize in intracellular signaling, and cooperate in inducing morphogenic responses. Here we show that the RON and MET oncogenes were expressed in 55% and 56% of human ovarian carcinomas, respectively, and were significantly coexpressed in 42% (P < 0.001). In ovarian carcinoma samples and cell lines we did not find mutations in RON and MET gene kinase domain, nor coexpression of RON and MET receptor ligands (MSP and HGF, respectively). We show that motility and invasiveness of ovarian cancer cells coexpressing MET and RON receptors were elicited by HGF and, to a lesser extent, by MSP. More interestingly, invasion of both reconstituted basement membrane and collagen gel was greatly enhanced by the simultaneous addition of the two ligands. These data suggest that coexpression of the MET and RON receptors confer a selective advantage to ovarian cancer cells and might promote ovarian cancer progression.

Hematopoietic growth factors (HGF) are essential for proliferation and differentiation of hematopoietic precursors and activate a distinct set of JAK-STAT (Janus kinases-signal transducers and activators of transcription) proteins. Previous results from our group have shown a strong expression of JAK-STAT proteins in primary acute myelogenous leukemia (AML) blasts and AML cell lines. Here, we asked whether a constitutive activation of the JAK-STAT pathway might be involved in the pathogenesis of AML. We could demonstrate a constitutive activation of STAT1, 3 and 5 by immunoprecipitation of the tyrosine phosphorylated proteins in different human AML cell lines. Three patterns of STAT activation were found: (I) activation of only STAT1, (II) activation of STAT1 in combination with STAT3, and (III) activation of STAT1, 3 and 5. Furthermore, STAT1 and 3 formed stable heterodimers only in cell lines with constitutive STAT3 activation. In all cell lines analyzed, tyrosine phosphorylation of the four known Janus kinases could not be detected, although JAK1 was stably associated with STAT3. To further analyze whether a constitutive activation of tyrosine kinases might contribute to the autonomous growth of AML blasts, inhibitor studies were performed. The tyrphostin AG490, an inhibitor of the JAK-STAT pathway, but not A1, an inactive tyrphostin induced a time- and dose-dependent growth arrest without overt morphological signs of differentiation in AML cell lines. Our results show that STAT transcription factors are constitutively activated in human AML cell lines and might contribute to the autonomous proliferation of AML blasts. Inhibition of this pathway might be of interest for the establishment of more specific antileukemic strategies.

We recently demonstrated a cardiac therapeutic regimen based on injection of bone marrow mesenchymal stem cells (MSCs) into the skeletal muscle. Although the injected MSCs were trapped in the local musculature, the extracardiac cell delivery approach repaired the failing hamster heart. This finding uncovers a tissue repair mechanism mediated by trophic factors derived from the injected MSCs and local musculature that can be explored for minimally invasive stem cell therapy. However, the trophic factors involved in cardiac repair and their actions remain largely undefined. We demonstrate here a role of MSC-derived IL-6-type cytokines in cardiac repair through engagement of the skeletal muscle JAK-STAT3 axis. The MSC IL-6-type cytokines activated JAK-STAT3 signaling in cultured C2C12 skeletal myocytes and caused increased expression of the STAT3 target genes hepatocyte growth factor (HGF) and VEGF, which was inhibited by glycoprotein 130 (gp130) blockade. These in vitro findings were corroborated by in vivo studies, showing that the MSC-injected hamstrings exhibited activated JAK-STAT3 signaling and increased growth factor/cytokine production. Elevated host tissue growth factor levels were also detected in quadriceps, liver, and brain, suggesting a possible global trophic effect. Paracrine actions of these host tissue-derived factors activated the endogenous cardiac repair mechanisms in the diseased heart mediated by Akt, ERK, and JAK-STAT3. Administration of the cell-permeable JAK-STAT inhibitor WP1066 abrogated MSC-mediated host tissue growth factor expression and functional improvement. The study illustrates that the host tissue trophic factor network can be activated by MSC-mediated JAK-STAT3 signaling for tissue repair.

Although hepatocyte growth factor (HGF) and its receptor tyrosine kinase MET are widely expressed in the developing and mature central nervous system, little is known about the role of MET signaling in the brain. We have used particle-mediated gene transfer in cortical organotypic slice cultures established from early postnatal mice to study the effects of HGF on the development of dendritic arbors of pyramidal neurons. Compared with untreated control cultures, exogenous HGF promoted a highly significant increase in dendritic growth and branching of layer 2 pyramidal neurons, whereas inactivation of endogenous HGF with function-blocking, anti-HGF antibody caused a marked reduction in size and complexity of the dendritic arbors of these neurons. Furthermore, pyramidal neurons transfected with an MET dominant-negative mutant receptor likewise had much smaller and less complex dendritic arbors than did control transfected neurons. Our results indicate that HGF plays a role in regulating dendritic morphology in the developing cerebral cortex.

Minibrain-related kinase (Mirk)/Dyrk1B is an arginine-directed serine/threonine kinase that is active in skeletal muscle development but is also expressed in various carcinomas. In the current study, the Met adaptor protein Ran-binding protein M (RanBPM) was identified as a Mirk-binding protein by yeast two-hybrid analysis. The Mirk-RanBPM association was confirmed by glutathione S-transferase pull-down assays, co-immunoprecipitation studies, and in vivo cross-linking. Met plays an important role in tumor cell invasion and cell migration. RanBPM has been reported to bind to the tyrosine kinase domain of the hepatocyte growth factor (HGF) receptor Met, enhance Met downstream signaling, and enhance HGF-induced A704 kidney carcinoma cell invasion (Wang, D., Li, Z., Messing, E. M., and Wu, G. (2002) J. Biol. Chem. 277, 36216-36222). We made a stable Mirk-inducible subline from nontransformed Mv1Lu lung epithelial cells and now demonstrate that induction of Mirk inhibited the migration of these cells in wounding experiments and inhibited their invasion through polycarbonate Transwell filters. Furthermore the ability of Mirk to inhibit Mv1Lu cell migration was attenuated when cells were exposed to HGF or to elevated levels of transiently expressed RanBPM. RanBPM inhibited the kinase activity of Mirk/Dyrk1B and Dyrk1A. In addition, RanBPM and HGF inhibited the function of Mirk as a transcriptional coactivator. Our findings suggest that Mirk plays a role in modulating cell migration through opposing the action of the Met signaling cascade adaptor protein RanBPM.

Mutations in the genes encoding for Met, Ret and Kit receptor tyrosine kinases invariably result in increased kinase activity and in the acquisition of transforming potential. However, the requirement of receptor ligands for the transformation process is still unclear. We have investigated the role of hepatocyte growth factor (HGF), the high-affinity ligand for Met, in mutant Met-mediated cell transformation. We provide evidence that the transforming potential displayed by mutant forms of Met found in human cancer is not only sensitive but entirely dependent on the presence of HGF, by showing that mutant Met transforms NIH3T3 fibroblasts, which produce endogenous HGF, but is not able to transform epithelial cells, unless exogenous HGF is supplied. Accordingly, mutant Met-induced transformation of NIH3T3 cells can be inhibited by HGF antagonists and increased by HGF stimulation. We also show that an engineered Met receptor which contains an oncogenic mutation but is impaired in its ability to bind HGF completely loses its transforming activity, which can be rescued by causing receptor dimerization using a monoclonal antibody. These results indicate that point mutations resulting in Met kinase activation are necessary but not sufficient to cause cell transformation, the latter being dependent on ligand-induced receptor dimerization. They also suggest that mutant Met-driven tumour growth depends on the availability and tissue distribution of active HGF, and provide proof-of-concept for the treatment of mutant-Met related pathologies by HGF-antagonizing drugs.

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

Diabetic retinopathy is considered one of the vision-threatening diseases among working-age population. The pathogenesis of the disease is regarded multifactorial and complex: capillary basement membrane thickening, loss of pericytes, microaneuryms, loss of endothelial cells, blood retinal barrier breakdown and other anatomic lesions might contribute to macular edema and/or neovascularization the two major and sight threatening complications of diabetic retinopathy. A number of proangiogenic, angiogenic and antiangiogenic factors are involved in the pathogenesis and progression of diabetic retinal disease, Vascular Endothelial Growth Factor (VEGF) being one of the most important. Other growth factors, which are known to participate in the pathogenesis of the disease, are: Platelet Derived Growth Factor (PDGF), Fibroblast Growth Factor (FGF), Hepatocyte Growth Factor (HGF), Transforming Growth Factor (TGF), Placental Endothelial Cell Growth Factor (PlGF), Connective Tissue Growth Factor (CTGF). Other molecules that are involved in the disease mechanisms are: intergrins, angiopoietins, protein kinase C (PKC), ephrins, interleukins, leptin, angiotensin, monocyte chemotactic protein (MCP), vascular cell adhesion molecule (VCAM), tissue plasminogen activator (TPA), and extracellular matrix metalloproteinases (ECM-MMPs). However, the intraocular concentration of angiogenic factors is counterbalanced by the ocular synthesis of several antioangiogenic factors such as pigment epithelial derived factor (PEDF), angiostatin, endostatin, thrombospondin, steroids, atrial natriuretic peptide (ANP), inteferon, aptamer, monoclonal antibodies, VEGF receptor blocker, VEGF gene suppressors, intracellular signal transduction inhibitors, and extracellular matrix antagonists. Growth stimulation or inhibition by these factors depends on the state of development and differentiation of the target tissue. The mechanisms of angiogenesis factor action are very different and most factors are multipotential; they stimulate proliferation or differentiation of endothelial cells. This review attempts to briefly outline the knowledge about peptide growth factor involvement in diabetic retinopathy. Further ongoing research may provide better understanding of molecular mechanisms, disease pathogenesis and therapeutic interactions.

To understand the physiological functions of exogenous hepatocyte growth factor (HGF) on normal adult animals, we delivered human HGF gene into mice by a hydrodynamics-based in vivo gene transfection approach using a naked plasmid vector. Systemic administration of naked plasmid containing HGF cDNA driven under cytomegalovirus promoter (pCMV-HGF) by rapid injection via the tail vein produced a remarkable level of human HGF protein in the circulation, beginning to appear at 4 hours and peaking at 12 hours following injection. Tissue distribution studies identified the liver as the organ with the highest level of transgene expression. Through weekly repeated injections of plasmid vector, we achieved sustained, long-term, high levels of exogenous HGF expression in mice for 8 weeks. Increases of more than 31% and 16% in liver and body weights were found, respectively, in the mice that received pCMV-HGF plasmid compared with that given the control vector for 8 weeks. Expression of exogenous HGF in vivo activated mitogen-activated protein kinases and induced proliferating cell nuclear antigen expression in normal adult liver and kidneys. These data suggest that systemic administration of naked plasmid vector is a convenient, safe, and highly efficient approach to introduce and maintain exogenous HGF gene expression in vivo in a controllable fashion. Our results also indicate that long-term expression of human HGF in mice markedly activates growth-related signal transduction events, promotes cell proliferation, and leads to liver and overall body growth in whole adult animals.

Heptic fibrosis/cirrhosis is a common hepatic disease characterized by the hyper-accumulation of connective tissue components, and hepatic necrosis. Chronic alcohol ingestion, viral infection, and metabolic disorders are contributing factors and there has been no effective treatment. Hepatocyte growth factor (HGF), originally identified as a potent mitogen for mature hepatocytes, is a long-sought hepatotrophic factor for liver regeneration. Administration of human recombinant HGF into rats with hepatic fibrosis/cirrhosis caused by dimethylnitrosamine (DMN) elicited mitogenic action for hepatocytes, stimulated hepatic collagenase activity, and prevented the onset and progression of hepatic fibrosis/cirrhosis. Accumulation of fibrous tissue components in the liver due to DMN-treatment were markedly decreased in HGF-injected rats. Moreover, HGF completely abrogated death caused by severe hepatic cirrhosis and dysfunction. We postulate that HGF may prove to be an effective treatment for human liver fibrosis/cirrhosis and for chronic hepatic failure.

Invasion of various carcinoma cells follows their interaction with stromal cells. Hepatocyte growth factor (HGF), four-kringle-containing growth factor, is a mesenchymal or stromal-derived mediator which affects the growth and the invasiveness of carcinoma cells. We now have evidence that a four-kringle-containing antagonist for HGF, HGF/NK4 inhibits invasion of tumors in vivo, as well as in vitro. HGF/NK4 competitively inhibited the binding of HGF to Met/ HGF receptors on GB-d1 human gallbladder carcinoma cells. HGF induced invasion of the cells through Matrigel basement membrane components and into collagen gels, but HGF-induced invasion was inhibited by HGF/NK4. Invasion of GB-d1 cells was induced by co-cultivation with stromal fibroblasts, which mimics tumor-stromal interaction, but it was almost completely suppressed by HGF/NK4. Likewise, invasive growth induced by HGF in collagen gels in GB-dl cells, HuCC-T1 human cholangiocarcinoma cells, and ME-180 human uterus cervical carcinoma cells was also strongly inhibited by HGF/NK4. When GB-d1 cells were implanted subcutaneously into nude mouse, tumor cells invaded muscular tissue, but the infusion of HGF/NK4 inhibited this invasion. Furthermore, HGF/NK4 increased apoptotic cell death of GB-d1 cells and inhibited tumor growth in vivo. These results indicate that HGF/ NK4 may inhibit growth and invasion of carcinoma cells, as mediated by HGF during tumor-stromal interactions. We propose that there is a unique therapeutic potential for HGF/NK4 to prevent tumor invasion and perhaps even metastasis.

Activation of the HGF receptor, encoded by the c-MET protooncogene (Met receptor), triggers motility, matrix-invasion and branching morphogenesis in epithelial cells. It has recently been shown that the Met receptor interacts with Gab-1, an IRS-like adaptor protein, via the docking site (Y1349VHVNATY1356VNV) known to bind Grb2 and multiple SH2-containing signal transducers. Here we show that Gab1 is the major phosphorylation-substrate of the Met receptor and of its oncogenic variant Tpr-Met. A series of point mutations in the docking site established a direct correlation between the ability to recruit and phosphorylate Gab1 and the transforming potential. Interestingly, the mutations of either Y1356 or N1358 abolished the binding of both Grb2 and Gab1 in intact cells. Furthermore, peptides designed to block either the SH2 or the SH3 domains of Grb2 interfered with the receptor-Gab1 interaction. These data indicate that Gab1 coupling to the Met receptor requires binding of Grb2 and correlates with the transforming potential of Tpr-Met.

The c-met oncogene encodes the receptor for hepatocyte growth factor/scatter factor (HGF/SF), a multifunctional cytokine able to mediate morphogenesis as well as mitogenesis, motogenesis and invasiveness of epithelial cells. HGF/SF has been implicated in branching tubulogenesis of the developing kidney and in regeneration after renal injury and nephrectomy. We have examined the expression of the met/HGF receptor in normal human kidney and tissues of the genito-urinary tract, and in 50 kidney neoplasms of different histotypes, using monoclonal antibodies (MAbs) against the met/HGF receptor and immunohistochemistry. In normal kidneys, weak staining restricted to the distal tubules was observed. Transitional cell carcinomas were consistently negative, whereas increased expression at various levels was found in 87% of renal cell carcinomas with different cytological features and histological patterns. Western blot analysis of samples showed that the met/HGF receptor found in the malignant cells exhibits features of the normal receptor. The met/HGF receptor is also overexpressed in a renal cell carcinoma cell line, whose motility is triggered by HGF/SF. Our data suggest that expression of the met/HGF receptor may be involved in the onset and progression of renal cell carcinomas.

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.