Hypoxia is a condition always present in tumor environment owing to the fast growth of tumor cells not supported by adequate blood supply. There is increasing evidence that hypoxia plays an important role in cancer dormancy and cancer metabolism, increasing stemness activity and bringing about cancer initiation and progression . This condition may influence the production of hypoxia inducible factor (HIF) a helix transcription factor which is involved in carcinogenesis and tumor growth through the regulation of genes involved in angiogenesis, glycolytic metabolism and other biological mechanisms. In normoxia condition HIF is inactivated by prolyl hydroxylase enzymes (EGLN 1-3, also known as PHD 1-3) using oxygen as a substrate. Once hydroxilated it binds to a protein called Von Hippel Lindau protein (VHL) for its degradation, whereas in hypoxia condition stabilization and nuclear translocation occur, leading to oncogenes activation. It has got three isoforms HIF-1 HIF-2 and HIF-3. The most studied factor is HIF-1 which is a heterodimer consisting of two forms, the form α is expressed in manner oxygen dependent, the form β is expressed constitutively. Its presence in tumor microenvironment could foster among other the expression of VEGF, HGF, Met protoncogene which induces degradation of the extracellular matrix and TWIST gene, which is in turn involved in a mechanism of cancer cell metastasis called epithelial-mesenchimal transition(EMT). In this review, we summarize the most important findings in HIF action in different types of cancer focusing on its properties to induce tumor cell growth and highlighting its poor prognostic value in different cancers sites.

The generation of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β fuel the acute phase response (APR). To maintain body homeostasis, the increase of inflammatory proteins is resolved by acute phase proteins via presently unknown mechanisms. Hepatocyte growth factor (HGF) is transcribed in response to IL-6. Since IL-6 production promotes the generation of HGF and induces the APR, we posited that accumulating HGF might be a likely candidate for quelling excess inflammation under non-pathological conditions. We sought to assess the role of HGF and how it influences the regulation of inflammation utilizing a well-defined model of inflammatory activation, lipopolysaccharide (LPS)-stimulation of bone marrow derived macrophages (BMM). BMM were isolated from C57BL6 mice and were stimulated with LPS in the presence or absence of HGF. When HGF was present, there was a decrease in production of the pro-inflammatory cytokine IL-6, along with an increase in the anti-inflammatory cytokine IL-10. Altered cytokine production correlated with an increase in phosphorylated GSK3β, increased retention of the phosphorylated NFκB p65 subunit in the cytoplasm, and an enhanced interaction between CBP and phospho-CREB. These changes were a direct result of signaling through the HGF receptor, MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice. Combined, these data provide compelling evidence that under normal circumstances, HGF acts to suppress the inflammatory response.

We hypothesized that combined transgenic overexpression of hepatocyte growth factor (HGF) and placental lactogen in islets would lead to even greater increases in beta-cell mass and replication than either growth factor alone. This did not occur, suggesting that beta-cell replication is saturable or subject to molecular restraint. We therefore performed the first comprehensive G(1)/S cell cycle survey in islets, cataloguing the broad range of kinases, cyclins, and kinase inhibitors that control the G(1)/S transition in islets from normal, HGF, placental lactogen, and doubly transgenic mice. Many of the G(1)/S checkpoint regulators (E2Fs; pRb; p107; p130; cyclins D(1),(2),(3), A, and E; cdk-2; cdk-4; p15; p16; p18; p19; p21; p27; MDM2; p53; c-Myc; and Egr-1) are present in the murine islet. Most of these proteins were unaltered by overexpression of HGF or placental lactogen, either alone or in combination. In contrast, p21(cip) was uniquely, dramatically, and reproducibly upregulated in placental lactogen and HGF islets. p21(cip) was also present in, and upregulated in, proliferating human islets, localizing specifically in beta-cells and translocating to the nucleus on mitogenic stimulation. Homozygous p21(cip) loss releases islets from growth inhibition, markedly enhancing proliferation in response to HGF and placental lactogen.

Invasion is an essential cellular response that plays an important role in a number of physiological and pathological processes. Matrix metalloproteinase (MMP) production and cell movement are diverse cellular responses integral to the process of invasion. The complexity of the invasive process suggests the necessity of coordinate activation of more than one signaling pathway in order to activate specific factors responsible for regulating these cellular responses. In this report, we demonstrate that cell movement and MMP-9 production are both directly dependent on the activation of endogenous ERK signaling in hepatocyte growth factor (HGF)-or epidermal growth factor (EGF)-stimulated human epidermal keratinocytes. The kinetic profiles of endogenous MEK and ERK activity suggest that prolonged activation of these signal transducers is an underlying mechanism involved in stimulating cell motility and MMP-9 production. In support of this finding, a transient MEK/ERK signal elicited by keratinocyte growth factor (KGF) or insulin-like growth factor-1 (IGF-1) fails to stimulate these invasion-related responses. Specific inhibition of MEK leads to suppression of ERK activation, marked reduction in steady-state levels of c-Fos, and inhibition of cell movement and MMP-9 production. This occurs despite continued activation of JNK and c-Jun signaling in the presence of MEK-specific inhibition. In contrast, when JNK activity is specifically inhibited in HGF-stimulated cells, AP-1 activity is suppressed but cell motility is not affected. This evidence suggests that while ERK and JNK activity are necessary for AP-1 activation, ERK but not JNK is sufficient in stimulating cell motility.

Intestinal epithelial cells rest on a fibroblast sheath. Thus, factors produced by these fibroblasts may influence epithelial function in a paracrine fashion. We examined modulation of intestinal epithelial function by one such fibroblast product, scatter factor/hepatocyte growth factor (HGF/SF). This effect was studied in vitro by using model T84 intestinal epithelial cells. When applied to confluent T84 monolayers, HGF/SF attenuates transepithelial resistance to passive ion flow in a dose-dependent manner (maximum fall at 300 ng/ml, 28% control monolayer resistance, P < 0.001, ED50 of 1.2 nM), t1/2 of 20 h. This functional effect of HGF/SF and distribution of its receptor, c-met, are polarized to the basolateral membranes of T84 intestinal epithelial cells. HGF/SF effects on resistance are not attributable to altered transcellular resistance (opening of Cl- and/or basolateral K+ channels), cytotoxicity, or enhanced cell proliferation; they therefore represent specific regulation of paracellular tight junction resistance. Analysis with biochemically purified rodent HGF/SF and Madin-Darby canine kidney cells reveals that effects on paracellular tight junctions also occur in other nontransformed epithelia. Binding of HGF/SF to its receptor in T84 intestinal epithelial cells is accompanied by tyrosine phosphorylation of the receptor. Because loosening of intercellular junctions between cells could facilitate separation, spreading, and migration of epithelial cells during physiologic processes such as wound resealing, we determined the effects of HGF/SF on intestinal epithelial wound resealing using our previously published in vitro model (Nusrat, A., C. Delp, and J. L. Madara. 1992. J. Clin. Invest. 89:1501-1511). HGF/SF markedly enhanced wound closure >> 450% increase in rate, P < 0.001) by influencing the migratory and spreading response in not only cells adjoining the wound but also cells many positions removed from the wound. We thus speculate that HGF/SF may serve as an important cytokine that influences epithelial parameters such as transepithelial resistance and wound resealing. Further pharmacological approaches to manipulate HGF/SF signaling pathways may provide novel therapeutic strategies to enhance repair of intestinal epithelial erosions/ulcerations.

Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes in vitro. The receptor for HGF has recently been characterized as the product of the proto-oncogene c-met. We have examined the possible involvement of HGF in hepatic growth and differentiation in the rat. The experimental systems used were acetylaminofluorene treatment combined with partial hepatectomy to induce proliferation and differentiation of oval cells in adult liver and the pre- and postnatal liver. In the acetylaminofluorene model, Northern blot analysis showed that level of HGF transcripts increased one day after partial hepatectomy, reached a peak by day 6, were maintained at that level until day 13, and then declined, reaching normal level at 20 days. The expression of c-met also increased gradually, reached a peak around 9 to 13 days after partial hepatectomy, at which time oval cell proliferation was most prominent. In the developing liver, an elevated level of HGF transcripts was found between 4 and 21 days after birth. The expression of c-met also slightly increased at the same time. In situ hybridization showed that the transcripts for HGF were localized in desmin-positive Ito cells, whereas the transcripts for c-met were strongly expressed by oval cells. We have shown earlier that Ito cells and oval cells proliferate simultaneously and exist in close proximity in the acetylaminofluorene model and that Ito cells are a primary source of growth factors such as transforming growth factor-alpha and acidic fibroblast growth factors. The data presented here suggest that HGF is, in combination with other growth factors, involved in the proliferation and differentiation of oval cells via a paracrine mechanism.

Hepatocyte Growth Factor (HGF)/Scatter Factor secreted from sinusoidal endothelial cells and Kupffer cells in liver activates the c-Met tyrosine kinase receptor expressed on hepatocytes. Here we report yet another possible communication system through a different ligand and tyrosine kinase receptor in an opposite direction. We isolated and determined the primary structure of the entire coding region of rat flt-1 (fms-like tyrosine kinase), a receptor for Vascular Endothelial Growth Factor (VEGF). Using rat flt-1 cDNA as a probe we found that the flt-1 mRNA was expressed at very high levels in sinusoidal endothelial cells in normal rat liver, but was hardly detectable in hepatocytes. The transcripts of another VEGF receptor KDR/Flk-1 structurally related to Flt-1 was also expressed specifically in sinusoidal endothelial cells. On the other hand, VEGF mRNA was expressed weakly in hepatocytes, but not in the nonparenchymal cell fraction. Furthermore, in an in vitro culture system, VEGF demonstrated a remarkably specific growth-stimulatory activity as well as maintenance activity on the sinusoidal endothelial cells. These results suggest that hepatocytes regulate the proliferation and survival of the sinusoidal endothelial cells in liver in a paracrine manner. Therefore two reciprocal communication systems, VEGF-Flt receptor family and HGF-Met receptor, may exist in hepatic tissue.

OBJECTIVE

The authors sought to determine how hepatocyte growth factor (HGF) receptor c-Met and epidermal growth factor receptor (EGFR) cross talk in response to injury in human ARPE-19 cells.

METHODS

A scratch wound was made on a cell monolayer of ARPE-19 cells using a sequence-comb or a pipet tip, and it was allowed to heal in the presence or absence of HGF and heparin-binding EGF-like growth factor (HB-EGF). The activation of EGFR was analyzed by immunoprecipitation with EGFR antibody, followed by Western blotting with phosphotyrosine-specific antibody. Phosphorylation of extracellular signal-regulated kinase (ERK) and AKT (a major substrate of phosphatidylinositol 3'-kinase (PI3K) was assessed by Western blotting. The release of c-Met ectodomain into the culture media was determined by Western blotting using an antibody against the extracellular region. Cell migration was assessed by Boyden chamber migration assay.

RESULTS

ARPE-19 cells underwent spontaneous wound healing in basal medium, and exogenously added HB-EGF and HGF significantly enhanced wound closure. Basal and growth factor-enhanced wound closures were attenuated but not slowed by hydroxyurea, a cell proliferation inhibitor. RPE cells expressed all four erbBs, and wounding induced EGFR transactivation and downstream ERK and PI3K phosphorylation in ARPE-19 cells. HGF also induced EGFR tyrosine phosphorylation. The EGFR kinase inhibitor AG1478 blocked wound- and HGF-stimulated EGFR transactivation and attenuated spontaneous and growth factor-induced wound closure. Wounding and EGFR ligands induced the release of c-Met into the culture media. Moreover, pretreatment of cells with HB-EGF impaired ARPE-19 migration toward HGF in a matrix metalloproteinase inhibitor-sensitive manner.

CONCLUSIONS

EGFR modulates HGF/c-Met activity by inducing c-Met ectodomain shedding, and HGF/c-Met transactivates EGFR, leading to an enhanced activation of downstream signaling pathways. Cross talk between EGFR and c-Met may play a key role in regulating RPE cell migration, proliferation, and wound healing.

Human islet expansion in monolayer culture leads to loss of function and senescence. By maintaining the 3-D configuration of islets in fibrin gels, it is feasible to expand beta-cells in response to hepatocyte growth factor (HGF) while preserving physiologic glucose responsiveness both in vitro and in vivo after transplantation into nude mice. Islets were cultured free floating with or without growth factors and nicotinamide and in fibrin gels with the same conditions. Proliferation was observed only in islets cultured in fibrin gels and the cocktail; total insulin increased by threefold, with a concomitant increase in beta-cell mass by morphometry. Insulin release after glucose challenge was also preserved. Islets in fibrin gels gave rise in vivo to large grafts rich in insulin and glucagon, and grafts from free-floating islets were smaller with fewer endocrine cells. Circulating human C-peptide levels were higher than in the mice receiving free-floating islets. In summary, fibrin allows for HGF-mediated cell proliferation while preserving glucose responsiveness in an environment that preserves cell-cell contacts. Limited islet ex vivo expansion under these conditions may improve recipient-donor tissue ratios to equal the functional results of whole-organ transplants.

As a rule, hepatocyte growth factor/scatter factor (HGF/SF) is produced by mesenchymal cells, while its receptor, the tyrosine kinase encoded by the met proto-oncogene, is expressed by the neighboring epithelial cells in a canonical paracrine fashion. In the present work we show that both HGF/SF and met are coexpressed by undifferentiated C2 mouse myoblasts. In growing cells, the autocrine loop is active as the receptor exhibits a constitutive phosphorylation on tyrosine that can be abrogated by exogenously added anti-HGF/SF neutralizing antibodies. The transcription of HGF/SF and met genes is downregulated when myoblasts stop proliferating and differentiate. The coexpression of HGF/SF and met genes is not exclusive to C2 cells since it has been assessed also in other myogenic cell lines and in mouse primary satellite cells, suggesting that HGF/SF could play a role in muscle development through an autocrine way. To analyze the biological effects of HGF/SF receptor activation, we stably expressed the constitutively activated receptor catalytic domain (p65(tpr-met)) in C2 cells. This active kinase determined profound changes in cell shape and inhibited myogenesis at both morphological and biochemical levels. Notably, a complete absence of muscle regulatory markers such as MyoD and myogenin was observed in p65(tpr-met) highly expressing C2 clones. We also studied the effects of the ectopic expression of human isoforms of met receptor (h-met) and of HGF/SF (h-HGF/SF) in stable transfected C2 cells. Single constitutive expression of h-met or h-HGF/SF does not alter substantially the growth and differentiation properties of the myoblast cells, probably because of a species-specific ligand-receptor interaction. A C2 clone expressing simultaneously both h-met and h-HGF/SF is able to grow in soft agar and shows a decrease in myogenic potential comparable to that promoted by p65(tpr-met) kinase. These data indicate that a met kinase signal released from differentiation-dependent control provides a negative stimulus for the onset of myogenic differentiation.

The met protooncogene tyrosine kinase receptor (Met) and its ligand, hepatocyte growth factor/scatter factor (HGF/SF), ordinarily constitute a paracrine signaling system in which cells of mesenchymal origin produce the ligand, which binds to the receptor that is predominantly expressed in cells of epithelial origin. However, mouse NIH/3T3 fibroblasts overexpressing Met induce tumor formation in nude mice via an autocrine mechanism (S. Rong et al., Mol. Cell. Biol., 12: 5152-5158, 1992). In this study, we report that human cell lines established from various sarcomas express high levels of activated Met receptor. HGF/SF is also detected in the human sarcoma cell lines but at a reduced level when compared to primary fibroblasts. These properties, high Met expression and reduced ligand levels, are indistinguishable from the properties of NIH/3T3 tumor explant cells overexpressing Met (S. Rong et al., Mol. Cell. Biol., 12: 5152-5158, 1992; S. Rong et al., Cell Growth & Differ., 4: 563-569, 1993). Moreover, paraffin-embedded sections of primary tumors from human osteosarcomas, chondrosarcomas, and leiomyosarcoma stain intensely for Met and/or HGF/SF and display extensive tumor cell heterogeneity with regard to both paracrine and autocrine stimulation. On the basis of these findings, we propose that Met-HGF/SF autocrine signaling may contribute to the tumorigenic process in human sarcomas.

BACKGROUND

Mesenchymal stem cells (MSCs) are multipotent stem cells able to differentiate into different cell lineages. However, MSCs represent a subpopulation of a more complex cell composition of stroma cells contained in mesenchymal tissue. Due to a lack of specific markers, it is difficult to distinguish MSCs from other more mature stromal cells such as fibroblasts, which, conversely, are abundant in mesenchymal tissue. In order to find more distinguishing features between MSCs and fibroblasts, we studied the phenotypic and functional features of human adipose-derived MSCs (AD-MSCs) side by side with normal human dermal fibroblasts (HNDFs) in vitro

METHODS

AD-MSCs and HNDFs were cultured, expanded and phenotypically characterized by flow cytometry (FC). Immunofluorescence was used to investigate cell differentiation. ELISA assay was used to quantify angiogenic factors and chemokines release. Cultures of endothelial cells (ECs) and a monocyte cell line, U937, were used to test angiogenic and anti-inflammatory properties.

RESULTS

Cultured AD-MSCs and HNDFs display similar morphological appearance, growth rate, and phenotypic profile. They both expressed typical mesenchymal markers-CD90, CD29, CD44, CD105 and to a minor extent, the adhesion molecules CD54, CD56, CD106 and CD166. They were negative for the stem cell markers CD34, CD146, CD133, CD117. Only aldehyde dehydrogenase (ALDH) was expressed. Neither AD-MSCs nor HNDFs differed in their multi-lineage differentiation capacity; they both differentiated into osteoblast, adipocyte, and also into cardiomyocyte-like cells. In contrast, AD-MSCs, but not HNDFs, displayed strong angiogenic and anti-inflammatory activity. AD-MSCs released significant amounts of VEGF, HGF and Angiopoietins and their conditioned medium (CM) stimulated ECs proliferation and tube formations. In addition, CM-derived AD-MSCs (AD-MSCs-CM) inhibited adhesion molecules expression on U937 and release of RANTES and MCP-1. Finally, after priming with TNFα, AD-MSCs enhanced their anti-inflammatory potential; while HNDFs acquired pro-inflammatory activity.

CONCLUSIONS

AD-MSCs cannot be distinguished from HNDFs in vitro by evaluating their phenotypic profile or differentiation potential, but only through the analysis of their anti-inflammatory and angiogenic properties. These results underline the importance of evaluating the angiogenic and anti-inflammatory features of MSCs preparation. Their priming with inflammatory cytokines prior to transplantation may improve their efficacy in cell-based therapies for tissue regeneration.

The ATP analog K252a is a potent inhibitor for receptor tyrosine kinases of the Trk family. Here we show that nanomolar concentrations of K252a prevent HGF-mediated scattering in MLP-29 cells (30 nM), reduce Met-driven proliferation in GTL-16 gastric carcinoma cells (100 nM), and cause reversion in NIH3T3 fibroblasts transformed by the oncogenic form of the receptor, Tpr-Met (75 nM). K252a inhibits Met autophosphorylation in cultured cells and in immunoprecipitates and prevents activation of its downstream effectors MAPKinase and Akt. Interestingly, K252a seems to be more effective at inhibiting the mutated form of Met (M1268T) found in papillary carcinoma of the kidney than the wild type receptor. Pretreatment of both Tpr-Met-transformed NIH3T3 fibroblasts and of GTL-16 gastric carcinoma cells with K252a results in loss of their ability to form lung metastases in nude mice upon injection into the caudal vein. These observations suggest that K252a derivatives, which are active in vivo as anti-cancer drugs in models of Trk-driven malignancies, should also be effective for treatment of Met-mediated tumors.

Porphyromonas gingivalis is a predominant periodontal pathogen, which expresses a number of potential virulence factors involved in the pathogenesis of periodontitis. Among them, fimbriae are a critical factor to mediate the bacterial interaction with host tissues, which promotes the bacterial adhesion to and invasion of the targeted sites. Fimbriae are capable of binding to human salivary components, commensal bacteria, and a variety of host cells including macrophages, epithelial cells, and fibroblasts. Human extracellular matrix (ECM) proteins such as vitronectin and fibronectin play important roles in cellular signal transduction via binding to receptor integrins. Fimbriae showed significant binding affinity to ECM proteins and clearly inhibited the molecular interactions between vitronectin/fibronectin and their receptor alphavbeta3 and alpha5beta1 integrins overexpressed on Chinese hamster ovary (CHO) cell strain. P. gingivalis fimbriae are likely to interrupt the cellular signaling via ECM proteins/integrins in periodontal regions. Fimbriae are also thought to be critically important in invasive events of the organism to host cells. The fimA genes, encoding FimA (a subunit of fimbriae), of P. gingivalis strains are classified into 5 types, I to V. Recent clinical investigations demonstrated the close relationship between the organisms with type II fimA and periodontitis development. Recombinant FimA (rFimA) proteins of types I to V were generated to compare their adhesion/invasion abilities to human gingival fibroblasts (HGF) and a human epithelial cell line (HEp-2 cells), respectively. There were no significant differences in the adhesion ability of microspheres (MS) coated with these rFimAs to HGF; however, the adhesion of type II rFimA-MS to HEp-2 cells was significantly greater than that of other rFimA types. It was also observed that the type II rFimA-MS markedly invaded the epithelial cells and accumulated around the nuclei. Collectively, these findings suggest that fimbriae of P. gingivalis, especially type II, are involved in the initiation and progression of human periodontitis.

Renal inflammation, characterized by the influx of inflammatory cells, is believed to play a critical role in the initiation and progression of a wide range of chronic kidney diseases. Here, we show that hepatocyte growth factor (HGF) inhibited renal inflammation and proinflammatory chemokine expression by disrupting nuclear factor (NF)-kappaB signaling. In vivo, HGF gene delivery inhibited interstitial infiltration of inflammatory T cells and macrophages, and suppressed expression of both RANTES (regulated on activation, normal T cell expressed and secreted) and monocyte chemoattractant protein-1 in a mouse model of obstructive nephropathy. In vitro, HGF abolished RANTES induction in human kidney epithelial cells, which was dependent on NF-kappaB signaling. HGF did not significantly affect the phosphorylation or degradation of IkappaBalpha; it also did not influence the phosphorylation or nuclear translocation of p65 NF-kappaB. However, HGF prevented p65 NF-kappaB binding to its cognate cis-acting element in the RANTES promoter. HGF action was dependent on the activation of the phosphoinositide 3-kinase/Akt pathway, which led to the phosphorylation and inactivation of glycogen synthase kinase (GSK)-3beta. Suppression of GSK-3beta activity mimicked HGF and abolished RANTES expression, whereas ectopic expression of GSK-3beta restored RANTES induction. HGF also induced renal GSK-3beta phosphorylation and inactivation after obstructive injury in vivo. These observations suggest that HGF is a potent anti-inflammatory cytokine that inhibits renal inflammation by disrupting NF-kappaB signaling and may be a promising therapeutic agent for progressive renal diseases.

The muscle wound healing occurs in three overlapping phases: (1) degeneration and inflammation, (2) muscle regeneration, and (3) fibrosis. Simultaneously to injury cellular infiltration by neutrophils and macrophages occur, as well as cellular 'respiratory burst' via activation of the enzyme NADPH oxidase. When skeletal muscle is stretched or injured, myogenic satellite cells are activated to enter the cell cycle, divide, differentiate and fuse with muscle fibers to repair damaged regions and to enhance hypertrophy of muscle fibers. This process depends on nitric oxide (NO) production, metalloproteinase (MMP) activation and release of hepatocyte growth factor (HGF) from the extracellular matrix. Generation of a fibrotic scar tissue, with partial loss of function, can also occur, and seems to be dependent, at least in part, on local TGF-beta expression, which can be downregulated by NO. Hence, regeneration the muscle depends on the type and severity of the injury, the appropriate inflammatory response and on the balance of the processes of remodeling and fibrosis. It appears that in all these phases NO exerts a significant role. Better comprehension of this role, as well as of the participation of other important mediators, may lead to development of new treatment strategies trying to tip the balance in favor of greater regeneration over fibrosis, resulting in better functional recovery.

Neural stem cell (NSC) transplantation has exhibited considerable therapeutic potential in spinal cord injury. However, most experiments in animals have been performed by injecting these cells directly into the injured spinal cord. A cardinal feature of NSCs is their exceptional migratory ability through the nervous system. Based on the migratory ability of NSCs, we investigated whether minimally invasive intravenous delivery of NSCs could facilitate their migration to the injured spinal cord and identified the chemo-attractants secreted by the lesions. Nude mice were injected intravenously with labelled human NSCs at 3, 7 and 10 days after the compression of the spinal cord at the T8 level. The migration of NSCs to the lesioned spinal cord was highest at 7 days after injury; this correlated with the peak of hepatocyte growth factor and stromal cell-derived factor-1 mRNA expressions in the lesion but not with the disruption of the blood-brain barrier. Finally, the grafted NSCs differentiated into neuronal and glial subpopulations at 21 days after transplantation. Our study suggests that intravenously administered NSCs can be employed as a renewable source for replacing lost cells for the treatment of spinal cord injuries.

It is well recognized that the majority of cancer related deaths is caused by metastatic diseases. Therefore, there is an urgent need for the development of therapeutic intervention specifically targeted to the metastatic process. In the last decade, significant progress has been made in this research field, and many new concepts have emerged that shed light on the molecular mechanism of metastasis cascade which is often portrayed as a succession of six distinct steps; localized invasion, intravasation, translocation, extravasation, micrometastasis and colonization. Successful metastasis is dependent on the balance and complex interplay of both the metastasis promoters and suppressors in each step. Therefore, the basic strategy of our interventions is aimed at either blocking the promoters or potentiating the suppressors in this disease process. Toward this goal, various kinds of antibodies and small molecules have been designed. These include agents that block the ligand-recepter interaction of metastasis promoters (HGF/c-Met), antagonize the metastasis-promoting enzymes (AMF, uPA and MMP) and inhibit the transcriptional activity of metastasis promoter (beta-Catenin). On the other hand, the intriguing roles of metastasis suppressors and their signal pathways have been extensively studied and various attempts have been made to potentiate these factors. Small molecules have been developed to restore the expression or mimic the function of metastasis-suppressor genes such as NM23, E-cadherin, Kiss-1, MKK4 and NDRG1, and some of them are under clinical trials. This review summarizes our current understanding of the molecular pathway of tumor metastasis and discusses strategies and recent development of anti-metastatic drugs.

Hepatocyte growth factor (HGF) and its receptor MET play an important role in cancer growth and metastasis. Activation of MET elicit multiple cellular responses regulating cell survival, morphogenesis, adhesion, migration, breakdown of extracellular matrix (ECM) and angiogenesis. Numerous disorders related to deregulation of HGF-MET axis have been reported. Thus, new therapeutic agents targeting HGF-MET signaling have been sought. Here, we will present data describing the role of HGF-MET axis in growth and metastasis of tumor cells together with the recent approaches to block this axis.

Recent reports have indicated that mesenchymal stromal cells (MSCs) from bone marrow have a potential in vascular remodeling and angiogenesis. Here, we report a unique phenomenon that under serum-deprived conditions MSCs survive and replicate. Secretome analysis of MSCs grown under serum-deprived conditions (SD-MSCs) identified a significant upregulation of prosurvival and angiogenic factors including VEGF-A, ANGPTs, IGF-1, and HGF. An ex vivo rat aortic assay demonstrated longer neovascular sprouts generated from rat aortic rings cultured in SD-MSC-conditioned media compared to neovascular sprouts from aortas grown in MSC-conditioned media. With prolonged serum deprivation, a subpopulation of SD-MSCs began to exhibit an endothelial phenotype. This population expressed endothelial-specific proteins including VEGFR2, Tie2/TEK, PECAM/CD31, and eNOS and also demonstrated the ability to uptake acetylated LDL. SD-MSCs also exhibited enhanced microtubule formation in an in vitro angiogenesis assay. Modified chick chorioallantoic membrane (CAM) angiogenesis assays showed significantly higher angiogenic potential for SD-MSCs compared to MSCs. Analysis of CAMs grown with SD-MSCs identified human-specific CD31-positive cells in vascular structures. We conclude that under the stress of serum deprivation MSCs are highly angiogenic and a population of these cells has the potential to differentiate into endothelial-like cells.

Liver regeneration depends on timely restoration of cellular mass while orchestrating structural matrix remodeling. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the extracellular matrix (ECM) turnover and, more recently, the processing of growth factors and cytokines. We have previously demonstrated that TIMP-1 inhibits preneoplastic hepatocyte proliferation by attenuating growth factor bioavailability. In the present study, we examined the role of TIMP-1 in de novo hepatocyte cell division during liver regeneration. Comprehensive real-time reverse-transcriptase polymerase chain reaction analyses of regenerating livers revealed significant inductions in the messenger RNA of TIMP-1, TIMP-3, TIMP-4, MMP-2, MMP-9, MMP-13, MMP-14, and MMP-24, while MMP-15 expression was significantly reduced. Induction of TIMP-1 occurred during the peak of hepatocyte DNA synthesis. Studies using genetically altered mice revealed that TIMP-1 loss of function accelerated hepatocyte cell cycle progression. This finding was demonstrated by earlier expression of cyclin D1, proliferating cell nuclear antigen, and phosphorylated histone H3, which mark the G(1)-S, S, and M phase, respectively. Conversely, TIMP-1 gain of function delayed cell cycle progression. MMP activity was increased in the absence of Timp-1. Examination of hepatocyte growth factor (HGF), and its receptor Met, both of which provide a mitogenic signal for hepatocyte division, showed increased HGF activity in Timp-1(-/-)-regenerating livers. HGF is released from the ECM and is proteolytically processed to its active form. Active HGF was elevated in Timp-1(-/-) mice, leading to increased immunostaining of phosphorylated Met as well as activation of a downstream effector, p38. In conclusion, TIMP-1 is a novel negative regulator of HGF activity during liver regeneration.

Occlusive cerebrovascular disease leads to brain ischemia that causes neurological deficits. Here we introduce a new strategy combining mesenchymal stromal cells (MSCs) and ex vivo hepatocyte growth factor (HGF) gene transferring with a multimutated herpes simplex virus type-1 vector in a rat transient middle cerebral artery occlusion (MCAO) model. Gene-transferred MSCs were intracerebrally transplanted into the rats' ischemic brains at 2 h (superacute) or 24 h (acute) after MCAO. Behavioral tests showed significant improvement of neurological deficits in the HGF-transferred MSCs (MSC-HGF)-treated group compared with the phosphate-buffered saline (PBS)-treated and MSCs-only-treated group. The significant difference of infarction areas on day 3 was detected only between the MSC-HGF group and the PBS group with the superacute treatment, but was detected among each group on day 14 with both transplantations. After the superacute transplantation, we detected abundant expression of HGF protein in the ischemic brain of the MSC-HGF group compared with others on day 1 after treatment, and it was maintained for at least 2 weeks. Furthermore, we determined that the increased expression of HGF was derived from the transferred HGF gene in gene-modified MSCs. The percentage of apoptosis-positive cells in the ischemic boundary zone (IBZ) was significantly decreased, while that of remaining neurons in the cortex of the IBZ was significantly increased in the MSC-HGF group compared with others. The present study shows that combined therapy is more therapeutically efficient than MSC cell therapy alone, and it may extend the therapeutic time window from superacute to acute phase.

The hypoxic environment in tumor is reported to play an important role in pancreatic cancer progression. The interaction between stromal and cancer cells also contributes to the malignant behavior of pancreatic cancer. In the present study, we investigated whether hypoxic stimulation affects stromal as well as pancreatic cancer cells. Our findings demonstrated that hypoxia remarkably elevated the HIF-1alpha expression in both pancreatic cancer (PK8) and fibroblast cells (MRC5). Hypoxic stimulation accelerated the invasive activity of PK8 cells, and invasiveness was thus further accelerated when the hypoxic PK8 cells were cultured with conditioned medium prepared from hypoxic MRC5 cells (hypoxic conditioned medium). MMP-2, MMP-7, MT1-MMP and c-Met expressions were increased in PK8 cells under hypoxia. Hypoxic stimulation also increased the hepatocyte growth factor (HGF) secretion from MRC5 cells, which led to an elevation of c-Met phosphorylation in PK8 cells. Conversely, the elevated cancer invasion, MMP activity and c-Met phosphorylation of PK8 cells were reduced by the removal of HGF from hypoxic conditioned medium. In immunohistochemical study, the HIF-1alpha expression was observed in surrounding stromal as well as pancreatic cancer cells, thus indicating hypoxia exists in both of cancer and stromal cells. Moreover, the stromal HGF expression was found to significantly correlate with not only the stromal HIF-1alpha expression but also the c-Met expression in cancer cells. These results indicate that the hypoxic environment within stromal as well as cancer cells activates the HGF/c-Met system, thereby contributing to the aggressive invasive features of pancreatic cancer.

Hepatocyte growth factor (HGF)/Met system is deregulated in tumors and is implicated in different aspects of invasive growth. Here, we report that in the highly aggressive MDA-MB231 breast carcinoma cells, Met cytosolic fragments [C-terminal fragment (CTF)] were present in the nuclei. They were constitutively active because tyrosine phosphorylated at regulatory and catalytic domains and endowed with transactivating activity independently of HGF exposure. In fact, various constructs containing juxtamembrane (Jxtm) Met fragments, fused with Gal4 DNA-binding domain, transactivated Gal4Luc activity. MDA-MB231 cells were devoid of WW domain-containing oxidoreductase (Wwox) tumor suppressor. Exogenous Wwox protein expression negatively regulated Jxtm3-transactivating activity and decreased spontaneous migration of MDA-MB231 cells. Also, we demonstrate that the lack of endogenous Wwox in MDA-MB231 cells represented a molecular mechanism for intranuclear Met-CTF accumulation and for the decrease of full-length Met stability. Yes-associated proteins maintained constitutively activated nuclear Met fragments that played a role as transcription factors regulating genes probably including those for motile phenotype. The difference with low invasive MCF-7 cells was evident because the latter did not show intranuclear Met and the transfected constructs-containing Jxtm fragments were inactive also in the presence of HGF. The constitutive activation of nuclear Met-signaling pathway in MDA-MB231 cells, possibly determined at genetic or epigenetic levels of WWOX gene, might participate in breast carcinoma progression influencing invasive/metastatic phenotype. Wwox/Met system can be suggested as a potential target to impair breast carcinoma progression.