Hypoxia-inducible factor-1alpha (HIF-1alpha) is widely considered to be one of the key regulators of tumor angiogenesis. The upstream regulation is complex and involves several growth factors, cytokines, and hypoxia. Herein, we have identified miR-519c as a hypoxia-independent regulator of HIF-1alpha, acting through direct binding to the HIF-1alpha 3' untranslated region and leading to reduced tumor angiogenesis. Overexpression of miR-519c resulted in a significant decrease of HIF-1alpha protein levels and reduced the tube formation of human umbilical vein endothelial cells; similarly, antagomir inhibition of miR-519c increased the level of HIF-1alpha protein and enhanced angiogenic activity, suggesting an important role of miR-519c in HIF-1alpha-mediated angiogenesis. Consistent with the overexpression of miR-519c in cancer patients with better prognosis, mice injected with miR-519c-overexpressing cells exhibited dramatically reduced HIF-1alpha levels, followed by suppressed tumor angiogenesis, growth, and metastasis. In addition, we found that hepatocyte growth factor (HGF), a known HIF-1alpha inducer, reduced the miR-519c levels through an Akt-dependent pathway. This regulation was posttranscriptional and may be mediated by suppression of miR-519c maturation. Taken together, our findings provide the first evidence that miR-519c is a pivotal regulator of tumor angiogenesis and that microenvironmental HGF contributes to regulating miR-519c biogenesis in cancer cells.

The vascular system that ensures an adequate blood flow is required to provide the cells with sufficient supply of nutrients and oxygen. Two different mechanisms of the formation of new vessels can be distinguished: vasculogenesis, the formation of the first primitive vascular plexus de novo and angiogenesis, the formation of new vessels from preexisting ones. Both processes are regulated by a delicate balance of pro- and anti-angiogenic factors. Physiologically, angiostatic mediators outweigh the angiogenic molecules and angiogenesis does not occur. Under certain conditions such as tumor formation or wound healing, the positive regulators of angiogenesis predominate and the endothelium becomes activated. Angiogenesis is initiated by vasodilatation and an increased permeability. After destabilization of the vessel wall, endothelial cells proliferate, migrate and form a tube, which is finally stabilized by pericytes and smooth muscle cells. Numerous soluble growth factors and inhibitors, cytokines and proteases as well as extracellular matrix proteins and adhesion molecules strictly control this multi-step process. The properties and interactions of angiogenic molecules such as VEGFs, FGFs, angiopoietins, PDGF, angiogenin, angiotropin, HGF, CXC chemokines with ELR motif, PECAM-1, integrins and VE-cadherin as well as angiostatic key players such as angiostatin, endostatin, thrombospondin, CXC chemokines without ELR motif, PEDF are discussed in this review with respect to their molecular impact on angiogenesis.

The c-MET oncogene encodes the receptor for the Hepatocyte Growth Factor/Scatter Factor (HGF), a cytokine that stimulates the invasive growth of normal and neoplastic cells. The Met/HGF receptor is expressed by epithelial cells and its ligand by cells of mesenchymal origin. Receptor-ligand interaction occurs via a paracrine circuit. We studied the expression of the Met/HGF receptor and of its ligand in mesenchymal human tumours by examining 39 clinical samples of bone tumours. The Met/HGF receptor was not detectable in the majority of bone tumours, as expected from their mesenchymal origin. Notably, the receptor was overexpressed in 60% of the osteosarcomas examined. In 12 osteosarcoma cell lines the Met/HGF receptor was overexpressed, phosphorylated by HGF stimulation and fully functional. HGF was detected in two out of seven clinical specimens of osteosarcoma. The ligand and the receptor are co-expressed in two clonal osteosarcoma cell lines. In these lines the Met/HGF receptor was constitutively phosphorylated; phosphorylation was suppressed by suramin treatment, a known blocker of autocrine loops. These data suggest that activation of the Met/HGF receptor by a paracrine or an autocrine mechanism might play a role in the particularly aggressive behaviour of osteosarcomas.

The development of an altered stromal microenvironment is a common feature of many tumours including squamous cell carcinoma (SCC), and there is increasing evidence that these changes in the stroma, which include increased expression of proteases and cytokines, may actually promote tumour progression. A common finding is that stromal fibroblasts become 'activated' myofibroblasts, expressing smooth muscle actin and secreting cytokines, proteases and matrix proteins. We show that myofibroblasts are commonly found in the stroma of oral SCC and are often concentrated at the invasive margin of the tumour. Using oral SCC cells and primary oral fibroblasts, we demonstrate that tumour cells directly induce a myofibroblastic phenotype, and that this transdifferentiation is dependent on SCC-derived TGF-beta1. In turn, myofibroblasts secrete significantly higher levels of hepatocyte growth factor/scatter factor compared with fibroblast controls, and this cytokine promotes SCC invasion through Matrigel, a mixture of basement membrane proteins. This is the first time that this double paracrine mechanism has been demonstrated between squamous carcinoma cells and fibroblasts, and emphasises that cancer invasion can be promoted indirectly by the release of tumour-induced host factors from stroma.

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.

OBJECTIVE

Cetuximab, an antibody targeting the epidermal growth factor receptor (EGFR), is active in colorectal cancer (CRC). However, response rates range from only 10% to 20%. Here, we investigate hepatocyte growth factor (HGF)-dependent mesenchymal-epithelial transition factor (MET) activation as a mediator of cetuximab resistance through signal diversification in CRC cell lines.

METHODS

DiFi, GEO, and LIM1215 cells were treated with varying concentrations and combinations of EGF, HGF, cetuximab, and PHA-665752 (a highly specific MET kinase inhibitor). Biological end points included proliferation, cell cycle arrest, and apoptosis. Proliferation was measured using WST-1 assays and synergy investigated via isobolograms. Expression and signaling were examined using immunoblotting.

RESULTS

EGFR and MET are coexpressed in these CRC cell lines, and dual receptor activation synergistically increased proliferation. Cetuximab inhibited cell growth by 60%-80% with an associated dephosphorylation of EGFR, MAPK, and/or AKT. Addition of HGF to cetuximab-treated cells phosphorylated MET, but not EGFR or ErbB3, restimulated the MAPK and AKT pathways, restored cell proliferation, and rescued cells from G1 arrest and apoptosis. Importantly, this effect could be abrogated by inhibiting MET activation with PHA-665752 or by downregulating MET expression with RNAi.

CONCLUSIONS

HGF-induced MET activation is a novel mechanism of cetuximab resistance in CRC. Inhibition of the HGF-MET pathway may improve response to EGFR inhibitors in CRC, and combination therapy should be further investigated.

Heart failure carries a poor prognosis with few treatment options. While myocardial stem cell therapeutic trials have traditionally relied on intracoronary infusion or intramyocardial injection routes, these cell delivery methods are invasive and can introduce harmful scar tissue, arrhythmia, calcification, or microinfarction in the heart. Given that patients with heart failure are at an increased surgical risk, the development of a noninvasive stem cell therapeutic approach is logistically appealing. Taking advantage of the trophic effects of bone marrow mesenchymal stem cells (MSCs) and using a hamster heart failure model, the present study demonstrates a novel noninvasive therapeutic regimen via the direct delivery of MSCs into the skeletal muscle bed. Intramuscularly injected MSCs and MSC-conditioned medium each significantly improved ventricular function 1 mo after MSC administration. MSCs at 4 million cells/animal increased fractional shortening by approximately 40%, enhanced capillary and myocyte nuclear density by approximately 30% and approximately 80%, attenuated apoptosis by approximately 60%, and reduced fibrosis by approximately 50%. Myocyte regeneration was evidenced by an approximately twofold increase in the expression of cell cycle markers (Ki67 and phosphohistone H(3)) and an approximately 13% reduction in mean myocyte diameter. Increased circulating levels of hepatocyte growth factor (HGF), leukemia inhibitory factor, and macrophage colony-stimulating factor were associated with the mobilization of c-Kit-positive, CD31-positive, and CD133-positive progenitor cells and a subsequent increase in myocardial c-Kit-positive cells. Trophic effects of MSCs further activated the expression of HGF, IGF-II, and VEGF in the myocardium. The work highlights a cardiac repair mechanism mediated by trophic cross-talks among the injected MSCs, bone marrow, and heart that can be explored for noninvasive stem cell therapy.

Multipotent mesenchymal stem cells (MSCs) in adult tissue are known to be less immunogenic and immunosuppressive. Previous study showed that primary cultures of human adipose-derived stem cells (ADSCs) shared their immunomodulatory properties with other MSCs. However, whether passaged human ADSCs can retain their immunomodulatory effect after in vitro expansion remains unknown. In addition, the mechanism of ADSC-mediated immunomodulatory effect remains to be elucidated. This study aimed to investigate these issues by using passaged human ADSCs as an in vitro study model. Flow cytometry showed that passaged ADSCs expressed human leukocyte antigen (HLA) class I but not class II molecules, which could be induced to express to a high level with interferon-gamma (IFN-gamma) treatment. The study found that passaged ADSCs could not elicit lymphocyte proliferation after co-culturing with them, even after IFN-gamma treatment. In addition, either IFN-gamma-treated or non-treated ADSCs could inhibit phytohemagglutinin (PHA)-stimulated lymphocyte proliferation. Moreover, passaged ADSCs could serve as the third-party cells to inhibited two-way mixed lymphocyte reaction (MLR). Further study using a transwell system also showed that this type of immunosuppressive effect was not cell-cell contact dependent. In defining possible soluble factors, we found that passaged ADSCs significantly increased their secretion of prostaglandin E2 (PGE2), but not transforming growth factor-beta (TGF-beta) and hepatocyte growth factor (HGF), when they were co-cultured with MLR. Furthermore, the result demonstrated that only PGE2 production inhibitor indomethacine, but not TGF-beta- and HGF-neutralizing antibodies, could significantly counteract ADSC-mediated suppression on allogeneic lymphocyte proliferation. These results indicated that in vitro expanded ADSCs retain low immunogenicity and immunosuppressive effect, and PGE2 might be the major soluble factor involved in the in vitro inhibition of allogeneic lymphocyte reaction.

The CCN (cyr61, ctgf, nov) family of modular proteins regulate diverse biological affects including cell adhesion, matrix production, tissue remodelling, proliferation and differentiation. Recent targeted gene disruption studies have demonstrated the CCN family to be developmentally essential for chondrogenesis, osteogenesis and angiogenesis. CCN2 is induced by agents such as angiotensin II, endothelin-1, glucocorticoids, HGF, TGFbeta, and VEGF, and by hypoxia and biomechanical and shear stress. Dysregulated expression of CCN2 has also been widely documented in many fibroproliferative diseases. This mini-review will focus on CCN2, and the recent progress in understanding CCN2 gene regulation in health and disease. That CCN2 should be considered a novel and informative surrogate clinical bio-marker for fibroproliferative disease is discussed.

Activation of the hepatocyte growth factor (HGF) receptor c-met results in the regulation of cell-matrix interactions, including the MAPK-dependent stimulation of epithelial cell morphogenesis. In the present study we demonstrate that HGF stimulates the localization of ERK to sites of cell-matrix interactions and that this is mediated by the tyrosine phosphorylation-dependent association of inactive ERK and the focal adhesion complex protein paxillin. In addition, paxillin was found to associate with the upstream MAP kinases Raf and MEK, resulting in a complex that can mediate localized ERK activation. Mutation of the ERK binding site in paxillin prevented HGF-stimulated ERK-paxillin association and eliminated HGF-induced cell spreading and branching process formation. These experiments reveal that paxillin-dependent ERK activation at sites of cell-matrix interaction is critical for HGF-stimulated epithelial morphogenesis.

Hypaxial skeletal muscles develop from migratory and non-migratory precursor cells that are generated by the lateral lip of the dermomyotome. Previous work shows that the formation of migratory precursors requires the c-Met and SF/HGF genes. We show here that in mice lacking c-Met or SF/HGF, the initial development of the dermomyotome proceeds appropriately and growth and survival of cells in the dermomyotome are not affected. Migratory precursors are also correctly specified, as monitored by the expression of Lbx1. However, these cells remain aggregated and fail to take up long range migration. We conclude that parallel but independent cues converge on the migratory hypaxial precursors in the dermomyotomal lip after they are laid down: a signal given by SF/HGF that controls the emigration of the precursors, and an as yet unidentified signal that controls Lbx1. SF/HGF and c-Met act in a paracrine manner to control emigration, and migratory cells only dissociate from somites located close to SF/HGF-expressing cells. During long range migration, prolonged receptor-ligand-interaction appears to be required, as SF/HGF is expressed both along the routes and at the target sites of migratory myogenic progenitors. Mice that lack c-Met die during the second part of gestation due to a placental defect. Rescue of the placental defect by aggregation of tetraploid (wild type) and diploid (c-Met-/-) morulae allows development of c-Met mutant animals to term. They lack muscle groups that derive from migratory precursor cells, but display otherwise normal skeletal musculature.

BACKGROUND

Scatter factor/hepatocyte growth factor (SF/HGF) is a pleiotropic growth factor that stimulates proliferation and migration of endothelial cells (ECs) via the c-Met receptor, present on ECs as well as other cell types, including smooth muscle cells (SMCs). We studied the effects of recombinant human (rh) SF/HGF in vitro and in vivo in a rabbit model of hindlimb ischemia. We further compared these effects with those of recombinant human vascular endothelial growth factor (rhVEGF165), an EC-specific mitogen.

RESULTS

In vitro, rhSF/HGF and rhVEGF165 exhibited similar effects on proliferation and migration of ECs. When both cytokines were administered together, the result was an additive effect on EC proliferation and a synergistic effect on EC migration. Application of rhSF/HGF to cultures of human SMCs resulted in the induction of VEGF mRNA and protein. In vivo, administration of rhSF/HGF (500 microg x 3) was associated with significant improvements in collateral formation (P<.001) and regional blood flow (P<.0005) and with a significant reduction in muscle atrophy (P<.0001). These effects were significantly more pronounced than those of rhVEGF165 administered according to the same protocol (P<.05). Neither remote angiogenesis nor other pathological sequelae were observed with either rhSF/HGF or rhVEGF165.

CONCLUSIONS

The pleiotropic effects of certain growth factors may potentiate angiogenesis via a combination of direct effects on EC proliferation and migration and indirect effects that result in the generation of other potent EC mitogens from non-EC populations. The synergistic effects demonstrated when SF/HGF and VEGF are administered together in vitro may be reproduced in vivo by SF/HGF-induced upregulation of VEGF in vascular SMCs.

Accumulating evidence suggests that progenitor cells may decrease destructive inflammation and reduce tissue loss by antiapoptotic mechanisms. However, they remain poorly characterized, and many questions remain regarding the mechanisms by which they may positively affect wound healing, tissue remodeling, or tissue regeneration. It has been speculated that various growth factors are responsible, but what components of the wound milieu stimulate progenitor cell production of growth factors and by what mechanisms? We hypothesized that tumor necrosis factor-alpha (TNF-alpha) stimulated progenitor cell secretion of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and insulin-like growth factor I (IGF-I) by a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. Human mesenchymal stem cells (hMSCs) and human adipose progenitor cells (hAPCs) were divided into four groups: control, p38 MAPK inhibitor (p38MKI), TNF, and TNF + p38MKI. After 24 h of incubation, supernatants were harvested for ELISA of VEGF, HGF, and IGF-I. Cells were collected for Western blot analysis of p38 MAPK activation. Secretion of VEGF, HGF, and IGF-I in hMSCs and hAPCs was significantly increased by stimulation with TNF and was associated with increased activation of p38 MAPK. The p38 MAPK inhibitor decreased production of TNF-stimulated VEGF, HGF, and IGF-I in hMSCs and hAPCs. However, p38 MAPK inhibitor alone had no effect on production of growth factors. These data demonstrate that progenitor cells are potent sources of VEGF, HGF, and IGF-I. TNF, a prominent tissue cytokine, strongly stimulated production of growth factors by hMSCs and hAPCs via a p38 MAPK-dependent mechanism.

Since the discovery that non-small cell lung cancer (NSCLC) is driven by epidermal growth factor receptor (EGFR) mutations, the EGFR tyrosine kinase inhibitors (EGFR-TKIs, e.g., gefitinib and elrotinib) have been effectively used for clinical treatment. However, patients eventually develop drug resistance. Resistance to EGFR-TKIs is inevitable due to various mechanisms, such as the secondary mutation (T790M), activation of alternative pathways (c-Met, HGF, AXL), aberrance of the downstream pathways (K-RAS mutations, loss of PTEN), impairment of the EGFR-TKIs-mediated apoptosis pathway (BCL2-like 11/BIM deletion polymorphism), histologic transformation, ATP binding cassette (ABC) transporter effusion, etc. Here we review and summarize the known resistant mechanisms to EGFR-TKIs and provide potential targets for development of new therapeutic strategies.

Several hematopoietic growth factors, including interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-beta1), promote the differentiation of tolerogenic dendritic cells (DCs). Hepatocyte growth factor (HGF) is a pleiotropic cytokine whose effects on human DC differentiation and function have not been investigated. Monocytes cultured with HGF (HGFMo) differentiated into accessory cells with DC-like morphology, released low amounts of IL-12p70 and up-regulated IL-10 both at the mRNA and at the protein level. Upon activation with HGFMo, allogeneic CD4+CD25- T cells expressed the T regulatory (Treg)-associated transcription factor FoxP3, proliferated poorly, and released high levels of IL-10. Interestingly, blockade of surface immunoglobulin-like transcript 3 (ILT3) on HGFMo or neutralization of secreted IL-10 translated into partial restoration of T-cell proliferation. Secondary stimulation of HGFMo-primed CD4+ T cells with immunogenic DCs differentiated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 from monocytes of the same donor resulted in measurable T-cell proliferation. HGFMo-primed CD4+ T cells significantly inhibited the proliferation of naive CD4+CD25- T cells in a cell-contact-dependent manner. Finally, DNA microarray analysis revealed a unique gene-expression profile of HGF-activated monocytes. Collectively, our findings point to a novel role for HGF in the regulation of monocyte/DC functions that might be exploited therapeutically.

Macrophage-stimulating protein (MSP) is a member of the hepatocyte growth factor-scatter factor (HGF-SF) family. Labeled MSP bound to Madin-Darby canine kidney (MDCK) cells transfected with complementary DNA encoding Ron, a cell membrane protein tyrosine kinase. Cross-linking of 125I-labeled MSP to transfected cells (MDCK-RE7 cells) and immunoprecipitation by antibodies to Ron revealed a 220-kilodalton complex, a size consistent with that of MSP (80 kilodaltons) cross-linked to the beta chain of Ron (150 kilodaltons). The binding of 125I-labeled MSP to MDCK-RE7 cells was inhibited by unlabeled MSP, but not by HGF-SF. MSP caused phosphorylation of the beta chain of Ron and induced migration of MDCK-RE7 cells. These results establish the ron gene product as a specific cell-surface receptor for MSP.

During bone growth, development, and remodeling, angiogenesis as well as osteogenesis are closely associated processes, sharing some essential mediators. Vascular endothelial growth factor (VEGF) was initially recognized as the best-characterized endothelial-specific growth factor, which increased vascular permeability and angiogenesis, and it is now apparent that this cytokine regulates multiple biological functions in the endochondral ossification of mandibular condylar growth, as well as long bone formation. The complexity of VEGF biology is paralleled by the emerging complexity of interactions between VEGF ligands and their receptors. This narrative review summarizes the family of VEGF-related molecules, including 7 mammalian members, namely, VEGF, placenta growth factor (PLGF), and VEGF-B, -C, -D, -E, and -F. The biological functions of VEGF are mediated by at least 3 corresponding receptors: VEGFR-1/Flt-1, VEGFR-2/Flk-1, VEGFR-3/Flt-4 and 2 co-receptors of neuropilin (NRP) and heparan sulfate proteoglycans (HSPGs). Current findings on endochondral ossification are also discussed, with emphasis on VEGF-A action in osteoblasts, chondroblasts, and chondroclasts/osteoclasts and regulatory mechanisms involving oxygen tension, and some growth factors and hormones. Furthermore, the therapeutic implications of recombinant VEGF-A protein therapy and VEGF-A gene therapy are evaluated. Abbreviations used: VEGF, Vascular endothelial growth factor; PLGF, placenta growth factor; NRP, neuropilin; HSPGs, heparan sulfate proteoglycans; FGF, fibroblast growth factor; TGF, transforming growth factor; HGF, hepatocyte growth factor; TNF, tumor necrosis factor; ECM, extracellular matrix; RTKs, receptor tyrosine kinases; ERK, extracellular signal kinases; HIF, hypoxia-inducible factor.

Although hepatocyte growth factor (HGF) can act synergistically or antagonistically with transforming growth factor-beta (TGF-beta) signaling, molecular mechanism of their crosstalk remains unknown. Using antibodies which selectively distinguished receptor-regulated Smads (R-Smads) phosphorylated at linker regions from those at C-terminal regions, we herein showed that either HGF or TGF-beta treatment of normal stomach-origin cells activated the JNK pathway, thereafter inducing endogenous R-Smads phosphorylation at linker regions. However, the phosphorylation at their C-terminal regions was not induced by HGF treatment. The activated JNK could directly phosphorylate R-Smads in vitro at the same sites that were phosphorylated in response to TGF-beta or HGF in vivo. Thus, the linker regions of R-Smads were the common phosphorylation sites for HGF and TGF-beta signaling pathways. The phosphorylation induced by simultaneous treatment with HGF and TGF-beta allowed R-Smads to associate with Smad4 and to translocate into the nucleus. JNK pathway involved HGF and TGF-beta-mediated infiltration potency since a JNK inhibitor SP600125 caused the reduction of invasive capacity induced by HGF and TGF-beta signals. Moreover, a combined treatment with HGF and TGF-beta led to a potent increase in plasminogen activator inhibitor type 1 transcriptional activity through Smad3 phosphorylation at the linker region. In contrast, HGF treatment reduced TGF-beta-dependent activation of p15INK4B promoter, in which Smad3 phosphorylation at the C-terminal region was involved. In conclusion, HGF and TGF-beta transmit the signals through JNK-mediated R-Smads phosphorylation at linker regions.

BACKGROUND

Mesenchymal stem cell (MSC)-based regenerative strategies were investigated to treat acute myocardial infarction and improve left ventricular function.

RESULTS

Murine AMI was induced by coronary ligation with subsequent injection of MSCs, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), or MSCs +HGF/VEGF into the border zone. Left ventricular ejection fraction was calculated using micro-computed tomography imaging after 6 months. HGF and VEGF protein injection (with or without concomitant MSC injection) significantly and similarly improved the left ventricular ejection fraction and reduced scar size compared with the MSC group, suggesting that myocardial recovery was due to the cytokines rather than myocardial regeneration. To provide sustained paracrine effects, HGF or VEGF overexpressing MSCs were generated (MSC-HGF, MSC-VEGF). MSC-HGF and MSC-VEGF showed significantly increased in vitro proliferation and increased in vivo proliferation within the border zone. Cytokine production correlated with MSC survival. MSC-HGF- and MSC-VEGF-treated animals showed smaller scar sizes, increased peri-infarct vessel densities, and better preserved left ventricular function when compared with MSCs transfected with empty vector. Murine cardiomyocytes were exposed to hypoxic in vitro conditions. The LDH release was reduced, fewer cardiomyocytes were apoptotic, and Akt activity was increased if cardiomyocytes were maintained in conditioned medium obtained from MSC-HGF or MSC-VEGF cultures.

CONCLUSIONS

This study showed that (1) elevating the tissue levels of HGF and VEGF after acute myocardial infarction seems to be a promising reparative therapeutic approach, (2) HGF and VEGF are cardioprotective by increasing the tolerance of cardiomyocytes to ischemia, reducing cardiomyocyte apoptosis and increasing prosurvival Akt activation, and (3) MSC-HGF and MSC-VEGF are a valuable source for increased cytokine production and maximize the beneficial effect of MSC-based repair strategies.

Dietary selenium has potent cancer prevention activity. Both low molecular weight selenocompounds and selenoproteins are implicated in this effect. Thioredoxin reductase 1 (TR1) is one of the major antioxidant and redox regulators in mammals that supports p53 function and other tumor suppressor activities. However, this selenium-containing oxidoreductase is also overexpressed in many malignant cells and has been proposed as a target for cancer therapy. To further assess the role of TR1 in the malignancy process, we used RNA interference technology to decrease its expression in mouse lung carcinoma (LLC1) cells. Stable transfection of LLC1 cells with a small interfering RNA construct that specifically targets TR1 removal manifested a reversal in the morphology and anchorage-independent growth properties of these cancer cells that made them similar to those of normal cells. The expression of at least two cancer-related protein mRNAs, Hgf and Opn1, were reduced dramatically in the TR1 knockdown cells. Mice injected with the TR1 knockdown showed a dramatic reduction in tumor progression and metastasis compared with those mice injected with the corresponding control vector. In addition, tumors that arose from injected TR1 knockdown cells lost the targeting construct, suggesting that TR1 is essential for tumor growth in mice. These observations provide direct evidence that the reduction of TR1 levels in malignant cells is antitumorigenic and suggest that the enzyme is a prime target for cancer therapy.

Mesenchymal stem cells (MSC) inhibit the response of allogeneic T lymphocytes in culture. Because the mechanisms of this effect may differ according to the existence of cell contact, we investigated the differences in gene expression of inhibitory molecules during MSC-T lymphocyte coculture when cell contact does and does not occur. Human MSC and T lymphocytes were cultured together in standard and transwell cultures. MSC gene expression was analyzed by semiquantitative real-time RT-PCR. MSC elicited a high dose-dependent inhibition of T lymphocytes in cultures with cell contact, but inhibition occurred even without cell contact. In both cases, we observed significant upregulation of IDO, LIF, and HLA-G, along with downregulation of HGF and SDF1. In cultures with cell contact, IL-10 and TGF-beta transcripts were expressed in a significantly higher level than in cultures without this contact. Furthermore, in the latter, the increased inhibition of T-cell proliferation was positively correlated with IDO gene expression and negatively correlated with SDF1 gene expression. MSC appear to induce T-cell tolerance by two distinct mechanisms. The first of these, which does not require cell contact, induces expression of the tolerogenic genes IDO, LIF, and HLA-G. The second mechanism, which is contact dependent, modulates IL-10 and TGF-beta gene expression. These two mechanisms probably play separate roles in MSC-induced tolerance in allogeneic hematopoietic stem cell transplantation.

Treatment of traumatic brain injury (TBI) with bone marrow stromal cells (MSCs) improves functional outcome in the rat. However, the specific mechanisms by which introduced MSCs provide benefit remain to be elucidated. Currently, the ability of therapeutically transplanted MSCs to replace injured parenchymal CNS tissue appears limited at best. Tissue replacement, however, is not the only possible compensatory avenue in cell transplantation therapy. Various growth factors have been shown to mediate the repair and replacement of damaged tissue, so trophic support provided by transplanted MSCs may play a role in the treatment of damaged tissue. We therefore investigated the temporal profile of various growth factors, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF), within cultures of human MSCs (hMSCs) conditioned with cerebral tissue extract from TBI. hMSCs were cultured with TBI extracts of rat brain in vitro and quantitative sandwich enzyme-linked immunosorbent assays (ELISAs) were performed. TBI-conditioned hMSCs cultures demonstrated a time-dependent increase of BDNF, NGF, VEGF, and HGF, indicating a responsive production of these growth factors by the hMSCs. The ELISA data suggest that transplanted hMSCs may provide therapeutic benefit via a responsive secretion of an array of growth factors that can foster neuroprotection and angiogenesis.

Neuropilin-1 (NRP1) functions as a coreceptor through interaction with plexin A1 or vascular endothelial growth factor (VEGF) receptor during neuronal development and angiogenesis. NRP1 potentiates the signaling pathways stimulated by semaphorin 3A and VEGF-A in neuronal and endothelial cells, respectively. In this study, we investigate the role of tumor cell-expressed NRP1 in glioma progression. Analyses of human glioma specimens (WHO grade I-IV tumors) revealed a significant correlation of NRP1 expression with glioma progression. In tumor xenografts, overexpression of NRP1 by U87MG gliomas strongly promoted tumor growth and angiogenesis. Overexpression of NRP1 by U87MG cells stimulated cell survival through the enhancement of autocrine hepatocyte growth factor/scatter factor (HGF/SF)/c-Met signaling. NRP1 not only potentiated the activity of endogenous HGF/SF on glioma cell survival but also enhanced HGF/SF-promoted cell proliferation. Inhibition of HGF/SF, c-Met and NRP1 abrogated NRP1-potentiated autocrine HGF/SF stimulation. Furthermore, increased phosphorylation of c-Met correlated with glioma progression in human glioma biopsies in which NRP1 is upregulated and in U87MG NRP1-overexpressing tumors. Together, these data suggest that tumor cell-expressed NRP1 promotes glioma progression through potentiating the activity of the HGF/SF autocrine c-Met signaling pathway, in addition to enhancing angiogenesis, suggesting a novel mechanism of NRP1 in promoting human glioma progression.

c-Met, a high-affinity receptor for hepatocyte growth factor (HGF), plays a critical role in cancer growth, invasion, and metastasis. Hepatocellular carcinoma (HCC) patients with an active HGF/c-Met signaling pathway have a significantly worse prognosis. Although targeting the HGF/c-Met pathway has been proposed for the treatment of multiple cancers, the effect of c-Met inhibition in HCC remains unclear. The human HCC cell lines Huh7, Hep3B, MHCC97-L, and MHCC97-H were used in this study to investigate the effect of c-Met inhibition using the small molecule selective c-Met tyrosine kinase inhibitor PHA665752. MHCC97-L and MHCC97-H cells demonstrate a mesenchymal phenotype with decreased expression of E-cadherin and increased expression of c-Met, fibronectin, and Zeb2 compared with Huh7 and Hep3B cells, which have an epithelial phenotype. PHA665752 treatment blocked phosphorylation of c-Met and downstream phosphoinositide 3-kinase/Akt and mitogen-activated protein kinase/Erk pathways, inhibited cell proliferation, and induced apoptosis in c-Met-positive MHCC97-L and MHCC97-H cells. In xenograft models, administration of PHA665752 significantly inhibited c-Met-positive MHCC97-L and MHCC97-H tumor growth, and PHA665752-treated tumors demonstrated marked reduction of both c-Met phosphorylation and cell proliferation. c-Met-negative Huh7 and Hep3B cells were not affected by c-Met inhibitor treatment in vitro or in vivo. In addition, c-Met-positive MHCC97-L and MHCC97-H cells demonstrated cancer stem cell-like characteristics, such as resistance to chemotherapy, tumor sphere formation, and increased expression of CD44 and ABCG2, and PHA665752 treatment suppressed tumor sphere formation and inhibited CD44 expression.

CONCLUSIONS

c-Met represents a potential target of personalized treatment for HCC with an active HGF/c-Met pathway.