The human melanoma associated protein melanotransferrin promotes endothelial cell migration and angiogenesis in vivo

Melanotransferrin is a member of the transferrin family, which is comprised of serum transferrin, lactoferrin and ovotransferrin, and is highly expressed on melanoma cells compared to normal melanocytes. Since melanoma is an highly vascularized tumour that expresses melanotransferrin at high levels, we tested purified recombinant melanotransferrin for its capability to induce angiogenesis in the chick chorioallantoic membrane. Macroscopic and microscopic evaluation of the vascular density demonstrated that melanotransferrin exerts an angiogenic response quantitatively similar to that elicited by fibroblast growth factor-2. Overexpression of vascular endothelial growth factor-receptor-2 was observed in newly formed vessels, suggesting that the angiogenic activity of melanotransferrin may depend on activation of endogenous vascular endothelial growth factor. In addition, when antibodies against vascular endothelial growth factor were included in the assay, the angiogenic response was inhibited by 50%. In a Boyden chamber assay purified recombinant melanotransferrin induced chemotactic migration of vascular cells, which was decreased in the the presence of anti-vascular endothelial growth factor antibodies suggesting an involvement of vascular endothelial growth factor present in endothelial cells also in this assay. However, melanotransferrin was found not to directly bind to integrin alphavbeta3 or the vascular endothelial growth factor-receptor-2 as assessed in a BlAcore assay. A possible correlation between vascularization occurring during melanoma progression and the expression of melanotransferrin and vascular endothelial growth factor was established by immunolocalization of the two factors in sections of melanoma at different clinical steps of melanoma progression. These latter data strongly imply that melanotransferrin may participate in the vascularization of solid tumours and that inhibition of melanotransferrin could form the basis for intervention in tumours which use this pathway.     

The molecular phenotype of heparan sulfate in the Hs2st-/- mutant mouse.

Heparan sulfate (HS) is a co-receptor for a number of growth factors, morphogens, and adhesion proteins. HS biosynthetic modifications may determine the strength and outcome of HS-ligand interactions. We previously described the phenotype of mice with a gene-trap mutation in Hs2st, encoding the key HS 2-O-sulfotransferase enzyme in HS polymer modification. In contrast to the early developmental failure of embryos lacking HS, the onset of abnormalities in the Hs2st(-/-) mice occurs only after midgestation, the most dramatic being the complete failure of kidney development. Uronate 2-O-sulfates were not detected in the mutant HS, indicating a complete loss of function of Hs2st. However, the domain structure of the mutant HS is conserved, and compensatory increases in N- and 6-O-sulfation maintain the overall charge density. The apparent affinities of the mutant HS for hepatocyte growth factor/scatter factor and fibronectin were unchanged but were reduced for fibroblast growth factor-1 and -2. Surprisingly, the Hs2st(-/-) cells were able to mount an apparently normal signaling response to fibroblast growth factor-1 and -2 as well as to hepatocyte growth factor/scatter factor.     

The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors

Sonic hedgehog (Shh) is a prototypical morphogen known to regulate epithelial/mesenchymal interactions during embryonic development. We found that the hedgehog-signaling pathway is present in adult cardiovascular tissues and can be activated in vivo. Shh was able to induce robust angiogenesis, characterized by distinct large-diameter vessels. Shh also augmented blood-flow recovery and limb salvage following operatively induced hind-limb ischemia in aged mice. In vitro, Shh had no effect on endothelial-cell migration or proliferation; instead, it induced expression of two families of angiogenic cytokines, including all three vascular endothelial growth factor-1 isoforms and angiopoietins-1 and -2 from interstitial mesenchymal cells. These findings reveal a novel role for Shh as an indirect angiogenic factor regulating expression of multiple angiogenic cytokines and indicate that Shh might have potential therapeutic use for ischemic disorders.     

Fibroblast growth factor-2 binding to the thrombospondin-1 type III repeats, a novel antiangiogenic domain

Thrombospondin-1, an antiangiogenic matricellular protein, binds with high affinity to the angiogenic fibroblast growth factor-2, affecting its bioavailability and activity. The present work aimed at further locating the fibroblast growth factor-2 binding site of thrombospondin-1 and investigating its activity, using recombinant thrombospondin-1 proteins. Only recombinant constructs containing the thrombospondin-1 type III repeats bound fibroblast growth factor-2, whereas other domains, including the known anti-angiogenic type I repeats, were inactive. Binding was specific and inhibited by the anti thrombospondin-1 monoclonal antibody B5.2. Surface plasmon resonance analysis on BIAcore revealed a binding affinity (K(d)) of 310nM for the type III repeats and 11nM for intact thrombospondin-1. Since the type III repeats bind calcium, the effect of calcium on thrombospondin-1 binding to fibroblast growth factor-2 was investigated. Binding was modulated by calcium, as thrombospondin-1 or the type III repeats bound to fibroblast growth factor-2 only in calcium concentrations <0.3mM. The type III repeats inhibited binding of fibroblast growth factor-2 to endothelial cells, fibroblast growth factor-2-induced endothelial cell proliferation in vitro and angiogenesis in the chorioallantoic membrane assay in vivo, thus indicating the antiangiogenic activity of the domain. In conclusion, this study demonstrates that the fibroblast growth factor-2 binding site of thrombospondin-1 is located in the type III repeats. The finding that this domain is active in inhibiting angiogenesis indicates that the type III repeats represent a novel antiangiogenic domain of thrombospondin-1.     

Histamine receptor 2-mediated growth-differentiation factor-15 expression is involved in histamine-induced melanogenesis

Vitiligo is a progressive depigmenting disorder. Histamine has been shown to induce melanogenesis via histamine receptor 2, suggesting the possibility of histamine as a repigmenting agent for the treatment of vitiligo. However, the role and signaling mechanism of histamine are still unclear in melanogenesis, especially in relation to growth-differentiation factor-15, which is a protein belonging to transforming growth factor beta and found to be overexpressed in metastatic or malignant melanoma. We found that histamine induces growth-differentiation factor-15 in melanoma cell lines such as SK-MEL-2, B16F10, and melan-a cells. Therefore, in the present study, the role of growth-differentiation factor-15 in histamine-induced melanogenesis was investigated using gene silencing or overexpression of growth-differentiation factor-15 and histamine related compounds such as histamine, amthamine, and cimetidine. Gene silencing of growth-differentiation factor-15 suppressed histamine-induced proliferation, melanin production, tyrosinase activity, and chemotactic migration of SK-MEL-2 cells. Histamine-induced expression of tyrosinase, tyrosinase-related protein 1, and tyrosinase-related protein 2 was also suppressed by growth-differentiation factor-15 gene silencing. On the other hand, overexpression of growth-differentiation factor-15 using a plasmid containing growth-differentiation factor-15 in SK-MEL-2 cells increased melanin production and chemotactic migration. Amthamine induced expression of growth-differentiation factor-15 in a time and concentration dependent manner. Amthamine-induced expression of growth-differentiation factor-15 was suppressed by cimetidine.Our results suggest that growth-differentiation factor-15 is a new player in histamine-induced melanogenesis, which can help researchers to extend the knowledge of the role of the transforming growth factor beta family in melanogenesis and in skin pigment disorders such as vitiligo.     

Mesenchymal cells potentiate vascular endothelial growth factor-induced angiogenesis in vitro

The role of soluble factors (including angiogenic cytokines) and extracellular matrix components in the regulation of angiogenesis is clearly established. However, the interrelationship between these factors and perivascular mesenchymal cells is not well understood. Here we have used a three-dimensional collagen gel coculture system to assess the effect of mesenchymal C3H10T1/2 cells on vascular endothelial growth factor-A (VEGF-A)- and fibroblast growth factor-2 (FGF-2)-induced angiogenesis in vitro. We found that coculture markedly potentiated the angiogenic activity of VEGF-A, irrespective of whether or not direct cell-to-cell contact occurred. In contrast, under conditions in which cell-to-cell contact was possible, FGF-2-induced angiogenesis was inhibited by cocultured 10T1/2 cells; this effect was not seen when cell-to-cell contact was prevented. Attempts to identify the molecules responsible for this effect allowed us to exclude FGF-2, transforming growth factorbeta1, platelet derived growth factor-BB, angiopoietin-1, and NO as possible mediators of the potentiating effect of coculture on VEGF-A-induced invasion. In the living organism, angiogenesis occurs in a three-dimensional microenvironment. Contrary to the inhibitory effect of 10T1/2 cells previously reported by others in two-dimensional cultures, our data demonstrate that the paracrine interaction between endothelial and mesenchymal cells potentiates angiogenesis in vitro and that this is cytokine-specific, i.e., it occurs with VEGF-A but not with FGF-2.     

Noninvasive dynamic fluorescence imaging of human melanomas reveals that targeted inhibition of bFGF or FGFR-1 in melanoma cells blocks tumor growth by apoptosis

BACKGROUND: Two prominent biological features of the advanced stages of human melanoma are their high degree of vascularity and high-level expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1). Given these characteristics, human melanoma serves as an ideal model to address an important question regarding the efficacy of angiogenesis-based cancer therapy. To induce tumor growth arrest and regression, does it suffice to block expression of bFGF and/or FGFR-1 in only the melanoma cells, or is it essential to inhibit expression of bFGF and/or FGFR-1 in both the melanoma cells and the melanoma cell-interspersing vasculature? MATERIALS AND METHODS: Primary and metastatic human melanomas, grown as subcutaneous tumors in nude mice, were injected twice a week with vector constructs containing the human tyrosinase promoter and antisense- oriented human bFGF or FGFR-1 cDNA. On alternating days, the bFGF and FGFR-1 antisense-targeted tumors received injections of cyanine fluorochrome-conjugated antibodies to a human melanoma and mouse blood vessel marker. Noninvasive, dynamic fluorescence imaging was used to document the cellular events that took place inside the tumors as the result of blocking expression of bFGF or FGFR-1 in the melanoma cells. RESULTS: In vivo, ex vivo, and in vitro fluorescence imaging of the bFGF and FGFR-1 antisense-targeted tumors demonstrated that inhibiting bFGF and FGFR-1 signaling in only the melanoma cells suffices to inhibit tumor growth due to massive induction of melanoma cell apoptosis. CONCLUSIONS: The investigations presented in this study document that inhibiting expression of bFGF or FGFR-1 in only the melanoma cells is as effective in blocking tumor growth as simultaneously inhibiting bFGF or FGFR-1 synthesis in the melanoma cells and the melanoma cell-interspersing vasculature. Furthermore, blocking expression of bFGF or FGFR-1 in the melanoma cells did not lead to activation or increased production of another angiogenic molecule, suggesting the absence of a "salvage pathway" that can circumvent or rescue the blockage of bFGF/FGFR-1 in the melanoma cells.     

Disassociation of met-mediated biological responses in vivo: the natural hepatocyte growth factor/scatter factor splice variant NK2 antagonizes growth but facilitates metastasis

Hepatocyte growth factor/scatter factor (HGF/SF) stimulates numerous cellular activities capable of contributing to the metastatic phenotype, including growth, motility, invasiveness, and morphogenetic transformation. When inappropriately expressed in vivo, an HGF/SF transgene induces numerous hyperplastic and neoplastic lesions. NK1 and NK2 are natural splice variants of HGF/SF; all interact with a common receptor, Met. Although both agonistic and antagonistic properties have been ascribed to each isoform in vitro, NK1 retains the full spectrum of HGF/SF-like activities when expressed as a transgene in vivo. Here we report that transgenic mice broadly expressing NK2 exhibit none of the phenotypes characteristic of HGF/SF or NK1 transgenic mice. Instead, when coexpressed in NK2-HGF/SF bitransgenic mice, NK2 antagonizes the pathological consequences of HGF/SF and discourages the subcutaneous growth of transplanted Met-containing melanoma cells. Remarkably, the metastatic efficiency of these same melanoma cells is dramatically enhanced in NK2 transgenic host mice relative to wild-type recipients, rivaling levels achieved in HGF/SF and NK1 transgenic hosts. Considered in conjunction with reports that in vitro NK2 induces scatter, but not other activities, these data strongly suggest that cellular motility is a critical determinant of metastasis. Moreover, our results demonstrate how alternatively structured ligands can be exploited in vivo to functionally dissociate Met-mediated activities and their downstream pathways.     

Synthesis and biological evaluation of aromatic enones related to curcumin

Curcumin, a natural product isolated from the spice turmeric, has been shown to exhibit a wide range of pharmacological activities including certain anti-cancer properties. It has been specifically shown to be an effective inhibitor of angiogenesis both in vitro and in vivo. Using curcumin as a lead compound for anti-angiogenic analog design, a series of structurally related compounds utilizing a substituted chalcone backbone have been synthesized and tested via an established SVR cell proliferation assay. The results have yielded a wide range of compounds that equal or exceed curcumin's ability to inhibit endothelial cell growth in vitro. Due to both their commercial availability and their fairly straightforward synthetic preparation, these low molecular weight compounds are attractive leads for developing future angiogenic inhibitors.     

Synthesis and biological evaluation of novel N, N'-disubstituted urea and thiourea derivatives as potential anti-melanoma agents

Two series of urea and thiourea derivatives (1a-11a, 1b-11b) have been synthesized; all the 22 compounds were reported for the first time. Their anti-proliferative activities against the melanoma cell line B16-F10 were evaluated. Among the compounds tested, compound 6b exhibited the most potent activity in melanoma cells growth inhibition (IC(50) = 0.33 μM). The bioassay tests showed that anti-proliferative activities of these novel compounds were possibly caused by inhibition of ERK1/2 phosphorylation level. Therefore, compound 6b can be a potential anti-melanoma agent and an inhibitor of ERK1/2 phosphorylation deserving further research.     

Regulation of the expression balance of angiopoietin-1 and angiopoietin-2 by Shh and FGF-2

Sonic hedgehog (Shh) is a typical morphogen to regulate epithelial–mesenchymal interactions during embryonic development. Shh is also an indirect angiogenic agent upregulating other angiogenic factors, including angiopoietin-1 (Ang-1). Recent studies revealed that angiogenesis induced by Shh is characterized by distinct large-diameter vessels with less branching. Ang-1 promotes blood vessel maturation, and angiopoietin-2 (Ang-2) counteracts Ang-1 activity and regulates vascular branching. Thus, we hypothesized that Shh-induced angiogenesis is affected by expression of Ang-1 and Ang-2, and we investigated the regulatory system of angiopoietins by Shh in vitro. Shh enhanced Ang-1 expression but did not enhance vascular endothelial growth factor in fibroblasts. The upregulation of Ang-1 expression by Shh was significantly decreased by fibroblast growth factor-2 (FGF-2), a potent angiogenic factor. Furthermore, FGF-2 increased the expression of Ang-2 in endothelial cells. These findings suggest that Shh and FGF-2 regulate the expression balance of vascular morphogens Ang-1 and Ang-2 and are involved in angiogenesis.     

Merlin is a negative regulator of human melanoma growth

Merlin is encoded by the neurofibromatosis type 2 (NF2) gene and is a member of the Band 4.1 protein family. This protein acts as a linker that connects cell surface proteins to the actin cytoskeleton. Defects caused by mutations of the NF2 gene give rise to NF2 disease, which is generally characterized by the formation of bilateral vestibular schwannomas and, to a lesser extent, meningiomas and ependymomas. In addition to these tumor types, NF2 is mutated and/or merlin expression is reduced or lost in numerous non-NF2 associated tumors, including melanoma. However, the role of merlin in human melanoma growth and the mechanism underlying its effect are currently unknown. In the present study, we show that merlin knockdown enhances melanoma cell proliferation, migration, and invasion in vitro and that decreased merlin expression promotes subcutaneous melanoma growth in immunocompromised mice. Concordantly, we find that increased expression of merlin in a metastatic melanoma cell line reduced their in vitro migration and proliferation, and diminished their ability to grow in an anchorage independent manner. Increased merlin expression also inhibits in vivo growth of these melanoma cells. Lastly, we demonstrate that higher merlin levels in human melanoma cells promote the H(2)O(2)-induced activation of MST1/2 Ser/Thr kinases, which are known tumor suppressors in the Hippo signaling pathway. Taken together, these results provide for the first time evidence that merlin negatively regulates human melanoma growth, and that loss of merlin, or impaired merlin function, results in an opposite effect. In addition, we show that increased merlin expression leads to enhanced activation of the MTS1/2 kinases, implying the potential roles of MST1/2 in mediating the anti-melanoma effects of merlin.     

1-Butyryl-glycerol: a novel angiogenesis factor secreted by differentiating adipocytes.

Differentiation of adipocytes is accompanied by secretion of molecules stimulating angiogenesis in vivo and endothelial cell growth and motility in vitro. We demonstrate that the angiogenic and motility-stimulating activities secreted by adipocytes are separable from the endothelial cell mitogenic activity by fractionation of adipocyte-conditioned medium. The major differentiation-dependent angiogenic molecule was purified and identified by GCMS as 1-butyryl-glycerol (monobutyrin). Monobutyrin levels increase at least 200-fold during adipocyte differentiation and represent a major fraction of the total angiogenic activity. Synthetic monobutyrin shows the same spectrum of biological activities as the adipocyte-derived factor: stimulation of angiogenesis in vivo and microvascular endothelial cell motility in vitro, with no effect on endothelial cell proliferation. Angiogenesis is stimulated at doses as low as 20 pg when tested in the chick chorioallantoic membrane assay. These results strongly suggest that monobutyrin is a key regulatory molecule in an angiogenic process linked to normal cellular and tissue development.     

Shp-1 mediates the antiproliferative activity of tissue inhibitor of metalloproteinase-2 in human microvascular endothelial cells

The tissue inhibitors of metalloproteinases (TIMPs) regulate matrix metalloproteinase activity required for cell migration/invasion associated with cancer progression and angiogenesis. TIMPs also modulate cell proliferation in vitro and angiogenesis in vivo independent of their matrix metalloproteinase inhibitory activity. Here, we show that TIMP-2 mediates G1 growth arrest in human endothelial cells through de novo synthesis of the cyclin-dependent kinase inhibitor p27Kip1. TIMP-2-mediated inhibition of Cdk4 and Cdk2 activity is associated with increased binding of p27Kip1 to these complexes in vivo. Protein-tyrosine phosphatase inhibitors or expression of a dominant negative Shp-1 mutant ablates TIMP-2 induction of p27Kip1. Finally, angiogenic responses to fibroblast growth factor-2 and vascular endothelial growth factor-A in "motheaten viable" Shp-1-deficient mice are resistant to TIMP-2 inhibition, demonstrating that Shp-1 is an important negative regulator of angiogenesis in vivo.     

Induction of angiogenic response by chemically stable prostacyclin analogs

Angiogenic activities of several chemically stable prostacyclin analogs (isocarbacyclins and 7-fluoro prostacyclin) were evaluated by the chick embryo chorioallantoic membrane assay. These compounds showed potent angiogenic activity at very low concentration (0.1 micrograms/egg 1.0 micrograms/egg), whereas naturally occurring prostaglandins such as prostacyclin and PGE1 were almost ineffective up to 1 microgram/egg. Pretreatment of chorioallantoic membranes with dexamethasone or indomethacin inhibited the angiogenic response induced by these chemically stable prostacyclin analogs. These results indicate that these prostacyclin analogs induce the angiogenic response of chick chorioallantoic membranes via a mechanism involving activation of inflammatory cells, as well as through their direct angiogenic activity.     

Two new asterosaponins from the antarctic starfish Diplasterias brucei. Structures and cytotoxic activities

Two new asterosaponins, diplasteriosides A and B, with the same oligosaccharide chains beta-D-Fucp-(1-->2)-beta-D-Galp-(1-->4)-[beta-D-Quip-(1-->2)]-beta-D-Quip-(1-->3)-beta-D-Quip-(1-->, linked to C6 of known genins, 3-O-sulfates of thornasterols A and B, respectively, were isolated along with the previously known asteriidoside A from the Antarctic starfish Diplasterias brucei. The structures of new compounds were elucidated by spectroscopic methods (mainly 2D NMR and mass spectrometry). The cytotoxicity of isolated asterosaponins against human colon cancer cell line HCT-116, human breast cancer cell line T-47D, and human melanoma cancer cell line RPMI-7951 was investigated.     

Developmental regulations of Perp in mice molar morphogenesis

Angiogenesis, a complex biologic process, is regulated by a large number of angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2). Whether Bone morphogenetic proteins-2 (BMP-2), the osteoinductive factor, could significantly reinforce the effect of VEGF and FGF-2 on angiogenesis has not been studied in detail. To study the positive effects of multiple growth factors on angiogenesis, HUVECs were treated with BMP-2, VEGF, or FGF-2 singly and in binary and ternary combinations. This study further investigates the optimal timing of the ternary combination of BMP-2, VEGF and FGF-2 for angiogenesis in the chorioallantoic membrane (FGF-2 CAM). Results of single applications of BMP-2, VEGF, or FGF-2 suggested that HUVECs angiogenesis could be promoted in a dose-dependent manner and that the optimal concentration of BMP, VEGF and FGF-2 was 10, 50 and 1 ng/mL, respectively. These results indicated that the angiogenic activity of VEGF and FGF-2 was amplified by combining with BMP-2. The ternary combination of BMP-2, VEGF and FGF-2 exhibited a positive and synergistic effect on HUVECs angiogenesis, with the lower concentrations of each factor (1 ng/mL of BMP-2, 25 ng/mL of VEGF and 0.1 ng/mL of FGF-2) being sufficient to show synergistic promotion. When VEGF and FGF-2 were added in the initial activation stage and BMP-2 was added in the maturation stage, both HUVECs angiogenesis in vitro and CAM angiogenesis in vivo could be enhanced more effectively. These results could provide a basis for the controlled release systems capable of delivering multiple factors sequentially to promote angiogenesis in tissue engineering.