Hyaluronate Receptors Mediating Glioma Cell Migration and Proliferation

The extracellular matrix (ECM) of the central nervous system (CNS) is enriched in hyaluronate (HA). Ubiquitous receptors for HA are CD44 and the Receptor for HA-Mediated Motility known as RHAMM. In the present study, we have investigated the potential role of CD44 and RHAMM in the migration and proliferation of human astrocytoma cells. HA-receptor expression in brain tumor cell lines and surgical specimens was determined by immunocytochemistry and western blot analyses. The ability of RHAMM to bind ligand was determined through cetylpyridinium chloride (CPC) precipitations of brain tumor lysates in HA-binding assays. The effects of HA, CD44 blocking antibodies, and RHAMM soluble peptide on astrocytoma cell growth and migration was determined using MTT and migration assays. Our results show that the expression of the HA-receptors, CD44, and RHAMM, is virtually ubiquitous amongst glioma cell lines, and glioma tumor specimens. There was a gradient of expression amongst gliomas with high grade gliomas expressing more RHAMM and CD44 than did lower grade lesions or did normal human astrocytes or non-neoplastic specimens of human brain. Specific RHAMM variants of 85- and 58-kDa size were shown to bind avidly to HA following CPC precipitations. RHAMM soluble peptide inhibited glioma cell line proliferation in a dose-dependent fashion. Finally, while anti-CD44 antibodies did not inhibit the migration of human glioma cells, soluble peptides directed at the HA-binding domain of RHAMM inhibited glioma migration both on and off an HA-based ECM. These data support the notion that HA-receptors contribute to brain tumor adhesion, proliferation, and migration, biological features which must be better understood before more effective treatment strategies for these tumors can be found.     

Fibroblast growth factors are required for efficient tumor angiogenesis

Although the function of vascular endothelial growth factor in the induction of tumor angiogenesis is well understood, the role of a second group of angiogenic factors, the fibroblast growth factors (FGFs), remains elusive. We used a recombinant adenovirus expressing soluble FGF receptor (AdsFGFR) to interfere with FGF function in tumor angiogenesis. AdsFGFR repressed endothelial cell proliferation in vitro and inhibited tumor angiogenesis in an ex vivo bioassay, in which endothelial cells were cocultured with angiogenic tumor biopsies in a collagen gel. Moreover, AdsFGFR repressed tumor angiogenesis and hence tumor growth in vivo in allograft transplantation experiments. Whereas adenoviral expression of a soluble form of VEGF receptor 1 (AdsFlt) predominantly affected the initiation of tumor angiogenesis, soluble FGF receptor (sFGFR) appeared to impair the maintenance of tumor angiogenesis. The combination of sFGFR and soluble Flt exhibited a synergistic effect in the repression of tumor growth. Finally, i.v. injection of AdsFGFR resulted in a dramatic repression of tumor growth in a transgenic mouse model of pancreatic beta cell carcinogenesis. Similar to control infections with AdsFlt, tumor-associated vessel density was decreased, indicating that the expression of sFGFR impaired tumor angiogenesis. These data indicate that FGFs play a critical role in tumor angiogenesis.     

Hyaluronan-independent lodgment of CD44+ lymphoma cells in lymphoid organs

We previously found that monoclonal antibodies (MAbs) directed against the constant region of the CD44 molecule block lymph node infiltration of a mouse LB T-cell lymphoma, suggesting a role for this glycoprotein in the LB cell dissemination process. In the present study, we investigated whether LB cells in the local tumor must undergo a change in the CD44 phenotype to be able to migrate to and invade the remote lymph nodes, and if hyaluronic acid (HA), the principal ligand of activated CD44, functions as a mediator in this process. We compared the in vivo behavior of a non-HA-binder LB cell line with that of a constitutive HA-binder HA9 subline. Our results show that the lymphoid organ-infiltrating LB cells express similar levels of pan-CD44 and V4- and V6-containing CD44 variants, as the corresponding cells in the local growth and the cultured LB cells. The tested CD44 phenotype of HA9 cells also remained unchanged during the metastatic process. Even after lymph node infiltration, LB cells remained incapable of binding HA, whereas the HA9 cells retained an efficient HA-binding capacity. The constitutive HA-binder HA9 cells that expressed an approximately 10-fold higher level of pan-CD44 than did the parental LB cells, as well as an elevated level of the V4 and V6 exon products formed a local tumor and invaded both lymph nodes and spleen, as did the parental LB cells, albeit at a much slower rate. Our finding indicates that there is no direct correlation between the amount of CD44 expressed on the cell surface, the HA-binding capacity and tumorigenicity. Moreover, interaction with HA is not obligatory for LB cell localization in the lymphoid organs, and a tight cell-HA interaction, as observed in HA9 cells, does not prevent tumor cell dissemination, although it may retard tumor spread. Int. J. Cancer 71:462-469, 1997. © 1997 Wiley-Liss Inc.     

CD36-mediated activation of endothelial cell apoptosis by an N-terminal recombinant fragment of thrombospondin-2 inhibits breast cancer growth and metastasis in vivo

Thus far the clinical benefits seen in breast cancer patients treated with drugs targeting the vascular endothelial growth factor (VEGF) pathway are only modest. Consequently, additional antiangiogenic approaches for treatment of breast cancer need to be investigated. Thrombospondin-2 (TSP-2) has been shown to inhibit tumor growth and angiogenesis with a greater potency than the related molecule TSP-1. The systemic effects of TSP-2 on tumor metastasis and the underlying molecular mechanisms of the antiangiogenic activity of TSP-2 have remained poorly understood. We generated a recombinant fusion protein consisting of the N-terminal region of TSP-2 and the IgG-Fc1 fragment (N-TSP2-Fc) and could demonstrate that the antiangiogenic activity of N-TSP2-Fc is dependent on the CD36 receptor. We found that N-TSP2-Fc inhibited VEGF-induced tube formation of human dermal microvascular endothelial cells (HDMEC) on matrigel in vitro and that concurrent incubation of anti-CD36 antibody with N-TSP2-Fc resulted in tube formation that was comparable to untreated control. N-TSP2-Fc potently induced apoptosis of HDMEC in vitro in a CD36-dependent manner. Moreover, we could demonstrate a CD36 receptor-mediated loss of mitochondrial membrane potential and activation of caspase-3 in HDMEC in vitro. Daily intraperitoneal injections of N-TSP2-Fc resulted in a significant inhibition of the growth of human MDA-MB-435 and MDA-MB-231 tumor cells grown in the mammary gland of immunodeficient nude mice and in reduced tumor vascularization. Finally, increased serum concentrations of N-TSP2-Fc significantly inhibited regional metastasis to lymph nodes and distant metastasis to lung as shown by quantitative real-time alu PCR. These results identify N-TSP2-Fc as a potent systemic inhibitor of tumor metastasis and provide strong evidence for an important role of the CD36 receptor in mediating the antiangiogenic activity of TSP-2.     

Inhibition of tumor growth by systemic treatment with thrombospondin-1 peptide mimetics

Many normal human cells produce thrombospondin-1 (TSP-1), a potent antiangiogenic protein that promotes vascular quiescence. In various organ systems, including the brain, breast and bladder and in fibroblasts, TSP-1 secretion is reduced during tumorigenesis, thereby allowing induction of the vigorous neovascularization required for tumor growth and metastasis. Full-length and short TSP-1-derived peptides inhibit angiogenesis by inducing endothelial cell apoptosis and thus disrupting the vasculature of the growing tumor. CD36 expressed on the surface of endothelial cells functions as the primary antiangiogenic receptor for TSP-1. A D-isoleucyl enantiomer of a TSP-1 heptapeptide specifically inhibits the proliferation and migration of capillary endothelial cells. DI-TSP, an approximately 1 kDa capped version of this peptide, is also antiangiogenic in vitro, with a specific activity approaching that of the 450 kDa parental molecule. Here, we show that DI-TSP delivered systemically dose-dependently inhibits the growth of murine melanoma metastases in syngeneic animals and that its more soluble isomer, DI-TSPa, similarly blocks the progression of primary human bladder tumors in an orthotopic model in immune-deficient mice. Like intact TSP-1, these peptide mimetics had no effect on cancer cells growing in vitro but markedly suppressed the growth of endothelial cells by inducing receptor-dependent apoptosis. Antibodies raised against CD36 blocked the ability of peptides to induce apoptosis in endothelial cells but had no effect on tumor necrosis factor-alpha-induced apoptosis. In vivo, the peptide mimetics were associated with a significantly reduced microvessel density and increased apoptotic indices in both the endothelial and tumor cell compartments. Such short peptides targeted to a specific antiangiogenic receptor, potent and easy to synthesize, show great promise as lead compounds in clinical antiangiogenic strategies.     

Melanoma differentiation-associated gene 7/interleukin (IL)-24 is a novel ligand that regulates angiogenesis via the IL-22 receptor

The melanoma differentiation-associated gene 7 (mda-7), also called interleukin (IL)-24, suppresses the growth of some cancers in vitro and in vivo as a result of the ectopic expression of its protein. However, the function of the secreted form of the protein in cancer has not been previously studied. The purpose of this study was to determine the antiangiogenic function of a secreted form of the MDA-7/IL-24 protein (sMDA-7/IL-24). In vitro, sMDA-7/IL-24 inhibited both endothelial cell differentiation and migration of endothelial cells induced by vascular endothelial growth factor and basic fibroblast growth factor. The sMDA-7/IL-24-mediated inhibitory effect was 10-50 times more potent than endostatin, IFN-gamma, and IFN-inducible protein 10 in vitro. Furthermore, the inhibitory effect was not mediated by IFN or IFN-inducible protein 10. IL-22 receptor mediated the antiangiogenic activity of sMDA-7/IL-24. Administration of a blocking antibody to IL-22 receptor in conjunction with sMDA-7/IL-24 led to abrogation of inhibition of endothelial differentiation. sMDA-7/IL-24 inhibited vascular endothelial growth factor-induced angiogenesis as evidenced by reduced vascularization and hemoglobin content in in vivo Matrigel plug assays. In vivo, the growth of human lung tumor cells was significantly inhibited, and vascularization was reduced when the cells were mixed with 293 cells stably expressing sMDA-7/IL-24. Systemic administration of sMDA-7/IL-24 inhibited lung tumor growth in a mouse xenograft model. Associated with tumor growth inhibition was decreased tumor microvessel density and hemoglobin content, indicating the presence of antiangiogenic activity. These data demonstrate that sMDA-7/IL-24 is a novel and potent antiangiogenic effector and support the development of MDA-7/IL-24-based therapeutics.     

Angiogenesis and radiation response modulation after vascular endothelial growth factor receptor-2 (VEGFR2) blockade

The formation of new blood vessels (angiogenesis) represents a critical factor in the malignant growth of solid tumors and metastases. Vascular endothelial cell growth factor (VEGF) and its receptor VEGFR2 represent central molecular targets for antiangiogenic intervention, because of their integral involvement in endothelial cell proliferation and migration. In the current study, we investigated in vitro and in vivo effects of receptor blockade on various aspects of the angiogenic process using monoclonal antibodies against VEGFR2 (cp1C11, which is human specific, and DC101, which is mouse specific). Molecular blockade of VEGFR2 inhibited several critical steps involved in angiogenesis. VEGFR2 blockade in endothelial cells attenuated cellular proliferation, reduced cellular migration, and disrupted cellular differentiation and resultant formation of capillary-like networks. Further, VEGFR2 blockade significantly reduced the growth response of human squamous cell carcinoma xenografts in athymic mice. The growth-inhibitory effect of VEGFR2 blockade in tumor xenografts seems to reflect antiangiogenic influence as demonstrated by vascular growth inhibition in an in vivo angiogenesis assay incorporating tumor-bearing Matrigel plugs. Further, administration of VEGFR2-blocking antibodies in endothelial cell cultures, and in mouse xenograft models, increased their response to ionizing radiation, indicating an interactive cytotoxic effect of VEGFR2 blockade with radiation. These data suggest that molecular inhibition of VEGFR2 alone, and in combination with radiation, can enhance tumor response through molecular targeting of tumor vasculature.     

Antiangiogenic and antitumor efficacy of EphA2 receptor antagonist

Tumor-associated angiogenesis is critical for tumor growth and metastasis and is controlled by various pro- and antiangiogenic factors. The Eph family of receptor tyrosine kinases has emerged as one of the pivotal regulators of angiogenesis. Here we report that interfering with EphA signaling resulted in a pronounced inhibition of angiogenesis in ex vivo and in vivo model systems. Administration of EphA2/Fc soluble receptors inhibited, in a dose-dependent manner, microvessel formation in rat aortic ring assay, with inhibition reaching 76% at the highest dose of 5000 ng/ml. These results were further confirmed in vivo in a porcine aortic endothelial cell-vascular endothelial growth factor (VEGF)/basic fibroblast growth factor Matrigel plug assay, in which administration of EphA2/Fc soluble receptors resulted in 81% inhibition of neovascularization. The additive effects of simultaneous inhibition of VEGF receptor 2 and EphA signaling pathways in aortic ring assay and antiangiogenic efficacy of EphA2/Fc soluble receptors against VEGF/basic fibroblast growth factor-mediated neovascularization in vivo indicated a critical and nonredundant role for EphA signaling in angiogenesis. Furthermore, in two independent experiments, we demonstrated that EphA2/Fc soluble receptors strongly (by approximately 50% versus controls) suppressed growth of ASPC-1 human pancreatic tumor s.c. xenografts. Inhibition of tumor growth was due to decreased proliferation of tumor cells. In an orthotopic pancreatic ductal adenocarcinoma model in mice, suppression of EphA signaling by i.p. administration of EphA2/Fc (30 micro g/dose, three times a week for 56 days) profoundly inhibited the growth of primary tumors and the development of peritoneal, lymphatic, and hepatic metastases. These data demonstrate a critical role of EphA signaling in tumor growth and metastasis and provide a strong rationale for targeting EphA2 receptors for anticancer therapies.     

Inhibition of VEGF-dependent multistage carcinogenesis by soluble EphA receptors

Elevated expression of Eph receptors has long been correlated with the growth of solid tumors. However, the functional role of this family of receptor tyrosine kinases in carcinogenesis and tumor angiogenesis has not been well characterized. Here we report that soluble EphA receptors inhibit tumor angiogenesis and tumor progression in vivo in the RIP-Tag transgenic model of vascular endothelial growth factor (VEGF)-dependent multistage pancreatic islet cell carcinoma. Soluble EphA receptors delivered either by a transgene or an osmotic minipump inhibited the formation of angiogenic islet, a premalignant lesion, and reduced tumor volume of solid islet cell carcinoma. EphA2-Fc or EphA3-Fc treatment resulted in decreased tumor volume but increased tumor and endothelial cell apoptosis in vivo. In addition, soluble EphA receptors inhibited VEGF and betaTC tumor cell-conditioned medium-induced endothelial cell migration in vitro and VEGF-induced cornea angiogenesis in vivo. A dominant negative EphA2 mutant inhibited--whereas a gain-of-function EphA2 mutant enhanced--tumor cell-induced endothelial cell migration, suggesting that EphA2 receptor activation is required for tumor cell-endothelial cell interaction. These data provide functional evidence for EphA class receptor regulation of VEGF-dependent tumor angiogenesis, suggesting that the EphA signaling pathway may represent an attractive novel target for antiangiogenic therapy in cancer.     

Interleukin-12 inhibits tumor growth in a novel angiogenesis canine hemangiosarcoma xenograft model

We established a canine hemangiosarcoma cell line derived from malignant endothelial cells comprising a spontaneous tumor in a dog to provide a renewable source of endothelial cells for studies of angiogenesis in malignancy. Pieces of the hemangiosarcoma biopsy were engrafted subcutaneously in a bg/nu/XID mouse allowing the tumor cells to expand in vivo. A cell line, SB-HSA, was derived from the xenograft. SB-HSA cells expressed vascular endothelial growth factor (VEGF) receptors 1 and 2, CD31, CD146, and alpha(v)beta(3) integrin, and produced several growth factors and cytokines, including VEGF, basic fibroblast growth factor, and interleukin (IL)-8 that are stimulatory to endothelial cell growth. These results indicated that the cells recapitulated features of mitotically activated endothelia. In vivo, SB-HSA cells stimulated robust angiogenic responses in mice and formed tumor masses composed of aberrant vascular channels in immunocompromised mice providing novel opportunities for investigating the effectiveness of antiangiogenic agents. Using this model, we determined that IL-12, a cytokine with both immunostimulatory and antiangiogenic effects, suppressed angiogenesis induced by, and tumor growth of, SB-HSA cells. The endothelial cell model we have described offers unique opportunities to pursue further investigations with IL-12, as well as other antiangiogenic approaches in cancer therapy.     

Inhibition of angiogenesis and vascular leakiness by angiopoietin-related protein 4

Angiopoietins and angiopoietin-related proteins (ARPs) have been shown to regulate angiogenesis, a process essential for various neovascular diseases including tumors. Here, we identify ARP4/fasting-induced adipose factor/peroxisome proliferator-activated receptor gamma angiopoietin-related as a novel antiangiogenic modulatory factor. We hypothesized that ARP4 may regulate angiogenesis. In vitro experiments using purified recombinant ARP4 protein revealed that ARP4 markedly inhibited the proliferation, chemotaxis, and tubule formation of endothelial cells. Moreover, using corneal neovascularization and Miles permeability assays, we found that both vascular endothelial growth factor-induced in vivo angiogenesis and vascular leakiness were significantly inhibited by the addition of ARP4. Finally, we found remarkable suppression of tumor growth within the dermal layer associated with decreased numbers of invading blood vessels in transgenic mice that express ARP4 in the skin driven by the keratinocyte promoter. These findings demonstrate that ARP4 functions as a novel antiangiogenic modulatory factor and indicate a potential therapeutic effect of ARP4 in neoplastic diseases.     

Metronomic low-dose chemotherapy boosts CD95-dependent antiangiogenic effect of the thrombospondin peptide ABT-510: a complementation antiangiogenic strategy.

Blocking angiogenesis is a promising approach in cancer therapy. Natural inhibitors of angiogenesis and derivatives induce receptor-mediated signals, which often result in the endothelial cell death. Low-dose chemotherapy, given at short regular intervals with no prolonged breaks (metronomic chemotherapy), also targets angiogenesis by obliterating proliferating endothelial cells and circulating endothelial cell precursors. ABT-510, a peptide derivative of thrombospondin, kills endothelial cell by increasing CD95L, a ligand for the CD95 death receptor. However, CD95 expression itself is unaffected by ABT-510 and limits its efficacy. We found that multiple chemotherapy agents, cyclophosphamide (cytoxan), cisplatin, and docetaxel, induced endothelial CD95 in vitro and in vivo at low doses that failed to kill endothelial cells (cytoxan > cisplatin > docetaxel). Thus, we concluded that some of these agents might complement each other and together block angiogenesis with maximal efficacy. As a proof of principle, we designed an antiangiogenic cocktail combining ABT-510 with cytoxan or cisplatin. Cyclophosphamide and cisplatin synergistically increased in vivo endothelial cell apoptosis and angiosuppression by ABT-510. This synergy required CD95, as it was reversible with the CD95 decoy receptor. In a mouse model, ABT-510 and cytoxan, applied together at low doses, acted in synergy to delay tumor take, to stabilize the growth of established tumors, and to cause a long-term progression delay of PC-3 prostate carcinoma. These antitumor effects were accompanied by major decreases in microvascular density and concomitant increases of the vascular CD95, CD95L, and apoptosis. Thus, our study shows a "complementation" design of an optimal cancer treatment with the antiangiogenic peptide and a metronomic chemotherapy.     

Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis

BACKGROUND: The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. METHODS: The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. RESULTS: GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. CONCLUSIONS: In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.     

Rationale for antiangiogenic cancer therapy with vaccination using epitope peptides derived from human vascular endothelial growth factor receptor 2

Angiogenesis is a critical mechanism for tumor progression. Multiple studies have suggested that tumor growth can be suppressed if tumor angiogenesis can be inhibited using various types of antiangiogenic agents. Recent studies in mouse systems have shown that tumor angiogenesis can also be inhibited if cellular immune response could be induced against vascular endothelial growth factor receptor 2 (VEGFR2), which is one of the key factors in tumor angiogenesis. In this study, we examined the possibility of developing this novel immunotherapy in clinical setting. We first identified the epitope peptides of VEGFR2 and showed that stimulation using these peptides induces CTLs with potent cytotoxicity in the HLA class I-restricted fashion against not only peptide-pulsed target cells but also endothelial cells endogenously expressing VEGFR2. In A2/Kb transgenic mice that express alpha1 and alpha2 domains of human HLA-A*0201, vaccination using these epitope peptides in vivo was associated with significant suppression of the tumor growth and prolongation of the animal survival without fatal adverse effects. In antiangiogenesis assay, tumor-induced angiogenesis was significantly suppressed with the vaccination using these epitope peptides. Furthermore, CTLs specific to the epitope peptides were successfully induced in cancer patients, and the specificities of the CTLs were confirmed using functional and HLA-tetramer analysis. These results in vitro and in vivo strongly suggest that the epitope peptides derived from VEGFR2 could be used as the agents for antiangiogenic immunotherapy against cancer in clinical settings.     

Epoxyquinol B shows antiangiogenic and antitumor effects by inhibiting VEGFR2, EGFR, FGFR, and PDGFR

Angiogenesis is the development of new blood vessels to provide oxygen and nutrients and is indispensable for solid tumor growth. Therefore, the inhibition of angiogenesis is an important modality for cancer chemotherapy. Here we report the antiangiogenic mechanism and antitumor effects of epoxyquinol B (EPQB), which was isolated from fungal metabolites. Short-term treatment of EPQB resulted in the reduction of tumor growth and the number of blood vessels directed to the tumor in a murine xenografts model. Furthermore, EPQB inhibited vascular endothelial growth factor (VEGF)-induced migration and tube formation in human umbilical vein endothelial cells (HUVECs) without cytotoxicity. VEGF-stimulated phosphorylation of VEGF receptor 2 (VEGFR2), phospholipase Cgamma-1 (PLCgamma1), and p44/42 MAP kinases (ERK) was inhibited by EPQB in a dose-dependent manner, and in vitro assay using kinase domain of VEGFR2 showed that EPQB covalently bound and inhibited the VEGFR2 kinase. Its binding site on VEGFR2 was different from SU5614, a well-known VEGFR2 kinase inhibitor. Interestingly, EPQB inhibited growth factor-induced activation of not only VEGFR2 but also epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), and platelet-derived growth factor receptor (PDGFR), suggesting that EPQB is a novel multiple kinase inhibitor. These findings suggest that EPQB would be a good lead compound for the development of potent antiangiogenic and antitumor drugs.     

HGF/NK4, a four-kringle antagonist of hepatocyte growth factor, is an angiogenesis inhibitor that suppresses tumor growth and metastasis in mice

We reported that NK4, composed of the N-terminal hairpin and subsequent four kringle domains of hepatocyte growth factor (HGF), acts as the competitive antagonist for HGF. We now provide the first evidence that NK4 inhibits tumor growth and metastasis as an angiogenesis inhibitor as well as an HGF antagonist. Administration of NK4 suppressed primary tumor growth and lung metastasis of Lewis lung carcinoma and Jyg-MC(A) mammary carcinoma s.c. implanted into mice, although neither HGF nor NK4 affected proliferation and survival of these tumor cells in vitro. NK4 treatment resulted in a remarkable decrease in microvessel density and an increase of apoptotic tumor cells in primary tumors, which suggests that the inhibition of primary tumor growth by NK4 may be achieved by suppression of tumor angiogenesis. In vivo, NK4 inhibited angiogenesis in chick chorioallantoic membranes and in rabbit corneal neovascularization induced by basic fibroblast growth factor (bFGF). In vitro, NK4 inhibited growth and migration of human microvascular endothelial cells induced by bFGF and vascular endothelial growth factor (VEGF) as well as by HGF. HGF and VEGF activated the Met/HGF receptor and the KDR/VEGF receptor, respectively, whereas NK4 inhibited HGF-induced Met tyrosine phosphorylation but not VEGF-induced KDR phosphorylation. NK4 inhibited HGF-induced ERK1/2 (p44/42 mitogen-activated protein kinase) activation, but allowed for bFGF- and VEGF-induced ERK1/2 activation. These results indicate that NK4 is an angiogenesis inhibitor as well as an HGF antagonist, and that the antiangiogenic action of NK4 is independent of its activity as HGF antagonist. The bifunctional properties of NK4 to act as an angiogenesis inhibitor and as an HGF antagonist raises the possibility that NK4 may prove therapeutic for cancer patients.     

Antiangiogenic mechanisms of PJ-8, a novel inhibitor of vascular endothelial growth factor receptor signaling

Angiogenesis occurs not only during tissue growth and development but also during wound healing and tumor progression. Angiogenesis is a balanced process controlled by proangiogenic and antiangiogenic molecules. As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for antiangiogenic and cancer therapeutic agents. In an effort to develop novel inhibitors to block VEGF signaling, we selected Pj-8, a benzimidazole derivative, and investigated its inhibitory mechanisms in human umbilical vascular endothelial cells (HUVECs). Pj-8 concentration-dependently inhibited VEGF-induced proliferation, migration and tube formation of HUVECs. Pj-8 also suppressed VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization of implanted matrigel plugs in vivo. Pj-8 inhibited VEGF-induced phosphorylation of VEGF receptor (VEGFR) 2 and the downstream protein kinases, including Akt, focal adhesion kinase, extracellular signal-regulated kinases and Src. Results from in vitro kinase assay further demonstrated that Pj-8 suppressed the kinase activity of 3-phosphoinositide-dependent kinase 1 (PDK1). Using xenograft tumor angiogenesis model, Pj-8 markedly eliminated tumor-associated angiogenesis. Taken together, our findings suggest that Pj-8 inhibits VEGF and tumor cells MDA-MB-231-induced angiogenesis, and it may be a potential drug candidate in anticancer therapy. Downregulation of VEGFR2-mediated signaling may contribute to its antiangiogenic actions.     

Vasculostatin, a proteolytic fragment of brain angiogenesis inhibitor 1, is an antiangiogenic and antitumorigenic factor

Brain angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with unknown function expressed primarily in normal but not tumoral brain. The finding of thrombospondin type 1 repeats in its extracellular domain suggested an antiangiogenic function, but the mechanisms by which a transmembrane receptor could inhibit angiogenesis remained unexplained. Here we demonstrate that BAI1 is proteolytically cleaved at a conserved G-protein-coupled receptor proteolytic cleavage site (GPS), releasing its 120 kDa extracellular domain. We named this secreted fragment Vasculostatin as it inhibited migration of endothelial cells in vitro and dramatically reduced in vivo angiogenesis. Both constitutive and doxycycline-induced expression of Vasculostatin elicited dose-dependent suppression of tumor growth and vascular density in mice, implicating Vasculostatin in the regulation of vascular homeostasis and tumor prevention. Generation of a soluble antiangiogenic factor by cleavage of a pre-existing transmembrane protein represents a novel mechanism for regulating vascular homeostasis and preventing tumorigenesis. Modulation of this cleavage or delivery of Vasculostatin may constitute novel treatment modalities for cancer and other diseases of aberrant angiogenesis, especially in the brain.     

A notch1 ectodomain construct inhibits endothelial notch signaling, tumor growth, and angiogenesis

Notch signaling is required for vascular development and tumor angiogenesis. Although inhibition of the Notch ligand Delta-like 4 can restrict tumor growth and disrupt neovasculature, the effect of inhibiting Notch receptor function on angiogenesis has yet to be defined. In this study, we generated a soluble form of the Notch1 receptor (Notch1 decoy) and assessed its effect on angiogenesis in vitro and in vivo. Notch1 decoy expression reduced signaling stimulated by the binding of three distinct Notch ligands to Notch1 and inhibited morphogenesis of endothelial cells overexpressing Notch4. Thus, Notch1 decoy functioned as an antagonist of ligand-dependent Notch signaling. In mice, Notch1 decoy also inhibited vascular endothelial growth factor-induced angiogenesis in skin, establishing a role for Notch receptor function in this process. We tested the effects of Notch1 decoy on tumor angiogenesis using two models: mouse mammary Mm5MT cells overexpressing fibroblast growth factor 4 (Mm5MT-FGF4) and NGP human neuroblastoma cells. Exogenously expressed FGF4 induced Notch ligand expression in Mm5MT cells and xenografts. Notch1 decoy expression did not affect tumorigenicity of Mm5MT-FGF4 cells in vitro but restricted Mm5MT-FGF4 xenograft growth in mice while markedly impairing neoangiogenesis. Similarly, Notch1 decoy expression did not affect NGP cells in vitro but disrupted vessels and decreased tumor viability in vivo. These results strongly suggest that Notch receptor signaling is required for tumor neoangiogenesis and provides a new target for tumor therapy.     

Antagonism of sphingosine-1-phosphate receptors by FTY720 inhibits angiogenesis and tumor vascularization

FTY720, a potent immunomodulator, becomes phosphorylated in vivo (FTY-P) and interacts with sphingosine-1-phosphate (S1P) receptors. Recent studies showed that FTY-P affects vascular endothelial growth factor (VEGF)-induced vascular permeability, an important aspect of angiogenesis. We show here that FTY720 has antiangiogenic activity, potently abrogating VEGF- and S1P-induced angiogenesis in vivo in growth factor implant and corneal models. FTY720 administration tended to inhibit primary and significantly inhibited metastatic tumor growth in a mouse model of melanoma growth. In combination with a VEGFR tyrosine kinase inhibitor PTK787/ZK222584, FTY720 showed some additional benefit. FTY720 markedly inhibited tumor-associated angiogenesis, and this was accompanied by decreased tumor cell proliferation and increased apoptosis. In transfected HEK293 cells, FTY-P internalized S1P1 receptors, inhibited their recycling to the cell surface, and desensitized S1P receptor function. Both FTY720 and FTY-P apparently failed to impede VEGF-produced increases in mitogen-activated protein kinase activity in human umbilical vascular endothelial cells (HUVEC), and unlike its activity in causing S1PR internalization, FTY-P did not result in a decrease of surface VEGFR2 levels in HUVEC cells. Pretreatment with FTY720 or FTY-P prevented S1P-induced Ca2+ mobilization and migration in vascular endothelial cells. These data show that functional antagonism of vascular S1P receptors by FTY720 potently inhibits angiogenesis; therefore, this may provide a novel therapeutic approach for pathologic conditions with dysregulated angiogenesis.