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To fully assess the role of VEGF-A in tumor angiogenesis, antibodies that can block all sources of vascular endothelial growth factor (VEGF) are desired. Selectively targeting tumor-derived VEGF overlooks the contribution of host stromal VEGF. Other strategies, such as targeting VEGF receptors directly or using receptor decoys, result in inhibiting not only VEGF-A but also VEGF homologues (e.g. placental growth factor, VEGF-B, and VEGF-C), which may play a role in angiogenesis. Here we report the identification of novel anti-VEGF antibodies, B20 and G6, from synthetic antibody phage libraries, which block both human and murine VEGF action in vitro. Their affinity-improved variants completely inhibit three human tumor xenografts in mice of skeletal muscle, colorectal, and pancreatic origins (A673, HM-7, and HPAC). Avastin, which only inhibits the tumor-derived human VEGF, is approximately 90% effective at inhibiting HM-7 and A673 growth but is <50% effective at inhibiting HPAC growth. Indeed, HPAC tumors contain more host stroma invasion and stroma-derived VEGF than other tumors. Thus, the functional contribution of stromal VEGF varies greatly among tumors, and systemic blockade of both tumor and stroma-derived VEGF is sufficient for inhibiting the growth of tumor xenografts.

The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to alpha(v)beta(3) and induced integrin alpha(v)beta(3)-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin alpha(v)beta(3) and suggest a possible role in the regulation of angiogenesis.

Vascular endothelial growth factor (VEGF) has been found to have various functions on endothelial cells, the most prominent of which is the induction of proliferation and differentiation. In this report we demonstrate that VEGF or a mutant, selectively binding to the Flk-1/KDR receptor, displayed high levels of survival activity, whereas Flt-1-specific ligands failed to promote survival of serum-starved primary human endothelial cells. This activity was blocked by the phosphatidylinositol 3'-kinase (PI3-kinase)-specific inhibitors wortmannin and LY294002. Endothelial cells cultured in the presence of VEGF and the Flk-1/KDR-selective VEGF mutant induced phosphorylation of the serine-threonine kinase Akt in a PI3-kinase-dependent manner. Akt activation was not detected in response to stimulation with placenta growth factor or an Flt-1-selective VEGF mutant. Furthermore, a constitutively active Akt was sufficient to promote survival of serum-starved endothelial cells in transient transfection experiments. In contrast, overexpression of a dominant-negative form of Akt blocked the survival effect of VEGF. These findings identify the Flk-1/KDR receptor and the PI3-kinase/Akt signal transduction pathway as crucial elements in the processes leading to endothelial cell survival induced by VEGF. Inhibition of apoptosis may represent a major aspect of the regulatory activity of VEGF on the vascular endothelium.

We investigated the possibility that vascular endothelial growth factor (VEGF) treatment could regulate KDR/Flk-1 receptor expression in endothelial cells. Bovine adrenal cortex endothelial cells were incubated with 200 pM rhVEGF165 for 0-7 days. Western blot analysis showed a 3-5-fold increase in total KDR protein following 4-day VEGF treatment. Scatchard analysis revealed that VEGF induced a 2-3-fold increase in high affinity receptor number (5.0 x 10(4)/cell versus 2. 4 x 10(4)/cell) without significantly affecting receptor binding affinity (Kd 76 pM versus 72 pM). Quantitative polymerase chain reaction analysis demonstrated a 3-fold increase in KDR mRNA levels following VEGF exposure. VEGF-induced KDR expression primarily occurred at the transcriptional level as demonstrated by a luciferase reporter assay system. Receptor selective mutants with wild-type KDR binding and decreased Flt-1 binding also induced KDR up-regulation; in contrast, mutants with decreased KDR binding and wild-type Flt-1 binding did not, suggesting that KDR receptor signaling mediated the increase in KDR expression. Inhibition of tyrosine kinase, Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase activities all blocked VEGF-induced KDR up-regulation. Finally, co-incubation of nitric-oxide synthase inhibitors with VEGF had no significant effect on KDR expression, but 100 microM sodium nitroprusside, a NO donor, significantly inhibited VEGF-induced KDR up-regulation, indicating that NO negatively regulates KDR expression. In conclusion, our data demonstrate that VEGF binding to the KDR receptor tyrosine kinase results in an increase in KDR receptor gene transcription and protein expression. Thus, KDR up-regulation induced by VEGF may represent an important positive feedback mechanism for VEGF action in tumor and ischemia-induced angiogenesis.

Tumor necrosis factor-alpha (TNF-alpha) modulates gene expression in endothelial cells and is angiogenic in vivo. TNF-alpha does not activate in vitro migration and proliferation of endothelium, and its angiogenic activity is elicited by synthesis of direct angiogenic inducers or of proteases. Here, we show that TNF-alpha up-regulates in a dose- and time-dependent manner the expression and the function of vascular endothelial growth factor receptor-2 (VEGFR-2) as well as the expression of its co-receptor neuropilin-1 in human endothelium. As inferred by nuclear run-on assay and transient expression of VEGFR-2 promoter-based reporter gene construct, the cytokine increased the transcription of the VEGFR-2 gene. Mithramycin, an inhibitor of binding of nuclear transcription factor Sp1 to the promoter consensus sequence, blocked activation of VEGFR-2, suggesting that the up-regulation of the receptor required Sp1 binding sites. TNF-alpha increased the cellular amounts of VEGFR-2 protein and tripled the high affinity 125I-VEGF-A165 capacity without affecting the Kd of ligand-receptor interaction. As a consequence, TNF-alpha enhanced the migration and the wound healing triggered by VEGF-A165. Since VEGFR-2 mediates angiogenic signals in endothelium, our data indicate that its up-regulation is another mechanism by which TNF-alpha is angiogenic and may provide insight into the mechanism of neovascularization as occurs in TNF-alpha-mediated pathological settings.

We examined the role of vascular endothelial growth factor (VEGF) in preventing apoptosis in primary human umbilical vein endothelial (HUVE) cells. VEGF was capable of preventing serum starvation-induced apoptosis at concentrations between 10 and 100 ng/ml. The addition of VEGF to serum-starved HUVE cells led to a 5. 2-fold induction of Bcl-2 after 36 h and to a transient, 2.4-fold induction of A1 after a 7-h incubation, as quantitated by real time reverse transcriptase-polymerase chain reaction analysis. Western blot analysis demonstrated a 2-3-fold induction of Bcl-2 protein after 18-36 h of exposure to VEGF and a transient induction of A1 after 7 h of VEGF stimulation. Moreover, overexpression of Bcl-2 by means of transient biolistic transfection experiments of HUVE cells was sufficient to prevent endothelial cells from apoptotic cell death in the absence of VEGF. These findings indicate that Bcl-2 plays an important role in mediating the survival activity of VEGF on endothelial cells.

Vascular endothelial growth factor (VEGF) and its two endothelial cell-specific receptor tyrosine kinases, Flk-1/KDR and Flt-1, play a key role in physiological and pathological angiogenesis. Hypoxia has been shown to be a major mechanism for up-regulation of VEGF and its receptors in vivo. When we exposed human umbilical vein endothelial cells to hypoxic conditions in vitro, we observed increased levels of Flt-1 expression. In contrast, Flk-1/KDR mRNA levels were unchanged or slightly repressed. These findings suggest a differential transcriptional regulation of the two receptors by hypoxia. To identify regulatory elements involved in the hypoxic response, promoter regions of the mouse Flt-1 and Flk-1/KDR genes were isolated and tested in conjunction with luciferase reporter gene. In transient transfection assays, hypoxia led to strong transcriptional activation of the Flt-1 promoter, whereas Flk-1/KDR transcription was essentially unchanged. Promoter deletion analysis demonstrated a 430-bp region of the Flt-1 promoter to be required for transcriptional activation in response to hypoxia. This region includes a heptamer sequence matching the hypoxia-inducible factor-1 (HIF) consensus binding site previously found in other hypoxia-inducible genes such as the VEGF gene and erythropoietin gene. We further narrowed down the element mediating the hypoxia response to a 40-base pair sequence including the putative HIF binding site. We show that this element acts like an enhancer, since it activated transcription irrespective of its location or orientation in the construct. Furthermore, mutations within the putative HIF consensus binding site lead to impaired transcriptional activation by hypoxia. These findings indicate that, unlike the KDR/Flk-1 gene, the Flt-1 receptor gene is directly up-regulated by hypoxia via a hypoxia-inducible enhancer element located at positions -976 to -937 of the Flt-1 promoter.