Expression of Soluble VEGF Receptor 2 and Characterization of Its Binding by Surface Plasmon Resonance

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, 75235, USA.
Biochemical and Biophysical Research Communications (Impact Factor: 2.3). 12/1998; 252(3):643-8. DOI: 10.1006/bbrc.1998.9717
Source: PubMed


Vascular endothelial growth factor (VEGF) is an endothelial cell specific mitogen that induces angiogenesis in several pathological conditions. To block angiogenesis, soluble VEGF receptor can be used. In this study, we describe a method for high yield expression of soluble VEGF receptor 2 (sFlk-1) in a baculovirus expression system (30 mg purified sFlk-1 per L of insect cell supernatant). We also determined the binding constants for both human and mouse VEGF to the recombinant receptor by surface plasmon resonance. In this cell-free assay, under the given experimental conditions, the on-rate ka was 0.5-2.2 x 10(6) M-1s-1 and the off-rate kd was 2-4 x 10(-4) s-1 (KD = 2-6 x 10(-10) M). To our knowledge this is the first study to report on- and off-rates for the VEGF:sFlk-1 interaction. Heparin was not required for the binding of VEGF to sFlk-1 in this assay. The obtained values will serve as baseline parameters for the design of improved versions of recombinant soluble VEGF receptor.

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    • "The soluble domain of mouse VEGFR2 was expressed in Sf9 insect cells and purified to homogeneity as previously described [22]. Normal, 6-week-old, female Lewis rats were purchased from Charles River (Wilmington, MA) and used for immunizations. "
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    ABSTRACT: We generated a panel of eight rat IgG(2a) monoclonal antibodies with high affinity for mouse VEGFR2 (KDR/Flk-1), the main receptor that mediates the angiogenic effect of VEGF-A. The antibodies (termed RAFL, R at Anti Flk) bound to dividing endothelial cells more strongly than they did to nondividing cells. Most of the RAFL antibodies blocked [(125)I]VEGF(165) binding to VEGFR2. Three of eight antibodies localized to VEGFR2-positive tumor endothelium after intravenous injection into mice bearing orthotopic MDA-MB-231 breast carcinomas, as judged by indirect immunohistochemistry. An average of 60% of vessels in the tumors was stained. The majority (50-80%) of vessels were also stained in a variety of other human and murine tumors growing in mice. The antibodies did not bind detectably to the vascular endothelium in normal heart, lung, liver, and brain cortex, whereas the vascular endothelium in kidney glomerulus and pancreatic islets was stained. Treatment of mice bearing orthotopic MDA-MB-231 tumors with RAFL-1 antibody inhibited tumor growth by an average of 48% and reduced vascular density by 65%, compared to tumors in mice treated with control IgG. Vascular damage was not observed in normal organs, including kidneys and pancreas. These studies demonstrate that anti-VEGFR2 antibodies have potential for vascular targeting and imaging of tumors in vivo.
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    • "Snap frozen tumors were sectioned, blocked with 20% Aquablock (East Coast Biologics, North Berwick, ME) and stained for markers of interest. Primary antibodies used include MECA-32 (DSHB; University of Iowa), endomucin (Santa Cruz Biotechnology®, Inc., Santa Cruz, CA), NG2 (Millipore®, Billerica, MA), smooth muscle actin (NeoMarkers, Fremont, CA), Lyve1, VEGFR2 (55B11) (Cell Signaling Technology®, Danvers, MA), rabbit anti-VEGFR2 T014 (purified in our laboratory) [4], [18], rat anti-VEGFR2 RAFL-2 [19], and insulin (Dako, Glostrup, Denmark). "
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    ABSTRACT: Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF:VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF:VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors.
    Full-text · Article · Aug 2010 · PLoS ONE
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    • "A more detailed description of the receptor dimerization schemes is contained in the Methods section. Both of these mechanisms are involved in VEGF receptor dimerization; VEGF can bind to receptor monomers [12] [13] [14] [15] [16], and VEGF receptors can associate in the absence of ligand [17], however it is not known to what extent each mechanism actually occurs. One pathway may dominate over the other, and so we examine the complete dimerization model as well as these two pathways alone. "
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    ABSTRACT: Vascular endothelial growth factor (VEGF) is a potent cytokine involved in the induction of neovascularization. Secreted as a cysteine-linked dimer, it has two binding sites at opposite poles through which it may bind VEGF receptors (VEGFRs), receptor tyrosine kinases found on the surface of endothelial and other cells. The binding of a VEGF molecule to two VEGFR molecules induces transphosphorylation of the intracellular domains of the receptors, leading to signal transduction. The dominant mechanism of receptor dimerization is not clear: the receptors may be present in an inactive pre-dimerized form, VEGF binding first to one of the receptors, the second receptor then ideally located for dimerization; or VEGF may bind receptor monomers on the cell surface, which then diffuse and bind to available unligated receptor monomers to complete the activation. Both processes take place and one or other may dominate on different cell types. We demonstrate the impact of dimerization mechanism on the binding of VEGF to the cell surface and on the formation of active signaling receptor complexes. We describe two methods to determine which process dominates, based on binding and phosphorylation assays. The presence of two VEGF receptor populations, VEGFR1 and VEGFR2, can result in receptor heterodimer formation. Our simulations predict that heterodimers will comprise 10-50% of the active, signaling VEGF receptor complexes, and that heterodimers will form at the expense of homodimers of VEGFR1 when VEGFR2 populations are larger. These results have significant implications for VEGF signal transduction and interpretation of experimental studies. These results may be applicable to other ligand-receptor pairs, in particular PDGF.
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