Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase)

Flt-1 tyrosine kinase, vascular endothelial growth factor (VEGF) receptor-1, binds VEGF and a new VEGF-related ligand, placenta growth factor, but KDR/Flk-1 (VEGF receptor-2) binds only VEGF. To characterize the functional regions in the Flt-1 extracellular domain such as the ligand binding region and the dimer formation of the receptor, we constructed a series of mutants of the Flt-1 extracellular domain as soluble forms in a baculovirus system. We found that a region carrying the N-terminal 1st to 3rd immunoglobulin (Ig)-like domains of Flt-1 binds both ligands with high affinity. However, for dimer formation of soluble Flt-1, a region further downstream in the Flt-1 extracellular domain was required. Mutant Flt-1 receptors expressed in COS cells confirmed the requirement of the 4th to 7th Ig region for the activation of Flt-1 tyrosine kinase. Soluble Flt-1 carrying the N-terminal 1st to 3rd Ig region suppressed VEGF-dependent endothelial proliferation in vitro to the same level as the larger forms of soluble Flt-1, suggesting that the binding of one soluble Flt-1 molecule to one subunit of the VEGF homodimer may be sufficient to block the VEGF activity.     

Mapping the charged residues in the second immunoglobulin-like domain of the vascular endothelial growth factor/placenta growth factor receptor Flt-1 required for binding and structural stability

Flt-1 is one of two receptor tyrosine kinases through which the angiogenic factor vascular endothelial growth factor (VEGF) functions. Placenta growth factor (PlGF) is an additional ligand for Flt-1. The second immunoglobulin-like domain in the extracellular domain of Flt-1 has previously been identified as the region containing the critical ligand-binding determinants. We analyzed the contribution of charged residues within the first three domains of Flt-1 to ligand binding by alanine-scanning mutagenesis. Domain 2 residues Arg159, Glu208 and His223-Arg224 (together) affect both VEGF and PlGF binding, while Glu137, Lys171, His223, and Arg224 affect PlGF but not VEGF. Several charged residues, especially Asp187, are important in maintaining the structural integrity of domain 2. In addition, some residues in domain 3 contribute to binding (Asp231) or provide for additional discrimination between ligands (Arg280-Asp283).     

A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E, mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor tyrosine kinases

The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E. VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer. VEGF-E and VEGF-A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1). VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2. These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis.     

Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis

Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation.     

A novel type of vascular endothelial growth factor, VEGF-E (NZ-7 VEGF), preferentially utilizes KDR/Flk-1 receptor and carries a potent mitotic activity without heparin-binding domain

Vascular endothelial growth factor (VEGF) mediates endothelial cell proliferation, angiogenesis, and vascular permeability via the endothelial cell receptors, KDR/Flk-1 and Flt-1. Recently, a gene encoding a polypeptide with about 25% amino acid identity to mammalian VEGF was identified in the genome of Orf virus (OV), a parapoxvirus that affects sheep and goats and occasionally, humans, to generate lesions with angiogenesis. In this study, we examined the biological activities and receptor of OV-derived NZ-7 VEGF (VEGF-E). VEGF-E was found to be a dimer of about 20 kDa with no basic domain nor affinity for heparin column, similar to VEGF121 subtype. VEGF121 has 10-100-fold less endothelial cell mitotic activity than VEGF165 due to lack of a heparin-binding basic region. Interestingly, however, VEGF-E showed almost equal levels of mitotic activity on primary endothelial cells and vascular permeability activity as VEGF165. Furthermore, VEGF-E bound KDR/Flk-1 (VEGFR-2) and induced its autophosphorylation to almost the same extent as VEGF165, but did not bind Flt-1 (VEGFR-1) nor induce autophosphorylation of Flt-1. These results indicate that VEGF-E is a novel type of endothelial growth factor, utilizing only one of the VEGF receptors, and carrying a potent mitogenic activity without affinity to heparin.     

Eicosapentaenoic acid enhances nitric oxide production by cultured human endothelial cells

Flt-1, a tyrosine kinase receptor for vascular endothelial growth factor (VEGF), plays important roles in the angiogenesis required for embryogenesis and in monocyte/macrophage migration. However, the signal transduction of Flt-1 is poorly understood due to its very weak tyrosine kinase activity. Therefore, we overexpressed Flt-1 in insect cells using the Baculovirus system in order to examine for autophosphorylation sites and association with adapter molecules such as phospholipase Cgamma-1 (PLCgamma). Tyr-1169 and Tyr-1213 on Flt-1 were found to be auto-phosphorylated, but only a phenylalanine mutant of Tyr-1169 strongly suppressed its association with PLCgamma. In Flt-1 overexpressing NIH3T3 cells, VEGF induced autophosphorylation of Flt-1, tyrosine-phosphorylation of PLCgamma and protein kinase C-dependent activation of MAP kinase. These results strongly suggest that Tyr-1169 on Flt-1 is a major binding site for PLCgamma and important for Flt-1 signal transduction within the cell.     

Fetal liver kinase 1 is a receptor for vascular endothelial growth factor and is selectively expressed in vascular endothelium

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, induces endothelial proliferation in vitro and vascular permeability in vivo. The human transmembrane c-fms-like tyrosine kinase Flt-1 has recently been identified as a VEGF receptor. Flt-1 kinase has seven immunoglobulin-like extracellular domains and a kinase insert sequence, features shared by two other human gene-encoded proteins, kinase insert domain-containing receptor (KDR) and FLT-4. In this study we show that the mouse homologue of KDR, Flk-1, is a second functional VEGF receptor. Flk-1 binds VEGF with high affinity, undergoes autophosphorylation, and mediates VEGF-dependent Ca2+ efflux in Xenopus oocytes injected with Flk-1 mRNA. We also demonstrate by in situ hybridization that Flk-1 protein expression in the mouse embryo is restricted to the vascular endothelium and the umbilical cord stroma. VEGF and its receptors Flk-1/KDR and Flt-1 may play a role in vascular development and regulation of vascular permeability.     

Expression of vascular endothelial growth factor and placenta growth factor in human placenta

Normal development and function of the placenta requires invasion of the maternal decidua by trophoblasts, followed by abundant and organized vascular growth. Little is known of the significance and function of the vascular endothelial growth factor (VEGF) family, which includes VEGF, VEGF-B, and VEGF-C, and of placenta growth factor (PIGF) in these processes. In this study we have analyzed the expression of VEGF and PIGF mRNAs and their protein products in placental tissue obtained from noncomplicated pregnancies. Expression of VEGF and PIGF mRNA was observed by in situ hybridization in the chorionic mesenchyme and villous trophoblasts, respectively. Immunostaining localized the VEGF and PIGF proteins in the vascular endothelium, which was defined by staining for von Willebrand factor and for the Tie receptor tyrosine kinase, an early endothelial cell marker. VEGF-B and VEGF-C mRNAs were strongly expressed in human placenta as evidenced by Northern blot analysis. These data imply that VEGF and PIGF are produced by different cells but that both target the endothelial cells of normal human term placenta.     

Flt-1, a receptor for vascular endothelial growth factor, has transforming and morphogenic potentials

A paradox of Flt-1, a tyrosine kinase receptor for vascular endothelial growth factor (VEGF), is that the ligand cannot activate the receptor to stimulate growth of cells that exogenously overexpress the receptor. In order to find Flt-1 kinase-dependent biological systems, we obtained for the first time activated forms of the Flt-1 kinase in a ligand-independent manner. Replacement of the ABL sequences in the human leukemia oncoprotein BCR-ABL with the cytoplasmic domain of Flt-1 (BCR-FLT) followed by a retroviral random mutagenesis scheme gave constitutively active artificial chimera BCR-FLTm with mutations within the Flt-1 sequence. Like BCR-ABL it could, but not the original BCR-FLT, transform Rat1 fibroblasts, abrogate cytokine dependence in Ba/F3 cells, and induce neurite-like structures in neuronal PC12 cells. Interestingly, Rat1 cells transformed by BCR-FLTm formed tube-like structures in basement membrane matrix. BCR-FLTm retroviruses may be a very useful tool to investigate an as yet uncovered functions of the Flt-1 kinase.     

Identification of vascular endothelial growth factor receptor-1 tyrosine phosphorylation sites and binding of SH2 domain-containing molecules

Receptor tyrosine phosphorylation is crucial for signal transduction by creating high affinity binding sites for Src homology 2 domain-containing molecules. By expressing the intracellular domain of Flt-1/vascular endothelial growth factor receptor-1 in the baculosystem, we identified two major tyrosine phosphorylation sites at Tyr-1213 and Tyr-1242 and two minor tyrosine phosphorylation sites at Tyr-1327 and Tyr-1333 in this receptor. This pattern of phosphorylation of Flt-1 was also detected in vascular endothelial growth factor-stimulated cells expressing intact Flt-1. In vitro protein binding studies using synthetic peptides and immunoblotting showed that phospholipase C-gamma binds to both Y(p)1213 and Y(p)1333, whereas Grb2 and SH2-containing tyrosine protein phosphatase (SHP-2) bind to Y(p)1213, and Nck and Crk bind to Y(p)1333 in a phosphotyrosine-dependent manner. In addition, unidentified proteins with molecular masses around 74 and 27 kDa bound to Y(p)1213 and another of 75 kDa bound to Y(p)1333 in a phosphotyrosine-dependent manner. SHP-2, phospholipase C-gamma, and Grb2 could also be shown to bind to the intact Flt-1 intracellular domain. These results indicate that a spectrum of already known as well as novel phosphotyrosine-binding molecules are involved in signal transduction by Flt-1.     

Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice

Receptor tyrosine kinases Flt-1 and Flk-1/KDR, and their ligand, the vascular endothelial growth factor (VEGF), were shown to be essential for angiogenesis in the mouse embryo by gene targeting. Flk-1/KDR null mutant mice exhibited impaired endothelial and hematopoietic cell development. On the other hand, Flt-1 null mutation resulted in early embryonic death at embryonic day 8.5, showing disorganization of blood vessels, such as overgrowth of endothelial cells. Flt-1 differs from Flk-1 in that it displays a higher affinity for VEGF but lower kinase activity, suggesting the importance of its extracellular domain. To examine the biological role of Flt-1 in embryonic development and vascular formation, we deleted the kinase domain without affecting the ligand binding region. Flt-1 tyrosine kinase-deficient homozygous mice (flt-1(TK-/-)) developed normal vessels and survived. However, VEGF-induced macrophage migration was strongly suppressed in flt-1(TK-/-) mice. These results indicate that Flt-1 without tyrosine kinase domain is sufficient to allow embryonic development with normal angiogenesis, and that a receptor tyrosine kinase plays a main biological role as a ligand-binding molecule.     

The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and vasculogenic mitogen. VEGF also plays a role in pathogenic vascularization which is associated with a number of clinical disorders, including cancer and rheumatoid arthritis. The development of VEGF antagonists, which prevent the interaction of VEGF with its receptor, may be important for the treatment of such disorders. VEGF is a homodimeric member of the cystine knot growth factor superfamily, showing greatest similarity to platelet-derived growth factor (PDGF). VEGF binds to two different tyrosine kinase receptors, kinase domain receptor (KDR) and Fms-like tyrosine kinase 1 (Flt-1), and a number of VEGF homologs are known with distinct patterns of specificity for these same receptors. The structure of VEGF will help define the location of the receptor-binding site, and shed light on the differences in specificity and cross-reactivity among the VEGF homologs. RESULTS: We have determined the crystal structure of the receptor-binding domain of VEGF at 1.93 A resolution in a triclinic space group containing eight monomers in the asymmetric unit. Superposition of the eight copies of VEGF shows that the beta-sheet core regions of the monomers are very similar, with slightly greater differences in most loop regions. For one loop, the different copies represent different snapshots of a concerted motion. Mutagenesis mapping shows that this loop is part of the receptor-binding site of VEGF. CONCLUSIONS: A comparison of the eight independent copies of VEGF in the asymmetric unit indicates the conformational space sampled by the protein in solution; the root mean square differences observed are similar to those seen in ensembles of the highest precision NMR structures. Mapping the receptor-binding determinants on a multiple sequence alignment of VEGF homologs, suggests the differences in specificity towards KDR and Flt-1 may derive from both sequence variation and changes in the flexibility of binding loops. The structure can also be used to predict possible receptor-binding determinants for related cystine knot growth factors, such as PDGF.     

Role of VEGF receptor-1 (Flt-1) in mediating calcium-dependent nitric oxide release and limiting DNA synthesis in human trophoblast cells

Vascular endothelial growth factor (VEGF) receptor KDR (kinase-insert-domain-containing receptor) is linked to endothelial cell proliferation, and VEGF receptor Flt-1 (fms-like tyrosine kinase) is essential for the organization of embryonic vasculature. Flt-1 is also known to be expressed on adult endothelial and trophoblast cells, although its function has not yet been established. Herein we report that human trophoblast and endothelial cells contain functional Flt-1 receptors for VEGF that trigger the synthesis and release of nitric oxide (NO) by the activation of constitutive NO synthase (cNOS). In first-trimester human trophoblast cells isolated by chorionic villous sampling, VEGF165 stimulated NO release in a concentration- and time-dependent manner, with a maximal increase of 60% (in comparison to basal release levels) occurring within 30 minutes (basal: 1342 pmol/ml; VEGF (10 ng/ml): 2162 pmol/ml; p < 0.001), as measured by an NO chemiluminescence analyzer. VEGF20, a peptide fragment that is composed of the first 20 amino acids at N-terminus, displayed properties of a partial agonist. VEGF165- and VEGF20-mediated NO biosynthesis was attenuated by NG-nitro-L-arginine in a concentration-dependent fashion, indicating NOS activation. VEGF-neutralizing anti-VEGF monoclonal antibody significantly inhibited VEGF-mediated NO release (p < 0.001), and the addition of a neutralizing anti-Flt-1 antibody inhibited the response by 79.6% +/- 7.59%, an effect found to be reversible with higher concentrations of VEGF. In contrast, anti-KDR antibody had no significant inhibitory effect. RT-PCR confirmed the presence of mRNA encoding the Flt-1 and KDR receptors as well as the endothelial form of cNOS in trophoblast cells. VEGF165-stimulated NO release was inhibited by genistein (5 microM; p < 0.001) as well as by the removal of calcium from the extracellular environment (p < 0.001), which suggests the contingency of this process on tyrosine phosphorylation and extracellular calcium, respectively. Addition of sodium nitroprusside, an NO donor, inhibited trophoblast DNA synthesis in a concentration-dependent manner, as measured by [3H]thymidine incorporation, without affecting cell viability. VEGF under maximal NO production had no mitogenic activity, suggesting that trophoblast-derived NO may limit trophoblast proliferation. Endogenous trophoblast DNA synthesis increased 3-fold in the presence of anti-Flt-1 antibody but not in the presence of anti-KDR antibody, suggesting that Flt-1 functions as a growth suppressive receptor to counteract the proliferative actions of KDR. Levels of immunoreactive endothelial cNOS were markedly increased in growth-restricted placentae (n = 4) in comparison to those of normal (n = 5) placentae, which may account for the relatively small-sized placentae associated with intrauterine growth restriction. VEGF165 stimulated NO release via phosphorylation of the Flt-1 receptor, indicating that VEGF may be an autocrine regulator of NO biosynthesis by aiding trophoblast penetration into spinal arterioles during the first trimester and preventing platelet aggregation within the placenta. Finally, the activation of Flt-1 receptor suppressed trophoblast DNA synthesis within the placenta via NO.     

Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.     

SCCA1 and SCCA2 are proteinase inhibitors that map to the serpin cluster at 18q21.3

Vascular endothelial growth factor (VEGF) is an essential molecule in the development and formation of mammalian blood vessels in health and disease. VEGF is also increasingly implicated in other biological processes including renal development and pathophysiology. The biological activities of VEGF in vivo and in its target cells in culture are mediated through two receptor protein tyrosine kinases, KDR/Flk-1 and Flt-1. KDR/Flk-1 is able to mediate the tyrosine phosphorylation of several cellular components as well as the generation of second messengers. Recent findings have revealed novel signaling mechanisms which may mediate the biological actions of VEGF. In contrast, the signal transduction mechanisms triggered by Flt-1 remain largely unknown.     

Requirements for binding and signaling of the kinase domain receptor for vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a dimeric hormone that controls much of vascular development through binding and activation of its kinase domain receptor (KDR). We produced analogs of VEGF that show it has two receptor-binding sites which are located near the poles of the dimer and straddle the interface between subunits. Deletion experiments in KDR indicate that of the seven IgG-like domains in the extracellular domain, only domains 2-3 are needed for tight binding of VEGF. Monomeric forms of the extracellular domain of KDR bind approximately 100 times weaker than dimeric forms showing a strong avidity component for binding of VEGF to predimerized forms of the receptor. Based upon these structure-function studies and a mechanism in which receptor dimerization is critical for signaling, we constructed a receptor antagonist in the form of a heterodimer of VEGF that contained one functional and one non-functional site. These studies establish a functional foundation for the design of VEGF analogs, mimics, and antagonists.     

2'-Fluoropyrimidine RNA-based aptamers to the 165-amino acid form of vascular endothelial growth factor (VEGF165). Inhibition of receptor binding and VEGF-induced vascular permeability through interactions requiring the exon 7-encoded domain

Vascular endothelial growth factor (VEGF) has been implicated in the pathological induction of new blood vessel growth in a variety of proliferative disorders. Using the SELEX process (systematic evolution of ligands by exponential enrichment), we have isolated 2'-F-pyrimidine RNA oligonucleotide ligands (aptamers) to human VEGF165. Representative aptamers from three distinct sequence families were truncated to the minimal sequence capable of high affinity binding to VEGF (23-29 nucleotides) and were further modified by replacement of 2'-O-methyl for 2'-OH at all ribopurine positions where the substitution was tolerated. Equilibrium dissociation constants for the interaction of VEGF with the truncated, 2'-O-methyl-modified aptamers range between 49 and 130 pM. These aptamers bind equally well to murine VEGF164, do not bind to VEGF121 or the smaller isoform of placenta growth factor (PlGF129), and show reduced, but significant affinity for the VEGF165/PlGF129 heterodimer. Cysteine 137 in the exon 7-encoded domain of VEGF165 forms a photo-inducible cross-link to a single uridine residue in each of the three aptamers. The aptamers potently inhibit the binding of VEGF to the human VEGF receptors, KDR and Flt-1, expressed by transfected porcine aortic endothelial cells. Furthermore, one of the aptamers is able to significantly reduce intradermal VEGF-induced vascular permeability in vivo.