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.     

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.     

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.     

Inhibition of vascular endothelial growth factor-induced receptor activation with anti-kinase insert domain-containing receptor single-chain antibodies from a phage display library

A single-chain antibody phage display library was constructed from spleen cells of mice immunized with a soluble form of a human vascular endothelial growth factor (VEGF) receptor, kinase insert domain-containing receptor (KDR). After two rounds of biopanning, >90% of the clones recovered were specifically reactive to KDR. Subsequent selection identified two clones that blocked VEGF binding to KDR. The clones were expressed in Escherichia coli and purified as soluble single-chain Fv (scFv) antibodies. The affinities of the scFv for binding to KDR were determined by BIAcore analysis (2.1 x 10(-9)-5.9 x 10(-9) M). One scFv, p1C11, was shown to inhibit VEGF-induced KDR phosphorylation and VEGF-stimulated DNA synthesis in human umbilical vein endothelial cells. There is much experimental evidence to suggest that the VEGF/KDR/Flk-1 pathway plays an important role in tumor angiogenesis, a process that is essential for tumor growth and metastasis. The antibodies discussed here, which block VEGF binding to KDR, have potential clinical application in the treatment of cancer and other diseases where pathological angiogenesis is involved.     

Differential mobilization of fatty acids from adipose tissue

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, and the permeability of blood vessels are regulated by vascular endothelial growth factor (VEGF) via its two known receptors Flt1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2). The Flt4 receptor tyrosine kinase is related to the VEGF receptors, but does not bind VEGF and its expression becomes restricted mainly to lymphatic endothelia during development. In this study, we have purified the Flt4 ligand, VEGF-C, and cloned its cDNA from human prostatic carcinoma cells. While VEGF-C is homologous to other members of the VEGF/platelet derived growth factor (PDGF) family, its C-terminal half contains extra cysteine-rich motifs characteristic of a protein component of silk produced by the larval salivary glands of the midge, Chironomus tentans. VEGF-C is proteolytically processed, binds Flt4, which we rename as VEGFR-3 and induces tyrosine autophosphorylation of VEGFR-3 and VEGFR-2. In addition, VEGF-C stimulated the migration of bovine capillary endothelial cells in collagen gel. VEGF-C is thus a novel regulator of endothelia, and its effects may extend beyond the lymphatic system, where Flt4 is expressed.     

Expression of vascular endothelial growth factor and its receptor, KDR, following retinal ischemia-reperfusion injury in the rat

PURPOSE: There is considerable evidence that vascular endothelial growth factor (VEGF) mediates ocular neovascularization in retinal vascular diseases. We investigated the time-dependent changes in the expression of VEGF and its receptor KDR/ Flk in a transient retinal ischemia-reperfusion injury model. METHODS: Transient retinal ischemia was induced by increasing the intraocular pressure in albino rats eyes for 45 min. In situ hybridization was used to identify the retinal cells synthesizing VEGF mRNA and KDR mRNA at various times following reperfusion. Immunohistochemical analysis was also carried out to detect VEGF immunoreactivity. RESULTS: In the control, non-ischemic retinas, signals for VEGF mRNA and KDR mRNA were observed in the cells of the ganglion cell layer. Immunoreactivity to VEGF was also found in the nerve fiber layer, the ganglion cell layer, and the retinal pigment epithelial (RPE) cell layer. Immediately and 6 h after reperfusion, VEGF and KDR mRNA expression was markedly decreased, but recovered by 24 h to the levels observed in normal retinas. Immunoreactivity for VEGF was also decreased immediately and 6 h after reperfusion, and was detected in the endothelial cells of the retinal vessels after 24 h. Immunoreactivity to VEGF recovered by 48 h after reperfusion. CONCLUSIONS: The hybridization pattern of VEGF and KDR mRNA in the ganglion cell layer strongly suggests that the ganglion cells are the major source of this growth factor. The decrease of VEGF mRNA, KDR/Flk mRNA and VEGF protein levels after ischemia and recovery after reperfusion suggest that transient hypoxia might mediate short-term down-regulation of VEGF and KDR mRNA.     

Identification of the extracellular domains of Flt-1 that mediate ligand interactions

Vascular Endothelial Growth Factor (VEGF) mediates its actions through the Flt-1 and KDR(Flk-1) receptor tyrosine kinases. To localize the extracellular region of Flt-1 that is involved in ligand interactions, we prepared secreted fusion proteins between various combinations of its seven extracellular IgG-like folds. Ligand binding studies show that in combination, domains one and two (amino acids 1-234) are sufficient to achieve VEGF165 interactions. Either domain alone is insufficient to achieve this effect. However, Scatchard analysis reveals that despite the binding capabilities of this construct, the Kd is five fold lower than ligand binding to the full extracellular domain. We find that addition of domain three to this minimal site restores high affinity receptor binding. Further, we show that domains one and two are sufficient to achieve interactions of Flt-1 with Placental Growth Factor (PIGF-1).     

Mapping of the sites involved in ligand association and dissociation at the extracellular domain of the kinase insert domain-containing receptor for vascular endothelial growth factor

The kinase insert domain-containing receptor (KDR) for vascular endothelial growth factor (VEGF) has been shown to be involved in vasculogenesis and angiogenesis. This receptor is characterized by seven immunoglobulin (Ig)-like domains within its extracellular region. To identify the domains involved in VEGF binding, we constructed various deletion mutants of the extracellular region fused with the crystallizable fragment portion of an IgG and then examined the binding affinity with VEGF by means of the BIAcore biosensor assay. Deletion of the COOH-terminal two or three Ig-like domains out of a total of seven affected ligand dissociation rather than association. Further deletion of the fourth domain caused a drastic decrease in the association rate. Binding ability was abolished completely with removal of the third domain. The mutant KDR proteins lacking the NH2-terminal Ig-like domain exhibited a slightly higher association rate compared with those of the mutants having this domain. Deletion of the first two NH2-terminal Ig-like domains caused a drastic reduction in the association rate, but affinity to VEGF was retained. These results suggest that the third Ig-like domain is critical for ligand binding, the second and fourth domains are important for ligand association, and the fifth and sixth domains are required for retention of the ligand bound to the receptor molecule. The first Ig-like domain may regulate the ligand binding.     

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.     

Oxidized alpha2-macroglobulin (alpha2M) differentially regulates receptor binding by cytokines/growth factors: implications for tissue injury and repair mechanisms in inflammation

Alpha2M binds specifically to TNF-alpha, IL-1beta, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), beta-nerve growth factor (beta-NGF), platelet-derived growth factor (PDGF), and TGF-beta. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of alpha2M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized alpha2M exhibits increased binding to TNF-alpha, IL-2, and IL-6 and decreased binding to beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Hypochlorite oxidation of methylamine-treated alpha2M (alpha2M*), an analogue of the proteinase/alpha2M complex, also results in decreased binding to bFGF, beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Concomitantly, we observed decreased ability to inhibit TGF-beta binding and regulation of cells by oxidized alpha2M and alpha2M*. We then isolated alpha2M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-beta compared with alpha2M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.     

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.     

Significance of vessel count and vascular endothelial growth factor and its receptor (KDR) in intestinal-type gastric cancer

Angiogenesis is essential for tumor growth and metastasis and depends on the production of angiogenic factors by host and/or tumor cells. The role of angiogenesis and angiogenic factor expression in intestinal- and diffuse-type gastric cancer are undefined. Archival specimens of 51 intestinal-type and 38 diffuse-type human gastric carcinomas were examined for tumor vessel counts, angiogenic factor expression, and the presence or absence of angiogenic factor receptors on tumor endothelium using antibodies against vascular endothelial growth factor (VEGF) and its receptors (KDR and flt-1), basic fibroblast growth factor (bFGF) and its receptors (bek and flg), and factor VIII (endothelial cells). Vessel count and VEGF and bFGF expression were higher in intestinal-type than in diffuse-type gastric cancers (P = 0.01, P < 0.001, and P < 0.001, respectively). Similarly, vessel count and VEGF expression were higher in patients with liver metastasis than in patients with peritoneal dissemination (P = 0.003 and P = 0.01, respectively). Vessel count correlated with VEGF expression and the presence of endothelial KDR in intestinal-type gastric cancer (P = 0.003 and P = 0.02, respectively) but not diffuse-type gastric cancer. Vessel count, VEGF expression, and presence of endothelial KDR increased with increasing stage of disease in intestinal-type gastric cancer but not diffuse-type gastric cancer. The expression of bFGF and its receptors did not correlate with vessel count in either cancer type. These findings suggest that the pattern of metastasis in intestinal-type gastric cancer is angiogenesis dependent. The correlation of VEGF expression and its endothelial receptor with vessel count and stage of disease suggests that VEGF is at least one of the factors responsible for the induction of angiogenesis in intestinal-type gastric cancer.     

Role of the neuronal histaminergic system in the regulation of somatotropic function: comparison between the neonatal and the adult rat

Some fibroangiogenic factors have recently been shown to play potential roles in fibrovascular diseases. The aim of this study was to examine whether there is any relationship between growth factors and pterygium genesis. Twenty-three primary pterygia and 4 normal conjunctiva specimens were analyzed by indirect immunohistochemistry using specific antibodies against basic fibroblast growth factor (b-FGF), platelet derived growth factor (PDGF), transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha). Positive immunostaining of these growth factors was located in the epithelial cells, endothelial cells of vessels, basement membranes of vessels and epithelium, fibroblasts and infiltrating inflammatory cells in the pterygium. In the normal conjunctiva, positive immunolabeling for TGF-beta and PDGF was much weaker than in the pterygium. We conclude that growth factors may interact directly or indirectly in the pathogenesis of pterygium although proof of this awaits further studies.     

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.     

Osteoarthritis in older adults

Perivascular glial cells are thought to be involved in physiologic vascularization and also in pathologic angiogenesis in the central nervous system. We have previously shown that astrocytes are a source of transforming growth factor-beta (TGF-beta) and another inhibiting factor, which block endothelial cell growth and induce their apoptosis. Astroglia are also known to express vascular endothelial growth factor (VEGF), which is up-regulated during hypoxia. Here we demonstrate the effects of hypoxia on the expression of both TGF-beta and VEGF by retinal glial cells. Muller cells isolated from rat retina were incubated under hypoxia or normoxia and the resulting conditioned media (H-MCM and N-MCM) were assayed for their effects on growth of bovine retinal capillary endothelial (BRE) and the TGF-beta-sensitive mink lung epithelial CCL cells. The expression and quantities of VEGF and TGF-beta (active vs. latent form) were determined by immuno-adsorption, Western or Northern blotting, and ELISA. N-MCM stimulated BRE cell growth by twofold but inhibited CCL cells under similar assay conditions, whereas H-MCM had a weak stimulating effect on BRE and substantial inhibitory activity on CCL cells. Adsorption of MCM by specific antibodies as well as Western and Northern blot analysis indicated that stimulating and inhibitory activities of MCM are due to the presence of VEGF and TGF-beta, respectively. ELISA revealed that the hypoxia condition converts latent TGF-beta into its active form. In N-MCM, TGF-beta is found predominantly in the latent form, but in hypoxia MCM it is mainly active. Furthermore, it was found that treatment of Muller cells with exogenous TGF-beta under either hypoxia or normoxia increases VEGF expression in a time- and dose-dependent fashion. TGF-beta activation may, therefore, be prerequisite for hypoxia-induced up-regulation of VEGF and stimulation of angiogenesis in vivo.     

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.     

Proteoglycan distribution in lesions of atherosclerosis depends on lesion severity, structural characteristics, and the proximity of platelet-derived growth factor and transforming growth factor-beta

The accumulation of proteoglycans (PGs) in atherosclerosis contributes to disease progression and stenosis and may partly depend on local regulation by growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF)-beta. In this study, the distribution of the major extracellular PGs is compared with that of PDGF and TGF-beta isoforms in developing lesions of atherosclerosis from hypercholesterolemic nonhuman primates. Strong immunostaining for decorin, biglycan, versican, and hyaluronan is observed in both intermediate and advanced lesions. Perlecan staining is weak in intermediate lesions but strong in advanced lesions in areas bordering the plaque core. Immunostaining for PDGF-B and TGF-beta1 is particularly prominent in macrophages in intermediate and advanced lesions. In contrast, TGF-beta2 and TGF-beta3 and PDGF-A are present in both macrophages and smooth muscle cells. Overall, PG deposits parallel areas of intense growth factor immunostaining, with trends in relative localization that suggest interrelationships among certain PGs and growth factors. Notably, decorin and TGF-beta1 are distributed similarly, predominantly in the macrophage-rich core, whereas biglycan is prominent in the smooth muscle cell matrix adjoining TGF-beta1-positive macrophages. Versican and hyaluronan are enriched in the extracellular matrix adjacent to both PDGF- and TGF-beta1-positive cells. These data demonstrate that PG accumulation varies with lesion severity, structural characteristics, and the proximity of PDGF and TGF-beta.