Vascular endothelial growth factor 165 (VEGF(165)) activities are inhibited by carboxymethyl benzylamide dextran that competes for heparin binding to VEGF(165) and VEGF(165).KDR Complexes

We have previously shown that carboxymethyl dextran benzylamide (CMDB7), a heparin-like molecule, inhibits the growth of tumors xenografted in nude mice, angiogenesis, and metastasis by altering the binding of angiogenic growth factors, including platelet-derived growth factor, transforming growth factor beta, and fibroblast growth factor 2, to their specific receptors. In this study, we explore the effect of CMDB7 on the most specific angiogenic growth factor, vascular endothelial growth factor 165 (VEGF(165)). We demonstrate here that CMDB7 inhibits the mitogenic effect of VEGF(165) on human umbilical vein endothelial cells (HUV-ECs) by preventing the VEGF(165)-induced VEGF receptor-2 (KDR) autophosphorylation and consequently a specific intracellular signaling. In competition experiments, the binding of (125)I-VEGF(165) to HUV-ECs is inhibited by CMDB7 with an IC(50) of 2 microm. Accordingly, CMDB7 inhibits the cross-linking of (125)I-VEGF(165) to the surface of HUV-ECs, causing the disappearance of both labeled complexes, 170-180 and 240-250 kDa. We show that CMDB7 increases the electrophoretic mobility of VEGF(165), thus evidencing formation of a stable complex with this factor. Moreover, CMDB7 reduces the (125)I-VEGF(165) binding to coated heparin-albumin and prevents a heparin-induced increase in iodinated VEGF(165) binding to soluble (125)I-KDR-Fc chimera. Concerning KDR, CMDB7 has no effect on (125)I-KDR-Fc electrophoretic migration and does not affect labeled KDR-Fc binding to coated heparin-albumin. In the presence of VEGF(165), (125)I-KDR-Fc binding to heparin is enhanced, and under these conditions, CMDB7 interferes with KDR binding. These data indicate that CMDB7 effectively inhibits the VEGF(165) activities by interfering with heparin binding to VEGF(165) and VEGF(165).KDR complexes but not by direct interactions with KDR.     

Crystal structure of the stromelysin-3 (MMP-11) catalytic domain complexed with a phosphinic inhibitor mimicking the transition-state

Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP-11) whose proteolytic activity plays an important role in tumorigenicity enhancement. In breast cancer, ST3 is a bad prognosis marker: its expression is associated with a poor clinical outcome. This enzyme therefore represents an attractive therapeutic target.The topology of matrix metalloproteinases (MMPs) is remarkably well conserved, making the design of highly specific inhibitors difficult. The major difference between MMPs lies in the S(1)' subsite, a well-defined hydrophobic pocket of variable depth. The present crystal structure, the first 3D-structure of the ST3 catalytic domain in interaction with a phosphinic inhibitor mimicking a (d, l) peptide, clearly demonstrates that its S(1)' pocket corresponds to a tunnel running through the enzyme. This open channel is filled by the inhibitor P(1)' group which adopts a constrained conformation to fit this pocket, together with two water molecules interacting with the ST3-specific residue Gln215. These observations provide clues for the design of more specific inhibitors and show how ST3 can accommodate a phosphinic inhibitor mimicking a (d, l) peptide.The presence of a water molecule interacting with one oxygen atom of the inhibitor phosphinyl group and the proline residue of the Met-turn suggests how the intermediate formed during proteolysis may be stabilized. Furthermore, the hydrogen bond distance observed between the methyl of the phosphinic group and the carbonyl group of Ala182 mimics the interaction between this carbonyl group and the amide group of the cleaved peptidic bond. Our crystal structure provides a good model to study the MMPs mechanism of proteolysis.     

Molecular mapping and functional characterization of the VEGF164 heparin-binding domain

The longer splice isoforms of vascular endothelial growth factor-A (VEGF-A), including mouse VEGF164, contain a highly basic heparin-binding domain (HBD), which imparts the ability of these isoforms to be deposited in the heparan sulfate-rich extracellular matrix and to interact with the prototype sulfated glycosaminoglycan, heparin. The shortest isoform, VEGF120, lacks this highly basic domain and is freely diffusible upon secretion. Although the HBD has been attributed significant relevance to VEGF-A biology, the molecular determinants of the heparin-binding site are unknown. We used site-directed mutagenesis to identify amino acid residues that are critical for heparin binding activity of the VEGF164 HBD. We focused on basic residues and found Arg-13, Arg-14, and Arg-49 to be critical for heparin binding and interaction with extracellular matrix in tissue samples. We also examined the cellular and biochemical consequences of abolishing heparin-binding function, measuring the ability of the mutants to interact with VEGF receptors, induce endothelial cell gene expression, and trigger microvessel outgrowth. Induction of tissue factor expression, vessel outgrowth, and binding to VEGFR2 were unaffected by the HBD mutations. In contrast, the HBD mutants showed slightly decreased binding to the NRP1 (neuropilin-1) receptor, and analyses suggested the heparin and NRP1 binding sites to be distinct but overlapping. Finally, mutations that affect the heparin binding activity also led to an unexpected reduction in the affinity of VEGF164 binding specifically to VEGFR1. This finding provides a potential basis for previous observations suggesting enhanced potency of VEGF164 versus VEGF120 in VEGFR1-mediated signaling in inflammatory cells.     

Solution structure of the Grb2 SH2 domain complexed with a high-affinity inhibitor

The solution structure of the growth factor receptor-bound protein 2 (Grb2) SH2 domain complexed with a high-affinity inhibitor containing a non-phosphorus phosphate mimetic within a macrocyclic platform was determined by nuclear magnetic resonance (NMR) spectroscopy. Unambiguous assignments of the bound inhibitor and intermolecular NOEs between the Grb2 SH2 domain and the inhibitor was accomplished using perdeuterated Grb2 SH2 protein. The well-defined solution structure of the complex was obtained and compared to those by X-ray crystallography. Since the crystal structure of the Grb2 SH2 domain formed a domain-swapped dimer and several inhibitors were bound to a hinge region, there were appreciable differences between the solution and crystal structures. Based on the binding interactions between the inhibitor and the Grb2 SH2 domain in solution, we proposed a design of second-generation inhibitors that could be expected to have higher affinity.     

Distinct heparin binding sites on VEGF165 and its receptors revealed by their interaction with a non sulfated glycoaminoglycan (NaPaC)

We previously demonstrated that a non sulfated analogue of heparin, phenylacetate carboxymethyl benzylamide dextran (NaPaC) inhibited angiogenesis. Here, we observed that NaPaC inhibited the VEGF165 binding to both VEGFR2 and NRP-1 and abolished VEGFR2 activity. Further, we explored the effects of NaPaC on VEGF165 interactions with its receptors, VEGFR2 and NRP-1, co-receptor of VEGFR2. Surface plasmon resonance and affinity gel electrophoresis showed that NaPaC interacted directly with VEGF165, VEGFR2 and NRP-1 but not with heparin-independent factor such as VEGF121. NaPaC completely inhibited the heparin binding to VEGF165, NRP-1 and VEGFR2. We found that NaPaC bound to all three molecules, VEGF165, VEGFR2 and NRP-1, but was more effective in inhibiting heparin binding to VEGF165. These results suggested that heparin binding sites of VEGFR2 and NRP-1 were different from those of VEGF165.     

Effects of VEGF(165) and VEGF(121) on vasculogenesis and angiogenesis in cultured embryonic quail hearts

It has been documented that hypoxia enhances coronary vasculogenesis and angiogenesis in cultured embryonic quail hearts via the upregulation of vascular endothelial growth factor (VEGF). In this study, we compared the functions of two VEGF splice variants. Ventricles from 6-day-old embryonic quail hearts were cultured on three-dimensional collagen gels. Recombinant human VEGF(121) or VEGF(165) were added to the culture medium for 48 h, and vascular growth was visualized by immunostaining with a quail-specific endothelial cell (EC) marker, QH1. VEGF(165) enhanced vascular growth in a dose-dependent manner: 5 ng/ml of VEGF(165) slightly increased the number of ECs, 10 ng/ml of VEGF(165) increased the incorporation of ECs into tubular structures, and at 20 ng/ml of VEGF(165) wider tubes were formed. This pattern plateaued at the 50 ng/ml dose. In contrast, VEGF(121) did not enhance either the number of ECs or tube formation at these or higher dosages. Combined effects of hypoxia and exogenous VEGF(165) were then compared. Tube formation from the heart explants treated with both hypoxia and 50 ng/ml of VEGF(165) had a morphology intermediate to those treated with hypoxia or VEGF(165) alone. Immunocytochemistry study revealed EC lumenization under all culture conditions. However, the addition of VEGF(165) stimulated the coalescence of ECs to form larger vessels. We conclude the following: 1) VEGF(121) and VEGF(165) induced by hypoxia have different functions on coronary vascular growth, 2) unknown factors induced by hypoxia can modify the effect of VEGF(165), and 3) EC lumenization observed in the heart explant culture closely mimics in vivo coronary vasculogenesis.     

Identification of the heparin-binding region of snake venom vascular endothelial growth factor (VEGF-F) and its blocking of VEGF-A165

VEGF-A165 displays multiple effects through binding to KDR (VEGFR-2). Heparin/heparan sulfate-like molecules are known to greatly modulate their interaction. In fact, VEGF-A lacking a C-terminal heparin-binding region exhibits significantly reduced mitogenic activity. We recently found novel heparin-binding VEGFs in snake venom, designated VEGF-Fs, which specifically recognize KDR, rather than other VEGF receptors. VEGF-Fs virtually lack the C-terminal heparin-binding region when compared with other heparin-binding VEGF subtypes, despite their heparin-binding potential. The C-terminal region does not exhibit any significant homology with other known proteins or domains. In this study, we attempted to identify the heparin-binding region of VEGF-F using synthetic peptides. The C-terminal peptide of vammin (one of the VEGF-Fs, 19 residues) bound to heparin with similar affinity as native vammin. We then evaluated the effects of this peptide on the biological activity of VEGF-A165. The C-terminal peptide of VEGF-F exhibited specific blockage of VEGF-A165 activity both in vitro and in vivo. These observations demonstrate that the short C-terminal region of VEGF-F functions fully as the active heparin-binding domain and the corresponding peptide specifically blocks VEGF-A165, thus suggesting that the C-terminal heparin-binding region of VEGF-F recognizes similar heparin/heparan sulfate molecules as VEGF-A165. The present results will provide novel insight into VEGF-heparin interaction and may facilitate the design of new anti-VEGF drugs based on novel strategies.     

Expression and protein chemistry yielding crystallization of the catalytic domain of ADAM17 complexed with a hydroxamate inhibitor

The membrane-anchored metalloproteinase ADAM17 (TNF-alpha converting enzyme; TACE; EC 3.4.24.86) continues to be an attractive drug target in inflammatory diseases and cancer. Cocrystallization of its catalytic domain with a lead compound was complicated by the tenacious retention of the prodomain that has been shown to be enhanced if ADAM17 is expressed without the disintegrin/cysteine-rich domain that normally follows the N-terminal metalloproteinase. When a truncated form of ADAM17 composed of the signal peptide with the pro- and catalytic domains was expressed in baculovirus-infected insect cells, the major secreted product was a ternary complex of two prodomain fragments with the catalytic domain. The component polypeptides of the ternary complex were characterized by N-terminal analysis and mass spectrometry. Internal cleavage of the propeptide occurred following Arg-58, and a carboxypeptidase variably removed up to three basic residues from the newly created C-terminus. Cleavage at the C-terminus of the propeptide occurred after Arg-214. To prepare ADAM17 for crystal growth, a drug-like inhibitor was used to displace the propeptide and the complex of the catalytic domain with the inhibitor was isolated by size-exclusion chromatography and crystallized.     

Suppression of the biological activities of the epidermal growth factor (EGF)-like domain by the heparin-binding domain of heparin-binding EGF-like Growth Factor

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that has a high affinity for heparin and heparan sulfate. While interactions with heparin are thought to modulate the biological activity of HB-EGF, the precise role of the heparin-binding domain has remained unclear. We analyzed the activity of wild-type HB-EGF and a mutant form lacking the heparin-binding domain (DeltaHB) in the presence or absence of heparin. The activity of the EGF-like domain of HB-EGF was determined by measuring binding to diphtheria toxin (DT) as well as the growth factor activity in EGF receptor-expressing cells. The binding affinity of DeltaHB for DT was much higher than that of wild-type HB-EGF in the absence of heparin. The binding affinity of HB-EGF for DT was increased by addition of exogenous heparin and reached the level close to the affinity of DeltaHB, whereas that of DeltaHB was not affected. Moreover, the growth factor activity of DeltaHB was much higher than that of wild-type HB-EGF in the absence of heparin but was not affected by addition of exogenous heparin, whereas HB-EGF had increased growth factor activity with added heparin. These results indicate that the heparin-binding domain suppresses the activity of the EGF-like domain of HB-EGF and that association of heparin with HB-EGF via this domain removes the suppressive effect. Thus, we conclude that the heparin-binding domain serves as a negative regulator of this growth factor.     

Clinical improvement after treatment with VEGF(165) in patients with severe chronic lower limb ischaemia

The present study focuses on the application of a therapeutic strategy in patients with chronic severe lower limb ischaemia using a plasmid vector encoding the vascular endothelial growth factor (phVEGF₁₆₅). It has been shown that VEGF promotes neo-vascularization and blood vessel network formation and thus might have the ability to improve blood-flow at the level of the affected limbs. However, little information is available regarding the necessary level of expression of VEGF and its possible related adverse effects. We have subcloned VEGF ₁₆₅ isoform into pCMV-Script expression vector (Stratagene) under the control of the CMV promoter. Three patients with chronic ischaemia of the lower limb, considered as not suitable for surgical re-vascularization, received intramuscular injection with 0.5 ml saline solution containing 10¹¹ copies of VEGF ₁₆₅ plasmid. The clinical evolution has been monitored by angiography and estimated by walking time on the rolling carpet (Gardner protocol). Two months after therapy, all three patients showed complete relief of rest pain, improvement of ischaemic ulcer lesions and increased walking distance on the rolling carpet most probably due to appearance of newly formed collateral vessels.     

The structure of a synthetic pepsin inhibitor complexed with endothiapepsin

The conformation of a synthetic polypeptide inhibitor, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 0.20-nm resolution and refined to an agreement factor of 0.20. The inhibitor: Pro Thr Glu Phe-R-Phe Arg Glu (R = -CH2NH-) is based on a chromogenic substrate of pepsin (EC 3.4.23.1). It has, in place of the scissile bond, a reduced peptide group which is resistant to hydrolysis and mimics the tetrahedral transition state. The inhibitor binds in an extended conformation with the reduced bond close to the essential aspartate side-chains of the enzyme. The hydrogen bonds and hydrophobic interactions between the enzyme and the inhibitor do not induce large conformational changes.     

Covalently grafted VEGF(165) in hydrogel models upregulates the cellular pathways associated with angiogenesis

Angiogenesis is an important biological response known to be involved in many physiological and pathophysiological situations. Cellular responses involved in the formation of new blood vessels, such as increases in endothelial cell proliferation, cell migration, and the survival of apoptosis-inducing events, have been associated with vascular endothelial growth factor isoform 165 (VEGF(165)). Current research in the areas of bioengineering and biomedical science has focused on developing polyethylene glycol (PEG)-based systems capable of initiating and sustaining angiogenesis in vitro. However, a thorough understanding of how endothelial cells respond at the molecular level to VEGF(165) incorporated into these systems has not yet been established in the literature. The goal of the current study was to compare the upregulation of key intracellular proteins involved in angiogenesis in human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMEC) seeded on PEG hydrogels containing grafted VEGF(165) and adhesion peptides Arg-Gly-Asp-Ser (RGDS). Our data suggest that the covalent incorporation of VEGF(165) into PEG hydrogels encourages the upregulation of signaling proteins responsible for increases in endothelial cell proliferation, cell migration, and the survival after apoptosis-inducing events.     

Solution structure and backbone dynamics of the catalytic domain of matrix metalloproteinase-2 complexed with a hydroxamic acid inhibitor

MMP-2 is a member of the matrix metalloproteinase family that has been implicated in tumor cell metastasis and angiogenesis. Here, we describe the solution structure of a catalytic domain of MMP-2 complexed with a hydroxamic acid inhibitor (SC-74020), determined by three-dimensional heteronuclear NMR spectroscopy. The catalytic domain, designated MMP-2C, has a short peptide linker replacing the internal fibronectin-domain insertion and is enzymatically active. Distance geometry-simulated annealing calculations yielded 14 converged structures with atomic root-mean-square deviations (r.m.s.d.) of 1.02 and 1.62 A from the mean coordinate positions for the backbone and for all heavy atoms, respectively, when 11 residues at the N-terminus are excluded. The structure has the same global fold as observed for other MMP catalytic domains and is similar to previously solved crystal structures of MMP-2. Differences observed between the solution and the crystal structures, near the bottom of the S1' specificity loop, appear to be induced by the large inhibitor present in the solution structure. The MMP-2C solution structure is compared with MMP-8 crystal structure bound to the same inhibitor to highlight the differences especially in the S1' specificity loop. The finding provides a structural explanation for the selectivity between MMP-2 and MMP-8 that is achieved by large inhibitors.     

Primary structure of a DNA- and heparin-binding domain (Domain III) in human plasma fibronectin

The complete amino acid sequence of a DNA- and heparin-binding domain isolated by limited thermolysin digestion of human plasma fibronectin has been obtained. The domain contains 90 amino acids with a calculated molecular weight of 10,225. The apparent molecular mass of this domain is 14 kDa when analyzed by sodium dodecyl sulfate-gel electrophoresis. The anomalously high molecular size estimation may be due to the inaccuracy of this method in the low range. The structure was established from microsequence analysis of the chymotryptic, tryptic, and Staphylococcus aureus protease peptides. The molecular ion of each of the chymotryptic peptides was obtained by fast atom bombardment mass spectrometry. The domain has a preponderance of basic residues with a net charge of +5 at neutral pH. The basic nature of the domain may account for its affinity for the polyanions, DNA and heparin. The predicted secondary structure is beta-sheet, in common with all of the type III internal sequence homology structures obtained for fibronectin so far. The location of the domain in fibronectin was made possible by limited thermolysin digestion and identification of the fragments and by comparison of the sequence of the 14-kDa fragment with the partial structure of bovine plasma fibronectin. The domain comprises residues 585-675 and defines a region immediately adjacent to the collagen-binding domain. Numbering domains beginning at the amino terminus, this domain is Domain III after the fibrin/heparin/actin/S. aureus binding Domain I and the collagen-binding Domain II. The domain was obtained from a larger precursor (56 kDa) which bound heparin, DNA, and gelatin. Further digestion of the 56-kDa fragment gave rise to a 40-kDa fragment which only bound gelatin, and a 14-kDa fragment which only bound heparin or DNA. The 14-kDa fragment (Domain III) marks the beginning of the type III homology region in fibronectin, for there may be up to 15 repeats of 90 amino acids. The size of this domain corresponds to one repeat of 90 amino acids and it has some sequence homology to the other type III sequences found thus far in fibronectin.     

Specific interaction of VEGF165 with beta-amyloid, and its protective effect on beta-amyloid-induced neurotoxicity

beta-amyloid (Abeta) is a major component of senile plaques that is commonly found in the brain of Alzheimer's disease (AD) patient. In the previous report, we showed that an important angiogenic factor, vascular endothelial growth factor (VEGF) interacts with Abeta and is accumulated in the senile plaques of AD patients' brains. Here we show that Abeta interacts with VEGF(165) isoform, but not with VEGF(121). Abeta binds to the heparin-binding domain (HBD) of VEGF(165) with similar affinity as that of intact VEGF(165). Abeta binds mostly to the C-terminal subdomain of HBD, but with greatly reduced affinity than HBD. Therefore, the full length of HBD appears to be required for maximal binding of Abeta. Although Abeta binds to heparin-binding sequence of VEGF, it does not bind to other heparin-binding growth factors except midkine. Thus it seems that Abeta recognizes unique structural features of VEGF HBD. VEGF(165) prevents aggregation of Abeta through its HBD. We localized the core VEGF binding site of Abeta at around 26-35 region of the peptide. VEGF(165) and HBD protect PC12 cells from the Abeta-induced cytotoxicity. The mechanism of protection appears to be inhibition of both Abeta-induced formation of reactive oxygen species and Abeta aggregation.     

Structural features in heparin that interact with VEGF165 and modulate its biological activity.

The 165 amino acid form of vascular endothelial growth factor (VEGF165) is a heparin-binding growth factor with mitogenic activity for vascular endothelial cells. We examined activities of various heparin derivatives toward their interactions with VEGF165 using an enzyme-linked immunosorbent assay and elucidated the structural features in heparin for the interactions. Native heparin interacted with VEGF165, whereas N-desulfated, N-acetylated (N-DS, N-Ac-) heparin, and 6-O-desulfated (6-O-DS-) heparin did not. The 2-O-desulfated (2-O-DS-) heparin retained the ability for the interaction with VEGF165. In contrast, the 2-O-DS-heparin exhibited no ability for the interaction with FGF-2 and HGF. Thus, structural requirements in heparin for the specific interaction with VEGF165 are distinct from those with FGF-2 and HGF which require a high content of 2-O-sulfate groups. In a cell proliferation assay, native heparin and 2-O-DS-heparin exhibited inhibitory abilities for VEGF165-induced proliferation of human umbilical vein endothelial cells (HUVECs) with their high concentrations (more than 64 microg/ml), while only native heparin could enhance the proliferation of the chlorate-treated cells. These results suggested that a high content of 2-O-sulfate groups is not required for the specific interaction with VEGF165alone, although it is essential for the mitogenic activity of the growth factor.     

X-ray structure of plasmepsin II complexed with a potent achiral inhibitor

The malaria parasite Plasmodium falciparum degrades host cell hemoglobin inside an acidic food vacuole during the blood stage of the infectious cycle. A number of aspartic proteinases called plasmepsins (PMs) have been identified to play important roles in this degradation process and therefore generated significant interest as new antimalarial targets. Several x-ray structures of PMII have been described previously, but thus far, structure-guided drug design has been hampered by the fact that only inhibitors comprising a statine moiety or derivatives thereof have been published. Our drug discovery efforts to find innovative, cheap, and easily synthesized inhibitors against aspartic proteinases yielded some highly potent non-peptidic achiral inhibitors. A highly resolved (1.6 A) x-ray structure of PMII is presented, featuring a potent achiral inhibitor in an unprecedented orientation, contacting the catalytic aspartates indirectly via the "catalytic" water. Major side chain rearrangements in the active site occur, which open up a new pocket and allow a new binding mode of the inhibitor. Moreover, a second inhibitor molecule could be located unambiguously in the active site of PMII. These newly obtained structural insights will further guide our attempts to improve compound properties eventually leading to the identification of molecules suitable as antimalarial drugs.     

Site-directed mutagenesis in the B-neuropilin-2 domain selectively enhances its affinity to VEGF165, but not to semaphorin 3F

Neuropilins (NRPs) are 130-kDa receptors that bind and respond to the class 3 semaphorin family of axon guidance molecules (SEMAs) and to members of the vascular endothelial growth factor (VEGF) family of angiogenic factors. Two NRPs have been reported so far, NRP1 and NRP2. Unlike NRP1, little is known about NRP2 interactions with its ligands, VEGF165 and SEMA3F. Cell binding studies reveal that VEGF165 and SEMA3F bind NRP2 with similar affinities, 5.2 and 3.9 nM, respectively, and are competitive NRP2 ligands. Immunoprecipitation studies show that the B (b1b2) extracellular domain of NRP2 is sufficient for VEGF165 binding, whereas SEMA3F requires both the A (a1a2) and B domains. To identify residues of B-NRP2 involved in VEGF165 binding, point mutations were introduced by site-directed mutagenesis. VEGF165 is a basic protein. Reduction of the electronegative potential of B-NRP2 by exchanging acidic residues for uncharged alanine (B-NRP2 E284A,E291A) in the 280-290 b1-NRP2 loop resulted in a 2-fold reduction in VEGF165 affinity. Conversely, enhancing the electronegative potential (B-NRP2 R287E,N290D and R287E,N290S) significantly increased VEGF165 affinity for B-NRP2 by 8- and 6.6-fold, respectively. The mutagenesis did not affect SEMA3F/B-NRP2 interactions. These results demonstrate that it is possible to alter VEGF165 affinity for NRP2 without affecting SEMA3F affinity. They also identify NRP2 residues involved in VEGF165 binding and suggest that modifications of B-NRP2 could lead to potentially high affinity selective inhibitors of VEGF165/NRP2 interactions.     

The fourth immunoglobulin-like loop in the extracellular domain of FLT-1, a VEGF receptor, includes a major heparin-binding site.

We found that heparin at low concentrations increases the vascular endothelial growth factor (VEGF)-induced proliferation of human umbilical vein endothelial cells (HUVECs) but at high concentrations decreases it. To examine whether FLT-1, a VEGF receptor, interacts with heparin and which domain of FLT-1 binds to heparin, various extracellular domains of FLT-1 were expressed in a baculovirus/insect cell system: sFLT-1(1-7), sFLT-1(1-4), sFLT-1(1-3), and sFLT-1(1-2) containing immunoglobulin (Ig)-like loop one to seven, one to four, one to three, and one to two, respectively. The sFLT-1(1-7) and sFLT-1(1-4) readily bound heparin at the physiological salt concentration and half-dissociated from heparin at 0.65 M and 0.57 M NaCl, respectively. In contrast, the sFLT-1(1-3) and sFLT-1(1-2) poorly bound heparin at the physiological salt concentration. In addition, the interaction of sFLT-1(1-7) with heparin was not affected by EDTA up to 80 mM. We thus concluded that the fourth Ig-like loop of FLT-1 is a major heparin-binding site and divalent cations are not involved in the interaction of FLT-1 and heparin.