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.     

Synergistic Binding of Vascular Endothelial Growth Factor-A and Its Receptors to Heparin Selectively Modulates Complex Affinity

Angiogenesis is a highly regulated process orchestrated by the VEGF system. Heparin/heparan sulfate proteoglycans and neuropilin-1 (NRP-1) have been identified as co-receptors, yet the mechanisms of action have not been fully defined. In the present study, we characterized molecular interactions between receptors and co-receptors, using surface plasmon resonance and in vitro binding assays. Additionally, we demonstrate that these binding events are relevant to VEGF activity within endothelial cells. We defined interactions and structural requirements for heparin/HS interactions with VEGF receptor (VEGFR)-1, NRP-1, and VEGF₁₆₅ in complex with VEGFR-2 and NRP-1. We demonstrate that these structural requirements are distinct for each interaction. We further show that VEGF₁₆₅, VEGFR-2, and monomeric NRP-1 bind weakly to heparin alone yet show synergistic binding to heparin when presented together in various combinations. This synergistic binding appears to translate to alterations in VEGF signaling in endothelial cells. We found that soluble NRP-1 increases VEGF binding and activation of VEGFR-2 and ERK1/2 in endothelial cells and that these effects require sulfated HS. These data suggest that the presence of HS/heparin and NRP-1 may dictate the specific receptor type activated by VEGF and ultimately determine the biological output of the system. The ability of co-receptors to fine-tune VEGF responsiveness suggests the possibility that VEGF-mediated angiogenesis can be selectively stimulated or inhibited by targeting HS/heparin and NRP-1.     

Neuropilin-1 forms complexes with vascular endothelial growth factor receptor-2 during megakaryocytic differentiation of UT-7/TPO cells

We investigated whether the gene expression of vascular endothelial growth factor (VEGF) and its receptors (VEGFR and neuropilin-1 [NRP-1]) could be specifically regulated during the megakaryocytic differentiation of human thrombopoietin (TPO)-dependent UT-7/TPO cells. Undifferentiated UT-7/TPO cells expressed a functional VEGFR-2, leading to VEGF binding and VEGF₁₆₅-induced tyrosine phosphorylation, cell proliferation, and apoptosis inhibition. The megakaryocytic differentiation of UT-7/TPO cells on treatment with phorbol myristate acetate (PMA) was accompanied by a marked up-regulation of NRP-1 mRNA and protein expression and by an increase in VEGF-binding activity, which was mainly mediated by VEGFR-2. VEGF₁₆₅ promoted the formation of complexes containing NRP-1 and VEGFR-2 in undifferentiated UT-7/TPO cells in a dose-dependent manner. Unlike human umbilical vein endothelial cells, PMA-differentiated UT-7/TPO cells exhibited complex formation between NRP-1 and VEGFR-2 even in the absence of VEGF₁₆₅. These findings suggest that NRP-1-VEGFR-2-complex formation may contribute to effective cellular functions mediated by VEGF₁₆₅ in megakaryocytic cells.     

Neuropilin-1 regulates a new VEGF-induced gene, Phactr-1, which controls tubulogenesis and modulates lamellipodial dynamics in human endothelial cells

Recently, we identified a new Vascular Endothelial Growth Factor (VEGF)-A₁₆₅-induced gene Phactr-1, (Phosphatase Actin Regulator-1). We reported that Phactr-1 gene silencing inhibited tube formation in human umbilical endothelial cells (HUVECs) indicating a key role for Phactr-1 in tubulogenesis in vitro. In this study, we investigated the role of Phactr-1 in several cellular processes related to angiogenesis. We found that neuropilin-1 (NRP-1) and VEGF-R1 depletion inhibited Phactr-1 mRNA expression while NRP-2 and VEGF-R2 depletion had no effect. We described a new interaction site of VEGF-A₁₆₅ to VEGF-R1 in peptides encoded by exons 7 and 8 of VEGF-A₁₆₅. The specific inhibition of VEGF-A₁₆₅ binding on NRP-1 and VEGF-R1 by ERTCRC and CDKPRR peptides decreased the Phactr-1 mRNA levels in HUVECs indicating that VEGF-A₁₆₅-dependent regulation of Phactr-1 expression required both NRP-1 and VEGF-R1 receptors. In addition, upon VEGFA₁₆₅-stimulation Phactr-1 promotes formation and maintenance of cellular tubes through NRP-1 and VEGFR1. Phactr-1 was previously identified as protein phosphatase 1 (PP1) α-interacting protein that possesses actin-binding domains. We showed that Phactr-1 depletion decreased PP1 activity, disrupted the fine-tuning of actin polymerization and impaired lamellipodial dynamics. Taken together our results strongly suggest that Phactr-1 is a key component in the angiogenic process.     

Structure-activity relationship study of tetrapeptide inhibitors of the Vascular Endothelial Growth Factor A binding to Neuropilin-1

Neuropilin-1 is considered as one of the key receptors responsible for signaling pathways involved in pathological angiogenesis necessary for tumor progression, therefore targeting of VEGF₁₆₅ binding to NRP-1 could be a relevant strategy for antiangiogenic treatment. It was shown before that the VEGF₁₆₅/NRP-1 interaction can be inhibited by short tetrapeptides with K/RXXR sequence.Here, we present a structure–activity relationship study of the systematic optimization of amino acid residues in positions 1–3 in the above tetrapeptides. All the 13 synthesized analogs possessed C-terminal arginine that is a necessary element for interaction with NRP-1. The obtained results of the inhibitory activity and modeling by molecular dynamics indicate that simultaneous interactions of the basic amino acid residues in position 1 and 4 (Arg) with Neuropilin-1 are crucial and their cooperation strongly affects the inhibitory activity. In addition, the binding strength is modulated by the flexibility of the peptide backbone (in the central part of the peptide), and the nature of the side chain of the amino acids at the second or third position. A dramatic decrease in the activity to the receptor is observed in flexible derivatives that are missing proline residues. The results described in this paper should prove useful for future studies aimed at establishing the best pharmacophore for inhibitors of VEGF₁₆₅ binding to NRP-1.     

Discovery of novel inhibitors of vascular endothelial growth factor-A–Neuropilin-1 interaction by structure-based virtual screening

Neuropilin-1 (NRP-1), one of the most important co-receptors of vascular endothelial growth factor-A (VEGF-A), increases its angiogenic action in several chronic diseases including cancer by increasing the activity of associated tyrosine kinase receptors, VEGFR1 and VEGFR2. Binding of VEGF-A to NRP-1 plays a critical role in pathological angiogenesis and tumor progression. Today, targeting this interaction is a validated approach to fight against angiogenesis-dependent diseases. Only anti-NRP-1 antibodies, peptide and peptidomimetic drug-candidates or hits have been developed thus far. In order to identify potent orally active small organic molecules various experimental and in silico approaches can be used. Here we report, novel promising small drug-like molecules disrupting the binding of VEGF-A₁₆₅ to NRP-1. We carried out structure-based virtual screening experiments using the ChemBridge compound collection on the VEGF-A₁₆₅ binding pocket of NRP-1. After docking and two rounds of similarity search computations, we identified 4 compounds that inhibit the biotinylated VEGF-A₁₆₅ binding to recombinant NRP-1 with K_i of about 10 μM. These compounds contain a common chlorobenzyloxy alkyloxy halogenobenzyl amine scaffold that can serve as a base for further development of new NRP-1 inhibitors.     

Investigation of the interaction between split aptamer and vascular endothelial growth factor 165 using single molecule force spectroscopy

Understanding the binding of split aptamer/its target could become a breakthrough in the application of split aptamer. Herein, vascular endothelial growth factor (VEGF), a major biomarker of human diseases, was used as a model, and its interaction with split aptamer was explored with single molecule force spectroscopy (SMFS). SMFS demonstrated that the interaction force of split aptamer/VEGF₁₆₅ was 169.44 ± 6.59 pN at the loading rate of 35.2 nN/s, and the binding probability of split aptamer/VEGF₁₆₅ was dependent on the concentration of VEGF₁₆₅. On the basis of dynamic force spectroscopy results, one activation barrier in the dissociation process of split aptamer/VEGF₁₆₅ complexes was revealed, which was similar to that of the intact aptamer/VEGF₁₆₅. Besides, the dissociation rate constant (k_off) of split aptamer/VEGF₁₆₅ was close to that of intact aptamer/VEGF₁₆₅, and the interaction force of split aptamer/VEGF₁₆₅ was higher than the force of intact aptamer/VEGF₁₆₅. It indicated that split aptamer also possessed high affinity with VEGF₁₆₅. The work can provide a new method for exploring the interaction of split aptamer/its targets at single‐molecule level.     

Sugar-based peptidomimetics as potential inhibitors of the vascular endothelium growth factor binding to neuropilin-1

Neuropilin-1 (NRP-1) is a co-receptor of VEGFR₁₆₅ and molecules interfering with VEGF₁₆₅ binding to NRP-1 seem to be promising candidates as new angiogenesis modulators. Based on the minimal four amino acid sequence of peptidic ligands known to bind NRP-1, we describe here the design, synthesis and biological evaluation of series of original sugar-based peptidomimetics using a C-glycosyl compound, derived from d-gulonolactone, as a scaffold, which was functionalized with side chains of the amino-acids arginine, and tryptophane or threonine. At 100 μM, all compounds exhibited a weak affinity for NRP-1, the most efficient being the bis-guanidinylated compound 32 (IC₅₀ = 92 μM) which could be considered as a new NRP-1 non-peptidic ligand.     

Antiangiogenic and antitumor activities of peptide inhibiting the vascular endothelial growth factor binding to neuropilin-1

Neuropilin-1 (NRP-1), a non-tyrosine kinase receptor of vascular endothelial growth factor-165 (VEGF165), was found expressed on endothelial and some tumor cells. Since its overexpression is correlated with tumor angiogenesis and progression, the targeting of NRP-1 could be a potential anti-cancer strategy. To explore this hypothesis, we identified a peptide inhibiting the VEGF165 binding to NRP-1 and we tested whether it was able to inhibit tumor growth and angiogenesis. To prove the target of peptide action, we assessed its effects on binding of radiolabeled VEGF165 to recombinant receptors and to cultured cells expressing only VEGFR-2 (KDR) or NRP-1. Antiangiogenic activity of the peptide was tested in vitro in tubulogenesis assays and in vivo in nude mice xenotransplanted in fat-pad with breast cancer MDA-MB-231 cells. Tumor volumes, vascularity and proliferation indices were determined. The selected peptide, ATWLPPR, inhibited the VEGF165 binding to NRP-1 but not to tyrosine kinase receptors, VEGFR-1 (flt-1) and KDR; nor did it bind to heparin. It diminished the VEGF-induced human umbilical vein endothelial cell proliferation and tubular formation on Matrigel and in co-culture with fibroblasts. Administration of ATWLPPR to nude mice inhibited the growth of MDA-MB-231 xenografts, and reduced blood vessel density and endothelial cell area but did not alter the proliferation indices of the tumor. In conclusion, ATWLPPR, a previously identified KDR-interacting peptide, was shown to inhibit the VEGF165 interactions with NRP-1 but not with KDR and to decrease the tumor angiogenesis and growth, thus validating, in vivo, NRP-1 as a possible target for antiangiogenic and antitumor agents.     

Soluble Fas ligand inhibits angiogenesis in rheumatoid arthritis

The characteristics of rheumatoid arthritis (RA) pathology include the infiltration of inflammatory leukocytes, the proliferation of synovial cells, and the presence of extensive angiogenesis, referred to as rheumatoid pannus. Fas ligand is critical to the homeostatic regulation of the immune response, but its role in the angiogenic process of RA remains to be defined. In this study, we investigated whether soluble Fas ligand (sFasL) induces synoviocyte apoptosis and regulates angiogenesis of endothelial cells in RA. The levels of sFasL were elevated in the synovial fluids of RA patients when compared to those of osteoarthritis (OA) patients, and they correlated inversely with vascular endothelial growth factor₁₆₅ (VEGF₁₆₅) concentrations. sFasL, ranging from 10 to 100 ng/ml, induced the apoptosis of RA fibroblast-like synoviocytes (FLS) in vitro, and thereby decreased VEGF₁₆₅ production. In addition, sFasL inhibited VEGF₁₆₅-induced migration and chemotaxis of endothelial cells to basal levels in a manner independent of the Fas-mediated cell death. sFasL dose-dependently suppressed the VEGF₁₆₅-stimulated increase in pAkt expression in endothelial cells, which might be associated with its anti-migratory effect on endothelial cells. Moreover, sFasL strongly inhibited neovascularization in the Matrigel plug in vivo. Our data suggest that sFasL shows anti-angiogenic activity within RA joints not only by inducing apoptosis of VEGF₁₆₅-producing cells but also by blocking VEGF₁₆₅-induced migration of endothelial cells, independent of Fas-mediated apoptosis.     

Effects of transforming growth factor-β1 and vascular endothelial growth factor 165 gene transfer on Achilles tendon healing

Repaired Achilles tendons typically take weeks before they are strong enough to handle physiological loads. Gene therapy is a promising treatment for Achilles tendon defects. The aim of the present study was to evaluate the histological/biomechanical effects of Transforming growth factor-β1 (TGF-β1) and vascular endothelial growth factor 165 (VEGF₁₆₅) gene transfer on Achilles tendon healing in rabbits. Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs) were transduced with adenovirus carrying human TGF-β1 cDNA (Ad-TGF-β1), human VEGF₁₆₅ cDNA (Ad-VEGF₁₆₅), or both (PIRES-TGF-β1/VEGF₁₆₅) Viruses, no cDNA (Ad-GFP), and the BMSCs without gene transfer and the intact tendon were used as control. BMSCs were surgically implanted into the experimentally injured Achilles tendons. TGF-β1 distribution, cellularity, nuclear aspect ratio, nuclear orientation angle, vascular number, collagen synthesis, and biomechanical features were measured at 1, 2, 4, and 8 weeks after surgery. The TGF-β1 and TGFβ1/VEGF₁₆₅ co-expression groups exhibited improved parameters compared with other groups, while the VEGF₁₆₅ expression group had a negative impact. In the co-expression group, the angiogenesis effects of VEGF₁₆₅ were diminished by TGF-β1, while the collagen synthesis effects of TGF-β1 were unaltered by VEGF₁₆₅. Thus treatment with TGF-β1 cDNA-transduced BMSCs grafts is a promising therapy for acceleration and improvement of tendon healing, leading to quicker recovery and improved biomechanical properties of Achilles tendons.     

Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors

Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF₁₆₅ elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF₁₆₅ resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.     

Molecular modelling,synthesis and biological evaluation of peptide inhibitors as anti-angiogenic agent targeting neuropilin-1 for anticancer application

Vascular endothelial growth factor (VEGF) and its co-receptor neuropilin-1 (NRP-1) are important targets of many pro-angiogenic factors. In this study, nine peptides were synthesized and evaluated for their molecular interaction with NRP-1 and compared to our previous peptide ATWLPPR. Docking study showed that the investigated peptides shared the same binding region as shown by tuftsin known to bind selectively to NRP-1. Four pentapeptides (DKPPR, DKPRR, TKPPR and TKPRR) and a hexapeptide CDKPRR demonstrated good inhibitory activity against NRP-1. In contrast, peptides having arginine residue at sites other than the C-terminus exhibited low activity towards NRP-1 and this is confirmed by their inability to displace the VEGF₁₆₅ binding to NRP-1. Docking study also revealed that replacement of carboxyl to amide group at the C-terminal arginine of the peptide did not affect significantly the binding interaction to NRP-1. However, the molecular affinity study showed that these peptides have marked reduction in the activity against NRP-1. Pentapeptides having C-terminal arginine showed strong interaction and good inhibitory activity with NRP thus may be a good template for anti-angiogenic targeting agent.     

Synthesis and structure–activity relationship of non-peptidic antagonists of neuropilin-1 receptor

Neuropilins (NRPs) are VEGF-A₁₆₅ co-receptors over-expressed in tumor cells, and considered as targets in angiogenic-related pathologies. We previously identified compound 1, the first non-peptidic antagonist of the VEGF-A₁₆₅/NRP binding, which exhibits in vivo anti-angiogenic and anti-tumor activities. We report here the synthesis and biological evaluations of new antagonists structurally-related to compound 1. Among these molecules, 4a, 4c and 4d show cytotoxic effects on HUVEC and MDA-MB-31 cells, and antagonize VEGF-A₁₆₅/NRP-1 binding. This study confirmed our key structure–activity relationships hypothesis and paved the way to compound 1 ‘hit to lead’ optimization.     

Anti-angiogenic action of PPAR�� ligand in human umbilical vein endothelial cells is mediated by PTEN upregulation and VEGFR-2 downregulation

Peroxisome proliferator-activated receptor ?? (PPAR??) activation has anti-angiogenic and apoptotic effects in endothelial cells. Here, we investigated the mechanisms of the anti-angiogenic action of a novel PPAR?? ligand, KR-62980. KR-62980 inhibited in vitro basal tube formation and in vivo neovascularization in mice induced by Matrigel containing vascular endothelial growth factor (VEGF₁₆₅, 5 ng/ml). VEGF₁₆₅-induced cell proliferation and chemotactic migration in human umbilical vein endothelial cells (HUVECs) were also suppressed by KR-62980, in a mechanism accompanied by apoptotic cell death. KR-62980 downregulated the VEGF₁₆₅-induced VEGFR-2 expression but increased the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression in parallel with reduced phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2), PI3K p85??, and p38 MAPK. The knockdown of PTEN expression abolished KR-62980-suppressed cell proliferation and angiogenesis. All of the effects of KR-62980 disappeared with pretreatment of bisphenol A diaglycidyl ether (BADGE), a PPAR?? antagonist. In summary, KR-62980 inhibited VEGF₁₆₅-induced angiogenesis in HUVECs by PPAR??-mediated dual mechanisms: VEGFR-2 downregulation and PTEN upregulation.     

Post-ExSELEX stabilization of an unnatural-base DNA aptamer targeting VEGF165 toward pharmaceutical applications

A new technology, genetic alphabet expansion using artificial bases (unnatural bases), has created high-affinity DNA ligands (aptamers) that specifically bind to target proteins by ExSELEX (genetic alphabet Expansion for Systematic Evolution of Ligands by EXponential enrichment). We recently found that the unnatural-base DNA aptamers can be stabilized against nucleases, by introducing an extraordinarily stable, unique hairpin DNA (mini-hairpin DNA) and by reinforcing the stem region with G–C pairs. Here, to establish this aptamer generation method, we examined the stabilization of a high-affinity anti-VEGF₁₆₅ unnatural-base DNA aptamer. The stabilized aptamers displayed significantly increased thermal and nuclease stabilities, and furthermore, exhibited higher affinity to the target. As compared to the well-known anti-VEGF₁₆₅ RNA aptamer, pegaptanib (Macugen), our aptamers did not require calcium ions for binding to VEGF₁₆₅. Biological experiments using cultured cells revealed that our stabilized aptamers efficiently inhibited the interaction between VEGF₁₆₅ and its receptor, with the same or slightly higher efficiency than that of the pegaptanib RNA aptamer. The development of cost-effective and calcium ion-independent high-affinity anti-VEGF₁₆₅ DNA aptamers encourages further progress in diagnostic and therapeutic applications. In addition, the stabilization process provided additional information about the key elements required for aptamer binding to VEGF₁₆₅.     

Synthetic Heparan Sulfate Oligosaccharides Inhibit Endothelial Cell Functions Essential for Angiogenesis

Background

Heparan sulfate (HS) is an important regulator of the assembly and activity of various angiogenic signalling complexes. However, the significance of precisely defined HS structures in regulating cytokine-dependent angiogenic cellular functions and signalling through receptors regulating angiogenic responses remains unclear. Understanding such structure-activity relationships is important for the rational design of HS fragments that inhibit HS-dependent angiogenic signalling complexes.

Methodology/Principal Findings

We synthesized a series of HS oligosaccharides ranging from 7 to 12 saccharide residues that contained a repeating disaccharide unit consisting of iduronate 2-O-sulfate linked to glucosamine with or without N-sulfate. The ability of oligosaccharides to compete with HS for FGF2 and VEGF₁₆₅ binding significantly increased with oligosaccharide length and sulfation. Correspondingly, the inhibitory potential of oligosaccharides against FGF2- and VEGF₁₆₅-induced endothelial cell responses was greater in longer oligosaccharide species that were comprised of disaccharides bearing both 2-O- and N-sulfation (2SNS). FGF2- and VEGF₁₆₅-induced endothelial cell migration were inhibited by longer 2SNS oligosaccharide species with 2SNS dodecasaccharide activity being comparable to that of receptor tyrosine kinase inhibitors targeting FGFR or VEGFR-2. Moreover, the 2SNS dodecasaccharide ablated FGF2- or VEGF₁₆₅-induced phosphorylation of FAK and assembly of F-actin in peripheral lamellipodia-like structures. In contrast, FGF2-induced endothelial cell proliferation was only moderately inhibited by longer 2SNS oligosaccharides. Inhibition of FGF2- and VEGF₁₆₅-dependent endothelial tube formation strongly correlated with oligosaccharide length and sulfation with 10-mer and 12-mer 2SNS oligosaccharides being the most potent species. FGF2- and VEGF₁₆₅-induced activation of MAPK pathway was inhibited by biologically active oligosaccharides correlating with the specific phosphorylation events in FRS2 and VEGFR-2, respectively.

Conclusion/Significance

These results demonstrate structure-function relationships for synthetic HS saccharides that suppress endothelial cell migration, tube formation and signalling induced by key angiogenic cytokines.     

Regulation of Vascular Endothelial Growth Factor (VEGF) Splicing from Pro-angiogenic to Anti-angiogenic Isoforms: A NOVEL THERAPEUTIC STRATEGY FOR ANGIOGENESIS*

Vascular endothelial growth factor (VEGF) is produced either as a pro-angiogenic or anti-angiogenic protein depending upon splice site choice in the terminal, eighth exon. Proximal splice site selection (PSS) in exon 8 generates pro-angiogenic isoforms such as VEGF₁₆₅, and distal splice site selection (DSS) results in anti-angiogenic isoforms such as VEGF₁₆₅b. Cellular decisions on splice site selection depend upon the activity of RNA-binding splice factors, such as ASF/SF2, which have previously been shown to regulate VEGF splice site choice. To determine the mechanism by which the pro-angiogenic splice site choice is mediated, we investigated the effect of inhibition of ASF/SF2 phosphorylation by SR protein kinases (SRPK1/2) on splice site choice in epithelial cells and in in vivo angiogenesis models. Epithelial cells treated with insulin-like growth factor-1 (IGF-1) increased PSS and produced more VEGF₁₆₅ and less VEGF₁₆₅b. This down-regulation of DSS and increased PSS was blocked by protein kinase C inhibition and SRPK1/2 inhibition. IGF-1 treatment resulted in nuclear localization of ASF/SF2, which was blocked by SPRK1/2 inhibition. Pull-down assay and RNA immunoprecipitation using VEGF mRNA sequences identified an 11-nucleotide sequence required for ASF/SF2 binding. Injection of an SRPK1/2 inhibitor reduced angiogenesis in a mouse model of retinal neovascularization, suggesting that regulation of alternative splicing could be a potential therapeutic strategy in angiogenic pathologies.     

KDR activation is crucial for VEGF165-mediated Ca2+ mobilization in human umbilical vein endothelial cells

We have prepared apolyclonal mouse antibody directed against the first threeimmunoglobulin-like domains of the kinase insert domain-containingreceptor (KDR) tyrosine kinase. It possesses the ability to inhibitbinding of the 165-amino acid splice variant of vascular endothelialcell growth factor (VEGF₁₆₅) torecombinant KDR in vitro as well as to reduceVEGF₁₆₅ binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the firstthree immunoglobulin-like domains of KDR are involved in VEGF₁₆₅ interactions. The anti-KDRantibody is able to completely blockVEGF₁₆₅-mediated intracellularCa²⁺ mobilization in HUVEC.Therefore, it appears that binding of VEGF₁₆₅ to the fms-like tyrosinekinase (Flt-1) in these cells does not translate into aCa²⁺ response. This is furtherexemplified by the lack of response to placental growth factor (PlGF),an Flt-1-specific ligand. Additionally, PlGF is unable to potentiatethe effects of submaximal concentrations ofVEGF₁₆₅. Surprisingly, theVEGF-PlGF heterodimer was also very inefficient at eliciting aCa²⁺ signaling event in HUVEC. Weconclude that KDR activation is crucial for mobilization ofintracellular Ca²⁺ in HUVEC inresponse to VEGF₁₆₅.