Kinase insert domain receptor (KDR) extracellular immunoglobulin-like domains 4-7 contain structural features that block receptor dimerization and vascular endothelial growth factor-induced signaling

The vascular endothelial growth factor (VEGF) receptor tyrosine kinase subtype kinase insert domain receptor (KDR) contains seven extracellular Ig-like domains, of which the three most amino-terminal contain the necessary structural features required for VEGF binding. To clarify the functional role of KDR Ig-like domains 4-7, we compared VEGF-induced signaling in human embryonic kidney and porcine aortic endothelial cells expressing native versus mutant receptor proteins in which Ig-like domains 4-7, 4-6, or 7 had been deleted. Western blotting using an anti-receptor antibody indicated equivalent expression levels for each of the recombinant proteins. As expected, VEGF treatment robustly augmented native receptor autophosphorylation. In contrast, receptor autophosphorylation, as well as downstream signaling events, were VEGF-independent for cells expressing mutant receptors. (125)I-VEGF(165) bound with equal or better affinity to mutant versus native receptor, although the number of radioligand binding sites was significantly reduced because a significant percentage of mutant, but not native, receptors were localized to the cell interior. As was the case for native KDR, (125)I-VEGF(165) binding to the mutant receptors was dependent upon cell surface heparan sulfate proteoglycans, and (125)I-VEGF(121) bound with an affinity equal to that of (125)I-VEGF(165) to the native and mutant receptors. It is concluded that KDR Ig-like domains 4-7 contain structural features that inhibit receptor signaling by a mechanism that is independent of neuropilin-1 and heparan sulfate proteoglycans. We speculate that this provides a cellular mechanism for blocking unwanted signaling events in the absence of VEGF.     

Low molecular weight fucoidan increases VEGF165-induced endothelial cell migration by enhancing VEGF165 binding to VEGFR-2 and NRP1

Therapeutic induction of angiogenesis is a potential treatment for chronic ischemia. Heparan sulfate proteoglycans are known to play an important role by their interactions with proangiogenic growth factors such as vascular endothelial growth factor (VEGF). Low molecular weight fucoidan (LMWF), a sulfated polysaccharide from brown seaweeds that mimic some biological activities of heparin, has been shown recently to promote revascularization in rat critical hindlimb ischemia. In this report, we first used cultured human endothelial cells (ECs) to investigate the possible ability of LMWF to enhance the actions of VEGF(165). Data showed that LMWF greatly enhances EC tube formation in growth factor reduced matrigel. LMWF is a strong enhancer of VEGF(165)-induced EC chemotaxis, but not proliferation. In addition, LMWF has no effect on VEGF(121)-induced EC migration, a VEGF isoform that does not bind to heparan sulfate proteoglycans. Then, with binding studies using (125)I-VEGF(165), we observed that LMWF enhances the binding of VEGF(165) to recombinant VEGFR-2 and Neuropilin-1 (NRP1), but not to VEGFR-1. Surface plasmon resonance analysis showed that LMWF binds with high affinity to VEGF(165) (1.2 nm) and its receptors (5-20 nm), but not to VEGF(121). Pre-injection of LMWF on immobilized receptors shows that VEGF(165) has the highest affinity for VEGFR-2 and NRP1, as compared with VEGFR-1. Overall, the effects of LMWF were much more pronounced than those of LMW heparin. These findings suggested an efficient mechanism of action of LMWF by promoting VEGF(165) binding to VEGFR-2 and NRP1 on ECs that could help in stimulating therapeutic revascularization.     

VEGF165 mediates formation of complexes containing VEGFR-2 and neuropilin-1 that enhance VEGF165-receptor binding

Co-expression of NRP1 and (VEGFR-2) KDR on the surface of endothelial cells (EC) enhances VEGF165 binding to KDR and EC chemotaxis in response to VEGF165. Overexpression of NRP1 by prostate tumor cells in vivo results in increased tumor angiogenesis and growth. We investigated the molecular mechanisms underlying NRP1-mediated angiogenesis by analyzing the association of NRP1 and KDR. An intracellular complex containing NRP1 and KDR was immunoprecipitated from EC by anti-NRP1 antibodies only in the presence of VEGF165. In contrast, VEGF121, which does not bind to NRP1, did not support complex formation. Complexes containing VEGF165, NRP1, and KDR were also formed in an intercellular fashion by co-culture of EC expressing KDR only, with cells expressing NRP1 only, for example, breast carcinoma cells. VEGF165 also mediated the binding of a soluble NRP1 dimer to cells expressing KDR only, confirming the formation of such complexes. Furthermore, the formation of complexes containing KDR and NRP1 markedly increased 125I-VEGF165 binding to KDR. Our results suggest that formation of a ternary complex of VEGF165, KDR, and NRP1 potentiates VEGF165 binding to KDR. These complexes are formed on the surface of EC and in a juxtacrine manner via association of tumor cell NRP1 and EC KDR.     

Vascular endothelial growth factor receptor-2 and neuropilin-1 form a receptor complex that is responsible for the differential signaling potency of VEGF(165) and VEGF(121)

The two most abundant secreted isoforms of vascular endothelial growth factor A (VEGF(165) and VEGF(121)) are formed as a result of differential splicing of the VEGF-A gene. VEGF(165) and VEGF(121) share similar affinities at the isolated VEGF receptor (VEGFR)-2 but have been previously demonstrated to have differential ability to activate VEGFR-2-mediated effects on endothelial cells. Herein we investigate whether the recently described VEGF(165) isoform-specific receptor neuropilin-1 (Npn-1) is responsible for the difference in potency observed for these ligands. We demonstrate that although VEGFR-2 and Npn-1 form a complex, this complex does not result in an increase in VEGF(165) binding affinity. Therefore, the differential activity of VEGF(165) and VEGF(121) cannot be explained by a differential binding affinity for the complex. Using an antagonist that competes for VEGF(165) binding at the VEGFR-2.Npn-1 complex, we observe specific antagonism of VEGF(165)-meditated phosphorylation of VEGFR-2 without affecting the VEGF(121) response. These data indicate that the formation of the complex is responsible for the increased potency of VEGF(165) versus VEGF(121). Taken together, these data suggest a receptor-clustering role for Npn-1, as opposed to Npn-1 behaving as an affinity-converting subunit.     

Neuropilin-2 is a receptor for the vascular endothelial growth factor (VEGF) forms VEGF-145 and VEGF-165 [corrected

Neuropilin-1 (np-1) and neuropilin-2 (np-2) are receptors for axon guidance factors belonging to the class 3 semaphorins. np-1 also binds to the 165-amino acid heparin-binding form of VEGF (VEGF(165)) but not to the shorter VEGF(121) form, which lacks a heparin binding ability. We report that human umbilical vein-derived endothelial cells express the a17 and a22 splice forms of the np-2 receptor. Both np-2 forms bind VEGF(165) with high affinity in the presence of heparin (K(D) 1.3 x 10(-10) m) but not VEGF(121). np-2 also binds the heparin-binding form of placenta growth factor. These binding characteristics resemble those of np-1. VEGF(145) is a secreted heparin binding VEGF form that contains the peptide encoded by exon 6 of VEGF but not the peptide encoded by exon 7, which is present in VEGF(165). VEGF(145) binds to np-2 with high affinity (K(D) 7 x 10(-10) m). Surprisingly, VEGF(145) did not bind to np-1. Indeed, VEGF(145) does not bind to MDA-MB-231 breast cancer cells, which predominantly express np-1. By contrast, VEGF(145) binds to human umbilical vein-derived endothelial cells, which express both np-1 and np-2. The binding of VEGF(165) to porcine aortic endothelial cells expressing recombinant np-2 did not affect the proliferation or migration of the cells. Nevertheless, it is possible that VEGF-induced np-2-mediated signaling will take place only in the presence of other VEGF receptors such as VEGF receptor-1 or VEGF receptor-2.     

Neuropilin-1 binds vascular endothelial growth factor 165, placenta growth factor-2, and heparin via its b1b2 domain

Neuroplin-1 (NRP1), a receptor for vascular endothelial growth factor (VEGF) family members, has three distinct extracellular domains, a1a2, b1b2, and c. To determine the VEGF(165) and placenta growth factor 2 (PlGF-2)-binding sites of NRP1, recombinant NRP1 domains were expressed in mammalian cells as Myc-tagged, soluble proteins, and used in co-precipitation experiments with 125I-VEGF165 and 125I-PlGF-2. Anti-Myc antibodies immunoprecipitated 125I-VEGF165 and 125I-PlGF-2 in the presence of the b1b2 but not of the a1a2 and c domains. Neither b1 nor b2 alone was capable of binding 125I-VEGF165. In competition experiments, VEGF165 competed PlGF-2 binding to the NRP1 b1b2 domain, suggesting that the binding sites of VEGF165 and PlGF-2 overlap. The presence of the a1a2 domain greatly enhanced VEGF165, but not PlGF-2 binding to b1b2. Heparin enhanced the binding of both 125I-VEGF165 and 125I-PlGF-2 to the b1b2 domain by 20- and 4-fold, respectively. A heparin chain of at least 20-24 monosaccharides was necessary for binding. In addition, the b1b2 domain of NRP1 could bind heparin directly, requiring heparin oligomers of at least 8 monosaccharide units. It was concluded that an intact b1b2 domain serves as the VEGF165-, PlGF-2-, and heparin-binding sites in NRP1, and that heparin is a critical component for regulating VEGF165 and PlGF-2 interactions with NRP1 by physically interacting with both receptor and ligands.     

Vascular endothelial growth factor (VEGF)-A165-induced prostacyclin synthesis requires the activation of VEGF receptor-1 and -2 heterodimer

We previously reported that vascular endothelial growth factor (VEGF)-A(165) inflammatory effect is mediated by acute platelet-activating factor synthesis from endothelial cells upon the activation of VEGF receptor-2 (VEGFR-2) and its coreceptor, neuropilin-1 (NRP-1). In addition, VEGF-A(165) promotes the release of other endothelial mediators including nitric oxide and prostacyclin (PGI(2)). However, it is unknown whether VEGF-A(165) is mediating PGI(2) synthesis through VEGF receptor-1 (VEGFR-1) and/or VEGF receptor-2 (VEGFR-2) activation and whether the coreceptor NRP-1 potentiates VEGF-A(165) activity. In this study, PGI(2) synthesis in bovine aortic endothelial cells (BAEC) was assessed by quantifying its stable metabolite (6-keto prostaglandin F(1alpha), 6-keto PGF(1alpha)) by enzyme-linked immunosorbent assay. Treatment of BAEC with VEGF analogs, VEGF-A(165) (VEGFR-1, VEGFR-2 and NRP-1 agonist) and VEGF-A(121) (VEGFR-1 and VEGFR-2 agonist) (up to 10(-9) m), increased PGI(2) synthesis by 70- and 40-fold within 15 min. Treatment with VEGFR-1 (placental growth factor and VEGF-B) or VEGFR-2 (VEGF-C) agonist did not increase PGI(2) synthesis. The combination of VEGFR-1 and VEGFR-2 agonists did not increase PGI(2) release. Pretreatment with a VEGFR-2 inhibitor abrogated PGI(2) release mediated by VEGF-A(165) and VEGF-A(121), and pretreatment of BAEC with antisense oligomers targeting VEGFR-1 or VEGFR-2 mRNA reduced PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121) up to 79%. In summary, our data demonstrate that the activation of VEGFR-1 and VEGFR-2 heterodimer (VEGFR-1/R-2) is essential for PGI(2) synthesis mediated by VEGF-A(165) and VEGF-A(121), which cannot be reproduced by the parallel activation of VEGFR-1 and VEGFR-2 homodimers with corresponding agonists. In addition, the binding of VEGF-A(165) to NRP-1 potentiates its capacity to promote PGI(2) synthesis.     

Vascular endothelial growth factor induces branching morphogenesis/tubulogenesis in renal epithelial cells in a neuropilin-dependent fashion

Vascular endothelial growth factor (VEGF) is well characterized for its role in endothelial cell differentiation and vascular tube formation. Alternate splicing of the VEGF gene in mice results in various VEGF-A isoforms, including VEGF-121 and VEGF-165. VEGF-165 is the most abundant isoform in the kidney and has been implicated in glomerulogenesis. However, its role in the tubular epithelium is not known. We demonstrate that VEGF-165 but not VEGF-121 induces single-cell branching morphogenesis and multicellular tubulogenesis in mouse renal tubular epithelial cells and that these morphogenic effects require activation of the phosphatidylinositol 3-kinase (PI 3-K) and, to a lesser degree, the extracellular signal-regulated kinase and protein kinase C signaling pathways. Further, VEGF-165-stimulated sheet migration is dependent only on PI 3-K signaling. These morphogenic effects of VEGF-165 require activation of both VEGF receptor 2 (VEGFR-2) and neuropilin-1 (Nrp-1), since neutralizing antibodies to either of these receptors or the addition of semaphorin 3A (which blocks VEGF-165 binding to Nrp-1) prevents the morphogenic response and the phosphorylation of VEGFR-2 along with the downstream signaling. We thus conclude that in addition to endothelial vasculogenesis, VEGF can induce renal epithelial cell morphogenesis in a Nrp-1-dependent fashion.     

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.     

The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules.

Vascular endothelial growth factor (VEGF) induces the proliferation of endothelial cells and is a potent angiogenic factor that binds to heparin. We have therefore studied the effect of heparin upon the interaction of VEGF with its receptors. Heparin, at concentrations ranging from 0.1 to 10 micrograms/ml, strongly potentiated the binding of 125I-VEGF to its receptors on endothelial cells. Scatchard analysis of 125I-VEGF binding indicates that 1 microgram/ml heparin induces an 8-fold increase in the apparent density of high affinity binding sites for VEGF, but does not significantly affect the dissociation constant of VEGF. Cross-linking experiments showed that heparin strongly potentiates the formation of the 170-, 195- and 225-kDa 125I-VEGF-receptor complexes on endothelial cells. At high 125I-VEGF concentrations (4 ng/ml), heparin preferentially enhanced the formation of the 170- and 195-kDa complexes. Preincubation of the cells with heparin, followed by extensive washes, produced a similar enhancement of subsequent 125I-VEGF binding. The binding of 125I-VEGF was completely inhibited following digestion of endothelial cells with heparinase and could be restored by the addition of exogenous heparin to the digested cells. The enhancing effect of heparin facilitated the detection of VEGF receptors on cell types that were not known previously to express such receptors. Our results suggest that cell surface-associated heparin-like molecules are required for the interaction of VEGF with its cell surface receptors.     

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.     

Tuftsin binds neuropilin-1 through a sequence similar to that encoded by exon 8 of vascular endothelial growth factor

Tuftsin, Thr-Lys-Pro-Arg (TKPR), is an immunostimulatory peptide with reported nervous system effects as well. We unexpectedly found that tuftsin and a higher affinity antagonist, TKPPR, bind selectively to neuropilin-1 and block vascular endothelial growth factor (VEGF) binding to that receptor. Dimeric and tetrameric forms of TKPPR had greatly increased affinity for neuropilin-1 based on competition binding experiments. On endothelial cells tetrameric TKPPR inhibited the VEGF(165)-induced autophosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) even though it did not directly inhibit VEGF binding to VEGFR-2. Homology between exon 8 of VEGF and TKPPR suggests that the sequence coded for by exon 8 may stabilize VEGF binding to neuropilin-1 to facilitate signaling through VEGFR-2. Given the overlap between processes involving neuropilin-1 and tuftsin, we propose that at least some of the previously reported effects of tuftsin are mediated through neuropilin-1.     

Interactions of VEGF isoforms with VEGFR-1, VEGFR-2, and neuropilin in vivo: a computational model of human skeletal muscle

The vascular endothelial growth factor (VEGF) family of cytokines is involved in the maintenance of existing adult blood vessels as well as in angiogenesis, the sprouting of new vessels. To study the proangiogenic activation of VEGF receptors (VEGFRs) by VEGF family members in skeletal muscle, we develop a computational model of VEGF isoforms (VEGF(121), VEGF(165)), their cell surface receptors, and the extracellular matrix in in vivo tissue. We build upon our validated model of the biochemical interactions between VEGF isoforms and receptor tyrosine kinases (VEGFR-1 and VEGFR-2) and nonsignaling neuropilin-1 coreceptors in vitro. The model is general and could be applied to any tissue; here we apply the model to simulate the transport of VEGF isoforms in human vastus lateralis muscle, which is extensively studied in physiological experiments. The simulations predict the distribution of VEGF isoforms in resting (nonexercising) muscle and the activation of VEGFR signaling. Little of the VEGF protein in muscle is present as free, unbound extracellular cytokine; the majority is bound to the cell surface receptors or to the extracellular matrix. However, interstitial sequestration of VEGF(165) does not affect steady-state receptor binding. In the absence of neuropilin, VEGF(121) and VEGF(165) behave similarly, but neuropilin enhances the binding of VEGF(165) to VEGFR-2. This model is the first to study VEGF tissue distribution and receptor activation in human muscle, and it provides a platform for the design and evaluation of therapeutic approaches.     

Glypican-1 is a VEGF165 binding proteoglycan that acts as an extracellular chaperone for VEGF165

Glypican-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparan sulfate proteoglycans implicated in the control of cellular growth and differentiation. The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for endothelial cells and a potent angiogenic factor in vivo. Heparin binds to VEGF165 and enhances its binding to VEGF receptors. However, native HSPGs that bind VEGF165 and modulate its receptor binding have not been identified. Among the glypicans, glypican-1 is the only member that is expressed in the vascular system. We have therefore examined whether glypican-1 can interact with VEGF165. Glypican-1 from rat myoblasts binds specifically to VEGF165 but not to VEGF121. The binding has an apparent dissociation constant of 3 x 10(-10) M. The binding of glypican-1 to VEGF165 is mediated by the heparan sulfate chains of glypican-1, because heparinase treatment abolishes this interaction. Only an excess of heparin or heparan sulfates but not other types of glycosaminoglycans inhibited this interaction. VEGF165 interacts specifically not only with rat myoblast glypican-1 but also with human endothelial cell-derived glypican-1. The binding of 125I-VEGF165 to heparinase-treated human vascular endothelial cells is reduced following heparinase treatment, and addition of glypican-1 restores the binding. Glypican-1 also potentiates the binding of 125I-VEGF165 to a soluble extracellular domain of the VEGF receptor KDR/flk-1. Furthermore, we show that glypican-1 acts as an extracellular chaperone that can restore the receptor binding ability of VEGF165, which has been damaged by oxidation. Taken together, these results suggest that glypican-1 may play an important role in the control of angiogenesis by regulating the activity of VEGF165, a regulation that may be critical under conditions such as wound repair, in which oxidizing agents that can impair the activity of VEGF are produced, and in situations were the concentrations of active VEGF are limiting.     

Homeostatic modulation of cell surface KDR and Flt1 expression and expression of the vascular endothelial cell growth factor (VEGF) receptor mRNAs by VEGF

Vascular endothelial cell growth factor (VEGF) is a potent angiogenic factor expressed during embryonic development, during wound healing, and in pathologies dependent on neovascularization, including cancer. Regulation of the receptor tyrosine kinases, KDR and Flt-1, to which VEGF binds on endothelial cells is incompletely understood. Chronic incubation with tumor-conditioned medium or VEGF diminished (125)I-VEGF binding to human umbilical vein endothelial cells, incorporation of (125)I-VEGF into covalent complexes with KDR and Flt1, and immunoreactive KDR in cell lysates. Receptor down-regulation desensitized VEGF activation of mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2) and p38 mitogen-activated protein kinase. Preincubation with VEGF or tumor-conditioned medium down-regulated cell surface receptor expression but up-regulated KDR and Flt-1 mRNAs, an effect abrogated by a neutralizing VEGF antibody. Removal of VEGF from the medium led to recovery of (125)I-VEGF binding and resensitization of human umbilical vein endothelial cells. Recovery of receptor expression was inhibited by cycloheximide, indicating that augmented VEGF receptor mRNAs, and not receptor recycling from a cytoplasmic pool, restored responsiveness. As the VEGF receptors promote endothelial cell survival, proliferation, and other events necessary for angiogenesis, the noncoordinate regulation of VEGF receptor proteins and mRNAs suggests that human umbilical vein endothelial cells are protected against inappropriate or prolonged loss of VEGF receptors by a homeostatic mechanism important to endothelial cell function.     

Cysteine-rich and basic domain HIV-1 Tat peptides inhibit angiogenesis and induce endothelial cell apoptosis

Previous findings suggest that both the Tat polypeptide encoded by HIV-1 and Tat-derived peptides can induce angiogenesis via activation of the KDR receptor for Vascular Endothelial Growth Factor (VEGF). We identified 20 amino acids and 12 amino acid peptides corresponding to the cysteine-rich and basic domains of HIV-1 Tat which inhibited (125)I-VEGF(165) binding to KDR and neuropilin-1 (NP-1) receptors in endothelial cells. Cysteine-rich and basic Tat peptides inhibited VEGF-induced ERK activation and mitogenesis in endothelial cells, and inhibited angiogenesis in vitro at concentrations similar to those which inhibited VEGF receptor binding. These peptides also inhibited proliferation, angiogenesis, and ERK activation induced by basic fibroblast growth factor with similar potency and efficacy. Surprisingly, we found that both cysteine-rich and basic domain Tat peptides strikingly induced apoptosis in endothelial cells, independent of their effects on VEGF and bFGF. Furthermore, we found no evidence for direct biological effects of recombinant Tat on VEGF receptor binding, ERK activation, endothelial cell survival, or mitogenesis. These findings demonstrate novel properties of Tat-derived peptides and indicate that their major effect in endothelial cells is apoptosis independent of specific inhibition of VEGF receptor activation.     

Expression of VEGF-C and activation of its receptors VEGFR-2 and VEGFR-3 in trophoblast

Placental villous development requires the co-ordinated action of angiogenic factors on both endothelial and trophoblast cells. Like vascular endothelial growth factor (VEGF), VEGF-C increases vascular permeability, stimulates endothelial cell proliferation and migration. In the present study, we investigated the expression of VEGF-C and its receptors VEGFR-3 and VEGFR-2 in normal and intrauterine growth-restricted (IUGR) placenta. Immunolocalisation studies showed that like VEGF and VEGFR-1, VEGF-C, VEGFR-3 and VEGFR-2 co-localised to the syncytiotrophoblast, to cells in the maternal decidua, as well as to the endothelium of the large placental blood vessels. Western blot analysis demonstrated a significant decrease in placental VEGF-C and VEGFR-3 protein expression in severe IUGR as compared to gestationally-matched third trimester pregnancies. Conditioned medium from VEGF-C producing pancreatic carcinoma (Suit-2) and endometrial epithelial (Hec-1B) cell lines caused an increased association of the phosphorylated extracellular signal regulated kinase (ERK) in VEGFR-3 immunoprecipitates from spontaneously transformed first trimester trophoblast cells. VEGF121 caused dose-dependant phosphorylation of VEGFR-2 in trophoblast cells as well as stimulating DNA synthesis. In addition, premixing VEGF165 with heparin sulphate proteoglycan potentiated trophoblast proliferation and the association of phospho-ERK with the VEGFR-2 receptor. VEGF165-mediated DNA synthesis was inhibited by anti-VEGFR-2 neutralising antibody. The results demonstrate functional VEGFR-2 and VEGFR-3 receptors on trophoblast and suggest that the decreased expression of VEGF-C and VEGFR-3 may contribute to the abnormal villous development observed in IUGR placenta.     

Platelet factor 4 disrupts the intracellular signalling cascade induced by vascular endothelial growth factor by both KDR dependent and independent mechanisms.

The mechanism by which the CXC chemokine platelet factor 4 (PF-4) inhibits endothelial cell proliferation is unclear. The heparin-binding domains of PF-4 have been reported to prevent vascular endothelial growth factor 165 (VEGF(165)) and fibroblast growth factor 2 (FGF2) from interacting with their receptors. However, other studies have suggested that PF-4 acts via heparin-binding independent interactions. Here, we compared the effects of PF-4 on the signalling events involved in the proliferation induced by VEGF(165), which binds heparin, and by VEGF(121), which does not. Activation of the VEGF receptor, KDR, and phospholipase Cgamma (PLCgamma) was unaffected in conditions in which PF-4 inhibited VEGF(121)-induced DNA synthesis. In contrast, VEGF(165)-induced phosphorylation of KDR and PLCgamma was partially inhibited by PF-4. These observations are consistent with PF-4 affecting the binding of VEGF(165), but not that of VEGF(121), to KDR. PF-4 also strongly inhibited the VEGF(165)- and VEGF(121)-induced mitogen-activated protein (MAP) kinase signalling pathways comprising Raf1, MEK1/2 and ERK1/2: for VEGF(165) it interacts directly or upstream from Raf1; for VEGF(121), it acts downstream from PLCgamma. Finally, the mechanism by which PF-4 may inhibit the endothelial cell proliferation induced by both VEGF(121) and VEGF(165), involving disruption of the MAP kinase signalling pathway downstream from KDR did not seem to involve CXCR3B activation.     

Semaphorin-3A and semaphorin-3F work together to repel endothelial cells and to inhibit their survival by induction of apoptosis

Semaphorin-3A (sema3A) is a neuropilin-1 (np1) agonist. It inhibits the binding of the 165-amino acid form of VEGF (VEGF(165)) to np1 and was reported to inhibit angiogenesis as a result. However, we find that sema3A concentrations that inhibit the mitogenic effects of VEGF(165) do not inhibit VEGF(165)-induced phosphorylation of VEGF receptor-2 (VEGFR-2). Furthermore, sema3A inhibits the biological effects of VEGF(121), a VEGF form that does not bind to neuropilins and basic fibroblast growth factor, a growth factor whose activity, unlike that of VEGF, is not inhibited by small interfering RNA directed against np1. Therefore, the mechanism by which sema3A inhibits VEGF(165) activity does not depend on competition with VEGF(165) for binding to np1. Sema3A induced rapid disappearance of focal contacts followed by collapse of the actin cytoskeleton in human umbilical vein-derived endothelial cells. HEK293 cells expressing sema3A repel human endothelial cells and at high concentrations induce their death by apoptosis. Furthermore, sema3A inhibited the formation of tubes from endothelial cells in an in vitro angiogenesis assay. Similar effects are induced by the neuropilin-2 (np2) agonist sema3F. These inhibitory effects are abrogated by small interfering RNAs directed against np1 or np2, respectively. The anti-proliferative effects of sema3A and sema3F are additive when the semaphorins are added as pure proteins. However, when sema3A and sema3F were co-expressed in HEK293 cells their pro-apoptotic and cell repellant activities appeared to be synergistic. These observations suggest that combinations of sema3A and sema3F may be able to inhibit tumor angiogenesis more effectively than single semaphorins.     

Signaling properties of VEGF receptor-1 and -2 homo- and heterodimers

Vascular endothelial growth factor (VEGF-A) exerts its effects through receptor tyrosine kinases VEGF receptor-1 (VEGFR-1) and VEGFR-2, which are expressed on most endothelial cell types in vitro and in vivo. We have examined VEGF-A-induced signal transduction in porcine aortic endothelial (PAE) cells individually expressing VEGFR-1 or VEGFR-2, and cells co-expressing both receptor types. We show that VEGF-A-stimulated PAE cells co-expressing VEGFR-1 and -2 contain receptor heterodimers. VEGF-A-stimulation of all three cell lines (expressing VEGFR-1, -2 and -1/2) resulted in signal transduction with different efficiencies. Thus, tyrosine phosphorylation of phospholipase Cgamma, and accumulation of inositol polyphosphates were efficiently transduced in the VEGFR-1/2 cells whereas cells expressing VEGFR-1 responded poorly in these assays. In contrast, VEGF-A-induced activation of phosphoinositide 3-kinase and induction of Ca2+ fluxes were transduced well by VEGFR-1 and VEGFR-2 homo- and heterodimers. The pattern of Ca2+ fluxes was unique for each type of VEGF receptor dimer. Our data show that signal transduction induced by VEGF-A is transduced in distinct manners by homo- and heterodimers of VEGF receptors.