Heparin induces changes in the synthesis of porcine aortic endothelial cell heparan sulfate proteoglycans

Heparin is known to bind to cultured endothelial cells. This report documents that addition of heparin to endothelial cells results in an alteration of the heparan sulfate proteoglycan synthetic pattern. Specifically, the addition of saturating amounts of heparin to confluent cultures of porcine aortic endothelial cells results in an increase in the amount of radiolabeled heparan sulfate proteoglycan secreted into the growth medium. The increase is apparent as early as 8 h after heparin administration. Although there is often a decrease in the amount of cell surface heparan sulfate proteoglycan produced, it is not sufficient to account for the increase in the secreted form. Of the other glycosaminoglycans tested, only dextran sulfate and commercial heparan sulfate induce changes in heparan sulfate proteoglycan synthesis and secretion. Chondroitin sulfate glycosaminoglycans do not elicit this synthetic change. These data indicate that endothelial cells can alter the synthesis of heparan sulfate proteoglycans in response to extracellular signals including heparin and related glycosaminoglycans.     

Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells

Evidence suggests that endothelial cell layer heparan sulfate proteoglycans include a variety of different sized molecules which most likely contain different protein cores. In the present report, approximately half of endothelial cell surface associated heparan sulfate proteoglycan is shown to be releasable with soluble heparin. The remaining cell surface heparan sulfate proteoglycan, as well as extracellular matrix heparan sulfate proteoglycan, cannot be removed from the cells with heparin. The heparin nonreleasable cell surface proteoglycan can be released by membrane disrupting agents and is able to intercalate into liposomes. When the heparin releasable and nonreleasable cell surface heparan sulfate proteoglycans are compared, differences in proteoglycan size are also evident. Furthermore, the intact heparin releasable heparan sulfate proteoglycan is closer in size to proteoglycans isolated from the extracellular matrix and from growth medium than to that which is heparin nonreleasable. These data indicate that cultured porcine aortic endothelial cells contain at least two distinct types of cell surface heparan sulfate proteoglycans, one of which appears to be associated with the cells through its glycosaminoglycan chains. The other (which is more tightly associated) is probably linked via a membrane intercalated protein core.Abbreviations ECM extracellular matrix - HSPG heparan sulfate proteoglycan - PAE porcine aortic endothelial - PBS phosphate buffered saline     

Regulation of pathologic retinal angiogenesis in mice and inhibition of VEGF-VEGFR2 binding by soluble heparan sulfate

Development of the retinal vascular network is strictly confined within the neuronal retina, allowing the intraocular media to be optically transparent. However, in retinal ischemia, pro-angiogenic factors (including vascular endothelial growth factor-A, VEGF-A) induce aberrant guidance of retinal vessels into the vitreous. Here, we show that the soluble heparan sulfate level in murine intraocular fluid is high particularly during ocular development. When the eyes of young mice with retinal ischemia were treated with heparan sulfate-degrading enzyme, the subsequent aberrant angiogenesis was greatly enhanced compared to PBS-injected contralateral eyes; however, increased angiogenesis was completely antagonized by simultaneous injection of heparin. Intraocular injection of heparan sulfate or heparin alone in these eyes resulted in reduced neovascularization. In cell cultures, the porcine ocular fluid suppressed the dose-dependent proliferation of human umbilical vein endothelial cells (HUVECs) mediated by VEGF-A. Ocular fluid and heparin also inhibited the migration and tube formation by these cells. The binding of VEGF-A and HUVECs was reduced under a high concentration of heparin or ocular fluid compared to lower concentrations of heparin. In vitro assays demonstrated that the ocular fluid or soluble heparan sulfate or heparin inhibited the binding of VEGF-A and immobilized heparin or VEGF receptor 2 but not VEGF receptor 1. The recognition that the high concentration of soluble heparan sulfate in the ocular fluid allows it to serve as an endogenous inhibitor of aberrant retinal vascular growth provides a platform for modulating heparan sulfate/heparin levels to regulate angiogenesis.     

Characterization of endostatin binding to heparin and heparan sulfate by surface plasmon resonance and molecular modeling: role of divalent cations

Endostatin (20 kDa) is a C-terminal proteolytic fragment of collagen XVIII that is localized in vascular basement membrane zones in various organs. It binds zinc, heparin/heparan sulfate, laminin, and sulfatides and inhibits angiogenesis and tumor growth. Here we determined the kinetics and affinity of the interaction of endostatin with heparin/heparan sulfate and investigated the effects of divalent cations on these interactions and on the biological activities of endostatin. The binding of human recombinant endostatin to heparin and heparan sulfate was studied by surface plasmon resonance using BIAcore technology and further characterized by docking and molecular dynamics simulations. Kinetic data, evaluated using a 1:1 interaction model, showed that heparan sulfate bound to and dissociated from endostatin faster than heparin and that endostatin bound to heparin and heparan sulfate with a moderate affinity (K(D) approximately 2 microm). Molecular modeling of the complex between endostatin and heparin oligosaccharides predicted that, compared with mutagenesis studies, two further arginine residues, Arg(47) and Arg(66), participated in the binding. The binding of endostatin to heparin and heparan sulfate required the presence of divalent cations. The addition of ZnCl(2) to endostatin enhanced its binding to heparan sulfate by approximately 40% as well as its antiproliferative effect on endothelial cells stimulated by fibroblast growth factor-2, suggesting that this activity is mediated by the binding of endostatin to heparan sulfate. In contrast, no increase in the antiangiogenic and anti-proliferative activities of endostatin promoted by vascular endothelial growth factor was observed upon the addition of zinc.     

Specificity for fibroblast growth factors determined by heparan sulfate in a binary complex with the receptor kinase.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the FGF receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation and supports the binding of activating FGF to self-associated FGFR. Here we show that in contrast to heparin, cellular heparan sulfate forms a binary complex with FGFR that discriminates between FGF-1 and FGF-2. FGFR type 4 (FGFR4) in liver parenchymal cells binds only FGF-1, whereas FGFR1 binds FGF-1 and FGF-2 equally. Cell-free complexes of heparin and recombinant FGFR4 bound FGF-1 and FGF-2 equally. However, in contrast to FGFR1, when recombinant FGFR4 was expressed back in epithelial cells by transfection, it failed to bind FGF-2 unless heparan sulfate was depressed by chlorate or heparinase treatment. Isolated heparan sulfate proteoglycan (HSPG) from liver cells in cell-free complexes with FGFR4 restored the specificity for FGF-1 and supported the binding of both FGF-1 and FGF-2 when complexed with FGFR1. In contrast, FGF-2 bound equally well to complexes of both FGFR1 and FGFR4 formed with endothelial cell-derived HSPG, but the endothelial HSPG was deficient for the binding of FGF-1 to both FGFR complexes. These data suggest that a heparan sulfate subunit is a cell type- and FGFR-specific determinant of the selectivity of the FGFR signaling complex for FGF. In a physiological context, the heparan sulfate subunit may limit the redundancy among the current 18 FGF polypeptides for the 4 known FGFR.     

Shedding of heparan sulfate proteoglycan by stimulated endothelial cells: Evidence for proteolysis of cell-surface molecules

Activation of endothelial cells by cytokines and endotoxin causes procoagulant and pro-inflammatory changes over a period of hours. We postulated that the same functional state might be achieved more rapidly by changes in the metabolism of heparan sulfate, which supports many of the normal functions of endothelial cells. We previously found that binding of anti-endothelial cell antibodies and activation of complement on endothelial cells causes the rapid shedding of endothelial cell heparan sulfate. Here we report the biochemical mechanism responsible for the release of the heparan sulfate. Stimulation of endothelial cells by anti-endothelial cell antibodies and complement resulted in the release of ³⁵S-heparan sulfate proteoglycan and partially degraded ³⁵S-heparan sulfate chains. Degradation of the ³⁵S-heparan sulfate chains was not necessary for release since heparin and suramin prevented cleavage of the heparan sulfate but did not inhibit release from stimulated endothelial cells. The ³⁵S-heparan sulfate proteoglycan released from endothelial cells originated from the cell surface and had a core protein similar in size (70.5 kD) to syndecan-1. Release was due to proteolytic cleavage of the protein core by serine and/or cysteine proteinases since the release of heparan sulfate was inhibited 87% by antipain and 53% by leupeptin. Release of heparan sulfate coincided with a decrease of ∼︁7 kD in the mass of the protein core and with a loss of hydrophobicity of the proteoglycan, consistent with the loss of the hydrophobic transmembrane domain. The cleavage and release of cell-surface ³⁵S-heparan sulfate proteoglycan might be a novel mechanism by which endothelial cells may rapidly acquire the functional properties of activated endothelial cells. © 1996 Wiley-Liss, Inc.     

Heparan sulfate regulates VEGF165- and VEGF121-mediated vascular hyperpermeability

VEGF was first described as vascular permeability factor, a potent inducer of vascular leakage. Genetic evidence indicates that VEGF-stimulated endothelial proliferation in vitro and angiogenesis in vivo depend on heparan sulfate, but a requirement for heparan sulfate in vascular hyperpermeability has not been explored. Here we show that altering endothelial cell heparan sulfate biosynthesis in vivo decreases hyperpermeability induced by both VEGF(165) and VEGF(121). Because VEGF(121) does not bind heparan sulfate, the requirement for heparan sulfate suggested that it interacted with VEGF receptors rather than the ligand. By applying proximity ligation assays to primary brain endothelial cells, we show a direct interaction in situ between heparan sulfate and the VEGF receptor, VEGFR2. Furthermore, the number of heparan sulfate-VEGFR2 complexes increased in response to both VEGF(165) and VEGF(121). Genetic or heparin lyase-mediated alteration of endothelial heparan sulfate attenuated phosphorylation of VEGFR2 in response to VEGF(165) and VEGF(121), suggesting that the functional VEGF receptor complex contains heparan sulfate. Pharmacological blockade of heparan sulfate-protein interactions inhibited hyperpermeability in vivo, suggesting heparan sulfate as a potential target for treating hyperpermeability associated with ischemic disease.     

Dermatan sulfate is the predominant antithrombotic glycosaminoglycan in vessel walls: implications for a possible physiological function of heparin cofactor II

The role of different glycosaminoglycan species from the vessel walls as physiological antithrombotic agents remains controversial. To further investigate this aspect we extracted glycosaminoglycans from human thoracic aorta and saphenous vein. The different species were highly purified and their anticoagulant and antithrombotic activities tested by in vitro and in vivo assays. We observed that dermatan sulfate is the major anticoagulant and antithrombotic among the vessel wall glycosaminoglycans while the bulk of heparan sulfate is a poorly sulfated glycosaminoglycan, devoid of anticoagulant and antithrombotic activities. Minor amounts of particular a heparan sulfate (< 5% of the total arterial glycosaminoglycans) with high anticoagulant activity were also observed, as assessed by its retention on an antithrombin-affinity column. Possibly, this anticoagulant heparan sulfate originates from the endothelial cells and may exert a significant physiological role due to its location in the interface between the vessel wall and the blood. In view of these results we discuss a possible balance between the two glycosaminoglycan-dependent anticoagulant pathways present in the vascular wall. One is based on antithrombin activation by the heparan sulfate expressed by the endothelial cells. The other, which may assume special relevance after vascular endothelial injury, is based on heparin cofactor II activation by the dermatan sulfate proteoglycans synthesized by cells from the subendothelial layer.     

Requirement for anticoagulant heparan sulfate in the fibroblast growth factor receptor complex.

A divalent cation-dependent association between heparin or heparan sulfate and the ectodomain of the fibroblast growth factor (FGF) receptor kinase (FGFR) restricts FGF-independent trans-phosphorylation between self-associated FGFR and determines specificity for and mediates binding of activating FGF. Here we show that only the fraction of commercial heparin or rat liver heparan sulfate which binds to immobilized antithrombin formed an FGF-binding binary complex with the ectodomain of the FGFR kinase. Conversely, only the fraction of heparin that binds to immobilized FGFR inhibited Factor Xa in the presence of antithrombin. Only the antithrombin-bound fraction of heparin competed with (3)H-heparin bound to FGFR in absence of FGF, whereas both antithrombin-bound and unretained fractions competed with radiolabeled heparin bound independently to FGF-1 and FGF-2. The antithrombin-bound fraction of heparin was required to support the heparin-dependent stimulation of DNA synthesis of endothelial cells by FGF-1. The requirement for divalent cations and the antithrombin-binding motif distinguish the role of heparan sulfate as an integral subunit of the FGFR complex from the wider range of effects of heparan sulfates and homologues on FGF signaling through FGFR-independent interactions with FGF.     

Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions

Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression.     

Basic fibroblast growth factor is released from endothelial extracellular matrix in a biologically active form

Basic fibroblast growth factor (bFGF) binds to heparin-like molecules present in the extracellular matrix (ECM) of transformed fetal bovine aortic endothelial GM 7373 cells. Binding of bFGF to ECM can be competed by heparin or heparan sulfate, and ECM-bound bFGF can be released by treating the cells with heparinase or heparatinase. After binding to ECM, bFGF is slowly released into the medium in a biologically active form, as shown by its capacity to induce an increase of cell-associated plasminogen activator activity and cell proliferation. The increase is prevented upon removal of ECM-bound bFGF by a neutral 2 M NaCl wash. Soluble heparin and heparan sulfate reduce the amount of ECM-bound bFGF released into the medium, possibly competing with ECM polysaccharides for heparinase-like enzymes produced by endothelial cells, suggesting that these enzymes are involved in the mobilization of ECM-bound bFGF.     

Basic fibroblast growth factor (bFGF) dissociates rapidly from heparan sulfates but slowly from receptors. Implications for mechanisms of bFGF release from pericellular matrix.

The effect of heparin on the rate of binding of basic fibroblast growth factor (bFGF) to high affinity (receptor) and low affinity (heparan sulfate) binding sites on endothelial cells and CHO cells transfected with FGF receptor-1 or FGF receptor-2 was investigated. Radiolabeled bFGF bound rapidly to both high and low affinity sites on all three types of cells. Addition of 10 micrograms/ml heparin eliminated binding to low affinity sites and decreased the rate of binding to high affinity sites to about 30% of the rate observed in the absence of heparin. However, the same amount of 125I-bFGF bound to high affinity sites at equilibrium in the presence and absence of heparin. The effect of heparin on the initial rate of binding to high affinity sites was related to the log of the heparin concentration. Depletion of the cells of heparan sulfates by treatment with heparinase also decreased the initial rate of binding to high affinity receptors. These results suggest that cell-surface heparan sulfates facilitate the interaction of bFGF with its receptor by concentrating bFGF at the cell surface. Dissociation rates for receptor-bound and heparan sulfate-bound bFGF were also measured. Dissociation from low affinity sites was rapid, with a half-time of 6 min for endothelial cell heparan sulfates and 0.5 min for Chinese hamster ovary heparan sulfates. In contrast, dissociation from receptors was slow, with a half-time of 46 min for endothelial cell receptors, 2.5 h for FGF receptor-1, and 1.4 h for FGF receptor-2. These results suggest that degradative enzymes may not be needed to release bFGF from the heparan sulfates in instances where receptors and heparan sulfate-bound bFGF are in close proximity because dissociation from heparan sulfates occurs rapidly enough to allow bFGF to bind to unoccupied receptors by laws of mass action.     

N-unsubstituted glucosamine in heparan sulfate of recycling glypican-1 from suramin-treated and nitrite-deprived endothelial cells. mapping of nitric oxide/nitrite-susceptible glucosamine residues to clustered sites near the core protein

We have analyzed the content of N-unsubstituted glucosamine in heparan sulfate from glypican-1 synthesized by endothelial cells during inhibition of (a) intracellular progression by brefeldin A, (b) heparan sulfate degradation by suramin, and/or (c) endogenous nitrite formation. Glypican-1 from brefeldin A-treated cells carried heparan sulfate chains that were extensively degraded by nitrous acid at pH 3.9, indicating the presence of glucosamines with free amino groups. Chains with such residues were rare in glypican-1 isolated from unperturbed cells and from cells treated with suramin and, surprisingly, when nitrite-deprived. However, when nitrite-deprived cells were simultaneously treated with suramin, such glucosamine residues were more prevalent. To locate these residues, chains were first cleaved at linkages to sulfated l-iduronic acid by heparin lyase and released fragments were separated from core protein carrying heparan sulfate stubs. These stubs were then cleaved off at sites linking N-substituted glucosamines to d-glucuronic acid. These fragments were extensively degraded by nitrous acid at pH 3.9. When purified proteoglycan isolated from brefeldin A-treated cells was incubated with intact cells, endoheparanase-catalyzed degradation generated a core protein with heparan sulfate stubs that were similarly sensitive to nitrous acid. We conclude that there is a concentration of N-unsubstituted glucosamines to the reducing side of the endoheparanase cleavage site in the transition region between unmodified and modified chain segments near the linkage region to the protein. Both sites as well as the heparin lyase-sensitive sites seem to be in close proximity to one another.     

Characterization of the recognition of tumor cells by the natural cytotoxicity receptor, NKp44

NKp44 is a natural cytotoxicity receptor expressed by human NK cells upon activation. In this study, we demonstrate that cell surface heparan sulfate proteoglycans (HSPGs), expressed by target cells, are involved in the recognition of tumor cells by NKp44. NKp44 showed heparan sulfate-dependent binding to tumor cells; this binding was partially blocked with an antibody to heparan sulfate. In addition, direct binding of NKp44 to heparin was observed, and soluble heparin/heparan sulfate enhanced the secretion of IFNgamma by NK92 cells activated with anti-NKp44 monoclonal antibody. Basic amino acids, predicted to constitute the putative heparin/heparan sulfate binding site of NKp44, were mutated. Tumor cell recognition of the mutated NKp44 proteins was significantly reduced and correlated with their lower recognition of heparin. We previously reported that NKp44 recognizes the hemagglutinin of influenza virus (IV). Nevertheless, the ability of the mutated NKp44 proteins to bind viral hemagglutinin expressed by IV-infected cells was not affected. Thus, we suggest that heparan sulfate epitope(s) are ligands/co-ligands of NKp44 and are involved in its tumor recognition ability.     

Lymphatic endothelial heparan sulfate deficiency results in altered growth responses to vascular endothelial growth factor-C (VEGF-C)

Growth and remodeling of lymphatic vasculature occur during development and during various pathologic states. A major stimulus for this process is the unique lymphatic vascular endothelial growth factor-C (VEGF-C). Other endothelial growth factors, such as fibroblast growth factor-2 (FGF-2) or VEGF-A, may also contribute. Heparan sulfate is a linear sulfated polysaccharide that facilitates binding and action of some vascular growth factors such as FGF-2 and VEGF-A. However, a direct role for heparan sulfate in lymphatic endothelial growth and sprouting responses, including those mediated by VEGF-C, remains to be examined. We demonstrate that VEGF-C binds to heparan sulfate purified from primary lymphatic endothelia, and activation of lymphatic endothelial Erk1/2 in response to VEGF-C is reduced by interference with heparin or pretreatment of cells with heparinase, which destroys heparan sulfate. Such treatment also inhibited phosphorylation of the major VEGF-C receptor VEGFR-3 upon VEGF-C stimulation. Silencing lymphatic heparan sulfate chain biosynthesis inhibited VEGF-C-mediated Erk1/2 activation and abrogated VEGFR-3 receptor-dependent binding of VEGF-C to the lymphatic endothelial surface. These findings prompted targeting of lymphatic N-deacetylase/N-sulfotransferase-1 (Ndst1), a major sulfate-modifying heparan sulfate biosynthetic enzyme. VEGF-C-mediated Erk1/2 phosphorylation was inhibited in Ndst1-silenced lymphatic endothelia, and scratch-assay responses to VEGF-C and FGF-2 were reduced in Ndst1-deficient cells. In addition, lymphatic Ndst1 deficiency abrogated cell-based growth and proliferation responses to VEGF-C. In other studies, lymphatic endothelia cultured ex vivo from Ndst1 gene-targeted mice demonstrated reduced VEGF-C- and FGF-2-mediated sprouting in collagen matrix. Lymphatic heparan sulfate may represent a novel molecular target for therapeutic intervention.     

Basic fibroblast growth factor is internalized through both receptor-mediated and heparan sulfate-mediated mechanisms.

Basic fibroblast growth factor (bFGF) was internalized at a rapid rate by Chinese hamster ovary (CHO) cells that do not express significant numbers of high affinity receptors for bFGF as well as CHO cells that have been transfected with cDNA encoding FGF receptor-1 or FGF receptor-2. Internalization of bFGF was completely blocked by the addition of 10 micrograms/ml heparin in the parental CHO cells but only partially inhibited in cells expressing transfected FGF receptors. Bovine aortic endothelial cells also exhibit heparin-sensitive and heparin-resistant internalization of bFGF. The internalization of bFGF through the heparin-resistant pathway in CHO cells was efficiently competed by addition of unlabeled bFGF, was proportional to the number of receptors expressed, and approached saturation, suggesting that the heparin-resistant internalization was due to high affinity receptors. Internalization of bFGF through the heparin-sensitive pathway was not efficiently competed by unlabeled bFGF and did not approach saturation at concentrations of bFGF up to 50 ng/ml, properties similar to the interaction of bFGF with low affinity heparan sulfate binding sites on the cell surface. Internalization of bFGF in CHO cells not expressing FGF receptors was inhibited by heparin, heparan sulfate, and dermatan sulfate, the same glycosaminoglycans that block binding to cell-surface heparin sulfates. Internalization of bFGF in the parental CHO cells was inhibited at the same concentrations of heparin that block binding to cell-surface heparan sulfates. Finally, inhibition of the sulfation of CHO cell heparan sulfates by the addition of chlorate or digestion of CHO cell heparan sulfates with heparinase inhibited bFGF internalization in the parental CHO cells. These results demonstrate that bFGF can be internalized through a direct interaction with cell-surface heparan sulfates. Thus, there are two pathways for internalization of bFGF: high affinity receptor-mediated and heparan sulfate-mediated.     

Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor

The radius of diffusion of basic FGF (bFGF) in the presence and in the absence of the glycosaminoglycans heparin and heparan sulfate was measured. Iodinated 125I-bFGF diffuses further in agarose, fibrin, and on a monolayer of bovine aortic endothelial (BAE) cells in the presence of heparin than in its absence. Heparan sulfates affected the diffusion of 125I-bFGF in a manner similar to, though less pronounced than, heparin. When applied at the center of a monolayer of BAE cells, bFGF plus heparin stimulated morphological changes at a 10-fold greater radius than bFGF alone. These results suggest that bFGF-heparin and/or heparan sulfate complexes may be more effective than bFGF alone in stimulating cells located away from the bFGF source because the bFGF-glycosaminoglycan complex partitions into the soluble phase rather than binding to insoluble glycosaminoglycans in the extracellular matrix. Thus, the complex of bFGF and glycosaminoglycan may represent one of the active forms of bFGF in vivo.     

Characterization of the heparin/heparan sulfate binding site of the natural cytotoxicity receptor NKp46

NKp46 is a member of a group of receptors collectively termed natural cytotoxicity receptors (NCRs) that are expressed by natural killer (NK) cells. NCRs are capable of mediating direct killing of tumor and virus-infected cells by NK cells. We have recently shown that NKp46 recognizes the heparan sulfate moieties of membranal heparan sulfate proteoglycans (HSPGs), thus enabling lysis of tumor cells by NK cells. In the current study, we further examined the residues in NKp46 that may be involved in heparan sulfate binding on tumor cells. On the basis of both the electrostatic potential map and comparison to the heparin binding site on human fibronectin, we predicted a continuous region containing the basic amino acids K133, R136, H139, R142, and K146 to be involved in NKp46 binding to heparan sulfate. Mutating these amino acids on NKp46D2 to noncharged amino acids retained its virus binding capacity but reduced its binding to tumor cells with a 10-100 fold lower K(D) when tested for direct binding to heparin. The minimal length of the heparin/heparan sulfate epitope recognized by NKp46 was eight saccharides as predicted from the structure and proven by testing heparin oligomers. Testing selectively monodesulfated heparin oligomers emphasized the specific contributions of O-sulfation, N-sulfation, and N-acetylation to epitope recognition by NKp46. The characterization of heparan sulfate binding region in NKp46 offers further insight into the identity of the ligands for NKp46 and the interaction of NK and cancers.     

Gekko-sulfated glycopeptide inhibits tumor angiogenesis by targeting basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) is a therapeutic target of anti-angiogenesis. Here, we report that a novel sulfated glycopeptide derived from Gekko swinhonis Guenther (GSPP), an anticancer drug in traditional Chinese medicine, inhibits tumor angiogenesis by targeting bFGF. GSPP significantly decreased the production of bFGF in hepatoma cells by suppressing early growth response-1. GSPP inhibited the release of bFGF from extracellular matrix by blocking heparanase enzymatic activity. Moreover, GSPP competitively inhibited bFGF binding to heparin/heparan sulfate via direct binding to bFGF. Importantly, GSPP abrogated the bFGF-stimulated proliferation and migration of endothelial cells, whereas it had no inhibitory effect on endothelial cells in the absence of bFGF. Further study revealed that GSPP prevented bFGF-induced neovascularization and inhibited tumor angiogenesis and tumor growth in a xenograft mouse model. These results demonstrate that GSPP inhibits tumor angiogenesis by blocking bFGF production, release from the extracellular matrix, and binding to its low affinity receptor, heparin/heparan sulfate.     

Heparin regulates vascular endothelial growth factor165-dependent mitogenic activity, tube formation, and its receptor phosphorylation of human endothelial cells. Comparison of the effects of heparin and modified heparins

Vascular endothelial growth factor (VEGF) is a family of glycoproteins with potent angiogenic activity. We reported previously that heparin has an affinity for VEGF165, the major isoform of VEGF, whereas 2-O-desulfated heparin and 6-O-desulfated heparin have weak but significant affinity (Ashikari-Hada, S., Habuchi, H., Kariya, Y., Itoh, N., Reddi, A. H., and Kimata, K. (2004) J. Biol. Chem. 279, 12346-12354). In this study, we first examined the effect of heparin and modified heparins (completely desulfated N-sulfated heparin, 2-O-desulfated heparin, and 6-O-desulfated heparin) on VEGF165-dependent mitogenic activity and tube formation on type I collagen gels of human umbilical vein endothelial cells. Both were enhanced by heparin, but not by modified heparins, suggesting that both the 2-O-sulfate group of hexuronic acid and the 6-O-sulfation group of N-sulfoglucosamine in heparin/heparan sulfate are necessary for VEGF165 activity. We then examined the activation of VEGF receptor (VEGFR) to understand the mechanism. We have made several new findings; 1) heparin yielded a 1.7-fold enhancement of VEGF165-induced phosphorylation of VEGFR-2; 2) depletion of cell surface heparan sulfate by heparinase/heparitinase treatment and preferential reduction of trisulfated disaccharide units of cell surface HS by sodium chlorate treatment resulted in the reduction of such phosphorylation, suggesting the involvement of a heparin-like domain in the phosphorylation of VEGFR-2; and 3) VEGF121, an isoform without the exon 7-encoded region, which has no capacity to bind to heparin, did not show these effects. It is therefore likely that a heparin-like domain of heparan sulfate/heparin forms a complex with VEGF165 and VEGFR-2 via the exon 7-encoded region, thereby enhancing VEGF165-dependent signaling.