2 Sigma-Tau Research Department, 0040 Pomezia, Rome, Italy; 3 G. Ronzoni Institute for Chemical and Biochemical Research, 20133 Milan, Italy; 4 Department of Human Anatomy and Histology, University of Bari, 70124 Bari, Italy; 5 Unit of General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, University of Brescia, 25123 Brescia, Italy
Received on May 28, 2004; revised on September 14, 2004; accepted on October 14, 2004
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: angiogenesis / endothelium / growth factor receptor / heparin / VEGF
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Vascular endothelial growth factor (VEGF) plays a major role in angiogenesis by acting via its tyrosine kinase receptors (VEGFRs) (Ferrara et al., 2003). VEGF/VEGFR antagonists affect tumor growth and vascularization (Margolin, 2002
), and the VEGF-specific antibody bevacizumab exerts antivascular effects in cancer patients (Willett et al., 2004
).
Among the five isoforms encoded by the VEGF gene (Ferrara and Davis-Smyth, 1997), VEGF165 plays a mayor role (Grunstein et al., 2000
; Yu et al., 2002
). Similar to several angiogenic growth factors, including fibroblast growth factors (FGFs) (Rusnati and Presta, 1996
), VEGF165 binds heparin and heparan sulfate (HS) proteoglycans (HSPGs) (Gitay-Goren et al., 1992
; Tessler et al., 1994
). Heparin/HS interaction modulates the activity of VEGF and its interaction with VEGFRs (Cohen et al., 1995
; Gitay-Goren et al., 1992
; Tessler et al., 1994
).
Heparin/HS derivatives and polyanionic heparin-like substances are endowed with antiangiogenic activity (Presta et al., 2003), a major mechanism of action being related to their capacity to bind heparin-binding growth factors and to prevent their productive interaction with signaling receptors (Presta et al., 2003
). Interestingly, low molecular weight (LMW) heparin inhibits the angiogenic activity exerted by VEGF165 (Norrby, 2000
; Norrby and Ostergaard, 1997
). Thus, the design of LMW heparin derivatives as VEGF antagonists can be envisaged.
Recently both thrombotic and hemorrhagic complications have been reported in cancer patients undergoing antiangiogenic therapy targeting the VEGF/VEGFR system (Daly et al., 2003). For instance, pulmonary hemorrhage has been observed in patients undergoing antiangiogenic therapy with the anti-VEGF bevacizumab. Even though the etiology of these complications remain unclear, these findings suggest that the use of antiangiogenic VEGF antagonists endowed with anticoagulant activity, for example, LMW heparin, should be approached with caution.
Recently we generated a novel nonanticoagulant antiangiogenic heparin derivative (ST1514) (Casu et al., 2002, 2004
). Sulfation gaps along the regular heparin sequences were generated by selectively removing 2-O-sulfate groups to reach a ratio of about 1:1 between sulfated and nonsulfated uronic acid residues. Next, the C(2)C(3) bonds of all nonsulfated uronic acid residues were split, generating flexible joints along the heparin chain while minimizing cleavage of glycosidic bonds. Because the splitting reaction also occurs at the level of the essential glucuronic acid residue of the active site for antithrombin, ST1514 was no longer anticoagulant, but it showed a potent FGF2 antagonist and angiostatic activity (Casu et al., 2002
, 2004
).
Here, we assess the capacity of ST2184, a LMW derivative of ST1514 (Casu et al., 2004), to interact with VEGF165 and act as a nonanticoagulant angiogenesis inhibitor.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
ST2184/VEGF165 interaction
Two experimental approaches were used to assess ST2184/VEGF165 interaction. First, 125I-VEGF165 was incubated with ST2184 or heparin and subjected to native polyacrilamide gel electrophoresis (Figure 2A). The mobility shift assay demonstrates that slow migrating 125I-VEGF165 complexes are formed starting from 60 ng or 600 ng/sample of heparin or ST2184, respectively. The slower migration rate of the heparin complex compared to the ST2184 complex is likely due to the smaller size and lower charge density of ST2184 compared to heparin.
|
On this basis, we evaluated the capacity of ST2184 to compete with immobilized heparin for the binding to VEGF165. ST2184 or heparin were preincubated with VEGF165 and injected onto the heparin-coated sensor chip. Both ST2184 and heparin caused a dose-dependent inhibition of immobilized heparin/VEGF165 interaction with ID50 values equal to 100 nM and 10 nM, respectively (Figure 2C). A LMWH preparation inhibited VEGF165/heparin interaction with an ID50 value equal to 300 nM (Fig. 2C). Thus ST2184 interacts with VEGF165 with an affinity 10 times lower than heparin but 3 times higher than LMW heparin.
Antiangiogenic VEGF165-antagonist activity of ST2184
As observed for bovine endothelial cells (Gitay-Goren et al., 1992), Scatchard plot analysis of 125I-VEGF165 binding to human umbilical vein endothelial (HUVE) cells revealed a high-affinity class of receptors (Kd = 20 pM; Bmax = 4 fmol/106 cells) and a low-affinity class (Kd = 0.25 nM; Bmax = 22 fmol/106 cells) (data not shown). Also, in keeping with previous observations (Gitay-Goren et al., 1992
), low doses of heparin (up to 110 µg/ml) caused a three- to fourfold increase in 125I-VEGF165 binding (Figure 3A). This effect is related to the capacity of heparin to bind directly to VEGFRs facilitating ligand interaction (Dougher et al., 1997
), as already observed for FGFreceptor interaction (Klagsbrun and Baird, 1991
). At higher doses, heparin caused a significant inhibition of 125I-VEGF165 binding instead.
|
Next, the anti-VEGF165 activity of ST2184 was evaluated on the chick embryo chorioallantoic membrane (CAM) (Casu et al., 2004). ST2184 caused a significant inhibition of the angiogenic response triggered by VEGF165, similar to its precursor ST1514 (Figure 4A,B). Histological analysis confirmed the inhibitory effect. In contrast, heparin did not affect the angiogenic activity of VEGF165.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Glycol-split heparin chains are more flexible than unmodified chains and conformationally driven to adopt geometries unfavorable for formation of ternary complexes with FGF2 and its receptor(s) (Casu et al., 2002, 2004
). This may also be the case for the formation of VEGFVEGFR complexes. Indeed, unlike heparin, ST2184 was unable to facilitate the interaction of VEGF165 with its receptors. Instead, at very high concentrations both ST2184 and heparin sequester the growth factor and inhibit ligand binding and cell proliferation. However, only ST2184 exerts a significant antiangiogenic activity in vivo. The incapacity to support VEGFVEGFR interaction may at least partly explain the angiostatic activity of ST2184.
Heparin is used in therapy as an anticoagulant and antithrombotic drug (Fareed et al., 2000). When administered to cancer patients, heparin increases survival times (Lebeau et al., 1994
; Zacharski et al., 2000
). Beneficial effects of heparin in cancer are though to be associated with the binding to and inhibition of one or more proteins overexpressed by tumor cells (Engelberg, 1999
). Heparin and its LMW derivatives are currently being investigated as antitumor agents (Lebeau et al., 1994
; Zacharski et al., 2000
), and therapeutic regimes have been proposed to exploit their antimetastatic activity (Varki and Varki, 2002
). However, the anticoagulant properties of heparin involve hemorrhagic risks, and nonanticoagulant variants of the polysaccharide endowed with potential antitumor properties are warranted (Lapierre et al., 1996
). This is of importance when considering that antiangiogenic anti-VEGF interventions may be associated with hemorrhagic complications in cancer patients (Daly et al., 2003
). Undersulfated, glycol-split ST1514 and its LMW derivative ST2184 are endowed with a negligible anticoagulant activity when compared to heparin. These compounds may therefore provide the basis for the design of novel nonanticoagulant LMW angiostatic molecules.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparation of LMW ST2184
ST2184 was prepared by controlled nitrous acid depolymerization of glycol-split ST1514 (Casu et al., 2004). Briefly, 4.0 g of ST1514 were dissolved in 65 ml H2O at 4°C. NaNO2 (75 mg) was added and the pH adjusted to 2.0 with 0.1 M HCl. The solution was stirred at 4°C for 20 min. The pH was then brought to 7.0. Solid NaBH4 (1.0 g) was added in several portions under stirring. After 23 h the pH was adjusted to 4.0 with HCl, and the solution was neutralized with NaOH. The product was isolated by ethanolic precipitation. The extent of glycol-splitting was evaluated by integration of 13C NMR signals at 106.5 and 102 ppm, corresponding to C1 of the split uronic residues and 2-O-sulfated iduronic residues, respectively. Yield: 75%. The average Mw, as evaluated by GPC, is 5800 (polydispersion 1.4) (Casu et al., 2004
). LMW heparin was prepared from unfractionated heparin as for ST2184, using 100 mg NaNO2 at 0°C. The relative content of terminal a.Man.ol residues (TR) with respect to total glucosamine residues was evaluated for both LMW compounds by integration of all anomeric 13C NMR signals at 98107 ppm (a) and those at 82, 85, and 87 ppm (b) corresponding to total C4, C2, and C5 of the terminal anhydromannitol unit (see Casu et al., 2004
). TR (= 2b/3a + 2b) was 0.14 and 0.13 for LMW heparin and ST2184, corresponding to average Mn values (calculated from ratios aM/total residues, including terminal aMan.ol) of 4200 and 3900 Da, respectively.
Plasma clotting assay
ST2184 or heparin were injected S.C. in BALB/c mice at 100 mg/kg. Mice were sacrificed by ether inhalation at different time intervals; blood was taken by intracardiac puncture using a plastic syringe containing 0.126 M sodium citrate (1:10, v/v) and platelet-poor plasma was obtained by centrifugation. APTT was measured using optimised Thrombofax reagent (Ortho Diagnostic Systems, Milan, Italy). Reference mouse platelet-poor plasma was used for comparison.
Gel mobility shift assay
125I-VEGF165 (105 cpm/2 ng) was preincubated at room temperature for 30 min with ST2184 or heparin. Samples were then loaded on a native 7% polyacrylamide gel. The gel buffer was 10 mM Tris (pH 7.4) and 1 mM ethylenediamine tetra-acetic acid (EDTA), and the electrophoresis buffer was 40 mM Tris (pH 8.0), 40 mM acetic acid, 1 mM EDTA (Wu et al., 2002). The gel was run at 6 V/cm for 2 h, transferred to 3MM paper, dried under vacuum, and autoradiographed.
BIAcore binding assay
A BIAcore F1 sensorchip was activated with streptavidin. Then, heparin biotinylated on its reducing end was allowed to react with the streptavidin-coated surface. VEGF165 (300 nM) alone or in the presence of heparin derivatives was dissolved in binding buffer (10 mM HEPES, 150 mM NaCl, 3.4 mM EDTA, 0.005% surfactant P20, pH 7.4) and injected over the heparin surface for 5 min. Then the sensor chip was washed with the same buffer until dissociation was observed. The surface plasmon resonance signal was expressed in terms of resonance units (RU).
125I-VEGF165 binding assay
HUVE cells (Clonetics, Cambrex, Milan, Italy) were seeded at 200,000/well in 24-well plates and incubated overnight in M199 medium (Invitrogen, Carlsbad, CA) containing 20% fetal bovine serum, 2 mM glutamine, and 2 ng/ml FGF2. For Scatchard plot analysis, cells were seeded. Then, cells were incubated for 2 h at 4°C with 01.26 nM 125I-VEGF165 and 900 ng/ml unlabeled VEGF165 dissolved in 0.2 ml M199 with 0.2% gelatin, 1.25 mM HEPES. Cells were then solubilized with 1% Triton X-100 and radioactivity measured using a -counter. Binding data were analyzed by the Scatchard procedure using PRISM software. For competition binding studies, cells were incubated with 125I-VEGF165 (4.0 ng/ml) in the presence of 101000 µg/ml of heparin or of ST2184 both in the absence or in the presence of 1.0 µg/ml heparin. Nonspecific binding in the presence of 900 ng/ml of unlabeled VEGF165 was subtracted from all the values.
HUVE cell proliferation assay
Cells were seeded at 20,000/cm2 and incubated overnight in complete EGM2 medium (BioWhittaker, Verviers, Belgium). Next, cells were treated for 72 h with fresh EGM2 medium in the presence of 0.11.0 mg/ml of ST2184 or heparin. Finally, cells were trypsinized and counted.
CAM assay
Gelatin sponges (1 mm3) were placed onto the CAM of fertilized chicken eggs on day 8 of development, immediately followed by topical administration (23 µl) of VEGF (500 ng/egg) in the presence of vehicle or of 100 µg/egg of heparin derivatives (Casu et al., 2004). Treatments were repeated daily for 4 days. At days 8 and 12, the number of macroscopic vessels converging toward the implant were assessed under a stereomicroscope. Also, angiogenesis was quantified at day 12 by a planimetric method of point counting on paraffin-embedded 8-mm sections (Casu et al., 2004
).
![]() |
Acknowledgements |
---|
![]() |
Abbreviations |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Casu, B., Guerrini, M., Naggi, A., Perez, M., Torri, G., Ribatti, D., Carminati, P., Giannini, G., Penco, S., Pisano, C., and others. (2002) Short heparin sequences spaced by glycol-split uronate residues are antagonists of fibroblast growth factor 2 and angiogenesis inhibitors. Biochemistry, 41, 1051910528.[CrossRef][ISI][Medline]
Casu, B., Guerrini, M., Guglieri, S., Naggi, A., Perez, M., Torri, G., Cassinelli, G., Ribatti, D., Carminati, P., Giannini, G., and others. (2004) Undersulfated and glycol-split heparins endowed with antiangiogenic activity. J. Med. Chem., 47, 838848.[CrossRef][ISI][Medline]
Cohen, T., Gitay-Goren, H., Sharon, R., Shibuya, M., Halaban, R., Levi, B.Z., and Neufeld, G. (1995) VEGF121, a vascular endothelial growth factor (VEGF) isoform lacking heparin binding ability, requires cell-surface heparan sulfates for efficient binding to the VEGF receptors of human melanoma cells. J. Biol. Chem., 270, 1132211326.
Daly, M.E., Makris, A., Reed, M., and Lewis, C.E. (2003) Hemostatic regulators of tumor angiogenesis: a source of antiangiogenic agents for cancer treatment? J. Natl Cancer Inst., 95, 16601673.
Dougher, A.M., Wasserstrom, H., Torley, L., Shridaran, L., Westdock, P., Hileman, R.E., Fromm, J.R., Anderberg, R., Lyman, S., Linhardt, R.J., and others. (1997) Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor. Growth Fact., 14, 257268.[ISI]
Engelberg, H. (1999) Actions of heparin that may affect the malignant process. Cancer, 85, 257272.[CrossRef][ISI][Medline]
Fareed, J., Hoppensteadt, D.A., and Bick, R.L. (2000) An update on heparins at the beginning of the new millennium. Semin. Thromb. Hemost., 26(suppl 1), 521.[CrossRef][ISI][Medline]
Ferrara, N. and Davis-Smyth, T. (1997) The biology of vascular endothelial growth factor. Endocr. Rev., 18, 425.
Ferrara, N., Gerber, H.P., and LeCouter, J. (2003) The biology of VEGF and its receptors. Nat. Med., 9, 669676.[CrossRef][ISI][Medline]
Gitay-Goren, H., Soker, S., Vlodavsky, I., and Neufeld, G. (1992) The binding of vascular endothelial growth factor to its receptors is dependent on cell surface-associated heparin-like molecules. J. Biol. Chem., 267, 60936098.
Grunstein, J., Masbad, J.J., Hickey, R., Giordano, F., and Johnson, R.S. (2000) Isoforms of vascular endothelial growth factor act in a coordinate fashion To recruit and expand tumor vasculature. Mol. Cell Biol., 20, 72827291.
Klagsbrun, M. and Baird, A. (1991) A dual receptor system is required for basic fibroblast growth factor activity. Cell, 67, 229231.[ISI][Medline]
Lapierre, F., Holme, K., Lam, L., Tressler, R.J., Storm, N., Wee, J., Stack, R.J., Castellot, J., and Tyrrell, D.J. (1996) Chemical modifications of heparin that diminish its anticoagulant but preserve its heparanase-inhibitory, angiostatic, anti-tumor and anti-metastatic properties. Glycobiology, 6, 355366.[Abstract]
Lebeau, B., Chastang, C., Brechot, J.M., Capron, F., Dautzenberg, B., Delaisements, C., Mornet, M., Brun, J., Hurdebourcq, J.P., and Lemarie, E. (1994) Subcutaneous heparin treatment increases survival in small cell lung cancer. "Petites Cellules" Group. Cancer, 74, 3845.[ISI][Medline]
Margolin, K. (2002) Inhibition of vascular endothelial growth factor in the treatment of solid tumors. Curr. Oncol. Rep., 4, 2028.[Medline]
Norrby, K. (2000) 2.5 kDa and 5.0 kDa heparin fragments specifically inhibit microvessel sprouting and network formation in VEGF165-mediated mammalian angiogenesis. Int. J. Exp. Pathol., 81, 191198.[CrossRef][ISI][Medline]
Norrby, K. and Ostergaard, P. (1997) A 5.0-kD heparin fraction systemically suppresses VEGF165-mediated angiogenesis. Int. J. Microcirc. Clin. Exp., 17, 314321.[ISI][Medline]
Presta, M., Leali, D., Stabile, H., Ronca, R., Camozzi, M., Coco, L., Moroni, E., Liekens, S., and Rusnati, M. (2003) Heparin derivatives as angiogenesis inhibitors. Curr. Pharm. Des., 9, 553566.[ISI][Medline]
Rusnati, M. and Presta, M. (1996) Interaction of angiogenic basic fibroblast growth factor with endothelial cell heparan sulfate proteoglycans. Biological implications in neovascularization. Int. J. Clin. Lab. Res., 26, 1523.[ISI][Medline]
Tessler, S., Rockwell, P., Hicklin, D., Cohen, T., Levi, B.Z., Witte, L., Lemischka, I.R., and Neufeld, G. (1994) Heparin modulates the interaction of VEGF165 with soluble and cell associated flk-1 receptors. J. Biol. Chem., 269, 1245612461.
Varki, N.M. and Varki, A. (2002) Heparin inhibition of selectin-mediated interactions during the hematogenous phase of carcinoma metastasis: rationale for clinical studies in humans. Semin. Thromb. Hemost., 28, 5366.[CrossRef][ISI][Medline]
Willett, C.G., Boucher, Y., di Tomaso, E., Duda, D.G., Munn, L.L., Tong, R.T., Chung, D.C., Sahani, D.V., Kalva, S.P., Kozin, S.V., and others. (2004) Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat. Med., 10, 145147.[CrossRef][ISI][Medline]
Wu, Z.L., Zhang, L., Beeler, D.L., Kuberan, B., and Rosenberg, R.D. (2002) A new strategy for defining critical functional groups on heparan sulfate. FASEB J., 16, 539545.
Yu, J.L., Rak, J.W., Klement, G., and Kerbel, R.S. (2002) Vascular endothelial growth factor isoform expression as a determinant of blood vessel patterning in human melanoma xenografts. Cancer Res., 62, 18381846.
Zacharski, L.R., Ornstein, D.L., and Mamourian, A.C. (2000) Low-molecular-weight heparin and cancer. Semin. Thromb. Hemost., 26(suppl 1), 6977.[CrossRef][ISI][Medline]