Fibroblast Growth Factor-2 Antagonist Activity and Angiostatic Capacity of Sulfated Escherichia coli K5 Polysaccharide

The angiogenic basic fibroblast growth factor (FGF2) interacts with tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Here, we report the FGF2 antagonist and antiangiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in Nand/or O-position after N-deacetylation. O-Sulfated and N,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to I-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG FGF2 FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cellcell attachment of FGFR1-overexpressing HSPGdeficient Chinese hamster ovary (CHO) cells to wildtype CHO cells. They also inhibited I-FGF2 binding to FGFR1-overexpressing HSPG-bearing CHO cells and adult bovine aortic endothelial cells. K5 derivatives also inhibited FGF2-mediated cell proliferation in endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective. Also, highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected endothelial FGF2-T-MAE cells in fibrin gel and spontaneous angiogenesis in vitro on Matrigel of FGF2-T-MAE and HUVE cells. Finally, the highly N,O-sulfated K5 derivative exerted a potent antiangiogenic activity on the chick embryo chorioallantoic membrane. These data demonstrate the possibility of generating FGF2 antagonists endowed with antiangiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds. Angiogenesis is the process of generating new capillary blood vessels. In the adult, the proliferation rate of endothelial cells is very low compared with that of many other cell types in the body. Physiological exceptions in which angiogenesis occurs under tight regulation are found in the female reproductive system and during wound healing. Uncontrolled endothelial cell proliferation is observed in tumor neovascularization and in angioproliferative diseases (1). Tumors cannot grow larger than a few square millimeters as a mass unless a new blood supply is induced (2). Hence the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy (3). Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors (4). Thus, the angiogenic switch represents the net result of the activity of angiogenic stimulators and inhibitors, suggesting that counteracting even a single major angiogenic factor could shift the balance toward inhibition. Major angiogenic factors include, among several others, basic fibroblast growth factor (FGF2) and vascular endothelial growth factor (VEGF). Heparin-binding FGF2 was one of the first of these factors to be characterized (5). It induces cell proliferation, chemotaxis, and protease production in cultured endothelial cells (5–7). In vivo, FGF2 shows angiogenic activity in different experimental models (8). In situ hybridization and immunolocalization experiments have shown the presence of FGF2 mRNA and/or protein in neoplastic cells, endothelial cells, and infiltrating cells within human tumors of different origin (9–13). Antisense FGF2 and fibroblast growth factor receptor (FGFR) 1 cDNAs inhibit neovascularization and growth of human melanomas in nude mice (14). Also, a secreted FGF-binding protein that mobilizes stored extracellular FGF2 can serve as an angiogenic switch for different tumor cell lines, including squamous cell carcinoma and colon cancer cells (15). Interestingly, targeting of the FGFbinding protein with specific ribozymes significantly reduces the growth and vascularization of xenografted tumors in mice (16), despite the high levels of VEGF produced by these cells (discussed in Ref. 17). These data are in keeping with the synergistic action exerted by the two growth factors in stimulating angiogenesis (18, 19) and with the observation that modulation of FGF2 expression may allow a fine tuning of the * This work was supported in part by grants from the European Community (“Biologically Active Novel Glycosaminoglycans” Contract Number QLK3-CT-1999-00536), Associazione Italiana per la Ricerca sul Cancro, Istituto Superiore di Sanità (AIDS Project), National Research Council (Target Project on Biotechnology), Ministero dell’Università e della Ricerca Scientifica e Tecnologica (Cofin 1999, Cofin 2000, and Centro di Eccellenza IDET to M. P. and Cofin 2000 to M. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom correspondence should be addressed: Unit of General Pathology and Immunology, Dept. of Biomedical Sciences and Biotechnology, via Valsabbina 19, 25123 Brescia, Italy. Tel.: 39-0303717311; Fax: 39-0303701157; E-mail: [email protected]. 1 The abbreviations used are: FGF2, basic fibroblast growth factor; CAM, chorioallantoic membrane; CHO, Chinese hamster ovary; FCS, fetal calf serum; FGFR, fibroblast growth factor receptor; GAG, glycosaminoglycan; Glc, glucosamine; GlcA, glucuronic acid; GlcNAc, Nacetyl-glucosamine; HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; IdoA, iduronic acid; VEGF, vascular endothelial growth factor; FCS, fetal calf serum; HUVE, human umbilical vascular endothelial; PBS, phosphate-buffered saline. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 41, Issue of October 12, pp. 37900–37908, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.