Fibroblast Growth Factor-2 Antagonist Activity and Angiostatic Capacity of Sulfated Escherichia coli K5 Polysaccharide Derivatives
2001; Elsevier BV; Volume: 276; Issue: 41 Linguagem: Inglês
10.1074/jbc.m105163200
ISSN1083-351X
AutoresDaria Leali, Mirella Belleri, Chiara Urbinati, Daniela Coltrini, Pasqua Oreste, G Zoppetti, Doménico Ribatti, Marco Rusnati, Marco Presta,
Tópico(s)Fibroblast Growth Factor Research
ResumoThe 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 inN- and/or O-position afterN-deacetylation. O-Sulfated andN,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to125I-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 cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wild-type CHO cells. They also inhibited125I-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 andN,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 highlyN,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 highlyN,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds. 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 inN- and/or O-position afterN-deacetylation. O-Sulfated andN,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to125I-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 cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wild-type CHO cells. They also inhibited125I-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 andN,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 highlyN,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 highlyN,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds. basic fibroblast growth factor chorioallantoic membrane Chinese hamster ovary fetal calf serum fibroblast growth factor receptor glycosaminoglycan glucosamine glucuronic acid N-acetyl-glucosamine heparan sulfate heparan sulfate proteoglycan iduronic acid vascular endothelial growth factor fetal calf serum human umbilical vascular endothelial phosphate-buffered saline 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 (1Folkman J. Nat. Med. 1995; 1: 27-31Crossref PubMed Scopus (7284) Google Scholar). Tumors cannot grow larger than a few square millimeters as a mass unless a new blood supply is induced (2Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7705) Google Scholar). Hence the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy (3Keshet E. Ben-Sasson S.A. J. Clin. Invest. 1999; 104: 1497-1501Crossref PubMed Scopus (129) Google Scholar). Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors (4Liotta L.A. Steeg P.S. Stetler-Stevenson W.G. Cell. 1991; 64: 327-336Abstract Full Text PDF PubMed Scopus (2671) Google Scholar). 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)1 and vascular endothelial growth factor (VEGF). Heparin-binding FGF2 was one of the first of these factors to be characterized (5Presta M. Moscatelli D. Joseph-Silverstein J. Rifkin D.B. Mol. Cell. Biol. 1986; 6: 4060-4066Crossref PubMed Scopus (233) Google Scholar). It induces cell proliferation, chemotaxis, and protease production in cultured endothelial cells (5Presta M. Moscatelli D. Joseph-Silverstein J. Rifkin D.B. Mol. Cell. Biol. 1986; 6: 4060-4066Crossref PubMed Scopus (233) Google Scholar, 6Gualandris A. Urbinati C. Rusnati M. Ziche M. Presta M. J. Cell. Physiol. 1994; 161: 149-159Crossref PubMed Scopus (60) Google Scholar, 7Ribatti D. Urbinati C. Nico B. Rusnati M. Roncali L. Presta M. Dev. Biol. 1995; 170: 39-49Crossref PubMed Scopus (152) Google Scholar). In vivo, FGF2 shows angiogenic activity in different experimental models (8Folkman J. Klagsbrun M. Science. 1987; 235: 442-447Crossref PubMed Scopus (4175) Google Scholar). In situhybridization 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 (9Schulze-Osthoff K. Risau W. Vollmer E. Sorg C. Am. J. Pathol. 1990; 137: 85-92PubMed Google Scholar, 10Zagzag D. Miller D.C. Sato Y. Rifkin D.B. Burstein D.E. Cancer Res. 1990; 50: 7393-7398PubMed Google Scholar, 11Ohtani H. Nakamura S. Watanabe Y. Mizoi T. Saku T. Nagura H. Lab. Invest. 1993; 68: 520-527PubMed Google Scholar, 12Takahashi J.A. Mori H. Fukumoto M. Igarashi K. Jaye M. Oda Y. Kikuchi H. Hatanaka M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5710-5714Crossref PubMed Scopus (255) Google Scholar, 13Statuto M. Ennas M.G. Zamboni G. Bonetti F. Pea M. Bernardello F. Pozzi A. Rusnati M. Gualandris A. Presta M. Int. J. Cancer. 1993; 53: 5-10Crossref PubMed Scopus (25) Google Scholar). Antisense FGF2 and fibroblast growth factor receptor (FGFR) 1 cDNAs inhibit neovascularization and growth of human melanomas in nude mice (14Wang Y. Becker D. Nat. Med. 1997; 3: 887-893Crossref PubMed Scopus (281) Google Scholar). 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 (15Aigner A. Butscheid M. Kunkel P. Krause E. Lamszus K. Wellstein A. Czubayko F. Int. J. Cancer. 2001; 92: 510-517Crossref PubMed Scopus (63) Google Scholar). Interestingly, targeting of the FGF-binding protein with specific ribozymes significantly reduces the growth and vascularization of xenografted tumors in mice (16Czubayko F. Liaudet-Coopman E.D.E. Aigner A. Tuveson A.T. Berchem G.J. Wellstein A. Nat. Med. 1997; 3: 1137-1140Crossref PubMed Scopus (219) Google Scholar), despite the high levels of VEGF produced by these cells (discussed in Ref. 17Rak J. Kerbel R.S. Nat. Med. 1997; 3: 1083-1084Crossref PubMed Scopus (66) Google Scholar). These data are in keeping with the synergistic action exerted by the two growth factors in stimulating angiogenesis (18Goto F. Goto K. Weindel K. Folkman J. Lab. Invest. 1993; 69: 508-517PubMed Google Scholar, 19Asahara T. Bauters C. Zheng L.P. Takeshita S. Bunting S. Ferrara N. Symes J.F. Isner J.M. Circulation. 1995; 92: 365-371Crossref Google Scholar) and with the observation that modulation of FGF2 expression may allow a fine tuning of the angiogenesis process even in the presence of significant levels of VEGF (20Konerding M.A. Fait E. Dimitropoulou C. Malkusch W. Ferri C. Giavazzi R. Coltrini D. Presta M. Am. J. Pathol. 1998; 152: 1607-1615PubMed Google Scholar). More recently, a recombinant adenovirus expressing soluble fibroblast growth factor receptor has been shown to repress tumor growth and angiogenesis (21Compagni A. Wilgenbus P. Impagnatiello M.A. Cotton M. Christofori G. Cancer Res. 2000; 60: 7163-7169PubMed Google Scholar). Finally, a significant correlation exists between the presence of FGF2 in cancer cells and advanced tumor stage in human pancreatic (22Yamanaka Y. Friess H. Buchler M. Beger H.G. Uchida E. Onda M. Kobrin M.S. Cancer Res. 1993; 53: 5289-5296PubMed Google Scholar), renal (23Slaton J.W. Inoue K. Perrotte P. El-Naggar A.K. Swanson D.A. Fidler I.J. Dinney C.P. Am. J. Pathol. 2001; 158: 735-743Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), and colon (24Takahashi Y. Ellis L.M. Wilson M.R. Bucana C.D. Kitadai Y. Fidler I.J. Oncol. Res. 1996; 8: 163-169PubMed Google Scholar) cancer. Also, interferon-α has been shown to inhibit FGF2 synthesis and to accelerate the regression of vascular tumors (25Chang E. Boyd A. Nelson C.C. Crowley D. Law T. Keough K.M. Folkman J. Ezekowitz R.A. Castle V.P. J. Pediatr. Hematol. Oncol. 1997; 19: 237-244Crossref PubMed Scopus (166) Google Scholar). Taken together, these data point to FGF2 as a target for antiangiogenic therapy in tumors. FGF2 exerts its biological activity on endothelial cells by interacting with high affinity tyrosine kinase FGFRs (26Johnson D.E. Williams L.T. Adv. Cancer Res. 1993; 60: 1-41Crossref PubMed Scopus (1190) Google Scholar) and low affinity proteoglycans containing heparan sulfate (HS) as polysaccharide (HSPGs) (27Schlessinger J. Lax I. Lemmon M. Cell. 1995; 83: 357-360Abstract Full Text PDF PubMed Scopus (455) Google Scholar). The physiological effects resulting from the interaction of FGF2 with cell-associated and free HSPGs are manifold. HSPGs protect FGF2 from inactivation in the extracellular environment and modulate the bioavailability of the growth factor (28Saksela O. Moscatelli D. Sommer A. Rifkin D.B. J. Cell Biol. 1988; 107: 743-751Crossref PubMed Scopus (683) Google Scholar, 29Edelman E.R. Nugent M.A. Karnovsky M.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1513-1517Crossref PubMed Scopus (247) Google Scholar). At the cell surface, free and cell-associated HSPGs may play contrasting roles in modulating the dimerization of FGF2 and its interaction with FGFRs. For instance, heparin induces FGF2-FGFR interaction in HS-deficient cells (30Yayon A. Klagsbrun M. Esko D. Leder P. Ornitz D.M. Cell. 1991; 64: 841-848Abstract Full Text PDF PubMed Scopus (2178) Google Scholar). This interaction relies on the capacity of the glycosaminoglycan (GAG) to form a ternary complex by interacting with both proteins (31Guimond S. Maccarana M. Olwin B.B. Lindahl U. Rapraeger A.C. J. Biol. Chem. 1993; 268: 23906-23914Abstract Full Text PDF PubMed Google Scholar,32Rusnati M. Coltrini D. Dell'Era P. Zoppetti G. Oreste P. Valsasina B. Presta M. Biochem. Biophys. Res. Commun. 1994; 203: 450-458Crossref PubMed Scopus (76) Google Scholar). In apparent contrast to these observations, heparin inhibits the binding of FGF2 to FGFRs when administered to cells bearing surface-associated HSPGs (33Ishihara M. Tyrrell D.J. Stauber G.B. Brown S. Cousens L.S. Stack R.J. J. Biol. Chem. 1993; 268: 4675-4683Abstract Full Text PDF PubMed Google Scholar). This is probably due to the competition of soluble GAGs with cell-associated HSPGs and FGFRs for binding to FGF2. Thus, the bioavailability and the biological activity of FGF2 on endothelial cells depend strictly on the extracellular GAG milieu, indicating the possibility of modulating the angiogenic activity of FGF2 in vivo by using exogenous GAGs. The capacity of systemically administered, low molecular weight heparin fragments to reduce the angiogenic activity of FGF2 supports this hypothesis (34Norrby K. Ostergaard P. Int. J. Microcirc. Clin. Exp. 1996; 16: 8-15Crossref PubMed Scopus (69) Google Scholar). A further implication of this hypothesis is that synthetic molecules and chemically modified heparins able to interfere with the HSPG-FGF2-FGFR interaction may act as angiogenesis inhibitors. In particular, heparin-mimicking, polyanionic compounds able to compete with HSPGs for growth factor interaction may be expected to hamper the binding of FGF2 to the endothelial cell surface, with consequent inhibition of its angiogenic capacity. Among such compounds are 6-O-desulfated heparin (35Lundin L. Larsson H. Kreuger J. Kanda S. Lindahl U. Salmivirta M. Claesson-Welsh L. J. Biol. Chem. 2000; 275: 24653-24660Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), suramin (36Pesenti E. Sola F. Mongelli N. Grandi M. Spreafico F. Br. J. Cancer. 1992; 66: 367-372Crossref PubMed Scopus (123) Google Scholar), several suramin analogues (37Firsching A. Nickel P. Mora P. Allolio B. Cancer Res. 1995; 55: 4957-4961PubMed Google Scholar), pentosan polysulfate (38Zugmaier G. Lippman M.E. Wellstein A. J. Natl. Cancer Inst. 1992; 84: 1716-1724Crossref PubMed Scopus (124) Google Scholar), and sulfonic acid polymers (39Liekens S. Leali D. Neyts J. Esnouf R. Rusnati M. Dell'Era P. Maudgal P.C. De Clercq E. Presta M. Mol. Pharmacol. 1999; 56: 204-213Crossref PubMed Scopus (94) Google Scholar). The capsular K5 polysaccharide from Escherichia coli has the same structure (→4)-β-d-GlcA-(1→4)-α-d-GlcNAc-1(1→)nas the heparin precursor N-acetyl heparosan (40Vann W.F. Schmidt M.A. Jann B. Jann K. Eur. J. Biochem. 1981; 116: 359-364Crossref PubMed Scopus (219) Google Scholar), in which GlcA is glucuronic acid, and GlcNAc is N-acetyl-glucosamine. Previous studies have shown the possibility of generating K5 derivatives by chemical sulfation in N- and/orO-positions (41Casu B. Grazioli G. Razi N. Guerrini M. Naggi A. Torri G. Oreste P. Tursi F. Zoppetti G. Lindahl U. Carbohydr. Res. 1994; 263: 271-284Crossref PubMed Scopus (99) Google Scholar). In the present study, we synthesized various N-, O-, andN,O-sulfated K5 derivatives with different degrees of sulfation. The compounds were then tested for their FGF2 antagonist activity and antiangiogenic capacity. The results demonstrate that a highly N,O-sulfated K5 derivative binds FGF2 and inhibits formation of the HSPG·FGF2·FGFR ternary complex, prevents 125I-FGF2 binding to endothelial cell surface HSPGs and FGFRs, inhibits FGF2-mediated mitogenesis, sprouting, and morphogenesis in cultured endothelial cells, and blocks angiogenesis in the developing chick embryo chorioallantoic membrane (CAM). Human recombinant FGF2 was from Pharmacia-Upjohn (Milan, Italy). Conventional heparin was obtained from a commercial batch preparation of unfractionated sodium heparin from beef mucosa (1131/900 from Laboratori Derivati Organici S.p.A., Milan, Italy) that was purified of contaminants (purity higher than 95%) according to previously described methodologies (42Andriuoli G. D'Altri G. Galimberti G. Sarret M. Zoppetti G. Casu B. Naggi A.M. Oreste P. Torri G. Ann. N. Y. Acad. Sci. 1989; 556: 416-418Crossref Scopus (8) Google Scholar). Selectively desulfated heparins were described previously (43Rusnati M. Coltrini D. Oreste P. Zoppetti G. Albini A. Noonan D. di Magagna F. Giacca M. Presta M. J. Biol. Chem. 1997; 272: 11313-11320Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and were a generous gift of Dr. B. Casu (Ronzoni Institute, Milan, Italy). Type I HS was from Opocrin (Corlo, Italy). Chondroitin 4-sulfate and dermatan sulfate were a gift of Dr. M. Del Rosso (University of Florence, Florence, Italy). All the K5 polysaccharide derivatives (Table I) were obtained from a single batch of K5 polysaccharide prepared from an E. coli cell culture grown for 16 h at 37 °C in a medium containing 2.0 g/liter defatted soy flour, 9.7 g/liter K2HPO4, 2.0 g/liter KH2PO4, 0.5 g/liter sodium citrate, 0.11 g/liter MgCl2, 1.0 g/liter ammonium sulfate, and 2.0 g/liter glucose, pH 7.3. Conditioned medium was collected and concentrated five times with a 10,000 Da cut-off ultrafiltration membrane. The polysaccharide was recovered after precipitation with 4 volumes of acetone, and proteins were hydrolyzed by a 90-min incubation at 37 °C with protease II from Aspergillus orizae (Sigma) in 0.1 m NaCl, 0.15 m EDTA, and 0.5% SDS, pH 8.0. K5 polysaccharide was finally recovered by ultrafiltration and precipitation.Table IChemical characterization of K5 derivativesSampleSO3−/COO−ratioGlc-NSO3−Glc-6SO3−GlcA-OSO3−1-aGlcA2SO3− or GlcA3SO3−.GlcA2,3SO3−Nonsulfated GlcAMolecular weight(%)(%)(%)(%)(%)K50000010030,000K5-NS1.0010000010015,000K5-OS(L)1.41090 9014,000K5-OS(H)3.7701000100011,000K5-N,OS(L)1.7010090 9013,000K5-N,OS(H)3.841001003070015,00013C NMR spectrum analysis, sulfate/carboxyl ratio (SO3−/COO−) analysis, and molecular weight determination were performed as described under "Experimental Procedures."1-a GlcA2SO3− or GlcA3SO3−. Open table in a new tab 13C NMR spectrum analysis, sulfate/carboxyl ratio (SO3−/COO−) analysis, and molecular weight determination were performed as described under "Experimental Procedures." One g of K5 polysaccharide was suspended at room temperature in 10 ml of anhydrousN,N-dimethylformamide, and then 15 ml ofN,N-dimethylformamide containing 2.4 or 0.7 g of pyridine-sulfotrioxyde complex were added to generate K5-OS(H) and K5-OS(L), respectively. Samples were incubated for 18 h at room temperature and recovered by precipitation with 16 ml of NaCl-saturated acetone. Pellets were dissolved in water and purified from salts by ultrafiltration. One g of K5 polysaccharide was dissolved in 100 ml of 2.0 nNaOH, incubated for 24 h at 60 °C, and cooled to room temperature, and the pH was adjusted to 7.0. The solution was warmed up to 40 °C, added in a single step to 1.6 g of sodium carbonate and 1.6 g of pyridine-sulfotrioxyde complex stepwise for 4 h, and incubated for an additional hour at the same temperature. The solution was then brought to room temperature, and the pH was adjusted to 7.5–8.0. TheN-deacetylated/N-sulfated K5 was purified from salts by ultrafiltration, and the sample was dried under a vacuum. N-Deacetylated/N-sulfated K5 was dissolved in 10 ml of water, run through a cation exchange column (IR-120 H+; Bio-Rad) at 10 °C, and neutralized with 15% tetrabutylammonium hydroxide in water. After concentration and freeze drying, the sample was dissolved in 40 ml ofN,N-dimethylformamide, and 0.7 or 3.5 g of pyridine-sulfotrioxyde complex in solid form was added to generate K5-N,OS(L) and K5-N,OS(H), respectively. Samples were incubated at 50 °C for 24 h and cooled to 4 °C, and NaCl-saturated acetone was added until precipitation was complete. After filtration, the precipitate was dissolved in 10 ml of water with NaCl added until 0.2 m concentration, and the pH was adjusted to 7.5–8.0 with 2.0 n NaOH. The product was recovered by acetone precipitation and ultrafiltration to obtain a 10% solution. Then, samples were warmed up to 40 °C, added in a single step to 1.6 g of sodium carbonate and 1.6 g of pyridine-sulfotrioxyde complex stepwise for 4 h, and incubated for an additional 24 h at the same temperature. Samples were then purified from salts by ultrafiltration. The 13C NMR spectrum analysis, the sulfate/carboxyl ratio analysis, and the molecular weight determination of the different compounds were performed as described previously (44Casu B. Diamantini G. Fedeli G. Mantovani M. Oreste P. Pescador R. Prino G. Torri G. Zoppetti G. Arzneim. Forsch. 1986; 36: 637-642PubMed Google Scholar, 45Casu B. Gennaro U. Carbohydr. Res. 1975; 39: 168-176Crossref PubMed Scopus (145) Google Scholar, 46Harenberg J. De Vries J.X. J. Chromatogr. 1983; 261: 287-292Crossref Scopus (75) Google Scholar, 47Coltrini D. Rusnati M. Zoppetti G. Oreste P. Grazioli G. Naggi A. Presta M. Biochem. J. 1994; 303: 583-590Crossref PubMed Scopus (41) Google Scholar). Human recombinant FGF2 was labeled with Na125I (37 GBq/ml; Amersham Pharmacia Biotech) using Iodogen (Pierce) as described previously (47Coltrini D. Rusnati M. Zoppetti G. Oreste P. Grazioli G. Naggi A. Presta M. Biochem. J. 1994; 303: 583-590Crossref PubMed Scopus (41) Google Scholar). Heparin was dissolved at 4.0 mg/ml in 25 mm Tris-HCl, pH 7.5, and 130 mm NaCl (TBS). Then, the heparin solution (50 μl/well) was incubated overnight at 37 °C in non-tissue culture plastic 96-well plates as described previously (48Rusnati M. Taraboletti G. Urbinati C. Tulipano G. Giuliani R. Molinari-Tosatti M.P. Sennino B. Giacca M. Tyagi M. Albini A. Noonan D. Giavazzi R. Presta M. FASEB J. 2000; 14: 1917-1930Crossref PubMed Scopus (28) Google Scholar). After washing with TBS, 125I-FGF2 was added at 20 ng/ml in the presence of increasing concentrations of the different K5 derivatives. After 2 h of incubation at 4 °C, bound radioactivity was collected by a 30-min incubation at 50 °C in 2% SDS and measured. Nonspecific binding to bovine serum albumin-coated wells was measured in the absence of any competitor and subtracted from all values. Adult bovine aortic endothelial cells were provided by M. Pepper (University of Geneva, Geneva, Switzerland) and grown in Eagle's minimal essential medium containing 10% fetal calf serum (FCS). Wild-type CHO cells, FGFR1-transfected CHO cells, wild-type CHO-K1 cells (a subclone of CHO cells from which HSPG-deficient mutants were originated (49Esko J.D. Curr. Opin. Cell Biol. 1991; 3: 805-816Crossref PubMed Scopus (187) Google Scholar)), and A745 CHO cell mutants (kindly provided by J. D. Esko, La Jolla, CA) were grown in Ham's F-12 medium supplemented with 10% FCS. A745 CHO cells harbor a mutation that inactivates the xylosyltransferase that catalyzes the first sugar transfer step in GAG synthesis (49Esko J.D. Curr. Opin. Cell Biol. 1991; 3: 805-816Crossref PubMed Scopus (187) Google Scholar). FGFR1-transfected CHO cells and the A745 CHOflg-1A clone, both bearing about 30,000 FGFR1 molecules/cell, were generated in our laboratory by transfection with the IIIc variant of murine FGFR1 cDNA (39Liekens S. Leali D. Neyts J. Esnouf R. Rusnati M. Dell'Era P. Maudgal P.C. De Clercq E. Presta M. Mol. Pharmacol. 1999; 56: 204-213Crossref PubMed Scopus (94) Google Scholar). Balb/c mouse aortic endothelial 22106 cells stably transfected with a human FGF2 cDNA (FGF2-T-MAE cells) (50Sola F. Gualandris A. Belleri M. Giuliani R. Coltrini D. Bastaki M. Molinari Tosatti M.P. Bonardi F. Vecchi A. Fioretti F. Giavazzi R. Ciomei M. Grandi M. Mantovani A. Presta M. Angiogenesis. 1997; 1: 102-116Crossref PubMed Scopus (23) Google Scholar) were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS in the presence of 500 μg/ml G418 sulfate (Sigma). Transformed fetal bovine aortic endothelial GM 7373 cells, corresponding to the described BFA-1c 1BPT multilayered transformed clone (47Coltrini D. Rusnati M. Zoppetti G. Oreste P. Grazioli G. Naggi A. Presta M. Biochem. J. 1994; 303: 583-590Crossref PubMed Scopus (41) Google Scholar), were obtained from the National Institute of General Medical Sciences, Human Genetic Mutual Cell Repository (Camden, NJ). Cells were grown in Eagle's minimal essential medium containing 10% FCS, vitamins, and essential and nonessential amino acids. Human umbilical vein endothelial (HUVE) cells at passage 3 (Clonetics) were grown in complete Endothelial Cell Growth Medium-2 (Clonetics). This assay was performed as described previously (51Richard C. Liuzzo J.P. Moscatelli D. J. Biol. Chem. 1995; 270: 24188-24196Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), with minor modifications. Briefly, wild-type CHO-K1 cells were seeded in 24-well plates at 52,000 cells/cm2. After 24 h, cell monolayers were washed with PBS and incubated with 3% glutaraldehyde in PBS for 2 h at 4 °C. Fixation was stopped with 0.1m glycine, and cells were washed extensively with PBS. Then, A745 CHO flg-1A cells (52,000 cells/cm2) were added to CHO-K1 monolayers in serum-free medium plus 10 mm EDTA with or without 30 ng/ml FGF2 in the absence or presence of increasing concentrations of the competitor being tested. After 2 h of incubation at 37 °C, unattached cells were removed by washing twice with PBS, and A745 CHO flg-1A cells bound to the wild-type CHO monolayer were counted under an inverted microscope at ×125 magnification. Adherent A745 CHO flg-1A cells have a rounded morphology and can be easily distinguished from the confluent wild-type CHO monolayer lying underneath on a different plane of focus. Data are expressed as the mean of the cell counts of three microscopic fields chosen at random. All experiments were performed in triplicate and repeated twice with similar results. Twenty-four h after plating in 24-well dishes at a density of 70,000 cells/cm2, bovine aortic endothelial cells or FGFR1-transfected CHO cells were washed three times with ice-cold PBS and incubated for 2 h at 4 °C in binding medium (serum-free medium containing 0.15% gelatin and 20 mm Hepes, pH 7.5) with 30 ng/ml 125I-FGF2 in the absence or presence of increasing concentrations of the compound being tested. Then, after a PBS wash, cells were washed twice with 2 m NaCl in 20 mm Hepes (pH 7.5) to remove 125I-FGF2 bound to low affinity HSPGs and twice with 2 m NaCl in 20 mm sodium acetate (pH 4.0) to remove 125I-FGF2 bound to high affinity FGFRs (47Coltrini D. Rusnati M. Zoppetti G. Oreste P. Grazioli G. Naggi A. Presta M. Biochem. J. 1994; 303: 583-590Crossref PubMed Scopus (41) Google Scholar). Nonspecific binding was measured in the presence of 300 μg/ml suramin and subtracted from all values. GM 7373 cells were seeded at 70,000 cells/cm2 in 24-well dishes. Plating efficiency was higher than 90%. After overnight incubation, cells were incubated in fresh medium containing 0.4% FCS and 10 ng/ml FGF2. After 8 h, increasing concentrations of the K5 derivative being tested were added to cell cultures without changing the medium. Sixteen h later, cells were trypsinized and counted in a Burker chamber. Under our experimental conditions, control cultures incubated in 0.4% FCS with or without 10 ng/ml FGF2 underwent 0.1–0.2 and 0.7–0.8 cell population doubling, respectively. Cells grown in 10% FCS underwent 1.0 cell population doubling (52Rusnati M. Urbinati C. Presta M. J. Cell. Physiol. 1993; 154: 152-161Crossref PubMed Scopus (88) Google Scholar). HUVE cells were seeded at 2,500 cells/well in 96-well plates in complete Endothelial Cell Growth Medium-2. After 24 h, all cell cultures were incubated in Endothelial Cell Growth Medium-2 plus 2% FCS without FGF2, VEGF, and heparin. Then 30 ng/ml FGF2 was added to wells in the absence or presence of K5 derivatives. After 3 days, cells were stained with crystal violet, and plates were read with a microplate reader at 595 nm. FGF2-T-MAE cell aggregates, prepared on agarose-coated plates exactly as described previously (53Gualandris A. Rusnati M. Belleri M. Nelli E.E. Bastaki M. Molinari-Tosatti M.P. Bonardi F. Parolini S. Albini A. Morbidelli L. Ziche M. Corallini A. Possati L. Vacca A. Ribatti D. Presta M. Cell Growth Differ. 1996; 7: 147-160PubMed Google Scholar), were seeded onto fibrin-coated 48-well plates. Immediately after seeding, 250 μl of calcium-free medium containing fibrinogen (2.5 mg/ml) and thrombin (250 milliunits/ml) were added to each well and allowed to gel for 5 min at 37 °C. Then, 500 μl of culture medium with or without K5 derivatives (all at 100 μg/ml) were added on the top of the gel. In all experiments, the fibrinolytic inhibitor trasylol was added to the gel and to the culture medium at 200 KIU/ml to prevent the dissolution of the substrate (53Gualandris A. Rusnati M. Belleri M. Nelli E.E. Bastaki M. Molinari-Tosatti M.P. Bonardi F. Parolini S. Albini A. Morbidelli L. Ziche M. Corallini A. Possati L. Vacca A. Ribatti D. Presta M. Cell Growth Differ. 1996; 7: 147-160PubMed Google Scholar). Formation of radially growing cell sprouts was observed during the next 1–2 days. Matrigel (Becton Dickinson, Milan, Italy) is an extracellular matrix extract of the murine EHS tumor grown in C57/bl6 mice. 150 μl/well Matrigel (10 mg/ml) was used to coat 48-well plates at 4 °C. After gelification at 37 °C, HUVE or FGF2-T-MAE cells were seeded onto Matrigel-coated dishes at 40,000 or 75,000 cells/cm2, respectively, in the absence or presence of K5 derivatives (all at 100 μg/ml). Newly formed endothelial cell "cords" and "tubes" were photographed using an inverted phase-contrast photomicroscope. Fertilized White Leghorn chick eggs were incubated under conditions of constant humidity at 37 °C. On the third day of incubation, a square window was opened in the egg shell after removal of 2–3 ml of albumen to detach the developing CAM from the shell. The window was sealed with a glass of the same size, and the eggs w
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