FGF2 belongs to the family of the heparin-binding growth factors (Basilico and Moscatelli, 1992). The single copy human FGF2 gene encodes multiple FGF2 isoforms with molecular weights ranging from 24 kD to 18 kD. High molecular weight isoforms (HMW-FGF2s) are colinear NH2-terminal extensions of the better characterized 18 kD protein (Florkiewicz and Sommer, 1989). Both low and high molecular weight FGF2s exert angiogenic activity in vivo and induce cell proliferation, protease production, and chemotaxis in endothelial cells in vitro (Gualandris et al., 1994). Also, FGF2 has been shown to stimulate endothelial cells to form capillary-like structures in collagen gels (Montesano et al., 1989) and to invade the amniotic membrane in vitro (Mignatti et al., 1989).

Moreover, the phenotype induced in vitro by FGF2 in endothelial cells includes modulation of integrin expression (Klein et al., 1993), gap-junctional intercellular communication (Pepper and Meda, 1992) and urokinase receptor upregulation (Mignatti et al., 1991a). Studies with neutralizing anti-FGF2 antibodies have implicated FGF2 in wound repair (Broadly et al., 1989), vascularization of the chorioallantoic membrane during chick embryo development (Ribatti et al., 1995), and tumor growth under defined experimental conditions (Baird et al., 1986; Gross et al., 1993).


3D structure of FGF2

Both low and high MW FGF2 isoforms interact with high affinity tyrosine-kinase FGF receptors (FGFRs) (Johnson and Williams, 1993) and low affinity proteoglycans containing heparan sulfate (HS) as polysaccharide (HSPGs) (Gualandris et al., 1994). The physiological effects of the interaction of FGF2 with cell-associated and free HSPGs are manyfold. HSPGs protect FGF2 from inactivation in the extracellular environment and modulate the bioavailability of the growth factor (Rusnati et al., 1996a). At the cell surface, free and cell-associated HSPGs may play contrasting roles in modulating the dimerization of FGF2 and its interaction with FGFRs.


HSPG-mediated dimerization of FGF2

For instance, free heparin induces FGF2-FGFR interaction in HS-deficient cells (Yayon et al., 1991). This relies on the capacity of the glycosaminoglycan (GAG) to form a ternary complex by interacting with both proteins (Guimond et al., 1993; Turnbull and Gallagher, 1993; Rusnati et al., 1994). Indeed, a heparin binding domain in FGFR-1/flg has been identified in the NH2-terminus of IgG-like domain II (Kan et al., 1993). In apparent contrast with these observations, free heparin inhibits the binding of FGF2 to FGFRs when administered to cells bearing surface-associated HSPGs (Ishihara et al., 1993; Coltrini et al., 1994). This is probably due to the competition of free GAGs with cell-associated HSPGs and FGFRs for the binding to FGF2.

The puzzling observation that heparin by itself can activate FGFR in the absence of the growth factor (Gao and Goldfarb, 1995) further increases the complexity of the GAG/FGF2/FGFR ternary interaction. Finally, HSPGs affect the internalization and the intracellular fate of FGF2, suggesting that FGF2-HSPG complexes are involved in the intracellular delivery of FGF2 (Gannoun-Zaky et al., 1991; Roghani and Moscatelli, 1993; Rusnati et al., 1993).

Thus, the bioavailability and the biological activity of FGF2 on endothelial cells strictly depend on the extracellular GAG milieu, indicating the possibility of modulating the angiogenic activity of FGF2 in vivo by using exogenous GAGs. Recent findings on the capacity of low molecular weight heparin fragments administered systemically to reduce the angiogenic activity of FGF2 support this hypothesis (Norrby and Ostergaard, 1996).

These observations raise the possibility that synthetic molecules able to interfere with HSPG/FGF2/FGFR interaction may act as angiogenesis inhibitors. In particular, heparin-mimicking, polyanionic compounds able to compete with HSPGs for growth factor interaction would hamper the binding of FGF2 to the endothelial cell surface with consequent inhibition of its angiogenic capacity. Among such compounds are suramin, several suramin analogues (Firsching et al., 1995; Ciomei et al., 1994), pentosan polysulfate (PPS) (Zugmaier et al., 1992), and the polycarboxylated compounds aurin tricarboxylic acid (Gagliardi et al., 1994) and RG-13577 (Miao et al., 1997).


Chemical structure of suramin

The most extensively studied, the polysulfonated naphtylurea suramin, has been shown to block the binding of several growth factors, including FGF2 to its receptors (Rusnati et al., 1996b; Braddock et al., 1994). In addition, suramin exerts a marked inhibitory effect on endothelial cell growth, migration, and urokinase-type plasminogen activator production in vitro (Takano et al., 1994) and angiogenesis and tumor growth in vivo (Gagliardi et al., 1992; Waltz et al., 1991). Finally, suramin has been used in clinical trials on cancer patients with some beneficial effects (Myers et al., 1992).

The sulfonic acid polymers PAMPS [poly(2-acrylamido-2-methyl-1-propanesulfonic acid)], PAS [poly(anetholesulfonic acid)], PSS [poly(4-styrenesulfonic acid)], and PVS [poly(vinylsulfonic acid)] suppress viral replication, including HIV replication (Mohan et al., 1992; Ikeda et al., 1994).

We recently reported on the potent inhibitory activity exerted by PAMPS, PAS, and PSS on neovascularization that occurs in the chick embryo chorioallantoic membrane (CAM) during development (Liekens et al., 1997). Also, these sulfonic acid polymers exerted an anti-angiogenic effect in the in vitro rat aorta-ring assay and inhibited FGF2-induced human umbilical vein endothelial cell proliferation. Interestingly, a significant correlation was found between the angiostatic activity of these compounds in the CAM assay in vivo and their capacity to inhibit the FGF2-induced mitogenic response in vitro, thus suggesting that FGF2 is a target for sulfonic acid polymers (Liekens et al., 1997).

We have investigated the capacity of sulfonic acid polymers to interact with FGF2 and to affect its biological activity in vitro and in vivo. The results indicate that sulfonic acid polymers mimic functional features of heparin/HS by binding to FGF2 and preventing its interaction with endothelial cell surface HSPGs and FGFRs. Also, the sulfonic acid polymers were evaluated for their capacity to prevent the formation of the HSPG/FGF2/FGFR ternary complex. For this purpose, we utilized an experimental model in which the disruption of the complex abolishes FGF2-mediated cell-cell attachment of HSPG-deficient CHO mutants transfected with FGFR-1 to a monolayer of wild type CHO-K1 cells bearing HSPGs but that express negligible amounts of FGFR (Richard et al., 1995).

FGF2-mediated cell-cell interaction. Left) FGF2 mediates the interaction of FGFR1-bearing cells with HSPGs of the cell monolayer. No interaction occurs in the absence of FGFR1 (Center) or of HSPGs (Right).
 

In this assay, all the molecules tested exerted an inhibitory activity, PSS being the most effective (ID50 equal to 0.01 mM, 0.06 mM, 0.12 mM, and 0.07 mM for PSS, PAMPS, PAS, and PVS, respectively).

Effect of polysulfated/polysulfonated compounds on
FGF2-mediated cell-cell interaction.

These data demonstrate the ability of sulfonic acid polymers to prevent the interaction of FGF2 with its low and high affinity receptors. Interestingly, PSS appears to be the most potent among the molecules studied, its activity being similar to that exerted by conventional heparin on a molar basis and at least 1,000 more potent than that exerted by suramin when tested under the same experimental conditions. Finally, both PAMPS and PAS inhibit FGF2-mediated angiogenesis in the rabbit cornea.

Inhibitory effect of PAMPS and PAS on FGF2-mediated angiogenesis


a The area of corneal neovascularization was determined 8 days after implantation by measuring the vessel length (L) from the limbus and the number of clock hours (C) of limbus involved.
A  formula was used to determine the area of a circular band segment: C/12 x 3.1416 [r2-(r-L)2], where r = 6 mm, the measured radius of the rabbit cornea.