1. Introduction

68 P. Auguste & A. Bikfalvi

Some of these unresolved issues were recently taken up by several investigators, thus FGFs becoming, once again the focus of angiogenesis research.

2. Molecular Mechanisms

The FGF/FGF receptor system comprises, to date, 23 FGF family members and four tyrosine kinase receptor prototypes (FGFRs). This repertoire is additionally increased by the presence of a number of isoforms and proteolytic processed derivatives within FGF family members and of spliced variants within FGFRs.3

FGF receptor activation requires heparan sulfate proteoglycans (HSPGs), such as syndecans or glypicans as co-receptors (Fig. 1). In one model, HSPGs induces ligand dimerization which in turn leads to FGFR dimerization and activation.4 Furthermore, a small sequence within FGF-2 spanning between amino acid 48–58 has been recently identified to participate in ligand dimerization.5 This domain seems to be functionally important since a peptide containing this sequence is able to inhibit ligand dimerization and biological activity. However, in another proposed model derived from crystallographic studies at 3A resolution of the FGF/FGFR complex with the heparan sulfate analogue heparin,6 heparin makes multiple contacts with FGF and FGFR and promotes receptor dimerization but not ligand dimerization. The reason for these differences is at present not known.

HSPGs may regulate FGF/FGFR interactions in a membranebound form or in a soluble form after shedding of heparan sulfates or HSPG fragments by heparanase or proteolytic enzymes. These different HSPGs forms may have distinct regulatory functions in FGF receptor activation. Indeed, it has been reported that membranebound or heparinolytically-shedded HSPGs enhance receptor activation whereas proteolytically-shedded HSPG are inhibitory.7 In perlecan heparan sulphate-deficient mice, FGF-2-induced corneal angiogenesis is severely impaired but mice develop normally.8 Membrane-associated gangliosides (GM1) were also added to the list of molecules able to regulate FGF/FGFR interactions.9 Treatment of endothelial cells with ganglioside biosynthesis inhibitors impairs FGF-2-induced endothelial cell proliferation.125I-FGF2 binds to cell membrane GM1 with high

Fig. 1. Signaling mechanisms regulated by FGFs. Fibroblast growth factors (FGFs) bind to FGF receptors (FGFR) and induce receptor dimerization. Heparan sulfate proteoglycans (HSPGs) and gangliosides (GM1) actively participate in the extracellular activation mechanism. Whether, ligand dimerization is involved is not clear. Furthermore, gangliosides (GM1) function also as co-receptors for FGF. FGF induces the mitogen-associated protein (MAP) kinase pathway, src and p38 MAP kinases. P38 kinase negatively regulates Jagged1. FGF activates PI3K/Akt pathway, but in endothelial cells it is not clear if FRS2/Grb2/Grab1 is implicated. Phospholipase C-γ (PLCγ p in FGF signaling in vascular cells is not clear. Sef, a transmembrane protein associates with FGFR and inhibits its activation (not represented). The HSPG syndecan-4 also participates in intracellular signaling and induces protein kinase C-α (PKCαp activation. Hypoxia induces hypoxia inducible factor-1α (HIF1α), transcriptionally activates the expression of 1,4-GlcNAc transferase (GlcNAcT-I) and of heparan sulfate 2-O sulfotransferase (HS2ST), the enzyme responsible for sulfation of iduronic acid (IdoA), ghich increases the expression of HSPGs at the cell surface. Proteolytic or heparinolytic cleavage of HSPGs generates modulators of FGF/FGFR interactions. Intracellular fibroblast growth factor-binding proteins (FIBPs) that interact with FGF have also been described. However, their role in the vasculature is not yet established.

affinity (Kd of 3 nM). FGF-2 binding to GM1 and its mitogenic activity are abrogated by cholera toxin-B, a ligand for gangliosides. This indicates that cell-associated gangliosides may act as functional FGF co-receptors.9

70 P. Auguste & A. Bikfalvi

FGFR1 is the main FGFR expressed in endothelial cells in vitro and has also been detected in activated endothelial cells in vivo.10 Small amounts of FGFR2 were also detected in endothelial cells.11 In capillary endothelial cell lines, stimulation of FGFR1 induces proliferation, migration, protease production and tubular morphogenesis, whereas FGFR2 only increases motility. It is at present not clear whether this observation is of more general significance and also applies to primary endothelial cells.

FGF receptor stimulation by FGFs activates the classical MAP kinase pathway and also recruits a number of binding partners such as FRS2 or GRB2 (Fig. 1 and Table 1). Furthermore, p38 MAP kinases seem to represent essential regulators of FGF’s signal transduction machinery in vascular endothelial cells.12 FGF2 induces tube formation, p38 kinase activation and expression of the notch ligand jagged1 in endothelial cells cultured on three-dimensional collagen gels. Inhibition of p38 kinase further increases the effect of FGF2 on tube formation, decreases apoptosis, stimulates DNA synthesis and further increases jagged1 expression. This correlates with the in vivo results in the chicken chorioallantoid membrane (CAM), where FGF-2 and SB202190, an inhibitor of p38 kinase, induce together endothelial cell hyperplasia and aberrant blood vessels.12 Thus, p38 may be part of a negative feedback loop providing an auto-inhibitory mechanism for FGF effects on the vasculature, or as a component of the vessel maturation pathway. A role for p38 in the vessel maturation pathway is also supported by gene inactivation studies in mice.13,14

Besides MAPK activation, a long-lasting PLCγ or PKC (downstream of PLCγ) activation is required for FGF-2’s full mitogenic activity in some endothelial cell lines.15 Nevertheless, FGF-2 is unable to activate PLCγ in HUVEC.16 In non-endothelial cells, PLCγ is required for FGFR internalization.17 This observation needs to be confirmed in vascular cells.

In non-vascular cells, it has been reported that FGFs induce phosphatidyl inositol (PI)-3 kinase activation via fibroblast growth factor receptor substrate-2 (FRS2)/Gab1.18 In culture, FGF-2 induces endothelial cell Akt phosphorylation resulting in an antiapoptotic effect and increased cell motility. This is confirmed in vivo

Table 1.. Molecular players involved in FGF-mediated effects on the vasculature.

where inhibition of the PI3K/Akt pathway inhibits FGF-2-induced angiogenesis.19

It has been recently reported that the transcription factor ETS- 1 is a downstream effector of FGF-2 signaling in FGF-2-induced

72 P. Auguste & A. Bikfalvi

angiogenesis in vivo in the mouse ear and in tumor angiogenesis20 (Table 1). Retroviral expression of a dominant-negative form of ETS- 1 lacking the transactivation domain abrogates the in vivo effects of FGF-2.

Other signaling modules involved in FGF-driven angiogenesis are Fyn, Src, Fes or Shb21−26 (Table 1).

Recently, HSPGs have also been reported to play an active role in the FGF-induced signaling.27 (Table 1). Phosphatidyl-inositol- 4,5-bisphosphate (PIP2) or postsynaptic density-95 (PDZ)-binding domain-mutated syndecan-4 overexpressed in vascular endothelial cells, inhibits protein kinase C-α (PKCα) activation. Furthermore, expression levels of HSPGs are modulated in endothelial cells during angiogenesis. Hypoxia increases the ratio of heparan sulfate to chondroitin sulfate at the endothelial cell surface and the binding of125I-FGF-2. Hypoxia upregulates heparan sulfate 2-O sulfotransferase (HS2ST) and 1,4-N-acetyl glucosamine (GlcNAc) transferase (GlcNAcT-I) through hypoxia inducible factor-1α (HIF1α)28 (Fig. 1). This increases the effect of FGF on the vasculature.

Several feedback inhibitors of FGFR activity have been identified by genetic screening in Drosophila or Zebrafish. One of them is Sef, a transmembrane protein that associates with FGFR1 at the level of the cytoplasmic domain.29 The human Sef homologue is expressed in HUVEC, interacts with FGFR1 and inhibits FGF-2-induced Erk activation30 (Table 1).

In addition to the paracrine effects of FGF, vascular cells also express different FGF forms which may act in an autocrine or intracellular manner. For example, FGF-2 exists as a cytoplasmic 18 kDa isoform and as four nuclear high molecular weight (HMW) isoforms. HMW isoforms but not 18 kDa FGF-2 has an N-terminal sequence responsible for nuclear targeting/retention signal. Dominant-negative strategies in cultured cells have demonstrated that HMW FGF-2 acts on cell growth by a cell surface receptor-independent mechanism.31,32 Intracellular FGFs may bind a number of molecules such as p34 for FGF-1,33 FGF interacting factor (FIF) or Translokin for FGF-234,35 may participate in intracellular effects (Table 1). The studies mentioned above have conducted in non vascular cells and it is, therefore, not firmly established