Fibroblast Growth Factor-2 Antagonist and ...

Current Pharmaceutical Design, 2009, 15, 3577-3589

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Fibroblast Growth Factor-2 Antagonist and Antiangiogenic Activity of Long-Pentraxin 3-Derived Synthetic Peptides D. Leali, P. Alessi, D. Coltrini#, M. Rusnati, L. Zetta§ and M. Presta* Unit of General Pathology and Immunology and #Unit of Histology, Department of Biomedical Sciences and Biotechnology, School of Medicine, University of Brescia, Italy; §Istituto Per lo Studio delle Macromolecole, Consiglio Nazionale delle Ricerche, 20133 Milan, Italy Abstract: Angiogenesis and inflammation are closely integrated processes. Fibroblast growth factor-2 (FGF2) is a prototypic angiogenesis inducer belonging to the family of the heparin-binding FGF growth factors. FGF2 exerts its proangiogenic activity by interacting with various endothelial cell surface receptors, including tyrosine kinase receptors, heparan-sulfate proteoglycans, and integrins. A tight cross-talk exists between FGF2 and the inflammatory response in the modulation of blood vessel growth. Pentraxins act as soluble pattern recognition receptors with a wide range of functions in various pathophysiological conditions. The long-pentraxin PTX3 shares the C-terminal pentraxin-domain with shortpentraxins and possesses a unique N-terminal domain. These structural features indicate that PTX3 may have distinct biological/ligand recognition properties when compared to short-pentraxins. Co-expression of PTX3 and FGF2 has been observed in different inflammation/angiogenesis-dependent diseases. PTX3 binds FGF2 with high affinity and specificity. The interaction prevents the binding of FGF2 to its cognate tyrosine kinase receptors, leading to inhibition of the angiogenic activity of the growth factor. This suggests that PTX3 may exert a modulatory function by limiting the angiogenic activity of FGF2. An integrated approach that utilized PTX3 fragments, monoclonal antibodies, and surface plasmon resonance analysis has identified the FGF2-binding domain in the unique N-terminal extension of PTX3. On this basis, PTX3-derived synthetic peptides have been designed endowed with a significant antiangiogenic activity in vitro and in vivo. They may provide the basis for the development of novel antiangiogenic FGF2 antagonists.

Keywords: Angiogenesis, FGF2, innate immunity, pentraxin, tumor, synthetic peptides, NMR, modeling. 1. FGF2: ANGIOGENESIS AND INFLAMMATION 1.1. FGF2 as an Angiogenic Growth Factor Angiogenesis, the process of new blood vessel formation from pre-existing ones, plays a key role in various physiological and pathological conditions, including embryonic development, wound repair, inflammation, and tumor growth [1]. Angiogenesis is a multi-step process that begins with the degradation of the basement membrane by activated endothelial cells (ECs) that will migrate and proliferate, leading to the formation of solid EC sprouts into the stromal space. Then, vascular loops are formed and capillary tubes develop with formation of tight junctions and deposition of a new basement membrane [2]. The local, uncontrolled release of angiogenic growth factors and/or alterations of the production of natural angiogenic inhibitors, with a consequent alteration of the angiogenic balance [3], are responsible for the uncontrolled EC proliferation that takes place during tumor neovascularization and in angiogenesis-dependent diseases [4]. Numerous inducers of angiogenesis have been identified, including the members of the vascular endothelial growth factor (VEGF) family, angiopoietins, transforming growth *Address correspondence to this author at the General Pathology and Immunology, Department of Biomedical Sciences and Biotechnology, Viale Europa 11, 25123 Brescia, Italy; Tel: ++39-30-3717311; Fax: ++39-30-3701157; E-mail: [email protected] 1381-6128/09 $55.00+.00

factor- and - (TGF- and -), platelet-derived growth factor (PDGF), tumor necrosis factor- (TNF-), interleukins (ILs), chemokines, and members of the fibroblast growth factor (FGF) family. Twenty-three structurally-related members of the FGF family have been identified [5]. FGFs are pleiotropic factors acting on different cell types, including ECs, following interaction with heparan-sulfate proteoglycans (HSPGs) and tyrosine kinase FGF receptors (FGFRs). FGFRs belong to the subclass IV of membrane-spanning receptors, are encoded by four distinct genes, and their structural variability is increased by alternative splicing [6]. FGFR1 [7], and less frequently FGFR2 [8] are expressed by ECs, whereas the expression of FGFR3 or FGFR4 has never been reported in endothelium. Among the FGF family members, FGF2 represents the prototypic and best characterized proangiogenic factor. FGF2 expression is augmented at sites of chronic inflammation [9-11], after tissue injury [12], and in different types of human cancer [13]. In vitro, FGF2 binds all FGFRs, with preferential activation of the alternative spliced IIIc form in FGFRs 1-3 [14]. FGF2/FGFR interaction causes receptor dimerization and autophosphorylation of specific tyrosine residues located in the intra-cytoplasmic tail of the receptor. This in turn leads to complex signal transduction pathways and activation of a “pro-angiogenic phenotype” in ECs (reviewed in [13]). In vivo, FGF2 has been shown to

© 2009 Bentham Science Publishers Ltd.

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induce neovascularization in a variety of animal models, including the chick embryo chorioallantoic membrane (CAM) assay, the rodent cornea assay, the subcutaneous (s.c.) Matrigel plug assay in mice, and the zebrafish yolk membrane (ZFYM) assay [13, 15]. FGF2 can exert its effects on ECs via a paracrine mode consequent to its release by tumor, stromal, and inflammatory cells and/or by mobilization from the ECM. On the other hand, endogenous FGF2 produced by ECs may play important autocrine, intracrine, or paracrine roles in angiogenesis and in the pathogenesis of vascular lesions, including Kaposi’s sarcoma and hemangiomas (see [16] and references therein). 1.2. FGF2-Dependent Angiogenesis and Inflammation Inflammation is the response of a vascularized tissue to sub-lethal injury, designed to destroy or inactivate invading pathogens, remove waste and debris, and permit restoration of normal function, either through resolution or repair. Angiogenesis and inflammation are closely integrated processes in a number of physiological and pathological conditions, including wound healing, psoriasis, diabetic retinopathy, rheumatoid arthritis, atherosclerosis, and cancer [1, 17, 18]. Inflammation may promote FGF2-dependent angiogenesis. Inflammatory cells, including mononuclear phagocytes [19, 20], CD4+ and CD8+ T lymphocytes [21, 22], and mast cells [23] can express FGF2. Moreover, osmotic shock and shear stress induce the release of FGF2 from ECs [24, 25]. FGF2 production and release from ECs are also triggered by interferon (IFN)- plus IL-2 [26], IL-1 [27], and nitric oxide (NO) [28]. Indeed, the pro-angiogenic effects exerted by NO and NO-inducing molecules are due, at least in part, to the NO-mediated FGF2 upregulation in ECs [29]. Similarly, prostaglandin E2-induced angiogenesis is mediated by the activation of EC-surface FGFR1 following mobilization of FGF2 sequestered by the ECM [30]. Thus, inflammatory mediators can activate the endothelium to synthesize and release FGF2 that, in turn, will stimulate angiogenesis by an autocrine mechanism of action. The inflammatory response may also cause cell damage, fluid and plasma protein exudation, and hypoxia. EC damage results in increased FGF2 production and release [31]; exudated fibrin(ogen) can bind FGF2 and enhances its biological activity [32, 33]; hypoxia upregulates the production of FGF2 in mononuclear phagocytes [19] and in vascular pericytes [34]. Furthermore, hypoxia increases EC responsiveness to FGF2 by promoting HSPG synthesis [35]. Conversely, by interacting with ECs, FGF2 may amplify both the inflammatory and the angiogenic responses by inducing vasoactive effects [36], vascular permeability [37], and the recruitment of an inflammatory infiltrate [38]. Moreover, FGF2-stimulated ECs upregulate the synthesis of various monocyte chemoattractants, including VEGF [39], the chemokine CCL2 [40, 41], and osteopontin (OPN) [42]. Relevant to this point, OPN induces neovascularization [42] by promoting the release of the pro-angiogenic cytokine IL1 from recruited monocytes/macrophages [43].

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In keeping with the existence of a tight cross-talk between inflammatory and angiogenic responses during FGF2-driven neovascularization, gene expression profiling of FGF2-stimulated murine microvascular ECs has revealed a pro-inflammatory signature characterized by the upregulation of pro-inflammatory cytokine/chemokines and their receptors, EC adhesion molecules, and members of the eicosanoid pathway [44]. Accordingly, we have observed that the early recruitment of mononuclear phagocytes precedes blood vessel formation in FGF2-driven angiogenesis in the s.c. Matrigel plug assay and that monocytes/macrophages play a functional, non-redundant early role in FGF2-driven angiogenesis [44]. It must be pointed out that, together with a proinflammatory signature, FGF2 upregulates also the expression of a variety of angiogenic growth factors in ECs, including FGF2 itself and VEGF [44]. This suggests that FGF2 is able to activate an autocrine loop of amplification of the angiogenic response that, together with the paracrine activity exerted by endothelium-derived cytokines/chemokines on inflammatory cells, will contribute to the modulation of the neovascularization process triggered by the growth factor. 1.3. FGF2 as a Target for Anti-Angiogenic Strategies A significant effort has been directed towards the development of anti-angiogenic agents that prevent the growth of new blood vessels, the monoclonal anti-VEGF antibody bevacizumab and the tyrosine kinase inhibitors sunitinib and sorafenib representing the first FDA approved anti-angiogenic drugs [45]. On the other hand, drug resistance to VEGF blockade may occur following reactivation of angiogenesis triggered by compensatory upregulation of the FGF2/FGFR system in experimental tumor models [46] and in cancer patients [47]. Thus, given its potent angiogenic activity, FGF2 may represent the target for the development of novel anti-angiogenic strategies. The various approaches based on the inhibition of FGF2 have been reviewed extensively elsewhere (see [48] and references therein). Briefly, the angiogenic activity of FGF2 can be neutralized by: i) inhibition of FGF2 production/ release; ii) inhibition of the expression of the various FGF2 receptors in ECs; iii) engagement of the various FGF2 receptors by selected antagonists; iv) sequestration of FGF2 in the extracellular environment; v) interruption of the signal transduction pathways triggered by FGF2 in ECs. Also, as stated above, FGF2 induces a complex “proinflammatory phenotype” in ECs whose blockage may result in the inhibition of FGF2-dependent angiogenesis [48]. Indeed, we have observed that FGF2-mediated angiogenesis is significantly reduced in the CAM assay by both steroidal (hydrocortisone) and non-steroidal (ketoprofen) anti-inflammatory drugs, further implicating inflammatory cells/ mediators in FGF2-dependent neovascularization [44]. Moreover, FGF2-induced neovascularization is inhibited also by M3 protein [44], a murine gammaherpesvirus 68 protein that binds with high affinity to human and mouse CC, CXC and CX3C chemokines and inhibits their activity [49, 50], with potential therapeutic implications in inflammatory conditions [51].

Antiangiogenic Activity of PTX3

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Interestingly, several ECM and serum components and/or their degradation products affect FGF-driven angiogenesis, including thrombospondin-1 (TSP-1) [52, 53], the fibronectin fragment fibstatin [54], the plasma protein 2macroglobulin [55, 56], PDGF-BB [57], and the chemokines CXCL4 [58] and CXCL13 [59]. In the search for effective FGF2 antagonists, numerous peptides derived from these natural FGF2-binders, FGFRs, and FGF2 itself have been demonstrated to exert an inhibitory activity on the FGF2/FGFR system (Table 1). 2. LONG-PENTRAXIN 3 2.1. PTX3 as a Member of the Pentraxin Family Pentraxins are a superfamily of evolutionarily conserved proteins originally named for their structural organization characterized by a radial pentameric structure. Pentraxins are Table 1.

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divided into two subfamilies (short-pentraxins and longpentraxins) sharing a C-terminal pentraxin domain that contains the HxCxS/TWxS pentraxin signature (where x is any amino acid) [60]. Long-pentraxins differ from shortpentraxins for the presence of an unrelated N-terminal domain coupled to the C-terminal domain [61] (Fig. 1A). C-reactive protein (CRP) and serum amyloid P component (SAP) constitute the short-pentraxin arm of the superfamily. These “classical” pentraxins are acute phase proteins in man and mouse, respectively, and are produced in liver in response to inflammatory mediators, most prominently IL-6 [62, 63]. They are involved in the innate resistance to microbes and scavenging of cellular debris and ECM components [60, 64-66]. Pentraxin 3 (PTX3, also called TSG-14) is the prototypic member of the long-pentraxin subfamily [67, 68] that

Synthetic Peptides Endowed with FGF2 Antagonist Activity

Protein of Origin

Peptide(s)

Target

References

FGF1

FGF1(112-147) and related peptides; FGF1(99-108); FGF1 mimetics (6 peptides studied)

FGFR

[117-119]

FGF2(48-58) (FREG)

FGF2

[120]

FGF2(38-61); FGF2(82-101); FGF2-derived DGR-containing peptides (4 peptides studied)

ND

[121]

FGF2(119-126)

FGF2

[122]

FGF2(68-77)

FGFR

[123]

FGF2(24-68) (Peptide D); FGF2(93-120) (Peptide N); FGF2(106-115)

FGFR

[124, 125]

FGF2(103-146)

FGFR

[126]

F2A4-K-NS

FGFR

[127]

16-24 mer peptides based on FGF2 molecular modeling

FGFR

[128, 129]

FGF5

FGF5(95-104) (peptide P3)

FGFR

[130]

FGFs (10-11 loop)

dekafins (homologous to the NCAM FGFR-binding region)

FGFR

[131]

Myelin Basic Protein

MBP(152-167)

FGFR

[132]

FGL

FGFR

[133-136]

FRM-10 and cyclic FRM-10. FRM-13

FGFR

[134, 136, 137]

DekaCAM

FGFR

[131, 134, 136]

BCL

FGFR

[134, 136, 138]

Encamin A, C, E

FGFR

[134, 139]

N-cadherin

Extracellular domain-4 mimetics (2 peptides studied)

FGFR

[119]

PF4 (CXCL4)

PF4(47-70)*

FGF2

[115, 140]

PTX3 N-terminus

PTX3(82-110) (and related peptides, including ARPCA*; see text for further details)

FGF2

[73, 111]

Epitope sequence, FGF2(13-18), FGF2(119-126), FGF2(120-125)

FGFR

[141]

Peptide P7*

FGF2

[116]

C19 (3 peptides studied)

FGFR

[142-144]

Peptide P2

FGFR

[145]

4N1K

ND

[146]

(type III repeats-derived peptides) (6 peptides studied)

FGF2

[147]

FGF2

NCAM(681-695)

Random phage epitope library

TSP-1 *peptide establishing hydrophobic interactions with FGF2. ND: mechanism of action not defined.


includes also guinea pig apexin, rat, human, and murine neuronal pentraxins 1 (NP1 or NPTX1) and NP2 (also called Narp or NPTX2), and the putative integral membrane pentraxin NRP [60] for a review]. Long-pentraxins have been found in Xenopus [61], Drosophila melanogaster [69], and zebrafish (Danio Rerio) [60]. The human PTX3 gene has been identified using differential screening of cDNA libraries constructed from IL1-stimulated human umbilical vein ECs [67] and from normal FS-4 fibroblasts stimulated by TGF- and TNF- [70]. Unlike the CRP and SAP genes that map on chromosome 1, the human PTX3 gene is localized on chromosome 3, band q25. The PTX3 gene comprises three exons, the first exon extending to nucleotide 197, the second one covering nucleotides 198-599, and the third one extending from nucleotide 600 to the 3’-terminus. The first two exons encode for the signal peptide and the N-terminal domain of PTX3 protein whereas the third exon encodes for its Cterminal domain, matching exactly the second exon of the short-pentraxin genes [71]. Thus, PTX3 appears to represent a fusion between regions encoding a specific N-terminal polypeptide of unknown function and a C-terminal polypeptide homologous to short-pentraxins. 2.2. PTX3 Protein Structure The human PTX3 protomer is a 381 amino acid glycoprotein, including a 17 amino acid signal peptide for secretion. The PTX3 primary sequence is highly conserved among animal species (human and murine PTX3 share 92% of conserved amino acid residues), suggesting a strong evolutionary pressure to maintain its structure-function relationships [60]. As the other members of the longpentraxin subfamily, PTX3 is composed of an unique Nterminal domain (spanning amino acid residues 1-178) and of a C-terminal 203 amino acid domain highly homologous among the various members of the pentraxin family (57% of conserved amino acids with short-pentraxins CRP and SAP). The human protein has a unique N-glycosylation site at Asn220 fully occupied by complex type-oligosaccharides, mainly fucosylated and sialylated biantennary sugars [72]. These negatively charged sialic acid residues may establish ionic interactions with polar and basic amino acids located far from the Asn220 N-glycosylation site [72]. No crystallographic data are available for the N-terminal portion of long-pentraxins. Consensus secondary structure prediction of PTX3 N-terminus has identified four -helix regions connected by short loops spanning amino acid residues 55-75 (A), 78-97 (B), 109-135 (C), and 144170 (D) [73]. Moreover, amino acid residues 85-91 in B helix form the structural heptad repeat motif (abcdefg), where a and d are hydrophobic residues and e and g represent charged residues [74]. Also, hydrophobic residues repeated with a period of one each three or six amino acids are present within the primary sequence of C and D helices. Thus, B, C, and D helices of the N-terminal PTX3 domain have propensity to be in a coiled-coil conformation [see [75] for further details]. The high similarity of the primary sequence of the PTX3 C-terminus with short-pentraxins allowed instead to produce

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a predicted structure for the C-terminal PTX3 region (residues 179-381) using the crystallographic structure of CRP as template [75]. The model of PTX3 C-terminus presents a hydrophobic core composed by two anti-parallel -sheets organised in a typical -jelly roll topology [76]. A similar model was described when PTX3 was modelled on the tertiary structure of SAP [63, 77]. The mature PTX3 protein contains nine cysteine residues: three residues are located within the N-terminal region (Cys47, Cys49, and Cys103) whereas six residues are in the C-terminal domain (Cys179, Cys210, Cys271, Cys317, Cys318, and Cys357). Cys210 and Cys271 are highly conserved among pentraxins and, based on the homology with CRP and SAP, are predicted to be engaged in an intrachain disulfide bond [61, 78]. The other Cys residues are involved in intra- and inter-chain disulfide bonds, thus determining the quaternary structure of PTX3 [76]. Indeed, PTX3 shows a complex quaternary structure with subunits assembled into high order oligomers [78]. Recently, the oligomeric assembly of PTX3 has been resolved [76], experimental data demonstrating that human recombinant PTX3 is mainly composed of covalently linked octamers (Fig. 1B). The multimeric organization of PTX3 is essential for the organization of the ECM of the cumulus oophorus, the tetramer being the smallest PTX3 oligomer still retaining full functionality in cumulus matrix assembly and stabilization [76]. 2.3. PTX3 Biological Functions and Interactions PTX3 is produced at the inflammatory site in response to inflammatory cytokines and bacterial components [67, 68, 79, 80]. Accordingly, the proximal 1317-bp promoter of the human PTX3 gene is responsive to TNF- and IL-1, but not to IL-6. Multiple binding sites for various transcription factors have been identified in this sequence, including NFIL-6, AP-1, Pu1, PEA-3, Ets-1, and Sp1 sites. Also, NF-kB sites are essential for induction by IL-1 and TNF- [81, 82]. Various cell types express PTX3, including ECs [67], macrophages [80], microglia [83], dendritic cells [84], adipocytes [85], fibroblasts [68], and myoblasts [77]. Also, PTX3 is upregulated during vasculitis [86], it is present in atherosclerotic plaques [87], and it is expressed by smooth muscle cells (SMCs) isolated from human arterial specimens [88]. Thus, when compared to the liver-produced shortpentraxin CRP, PTX3 may represent a rapid marker for primary local activation of inflammation as well as of innate immunity. Indeed, the levels of circulating PTX3 increase in humans during vasculitis [86], acute myocardial infarction [89], rheumatoid arthritis [90], and systemic inflammatory response syndrome/septic shock [91]. PTX3 is a soluble pattern recognition receptor that may serve as a mechanism of amplification of inflammation and innate immunity with unique non-redundant functions in various physiopathological conditions such as complement activation, protection against opportunistic pathogens, fertility, and angiogenesis [79]. The biological activity of PTX3 is related to its ability to interact with different ligands via its N-terminal or C-terminal domain as a consequence of the modular structure of the protein (see [75] for a review).



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upregulated rapidly in response to proinflammatory cytokines (e.g., IL-1 and TNF-). TSG-6 is comprised of a Link module and a CUB_C domain, both involved in the binding to different ECM components [93, 94]. The coordinated expression of PTX3 and TSG-6 has been described in inflammatory infiltrates and ECs in inflamed tissues [95] and in the cumulus oophorus where they are essential for in vivo fertilization [92, 96, 97]. At present, the possibility that TSG-6 may affect PTX3/FGF2 interaction is under investigation in our laboratory. 3. PTX3/FGF2 INTERACTION 3.1. Biochemical Interaction

Characterization

of

PTX3/FGF2

When assessed for the capacity to interact with a variety of extracellular signaling polypeptides, PTX3 was found to bind FGF2 with high specificity [98]. Under the same experimental conditions, PTX3 did not bind to a wide panel of cytokines, chemokines, and growth factors representative of different classes of soluble polypeptide mediators, showing only a limited interaction with FGF8 (but not with FGF1 or FGF4, all members of the FGF family) and with VEGF. PTX3/FGF2 interaction occurs with high affinity, with a Kd value ranging between 3.0 x 10–7 and 3.0 x 10-8 M depending upon the experimental model adopted [73, 98]. In agreement with the incapacity of short-pentraxins to bind FGF2 [98], an integrated approach that utilized recombinant N-terminal and C-terminal PTX3 fragments, monoclonal antibodies, and surface plasmon resonance analysis identified the FGF2-binding domain in the PTX3 Nterminus [73]. Indeed, the recombinant N-terminal fragment PTX3(1-178) binds FGF2, prevents PTX3/FGF2 interaction, and inhibits the mitogenic activity of FGF2 in ECs. Also, the monoclonal antibody mAb-MNB4, that recognizes the PTX3(87-99) epitope, prevents FGF2/PTX3 interaction and abolishes the FGF2 antagonist activity of PTX3 [73]. The capacity to bind FGF2 and to inhibit its biological activity represents a novel unanticipated function for the N-terminal extension of PTX3. Fig. (1). Schematic representation of PTX3. (A) The pentraxin superfamily: short- and long-pentraxins show a significant homology in their C-terminal pentraxin domain whereas longpentraxins are characterized by unique N-terminal extensions. The 8 amino acid-long pentraxin family signature is highlighted. (B) The PTX3 octamer: interchain disulfide bonds (dotted lines) connecting the N-terminal (white ellipse) or C-terminal (black bar) regions of the eight PTX3 monomers (a-h) are shown. See ref. [76] for details.

Recent observations have shown the ability of PTX3 to bind FGF2, thus acting as a FGF2 antagonist (see below). The coexpression of PTX3 and FGF2 may occur during inflammation, wound healing, atherosclerosis, and neoplasia, thus allowing a fine tuning of the neovascularization process via the production of both angiogenesis inhibitors and stimulators (Fig. 2). Interestingly, PTX3 has been shown to interact also with TSG-6 [92], a 35 kDa-secreted protein

As stated above, FGF2 acts on target cells by interacting with high affinity FGFRs [99] and low affinity HSPGs [100], leading to the formation of HSPG/FGF2/FGFR ternary complexes [101, 102]. PTX3 inhibits the formation of this complex in an experimental model in which the disruption of the complex abolishes FGF2-mediated cell-cell attachment of HSPG-deficient CHO mutants transfected with FGFR1 to wild-type CHO cells expressing HSPGs but not FGFRs [102] (Fig. 3A,B). Furthermore, surface plasmon resonance analysis has shown that PTX3 prevents the binding of FGF2 to the extracellular domain of FGFR1 immobilized to a BIAcore sensorchip (Fig. 3C) but not to immobilized heparin (Fig. 3D), thus suggesting that PTX3 may interact with the FGFR1-binding domain of FGF2. In keeping with these observations, PTX3 prevents the binding of 125I-FGF2 to FGFRs expressed on human SMCs. Also, the interaction of FGF2 with PTX3 or with a soluble form of FGFR1 are mutually exclusive [103]. Thus, as a consequence of PTX3


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Fig. (2). PTX3 as an antiangiogenic FGF2 antagonist. Different cell types produce PTX3 (1) and/or cytokines that stimulate PTX3 upregulation in ECs (2). PTX3 may then inhibit the autocrine (3) or paracrine (4) activity exerted by FGF2 on endothelium, thus modulating the angiogenic response.

Fig. (3). Effect of PTX3 on the formation of the HSPG/FGF2/FGFR1 ternary complex. (A) Schematic representation of the FGF2-mediated cell-cell adhesion assay. The interaction of FGFR1-bearing cells with HSPGs of the cell monolayer occurs in the presence (b) but not in the absence (a) of FGF2. FGF2-mediated cell-cell adhesion is prevented by the binding of PTX3 to FGF2 (c). (B) FGFR1-expressing cells were added to a monolayer of HSPG-expressing cells in the presence of FGF2 (30 ng/ml) and increasing concentrations of PTX3. After 2 h at 37°C, FGFR1-expressing cells bound to the monolayer were counted (see [102] for further experimental details). Data are expressed as percentage of FGF2-mediated cell-cell adhesion. (C) Plasmon resonance analysis of the effect of PTX3 on FGF2/FGFR1 and FGF2/heparin interactions. Injection of PTX3 (220 nM) prevents the binding of free FGF2 (50 nM) to the FGFR1-coated Biacore sensorchip (upper panel) but not the heparin-coated sensorchip (lower panel).


interaction, the angiogenic activity of FGF2 is inhibited both in vitro and in vivo. 3.2. Biological Consequences of PTX3/FGF2 Interaction PTX3 interaction inhibits the mitogenic activity exerted in vitro by FGF2 in bovine, murine, and human ECs. As stated above, this appears to be due to the capacity of PTX3 to prevent the binding of FGF2 to FGFRs rather than to a direct action of PTX3 on ECs. Indeed, PTX3 does not inhibit EC proliferation triggered by various mitogens (including serum, diacylglycerol, epidermal growth factor, phorbol ester, or VEGF) and PTX3 pretreatment does not affect EC responsiveness to FGF2 [98]. In keeping with the in vitro observations, PTX3 inhibits FGF2-driven neovascularization in different animal models, including the chick embryo CAM assay [98], the ZFYM assay performed on zebrafish embryos [15], and the Matrigel plug assay in mice (M. Presta, unpublished observations). Experimental evidences support the hypothesis that PTX3 may also inhibit FGF2-driven tumor angiogenesis. FGF2-overexpressing mouse aortic endothelial FGF2-TMAE cells are characterized by the capacity to generate opportunistic vascular lesions in nude mice [104]. When FGF2-T-MAE cells were stably transfected with an expression vector harboring the full length human PTX3 cDNA, these lesions showed a reduced rate of growth when compared to tumors originated by parental cells [98]. Similarly, PTX3 protein caused a significant inhibition of the angiogenic response elicited by FGF2-T-MAE cells in a zebrafish/tumor xenograft model in which injection of mammalian tumor cells into the perivitelline space induces


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the formation of tumor-driven neovessels sprouting from the sub-intestinal plexus of the embryo [105]. Accordingly, preliminary observations have shown that PTX3 overexpression decreases tumor growth and vascularization when PTX3 transfected murine melanoma B16/BL6 cells are embedded in Matrigel and grafted s.c. in syngeneic animals (Fig. 4). When considering the important cross-talk among innate immunity, inflammation, and tumor progression [106], these results raise the possibility that PTX3 may inhibit tumor growth and angiogenesis driven by FGF2. Relevant to this point, upregulation of PTX3 expression has been observed in human soft tissue liposarcoma [107] and in prostate cancer [108, 109]. It must be pointed out that PTX3 interacts with and inhibits the biological activity of FGF2 at doses comparable to those measured in the blood of patients affected by inflammatory diseases [86, 89]. Moreover, due to its capacity to accumulate in the ECM, the local concentration of PTX3 at the site of inflammation should be significantly higher than that measured in the blood stream, supporting the possibility that PTX3/FGF2 interaction may indeed occur and be biologically relevant in vivo. The putative effects of PTX3 on blood vessels are not limited to the angiogenic process during tumorigenesis. Indeed, PTX3 is present in atherosclerotic plaques [87] and is expressed by SMCs isolated from human arterial specimens [88]. Also, FGF2 is produced by SMCs and other cell types in the restenotic area, including ECs, macrophages, and T-cells [26]. Thus, FGF2 may exert autocrine and/or paracrine functions on FGFR-expressing SMCs of the injured vessel [22, 110]. In keeping with its FGF2 antagonist activity, PTX3 inhibits human coronary artery SMC prolife-

Fig. (4). PTX3 overexpression inhibits tumor growth and vascularization. Mock-transfected (upper panels) and PTX3-transfected (lower panels) murine melanoma B16/BL6 cells were implanted subcutaneously in syngeneic animals (3.0x105 cells in 400 μl of Matrigel). After 7 days, Matrigel plugs were harvested and snap-frozen. Sections were stained with haematoxylin and eosin (H&E) or double-immunostained with anti-CD31 and anti-PTX3 antibodies. Note the reduced cellularity and lack of CD31+ neovessels in PTX3-overexpressing plugs. Original magnification: x200.


ration and migration driven by endogenous and exogenous FGF2. Accordingly, PTX3 overexpression following recombinant adeno-associated virus gene transfer affects SMC proliferation and survival in vitro and intimal thickening after arterial injury in vivo [103]. 3.3. PTX3-Derived Antiangiogenic Peptides As described above, PTX3 binds FGF2 via its unique Nterminal extension [73]. To further define the FGF2-binding region in PTX3 N-terminus, the synthetic peptide PTX3(82110) and the overlapping peptides PTX3(82-96), PTX3(82101), and PTX3(97-110) were evaluated for their FGF2 antagonist activity together with three distinct peptides PTX3(31-60), PTX3(57-85), and PTX3(107-132) partially spanning the PTX3 N-terminus amino acid sequence (Fig. 5). The results demonstrated that the PTX3(82-110) peptide and the shorter PTX3(97-110) peptide show a similar FGF2 binding capacity and antagonist activity in vitro and in vivo, thus indicating that the amino acid linear sequence PTX3(97110) is responsible for PTX3/FGF2 interaction [73]. This region is predicted in an exposed loop of the PTX3 Nterminus that comprises the end of the B helix (Glu97), a turn on residues Ala104-Pro105-Gly106-Ala107, and the first two residues of the C helix (Ala109-Glu110) [73, 75]. On this basis, in an attempt to identify novel FGF2 antagonist(s), four acetylated (Ac) overlapping synthetic peptides based on the amino acid sequences PTX3(97-107), PTX3(100-113), PTX3(100-104), and PTX3(104-113) were compared with PTX3(97–110) peptide for their capacity to interact with FGF2 [111] (Fig. 5). Among them, the shortest pentapeptide Ac-ARPCA-NH2 [in single letter code, corresponding to amino acid sequence PTX3(100-104)] inhibits the

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interaction of FGF2 with PTX3 immobilized to a BIAcore sensorchip and suppresses FGF2-dependent proliferation in ECs. Also, Ac-ARPCA-NH2 inhibits angiogenesis triggered by FGF2 or by tumorigenic FGF2-T-MAE cells in chick and zebrafish embryos, respectively. Accordingly, the peptide hampers the binding of FGF2 to CHO cells overexpressing FGFR1 and to recombinant FGFR1 immobilized to a BIAcore sensorchip without affecting heparin interaction [111]. To assess the relevance of each amino acid residue of AcARPCA-NH2 pentapeptide for FGF2 interaction, a series of synthetic peptides carrying different amino acid substitutions were tested for their FGF2 antagonist activity. Scrambled Ac-PARAC-NH2 and Ac-APCRA-NH2 peptides showed a reduced inhibitory activity, pointing to the relevance of the relative position of each residue for the FGF2 antagonist capacity of the peptide. Also, the FGF2 antagonist activity was dramatically reduced for the non-acetylated H-ARPCANH2 peptide and for the Ac-ARPCG-NH2 and Ac-GRPCGNH2 peptides, but not for the Ac-GRPCA-NH2 peptide, indicating an essential role for the N-terminal blocking methyl group and for the methyl group of the side-chain of Ala5 residue in FGF2 interaction. The activity was lost also following amino acid substitution of residues Arg2, Pro3, or Cys4, thus underlying the role of the RPC amino acid sequence in Ac-ARPCA-NH2/FGF2 interaction. Interestingly, the FGF2 antagonist activity was lost also when the ARPCA region was mutated within synthetic peptides based on the amino acid sequences PTX3(97–110) and PTX3(82110), thus confirming the importance of the linear ARPCA sequence in PTX3/FGF2 interaction [111]. In order to map the peptide residues making direct contacts with FGF2, we applied Saturation Transfer Difference

Fig. (5). FGF2 antagonist activity of PTX3-derived peptides. Schematic representation of the synthetic peptides spanning the N-terminal domain of PTX3 and tested for their FGF2 antagonist activity [73, 98, 111]. Active peptide: grey bar; inactive peptide: open bar. All the active peptides contain the minimal linear FGF2-binding sequence PTX3(100-104) ARPCA (in black).


(STD) NMR methods [112] to a series of Ac-ARPCA-NH 2 mutants in the presence of FGF2 [111]. In particular, the STD spectrum of Ac-ARPCA-NH2 peptide in the presence of FGF2 proves that the methyl protons of Ala1, Ala5, and of the N-terminal blocking acetyl group receive saturation transfer from the protein, indicating that these groups are the main responsible for the direct contact with FGF2. On the other hand, the RPC sequence may play a conformational role in ARPCA/FGF2 interaction, helping to orient the methyl groups of the peptide for optimal interaction with the growth factor. The extracellular portion of FGFRs comprises three Iglike domains (D1, D2, and D3, with an acidic box between D1 and D2). Their ligand binding and specificity reside in D2, D3, and D2-D3 linker region. X-ray crystallography has shown that the interactions between FGF2 and D3 are of both hydrophobic and polar character whereas the interactions with the D2-D3 linker are mediated mainly via hydrogen bonds. At variance, hydrophobic interactions dominate the interface between FGF2 and D2 [113]. Indeed, hydrophobic residues from discontinuous regions in FGF2, including Tyr24, Phe31, Tyr103, Leu140, and Met142, form a flat solvent-exposed hydrophobic surface which interacts hydrophobically with Leu165, Ala167, Pro169, and Val248 of the D2 domain in FGFR1. These residues are well conserved among the four mammalian FGFRs, indicating that this hydrophobic interface represents a highly conserved interaction site for FGF family members [114]. On this basis, Ac-ARPCA-NH2 peptide may exert its FGF2 antagonist activity by mimicking the hydrophobic ligand-binding region of D2, thus establishing hydrophobic interactions with the receptor-binding domain of FGF2 and competing with FGFRs for the binding to the growth factor. Accordingly, when a model of the proposed interaction was built by conformational analysis, the superposition of the global


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minimum conformation of Ac-ARPCA-NH2 peptide to the -sheet region 164-170 of the hydrophobic domain D2 of FGFR1 indicated that the peptide could interact with FGF2 by mimicking this highly conserved FGF2-binding region of the receptor (Fig. 6). Hydrophobic interactions with FGF2 have been observed also for the C-terminal fragment of the FGF2 antagonist chemokine CXCL4 [115]. Moreover, an anti-angiogenic hydrophobic peptide has been identified by screening a phage display heptapeptide library following FGF2 biopanning [116]. At variance with Ac-ARPCA-NH2, this FGF2-binding peptide shares significant amino acid homology, charge distribution, and hydrophobic profile with the Ig-like domain D3 of FGFR1 and FGFR2. Thus, the complexity of FGF2/FGFR interaction is reflected by the possibility to generate various FGF2 antagonists endowed with the capacity to affect this interaction at different levels. 4. CONCLUDING REMARKS PTX3, a member of the pentraxin superfamily released locally by endothelial and inflammatory cells, binds the angiogenic growth factor FGF2 with high affinity and specificity, acting as a natural inhibitor of the autocrine and paracrine activity exerted by the growth factor on ECs. Thus, PTX3 may play an important role in modulating the crosstalk between inflammatory cells and endothelium in various physiopathological settings, including innate immunity, wound healing, restenosis, and atherosclerosis. Also, preliminary observations indicate that PTX3 overexpression may affect tumor growth via angiogenesis-dependent and independent mechanisms of action. Further experiments are required to clarify the impact of PTX3 on tumor progression and the possibility to design PTX3-derived anti-angiogenic and/or anti-neoplastic agents. To this respect, the identi-

Fig. (6). Hypothesis of interaction between Ac-ARPCA-NH2 and FGF2. The global minimum conformation of Ac-ARPCA-NH2 peptide (A) mimics the highly conserved -sheet portion 164-170 of the hydrophobic domain D2 of FGFR1 (B) interacting with FGF2 (PDB code: 1FQ9) [111]. Dotted spheres represent the hydrophobic groups of Ac-ARPCA-NH2 (N-terminal Ac, Ala1, and Ala5) and of FGFR1 (Leu165, Ala167, and Pro169) involved in the interaction with FGF2 (Tyr24, Phe31, Tyr103, Leu140, and Met142 in atom type).


fication of the acetylated Ac-ARPCA-NH2 pentapeptide as a short FGF2-binding peptide able to interfere with FGF2/ FGFR interaction and to exert a significant FGF2 antagonist activity may provide the basis for the design of novel PTX3derived peptidomimetic FGF2 antagonists.

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ACKNOWLEDGEMENTS [21]

This work was supported by grants from Istituto Superiore di Sanità (Oncotechnological Program), Ministero dell’Istruzione, Università e Ricerca (Centro di Eccellenza per l’Innovazione Diagnostica e Terapeutica, Cofin projects), Associazione Italiana per la Ricerca sul Cancro, Fondazione Berlucchi, NOBEL Project Cariplo; and Fondazione Cariplo (Grant 2008-2264) to MP. PA is supported by a FIRC Fellowship. REFERENCES [2] [3] [4] [5] [6]

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Received: July 14, 2009

Accepted: July 16, 2009

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