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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Selective recognition of fibroblast growth factor-2 by the long pentraxin
PTX3 inhibits angiogenesis
Marco Rusnati, Maura Camozzi, Emanuela Moroni, Barbara Bottazzi, Giuseppe Peri, Stefano Indraccolo,
Alberto Amadori, Alberto Mantovani, and Marco Presta
The long pentraxin PTX3 is a soluble
pattern recognition receptor produced by
monocytes and endothelial cells that
plays a nonredundant role in inflammation. Several pathologic conditions are
characterized by local production of both
PTX3 and the angiogenic fibroblast
growth factor-2 (FGF2). Here, solid-phase
binding assays demonstrated that PTX3
binds with high affinity to FGF2 but not to
a panel of cytokines and growth factors,
including FGF1, FGF4, and FGF8. Accordingly, PTX3 prevented 125I-FGF2 binding
to endothelial cell receptors, leading to
specific inhibition of FGF2-induced proliferation. PTX3 hampered also the motogenic activity exerted by endogenous
FGF2 on a wounded endothelial cell
monolayer. Moreover, PTX3 cDNA transduction in FGF2-transformed endothelial
cells inhibited their autocrine FGF2dependent proliferation and morphogenesis in vitro and their capacity to generate
vascular lesions when injected in nude
mice. Finally, PTX3 suppressed neovascularization triggered by FGF2 in the chick
embryo chorioallantoic membrane with
no effect on physiologic angiogenesis. In
contrast, the short pentraxin C-reactive
protein was a poor FGF2 ligand/antagonist. These results establish the selective
binding of a member of the pentraxin
superfamily to a growth factor. PTX3/
FGF2 interaction may modulate angiogenesis in various physiopathologic conditions driven by inflammation, innate
immunity, and/or neoplastic transformation. (Blood. 2004;104:92-99)
© 2004 by The American Society of Hematology
Introduction
Angiogenesis is the process of generating new capillary blood vessels.
In the adult, angiogenesis occurs under tight regulation in the female
reproductive system and during wound healing. Uncontrolled neovascularization is observed during tumor growth and atherosclerosis. Also,
angiogenesis plays a pivotal role in inflammation.1,2
Fibroblast growth factor-2 (FGF2) is a heparin-binding growth
factor that induces cell proliferation, chemotaxis, and protease
production in cultured endothelial cells (ECs)3 by interacting with
high-affinity tyrosine-kinase receptors (FGFRs) and low-affinity
heparan sulfate (HS) proteoglycans (HSPGs).4 FGF2 induces
angiogenesis in vivo5 and modulates neovascularization during
wound healing, inflammation, atherosclerosis, and tumor growth.6
Accordingly, preclinical studies demonstrate that FGF2 antagonists
inhibit tumor growth and vascularization.7,8 FGF2 is produced by
various tumor and normal cell types, including cells involved in
inflammation and immunity, such as mononuclear phagocytes,9 T
lymphocytes,10 and ECs.11 FGF2 lacks a classical signal peptide for
secretion. Nevertheless, it can be released by producing cells after
cell damage or by way of an alternative pathway for secretion.12
FGF2 production and release is modulated by inflammatory
mediators (eg, interleukin 1 [IL-1]13 and nitric oxide14), hypoxia,9
and cell damage.15 Finally, endogenous FGF2 can play an autocrine
role in ECs following its release and FGFR interaction.16-18
Angiogenesis is controlled by the balance between proangiogenic and antiangiogenic factors.19 Several molecules sequester
FGF2 in the extracellular environment, thus preventing its interaction with EC receptors. Free heparin and HS,4 sialo-gangliosides,20
␣-2 macroglobulin,21 platelet-derived growth factor BB,22 platelet
factor 4,23 perlecan,24 thrombospondin,25 and FGF-binding protein26 bind FGF2 and inhibit its angiogenic activity. Many of these
inhibitors are produced or released locally or systemically during
wound healing, inflammation, atherosclerosis, and tumor growth,
thus underlying the complex tuning of the angiogenesis process
that characterizes these physiopathologic conditions.
Pentraxin 3 (PTX3) is the prototypic member of the long
pentraxin family that includes also apexin,27 XL-PXN1,28 neuronal
pentraxins 1 and 2,29 and Narp.30 Originally cloned from IL-1–
stimulated ECs,31 PTX3 is a 45-kDa glycosylated protein predominantly assembled in 10 to 20 mer multimers that depend on
interchain disulfide bonds.32 The COOH-terminal 203-amino acid
pentraxin domain of PTX3 shares homology with the classic short
pentraxin C-reactive protein (CRP), whereas its NH2-terminal
From the Unit of General Pathology and Immunology, Department of
Biomedical Sciences and Biotechnology, University of Brescia, Brescia, Italy;
Mario Negri Institute, Milan, Italy; the Department of Oncology and Surgical
Sciences, University of Padova, Padova, Italy; and the Institute of General
Pathology, University of Milan, Milan, Italy.
di Sanità (AIDS Project) and Cofin 2003 (M.R.).
Submitted October 7, 2003; accepted January 30, 2004. Prepublished
online as Blood First Edition Paper, March 18, 2004; DOI 10.1182/blood2003-10-3433.
Supported partially by grants from Ministero dell’Istruzione, dell’Università e
della Ricerca (MIUR; Centro di Eccellenza “IDET,” FIRB, Cofin 2002),
Associazione Italiana per la Ricerca sul Cancro (M.P.), and by Istituto Superiore
92
M.R. and M.C. contributed equally to this work.
Reprints: Marco Presta, General Pathology, Department of Biomedical
Sciences and Biotechnology, Viale Europa 11, 25123 Brescia, Italy; e-mail:
[email protected].
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2004 by The American Society of Hematology
BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
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BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
178-amino acid portion does not show any significant homology
with any other known protein.31
Unlike CRP, which is produced by the liver in response to
inflammatory mediators, PTX3 is synthesized locally at the inflammatory site by mononuclear phagocytes and ECs in response to
IL-1␤,31 tumor necrosis factor ␣,33 and bacterial components.34
PTX3 expression increases in mice during inflammation,35,36
whereas PTX3 transgenic mice show improved rates of survival to
endotoxemia and sepsis.37 Also, the levels of circulating PTX3
increase in humans during vasculitides,38 acute myocardial infarction,39 rheumatoid arthritis,40 and systemic inflammatory response
syndrome or septic shock.41 High levels of PTX3 are found
associated to macrophages and ECs in atherosclerotic plaques.42
At the molecular level, PTX3 binds C1q, activating the
complement cascade.32 Also, PTX3 binds histones, suggesting a
role in the opsonization of intracellular residues during necrosis.32
Moreover, PTX3 binds to apoptotic cells regulating their clearance
by dendritic cells.43 In ECs, PTX3 up-regulates tissue-factor
expression, suggesting its action as a regulator of endothelium
during thrombogenesis and ischemic vascular disease.44 Taken
together, these data suggest that PTX3 may serve as a mechanism
of amplification of inflammation and innate immunity. Accordingly, PTX3⫺/⫺ mice show defective resistance against selected
pathogens45 and show an alteration in seizure-induced damage in
the central nervous system.36 Intriguingly, PTX3⫺/⫺ female mice
are also subfertile.46 Thus, available information suggests that
PTX3 is a soluble pattern recognition receptor with unique
nonredundant functions in various physiopathologic conditions.
In an effort to dissect the mechanisms responsible for the
complex functions of PTX3, we investigated its ability to interact
with cytokines and growth factors. Surprisingly, we found that
human PTX3 binds specifically to FGF2. PTX3/FGF2 interaction
neutralizes the capacity of the growth factor to bind FGFRs and
HSPGs in ECs, thus inhibiting its mitogenic activity in vitro and
angiogenic activity in vivo. PTX3 retains its FGF2-antagonist
activity in vitro and in vivo also when overexpressed by ECs. Thus,
PTX3 may contribute to the modulation of angiogenesis during
inflammation, wound healing, atherosclerosis, and neoplasia. This
represents the first demonstration that a member of the pentraxin
superfamily specifically recognizes a growth factor, a finding that
calls for similar studies with related long pentraxins.
PTX3/FGF2 INTERACTION
93
Solid-phase binding assays
Enzyme-linked immunosorbent assay (ELISA) microplates were incubated
for 16 hours at 4°C with 100 ␮L of 100 mM NaHCO3, pH 9.6 (carbonate
buffer) containing different concentrations of PTX3, FGF2, or various
control molecules. Then, wells were washed and incubated for 2 hours at
37°C with 1.0% bovine serum albumin (BSA) in carbonate buffer. For
binding experiments, 200-␮L aliquots of phosphate-buffered saline (PBS)
containing 125I-FGF2 (1.6 nM) were incubated for 2 hours at 37°C onto
PTX3-coated wells in the presence of different competitors. Then, wells
were washed, and bound radioactivity was solubilized by incubating the
wells for 30 minutes at 50°C with 2.0% sodium dodecyl sulfate (SDS) in
H2O, collected, and measured in a liquid scintillation counter. Alternatively,
100-␮L aliquots of PBS containing various concentrations of biotin-labeled
PTX3 (bPTX3) were incubated for 30 minutes at 37°C onto FGF2-coated
wells with or without different competitors. Then, wells were washed, and
the amount of bound bPTX3 was evaluated as described.32
Cell cultures
Fetal bovine aortic endothelial GM 7373 cells48 were obtained from the
N.I.G.M.S. Human Genetic Mutant Cell Repository (Institute for Medical
Research, Camden, NJ) and grown in Eagle minimal essential medium
(MEM) containing 10% fetal calf serum (FCS). Bovine aortic endothelial
(BAE) cells and murine endothelial E1G11 cells (provided by A. Vecchi,
Istituto Mario Negri, Milan, Italy) were grown in Dulbecco minimal
essential medium (DMEM) containing 10% heat-inactivated or 20% FCS,
respectively. Human umbilical vein endothelial (HUVE) cells at passage 3
(Clonetics, Biowhittaker, Walkersville, MA) were grown in complete
EGM-2 medium (Clonetics). Human embryonic kidney (EcoPack2-293)
packaging cells (Clontech, Palo Alto, CA) and Balb/c mouse aortic
endothelial 22106 cells stably transfected with a human FGF2 cDNA
(FGF2-T-MAE cells)49 were grown in DMEM containing 10% FCS plus
500 ␮g/mL G418 (Sigma).
PTX3 cDNA transfection
FGF2-T-MAE cells (3 ⫻ 105 cells/100-mm plates) were incubated for 48
hours with Lipofectamin (Invitrogen, San Diego, CA) and 10 ␮g of the
expression vector PLXSH harboring the 1470-bp human PTX3 cDNA
under the control of the 5⬘ long terminal repeat (LTR) promoter (PLXPTX3).50 Transfected clones were selected with hygromycin B (600
␮g/mL; Boehringer Mannheim GmbH, Mannheim, Germany) and tested
for FGF2 and PTX3 protein expression by ELISA of the cell extract and
supernatant, respectively.
PTX3 retroviral infection
Materials and methods
Chemicals
Human recombinant FGF2 and PTX3 were expressed in Escherichia coli
and Chinese hamster ovary cells, respectively, and purified as described.32,47 Recombinant FGF4 and FGF8 were provided by C. Basilico
(New York University Medical Center, New York, NY) and M. Jalkanen
(Biotie, Turku, Finland), respectively. Neutralizing anti-FGF2 antibodies
were from Upstate Biotech (Lake Placid, NY). Suramin was from FBA
(Bayer AG, Leverkusen, Germany). C1q, CRP, histone IIIS, and cytochrome C were from Sigma (St Louis, MO). 1,2-dioctanoyl-sn-glycerol
(DAG), epidermal growth factor (EGF), 12-O-tetradecanoyl phorbol 13acetate (TPA), and vascular endothelial growth factor (VEGF)165 isoform
were from Calbiochem (La Jolla, CA). FGF1, nerve growth factor, IL-4,
macrophage colony-stimulating factor, monocyte chemotactic protein 1,
IL-8, and lymphotactin were from Peprotech (London, United Kingdom).
Tumor necrosis factor (TNF) was from BASF (Ludwigshafen, Germany).
IL-6 was from Serono (Rome, Italy). IL-10 was from Schering-Plough
(Milan, Italy). IL-1 was obtained from F. Colotta (Dompè, L’Aquila, Italy).
IL-12 was from Genetics Institute (Andover, MA).
The cDNAs encoding for human PTX3 and for the enhanced green
fluorescent protein (EGFP) were obtained from PLX-PTX3 and pEGFP-C1
(Clontech) plasmids, respectively. The 2 fragments were cloned in the
pBABE retroviral vector,51 thus generating pBABE-PTX3 and pBABEEGFP that were used to transfect the EcoPack2-293 packaging cells in the
presence of Lipofectamin. Transduced cells were selected with puromycin
(1 ␮g/mL; Sigma) for 2 weeks. Clones with a viral titer higher than 105
cfu/mL were used for further experimentation. Confluent cultures of
FGF2-T-MAE cells were incubated for 24 hours with the conditioned
medium from pBABE-PTX3 and pBABE-EGFP packaging cells in the
presence of polybrene (8 ␮g/mL; Sigma). Infected cell populations were
selected for 7 days with puromycin and evaluated for FGF2 and PTX3
expression by ELISA. Observation of EGFP-infected cells by epifluorescence microscopy (Axiovert S100 microscope, ⫻10/0.25; Zeiss, Göttingen,
Germany) showed that retroviral infection efficiency was higher than 95%.
125I-FGF2
cell-binding assay
FGF2 was iodinated at a specific radioactivity equal to 800 cpm/fmol as
described.52 GM 7373 cells were incubated at 4°C in serum-free medium
containing 125I-FGF2 (0.55 nM), 0.15% gelatin, 20 mM HEPES (N-2hydroxyethylpiperazine-N⬘-2-ethanesulfonic acid) buffer (pH 7.5), and
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94
BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
RUSNATI et al
increasing concentrations of PTX3. After 2 hours, the amount of 125I-FGF2
bound to low- and high-affinity sites was evaluated as described.53
Cell proliferation assays
Cell proliferation assay on GM 7373 cells was performed as described.54
Briefly, 16 hours after seeding at 75 000 cells/cm2 in 24-well dishes, GM
7373 cells were incubated in fresh medium containing 0.4% FCS with or
without FGF2 (0.55 nM) and increasing concentrations of PTX3 or other
antagonists. After 24 hours, cells were trypsinized and counted in a Burker
chamber. HUVE cells (2500 cells/well in 96-well plates) were incubated for
24 hours in complete EGM-2 medium. After washing, cells were incubated
for another further 72 hours in EGM-2 medium plus 2.0% FCS without
FGF2, VEGF, and heparin. Finally, cells were added with FGF2 in the
presence of PTX3. After 3 days cells were trypsinized and counted in a
Burker chamber. E1G11 cells seeded at 8000 cells/cm2 in 1.0% gelatincoated 48-well plates and incubated for 24 hours in complete DMEM
medium were added with fresh medium containing 0.4% FCS and FGF2 in
the presence of PTX3. After 3 days of incubation, cells were trypsinized and
counted in a Burker chamber.
EC monolayer wound healing assay
Confluent BAE cell cultures on 1.5% gelatin-coated wells were wounded
with a rubber policeman. After extensive washing, wounded monolayers
were incubated at 37°C with DMEM containing 0.1% gelatin in the absence
or in the presence of anti-FGF2 antibodies or PTX3. Microphotographs
were taken under an inverted microscope (Olympus 1 ⫻ 51 microscope
with Camedia C-4040 digital camera, ⫻10/0.25; Olympus Biosystem,
Munich, Germany) to follow the repair process.
Subcutaneous tumor growth assay
Five-week-old nu/nu female mice (Harlan Nossan, S. Pietro al Natisone,
Italy) were injected subcutaneously in the dorsal scapular region with
1 ⫻ 106 parental, EGFP-infected, or PTX3-infected FGF2-T-MAE cells
(8 animals per group). The tumor volume was measured daily with calipers.
When killed, lesions were harvested, and FGF2 and PTX3 levels in the
tumor extracts were measured by ELISA.
Three-dimensional fibrin gel assay
Aggregates of parental or PTX3-transfected FGF2-T-MAE cells, prepared
on agarose-coated plates as described,18 were seeded onto fibrin-coated
48-well plates. Immediately after seeding, 250 ␮L calcium-free medium
containing fibrinogen (2.5 mg/mL) and thrombin (250 mU/mL) were added
to each well and allowed to gel for 5 minutes at 37°C. Then, 500 ␮L culture
medium was added on the top of the gel. In all the 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.18 Formation of
radially growing cell sprouts was observed during the next 1 to 2 days and
evaluated by computerized image analysis.
CAM assay
Fertilized white leghorn chick eggs were incubated at 37°C. At day 8 of
incubation, 1.0 mm3 sterilized gelatin sponges (Gelfoam; Upjohn Company, Kalamazoo, MI) adsorbed with FGF2 alone (0.028 nmoles/embryo)
or in the presence of PTX3 (0.11 nmoles/embryo) dissolved in 5 ␮L PBS
were implanted on the top of growing chorioallantoic membranes (CAMs).55
Sponges containing vehicle alone or PTX3 alone were used as controls. The
angiogenic response was graded at day 12 under a stereomicroscope
(STEMI-SR, ⫻ 2/0.12; Zeiss) on an arbitrary scale of 0 to 4⫹, with 0
representing no response and 4⫹ representing the strongest activity.
signaling polypeptides. To this purpose, a panel of cytokines,
chemokines, and growth factors representative of different classes
of soluble polypeptide mediators were immobilized onto plastic at
200 ng/well and compared with the PTX3 ligand C1q32 for their
capacity to bind bPTX3 (22 nM). As shown in Table 1, FGF2 binds
bPTX3 with high capacity, similarly to C1q. A limited binding is
also exerted by immobilized VEGF, whereas no significant interaction was observed for the other polypeptides tested, including
FGF1, a member of the FGF family structurally and functionally
related to FGF2. Similar results were obtained when plastic was
coated with higher concentrations of the proteins under test (1.0
␮g/well) and when the concentration of bPTX3 was increased to
220 nM (data not shown). Scatchard plot analysis of the binding of
increasing concentrations of bPTX3 (from 6.8 nM to 440 nM) to
immobilized FGF2 showed that PTX3/FGF2 interaction is dose
dependent and saturable (data not shown) with a Kd (dissociation
constant) equal to 30 nM. This value is close to that calculated for
PTX3/C1q interaction under the same experimental conditions.32
To better characterize PTX3/FGF2 interaction, PTX3 was
immobilized to plastic and evaluated for its capacity to bind free
125I-FGF2. As shown in Figure 1A, immobilized PTX3 binds
125I-FGF2 in a dose-dependent manner, and the interaction is
prevented by a monoclonal anti-PTX3 antibody. Under the same
experimental conditions, 125I-FGF2 does not bind to immobilized
BSA, fibronectin, gelatin, or laminin (Figure 1B). Unlabeled native
FGF2, but not heat-denatured FGF2, competes with 125I-FGF2 for
the binding to immobilized PTX3 (Figure 1C). Also, the PTX3
binder histone IIIS,32 but not cytochrome C, competes with
125I-FGF2 for the binding to immobilized PTX3 despite that both
proteins share with FGF2 a similar apparent molecular mass and a
highly positive charge. Specificity of the interaction was confirmed
further when FGF4 and FGF8, 2 other members of the FGF
family,56 were tested for their capacity to compete with 125I-FGF2
for PTX3 binding. FGF4 does not exert any competitive activity,
whereas FGF8 shows only a limited effect (Figure 1C).
Finally, Scatchard plot analysis of the binding of 125I-FGF2 to
immobilized PTX3 (Figure 1D) confirmed that PTX3/FGF2 interaction is a dose-dependent and saturable single component binding
with a Kd value equal to 8.5 ⫾ 7.2 nM, consistent with that
Table 1. Binding of bPTX3 to immobilized cytokines,
chemokines, and growth factors
Immobilized protein
None
PTX3/FGF2 interaction
In an effort to explore new molecular targets of PTX3, we
evaluated its capacity to interact with a variety of extracellular
0.015 ⫾ 0.008
C1q
0.434 ⫾ 0.034
Fibroblast growth factor 1
0.025 ⫾ 0.007
Fibroblast growth factor 2
0.596 ⫾ 0.048
Vascular endothelial growth factor
0.154 ⫾ 0.043
Nerve growth factor
Macrophage colony-stimulating factor
0.036 ⫾ 0.020
⫺0.017 ⫾ 0.007
Tumor necrosis factor
0.001 ⫾ 0.005
IL-1
0.028 ⫾ 0.003
IL-4
0.001 ⫾ 0.002
IL-6
0.002 ⫾ 0.004
IL-8
IL-10
Results
Bound bPTX3
(O.D. 405 nm)
0.016 ⫾ 0.002
⫺0.025 ⫾ 0.008
IL-12
0.004 ⫾ 0.004
Monocyte chemotactic protein 1
0.014 ⫾ 0.005
Lymphotactin
⫺0.015 ⫾ 0.004
ELISA plates were coated with the indicated protein (200 ng/well) and incubated
with bPTX3 (22 nM). Then, the amount of bPTX3 associated to immobilized proteins
was measured. Blank values obtained in the absence of bPTX3 were subtracted from
all the data that are the mean ⫾ SEM of 2 determinations in duplicate.
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BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
PTX3/FGF2 INTERACTION
95
To assess this possibility, PTX3 was evaluated for its capacity to
affect the binding of FGF2 to HSPGs and FGFRs present on EC
surface. As shown in Figure 2E, PTX3 inhibits the binding of
125I-FGF2 to both HSPGs and FGFRs expressed by endothelial GM
7373 cells in a dose-dependent manner.
In conclusion, PTX3 can sequester free FGF2, thus preventing
its binding to EC surface receptors and leading to the inhibition of
its mitogenic activity.
Interaction of CRP with FGF2
The COOH-terminal portion of PTX3 shares high homology with
the entire sequence of the classic short pentraxin CRP. To assess
whether also short pentraxins might interact with FGF2, we
compared unlabeled PTX3 and CRP for the capacity to compete
with bPTX3 for the binding to immobilized FGF2. As shown in
Figure 3, CRP exerted only a partial competition when compared
with unlabeled PTX3. Accordingly, CRP exerts only a limited
inhibition on the mitogenic activity triggered by FGF2 in GM 7373
cells (Figure 3).
Figure 1. Binding of 125I-FGF2 to immobilized PTX3. (A) PTX3-coated wells were
incubated with 125I-FGF2 in the absence (F) or in the presence (E) of anti-PTX3
antibody (5 ␮g/mL). (B) Wells coated with the indicated proteins (all at 440 nM) were
incubated with 125I-FGF2. (C) Wells coated with PTX3 (440 nM) were incubated with
125I-FGF2 in the absence (–) or in the presence of the indicated unlabeled proteins (all
at 111 nM; h.d. indicates heat denatured). (D) Increasing concentrations of 125I-FGF2
were incubated onto wells coated with PTX3 (100 nM). (D, inset) Scatchard plot
regression analysis of the 125I-FGF2 binding data to immobilized PTX3. For all the
experiments, bound radioactivity was measured after 2 hours at 37°C. In panels A-C,
each point is the mean ⫾ SEM of 3 to 4 determinations in duplicate, whereas the data
shown in panel D represent one experiment (similar results were obtained in a
second independent experiment).
PTX3 hampers the autocrine activity of endogenous
FGF2 in ECs
ECs express FGF2 in vitro and in vivo (see Gualandris et al18 and
references therein). Endogenous FGF2 plays an autocrine role in
calculated for the interaction of free bPTX3 with immobilized
FGF2 (as described earlier).
PTX3 inhibits the mitogenic activity of FGF2 in ECs
The capacity of PTX3 to bind FGF2 with high affinity prompted us
to assess its effect on the mitogenic activity exerted by the growth
factor on cultured ECs. PTX3 inhibits FGF2-dependent proliferation of endothelial GM 7373 cells in a dose-dependent manner, and
the effect was reverted by anti-PTX3 antibody but not by irrelevant
antibodies (Figure 2A). In contrast, PTX3 does not inhibit cell
proliferation triggered by serum, DAG, EGF, TPA, or VEGF, in
agreement with its limited capacity to bind this growth factor
(Figure 2B). In keeping with its ability to interact differently with
other members of the FGF family (Figure 1C), PTX3 does not
affect the mitogenic activity of FGF4 and exerted a partial
antagonist effect on that of FGF8 (Figure 2B).
The FGF2 antagonist activity of PTX3 was not restricted to GM
7373 cells. Indeed, PTX3 inhibits also cell proliferation driven by
FGF2 in mouse microvascular endothelial E1G11 cells and in
HUVE cells (Figure 2C).
The time dependency of the FGF2-antagonist activity of PTX3
was investigated. As shown in Figure 2D, 3-hour pretreatment with
PTX3 followed by its removal before FGF2 administration did not
affect the mitogenic activity of the growth factor. In contrast, PTX3
retained its inhibitory capacity when added to the culture medium
within 1 to 3 hours after the beginning of FGF2 treatment. Delayed
administration of PTX3 (ⱖ 6 hours) was instead ineffective.
These data indicate that PTX3 affects early event(s) exerted by
FGF2 in ECs, possibly related to the interaction of FGF2 with its
cell surface receptors.
Figure 2. Effect of PTX3 on FGF2 receptor interaction and mitogenic activity in
endothelial cells. (A) GM 7373 cells were treated with FGF2 and PTX3 in the
absence (F) or in the presence of anti-PTX3 (䡺) or irrelevant (‚) antibodies (both at
99 ␮g/mL). (B) GM 7373 cells were treated with 0.4% FCS with no addition (control),
FGF2 (1.66 nM), 10% FCS, DAG (15 ␮M), TPA (8.0 nM), EGF (0.6 nM), VEGF (0.7
nM), FGF4 (1.66 nM), or FGF8 (1.66 nM) in the absence (f) or in the presence (䡺) of
PTX3 (66 nM). (C) GM 7373 cells, E1G11 cells, or HUVE cells were incubated with
FGF2 (0.55 nM) in the absence (䡺) or in the presence (f) of PTX3 (222 nM). All
mitogens induced a statistically significant increase in the proliferation rate (Student t
test, P ⬍ .05). Each point is the mean ⫾ SEM of 3 to 7 determinations in duplicate.
(D) PTX3 (66 nM) was added to GM 7373 cells at the indicated periods of time before
or after the beginning of FGF2 treatment (T0). When FGF2 was added after PTX3,
cells were washed extensively before addition of the growth factor. Cells were
counted 24 hours after the beginning of FGF2 treatment. Two additional independent
experiments gave similar results. In panels A, C, and D, data are expressed as
percentage of the increase in cell number relative to control cells treated with the
different mitogens in the absence of PTX3. (E) GM 7373 cells were treated with
125I-FGF2 in the presence of PTX3. Then, 125I-FGF2 bound to HSPGs (E) and FGFRs
(F) was evaluated and expressed as percentage of the radioactivity measured in the
absence of PTX3. The arrow points to the inhibition measured in the presence of a
100-fold molar excess of unlabeled FGF2. Each point is the mean ⫾ SEM of 3
determinations in duplicate.
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96
RUSNATI et al
Figure 3. Interaction of short pentraxin CRP with FGF2. Wells coated with FGF2
(270 nM) were incubated with bPTX3 (22 nM) in the absence or in the presence of
increasing concentrations of unlabeled PTX3 (F) or CRP (E). The amount of bPTX3
bound to immobilized FGF2 was then measured and data were expressed as
percentage of binding measured in the absence of any competitor. In parallel, GM
7373 cells were treated with FGF2 in the absence or in the presence of increasing
concentrations of PTX3 (f) or CRP (䡺). Cells were then counted and data were
expressed as percentage of the increase in cell proliferation in respect to cells treated
with FGF2 alone. Each point is the mean ⫾ SEM of 3 determinations in duplicate.
ECs following its release by way of an alternative secretion
pathway or cell damage.12 To assess the capacity of PTX3 to
inhibit also the activity of endogenous FGF2 in ECs, confluent
BAE cell cultures, which express significant levels of FGF2,57
were scrape-wounded, and the effect of PTX3 or neutralizing
anti-FGF2 antibodies on the repair of the wounded monolayer
was followed. In keeping with previous findings,16,17 anti-FGF2
antibodies prevented EC repair. This was similarly suppressed
by PTX3 (Figure 4A).
To confirm these observations, we took advantage of a FGF2overexpressing mouse aortic endothelial cell line (FGF2-T-MAE
Figure 4. Effect of PTX3 on the autocrine activity exerted by endogenous FGF2
in ECs. (A) Wounded BAE cell monolayers were grown in the absence (i) or in the
presence (ii) of neutralizing anti-FGF2 antibodies (200 ␮g/mL) or PTX3 (666 nM) (iii).
Microphotographs (original magnification, ⫻ 10) were taken 16 hours after wounding.
White dotted lines mark the edge of the wound at the beginning of the experiment.
(B) FGF2-T-MAE cells were seeded at 10 000 cells/cm2 in 24-well plates. After 24
hours, the medium was replaced without (ctrl) or with the addition of neutralizing
anti-FGF2 antibody (␣FGF2-Ab) (10 ␮g/mL), suramin (300 ␮M), PTX3 (222 nM), or
CRP (222 nM). After 3 days the cells were counted and data were expressed as
percentage of cell proliferation with respect to untreated controls. Each point is the
mean ⫾ SEM of 4 determinations in duplicate. Student t test, **P ⬍ .01.
BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
Figure 5. Effect of PTX3 overexpression on endothelial FGF2-T-MAE cell
growth in vitro and in vivo. (A) Parental FGF2-T-MAE cells (F), PTX3overexpressing clones B4 (Œ), B8 (‚), B9 (f), and B20 (E), and mock-transfected
clones BVV6 (䉬) and BVV10 (䉫) seeded at 10 000 cells/cm2 in 24-well plates were
grown and counted at the indicated periods of time. Each point is the mean of 2
determinations in duplicate. (Inset) Linear regression of the number of cells
measured at day 8 versus the levels of PTX3 production (ng PTX3/5 ⫻ 106 cells/48
hours) (r ⫽ 0.88; P ⬍ .01). (B) Parental (F), EGFP-infected (f), and PTX3-infected
FGF2-T-MAE cells (E) were injected subcutaneously in nude mice, and the size of
the growing tumors was measured. Each point is the mean ⫾ SD of 8 animals.
*Statistically different from parental plus EGFP-infected FGF2-T-MAE lesions (Student t test, P ⬍ .05).
cells) generated in our laboratory.18,58 These cells are characterized
by a sustained rate of growth in vitro and by the capacity to
generate opportunistic vascular lesions in nude mice as the
consequence of an autocrine loop of stimulation triggered by the
release of the overexpressed growth factor.49 Accordingly, neutralizing anti-FGF2 antibodies or suramin, a well-known FGF2-binder/
antagonist,58 caused a 50% decrease in the rate of growth of
FGF2-T-MAE cells under basal culture conditions (Figure 4B).
Exogenously added PTX3 caused a similar inhibitory effect,
whereas CRP was ineffective (Figure 4B).
Taken together, these data indicate that exogenous PTX3
inhibits the autocrine motogenic and mitogenic activity exerted by
endogenous FGF2 on ECs.
Endogenous PTX3 inhibits EC responsiveness to FGF2
in vitro and in vivo
Because ECs represent a major local source of PTX3 during
inflammation,38 we investigated the FGF2 antagonist capacity of
PTX3 also when produced endogenously by FGF2-activated ECs.
To this purpose, FGF2-T-MAE cells were stably transfected with
an expression vector harboring the full-length human PTX3 cDNA.
Control cells were transfected with the empty vector. Mock and
PTX3 transfectants characterized by levels of FGF2 production
similar to those observed in parental FGF2-T-MAE cells (approximately 100 ng FGF2/␮g protein) were screened for PTX3 secretion
by ELISA. Four PTX3-producing clones, named B4, B8, B9, and
B20 cells, and 2 mock clones, named BVV6 and BVV10, were
selected and characterized further.
B4, B8, B9, and B20 PTX3 transfectants secrete 10.5, 22.0,
21.5, and 19.0 ng PTX3/5 ⫻ 106 cells/48 hours, respectively,
whereas parental cells, BVV6 cells, and BVV10 cells do not
release detectable amounts of PTX3. Mock and PTX3 transfectants
were then compared with parental FGF2-T-MAE cells for their rate
of growth under basal culture conditions. As shown in Figure 5A,
parental cells, BVV6 cells, and BVV10 cells are characterized by a
similar rate of growth. In contrast, PTX3-producing cells showed a
rate of growth significantly slower than that of FGF2-T-MAE cells
and inversely related to the amount of released PTX3 (inset to
Figure 5A). Similar results were observed when PTX3 overexpression (45 ng PTX3/5 ⫻ 106 cells/48 hours) was triggered in
FGF2-T-MAE cells by infection with a retrovirus harboring the
human PTX3 cDNA (data not shown).
In keeping with the in vitro data, vascular lesions originated by the
subcutaneous injection in nude mice of human PTX3-infected FGF2-TMAE cells showed a reduced rate of growth when compared with
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BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
tumors originated by parental or EGFP-infected cells (Figure 5B). All
xenografts expressed significant amounts of FGF2 (6.8 ⫾ 0.3 ng/mg
protein), whereas human PTX3 expression was detectable only in
PTX3-transduced lesions (15.0 ⫾ 2.6 ng/mg protein). No major morphologic differences were observed among the different lesions that retained
the characteristics of hemangioendothelioma-like tumors.49
FGF2-T-MAE cell aggregates have an invasive behavior when
embedded into 3D fibrin gels and form solid sprouts after 1 to 2
days in culture.18 Accordingly, mock-transfected BVV6 cells
formed radially growing sprouts in fibrin gel with an efficiency
similar to that of parental cells. In contrast, sprouting was strongly
inhibited in PTX3-overexpressing B8 and B9 clones (Figure 6).
These data indicate that also endogenously produced PTX3
inhibits the autocrine loop of stimulation exerted by FGF2 in
FGF2-T-MAE cells hampering EC proliferation and morphogenesis in vitro and tumorigenesis in vivo.
PTX3 inhibits the angiogenic activity of FGF2
To assess the capacity of PTX3 to affect FGF2-induced neovascularization in vivo, gelatin sponges adsorbed with FGF2 alone or
added with PTX3 were implanted on the top of 8-day-old chick
embryo CAMs.55 Sponges adsorbed with vehicle or PTX3 alone
were used as controls. As shown in Figure 7, PTX3 caused a
significant inhibition of FGF2-induced angiogenesis, whereas it did
not affect basal physiologic vascularization of the CAM.
Discussion
Here, we demonstrate that PTX3 binds FGF2 with high affinity and
selectivity. PTX3/FGF2 interaction prevents the binding of the
growth factor to FGFRs and HSPGs on EC surface, thus inhibiting
its angiogenic activity in vitro and in vivo and the capacity of
FGF2-transformed ECs to generate vascular lesions in nude mice.
PTX3 binds to FGF2 but it does not interact with a variety of
cytokines, chemokines, and growth factors (Table 1). In particular,
PTX3 does not bind or binds poorly to other members of the FGF
family, including FGF1, FGF4, and FGF8, indicating a high degree
of specificity for PTX3/FGF2 interaction.
X-ray crystallography has identified a “basic pocket” in the
FGF2 molecule that mediates the interaction of the growth factor
with heparin.59 However, a 100-molar excess of heparin does not
Figure 6. Effect of PTX3 overexpression on FGF2-T-MAE cell morphogenesis.
(A) Aggregates of parental FGF2-T-MAE cells (ctrl), PTX3-overexpressing clones B8
and B9, and mock-transfected clone BVV6 were seeded within 3D-fibrin gels. After 24
hours, aggregates were photographed and sprouts quantified by computerized
image analysis. Each point is the mean ⫾ SEM of 4 determinations in duplicate.
Student t test, **P ⬍ .01. (B) Representative images of aggregates of FGF2-T-MAE
(i), B8 (ii), and B9 (iii) cells. Original magnification, ⫻ 40.
PTX3/FGF2 INTERACTION
97
Figure 7. Effect of PTX3 on FGF2-induced neovascularization. (A) Chick embryo
CAMs were implanted with gelatin sponges adsorbed with vehicle, FGF2 (0.028
nmoles/embryo), PTX3 (0.11 nmoles/embryo), or both. At day 12, the angiogenic
response in each egg was graded. Bars represent the mean ⫾ SEM of 10 to 15
embryos per group. (B) Representative CAMs implanted with gelatin sponges
adsorbed with FGF2 alone (i) or FGF2 plus PTX3 (ii). Original magnification, ⫻ 5.
compete with bPTX3 for the binding to immobilized FGF2 (data
not shown), ruling out the possibility that the basic domain of FGF2
is involved in PTX3 interaction. In keeping with this hypothesis is
the overmentioned incapacity of other heparin-binding members of
the FGF family and chemokines to bind PTX3. Also, the observation that PTX3 does not recognize heat-denatured FGF2 or the
cationic cytochrome C indicates that the overall basic charge of
FGF2 is not sufficient for the interaction that requires a correct 3D
conformation of the growth factor. These data, together with the
incapacity of PTX3 to bind heparin (M.R., unpublished observations, December 14, 2002), suggest that PTX3 prevents the
interaction of FGF2 with cell-associated HSPGs by steric hindrance.
The short pentraxin CRP is an inefficient FGF2 binder/
antagonist despite its sequence homology with the 203-amino acid
COOH-terminal portion of PTX3.32 Similar to CRP, PTX3 multimers bind C1q by way of the pentraxin domain.32 However,
differences in binding properties of CRP and PTX3 have been
described.32 The capacity of PTX3, but not of CRP, to bind FGF2
suggests that the 178-amino acid NH2-terminal portion of PTX3,
absent in short pentraxins, mediates the binding to FGF2. Further
experiments are required to map the domains in PTX3 and FGF2
molecules responsible for the interaction.
PTX3 inhibits the mitogenic activity exerted by FGF2 in
bovine, murine, and human ECs in vitro. This appears to be due to
the capacity of PTX3 to prevent FGF2 binding to FGFRs rather
than to a direct action of PTX3 on ECs. Indeed, PTX3 pretreatment
does not affect EC responsiveness to FGF2. Also, PTX3 does not
inhibit EC proliferation triggered by various mitogens. It must be
pointed out that PTX3 interacts with and inhibits the biologic
activity of FGF2 at doses comparable to those measured in the
blood of patients affected by inflammatory diseases.38,39 Moreover,
the local concentration of PTX3 at the site of inflammation can be
significantly higher than that measured in the bloodstream, supporting the possibility that PTX3/FGF2 interaction may occur and be
biologically relevant in vivo. In keeping with this hypothesis is the
capacity of retroviral vector–transduced PTX3 to inhibit the growth
of vascular lesions caused by subcutaneous injection of FGF2activated endothelial FGF2-T-MAE cells in nude mice.
PTX3 is produced by macrophages,34 fibroblasts,33 myoblasts,60
microglia,35 and ECs,31 indicating that it may exert paracrine and
autocrine functions on endothelium. Similarly, various stimuli,
including inflammatory mediators IL-1 and nitric oxide14,61 induce
FGF2 expression in ECs that undergo an autocrine loop of
stimulation. Thus, ECs and other cell types can express both PTX3
and FGF2. The capacity of PTX3 to inhibit the repair driven by
endogenous FGF2 in wounded BAE cell monolayers, together with
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98
BLOOD, 1 JULY 2004 䡠 VOLUME 104, NUMBER 1
RUSNATI et al
the inhibitory effect exerted by PTX3 cDNA overexpression in
FGF2-T-MAE cells, indicates that both exogenous and endogenous
PTX3 can suppress the autocrine loop of stimulation triggered by
FGF2 in ECs, leading to a significant inhibition of cell migration,
proliferation, morphogenesis, and tumor growth. Thus, PTX3
produced by inflammatory cells or by ECs themselves may affect
the autocrine and paracrine activity exerted by FGF2 on endothelium in vitro and in vivo. This should allow a fine tuning of
neovascularization by way of the production of both angiogenesis
inhibitors and stimulators.
FGF2 is a pleiotropic growth factor that stimulates various cell
types of endodermal and mesodermal origin.62 Therefore, the role
exerted by FGF2 in various physiopathologic conditions may not
be limited to its angiogenic activity. For instance, FGF2 stimulates
the migration and proliferation of fibroblasts during wound healing
and of smooth muscle cells during atherosclerosis10,63 and restenosis.64 Also, it may favor neuronal cell survival and glia cell
proliferation in the injured central nervous system.65 In all these
conditions, the concomitant production of PTX336,42 may modulate
the activity exerted by FGF2 on these cells. Indeed, preliminary
experiments showed the capacity of PTX3 to inhibit FGF2dependent proliferation in smooth muscle cells (M.C., unpublished
data, March 2003).
In conclusion, we demonstrate for the first time that PTX3, a
member of the pentraxin superfamily released locally by inflammatory cells, binds the angiogenic growth factor FGF2 with high
affinity and specificity, acting as a natural angiogenesis inhibitor.
Thus, PTX3 may play an important role in modulating the
cross-talk between inflammatory cells and endothelium in various
physiopathologic settings.
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2004 104: 92-99
doi:10.1182/blood-2003-10-3433 originally published online
March 18, 2004
Selective recognition of fibroblast growth factor-2 by the long pentraxin
PTX3 inhibits angiogenesis
Marco Rusnati, Maura Camozzi, Emanuela Moroni, Barbara Bottazzi, Giuseppe Peri, Stefano
Indraccolo, Alberto Amadori, Alberto Mantovani and Marco Presta
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