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Blood First Edition
prepublished
online
March
18, 2004;
DOI 10.1182/blood-2003-10-3433
Selective recognition of fibroblast growth factor-2 by the long
pentraxin PTX3 inhibits angiogenesis
Running title: PTX3/FGF2 interaction.
Marco Rusnati1, Maura Camozzi1, Emanuela Moroni, Barbara Bottazzi2,
Giuseppe Peri2, Stefano Indraccolo3, Alberto Amadori3, Alberto Mantovani2,4,
and Marco Presta5
Unit of General Pathology and Immunology, Department of Biomedical Sciences and
Biotechnology, University of Brescia, 25123 Brescia, Italy. 2Mario Negri Institute, 20157 Milan,
Italy. 3Department of Oncology and Surgical sciences, University of Padova, 35128, Padova, Italy,
4
Institute of General Pathology, University of Milan, 20133 Milan, Italy.
1
The two authors contributed equally to this work
This work was partially supported by grants from MIUR (Centro di Eccellenza “IDET”, FIRB,
Cofin 2002), Associazione Italiana per la Ricerca sul Cancro to MP and by Istituto Superiore di
Sanità (AIDS Project) and Cofin 2003 to MR.
5
Corresponding author: Prof. Marco Presta,
General Pathology, Dept. Biomedical Sciences and Biotechnology Viale Europa 11 25123 Brescia,
Italy
Tel: ++39-30-3717311
Fax: ++ 39-30-3701157
E-mail: [email protected]
Total words in the text: 4767
Scientific category: Hemostasis, Thrombosis, and Vascular Biology
1
Copyright (c) 2004 American Society of Hematology
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Abstract
The long-pentraxin PTX3 is a soluble pattern recognition receptor produced by monocytes
and endothelial cells that plays a non-redundant role in inflammation. Several pathological
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
125
I-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 FGF2-dependent 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 physiological angiogenesis. In contrast, the shortpentraxin C-reactive protein was a poor FGF2 ligand/antagonist. These results report for the first
time the selective binding of a member of the pentraxin superfamily to a growth factor. PTX3/FGF2
interaction may modulate angiogenesis in various physiopathological conditions driven by
inflammation, innate immunity, and/or neoplastic transformation.
2
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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 vivo
5
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 like 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 via an alternative pathway for secretion 12. FGF2 production and release is
modulated by inflammatory mediators [e.g. IL 1 13 and nitric oxide 14], 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 pro- and anti-angiogenic 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 protein 26
bind FGF2 and inhibit its angiogenic activity. Many of these inhibitors are produced/released
locally and/or systemically during wound healing, inflammation, atherosclerosis, and tumor growth,
thus underlying the complex tuning of the angiogenesis process that characterizes these
physiopathological conditions.
Pentraxin 3 (PTX3) is the prototypic member of the long-pentraxin family that includes also
apexin 27, XL-PXN1
stimulated ECs
31
28
, neuronal pentraxins 1 and 2
29
, and Narp
30
. Originally cloned from IL-1-
, PTX3 is a 45 kD glycosylated protein predominantly assembled in 10-20 mer
multimers that depend upon 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 178 amino acid portion does not show any significant homology
with any other known protein 31.
3
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Unlike CRP, that 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 survival to endotoxemia and sepsis 37. Also, the levels of circulating PTX3 increase
in humans during vasculitides
38
, acute myocardial infarction
systemic inflammatory response syndrome/septic shock
41
39
, rheumatoid arthritis
40
, and
. 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
Moreover, PTX3 binds to apoptotic cells regulating their clearance by dendritic cells
43
32
.
. In ECs,
PTX3 upregulates tissue factor expression, suggesting its action as a regulator of endothelium
during thrombogenesis and ischemic vascular disease 44. Taken together, the data suggest that PTX3
may serve as a mechanism of amplification of inflammation and innate immunity. Accordingly,
PTX3-/- mice show defective resistance against selected pathogens
45
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 non-redundant functions in various physiopathological 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.
4
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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 Inc., Turku, Finland), respectively. Neutralizing anti-FGF2 antibodies were from
Upstate Biotech Inc. (NY, USA). Suramin was from FBA (Bayer AG, 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 13-acetate (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, UK). 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).
Solid phase binding assays
ELISA microplates were incubated for 16 h at 4°C with 100 µl 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 h at 37°C with 1.0% BSA in carbonate
buffer. For binding experiments, 200 µl aliquots of PBS containing
125
I-FGF2 (1.6 nM) were
incubated for 2 h 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 min at
50°C with 2.0% SDS in H2O, collected, and measured in a liquid scintillation counter.
Alternatively, 100 µl aliquots of PBS containing various concentrations of biotin-labeled PTX3
(bPTX3) were incubated for 30 min 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
5
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Fetal bovine aortic endothelial GM 7373 cells
48
were obtained from the N.I.G.M.S. Human
Genetic Mutant Cell Repository (Institute for Medical Research, Camden, NJ) and grown in Eagle's
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
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, CA, USA) 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
µg/ml of G418 (Sigma).
PTX3 cDNA transfection
FGF2-T-MAE cells (3x105 cells/100 mm plates) were incubated for 48 h with Lipofectamin
(Invitrogen, San Diego, CA) and 10 µg of the expression vector PLXSH harboring the 1470 bp
human PTX3 cDNA under the control of the 5' LTR promoter (PLX-PTX3) 50. Transfected clones
were selected with hygromycin B (600 µg/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
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 two
fragments were cloned in the pBABE retroviral vector
51
thus generating pBABE-PTX3 and
pBABE-EGFP that were used to transfect the EcoPack2-293 packaging cells in the presence of
Lipofectamin. Transduced cells were selected with puromycin (1 µg/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 h with the conditioned medium from pBABEPTX3 and pBABE-EGFP packaging cells in the presence of polybrene (8 µg/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 showed
that retroviral infection efficiency was higher than 95%.
125
I-FGF2 cell-binding assay
6
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FGF2 was iodinated at a specific radioactivity equal to 800 cpm/fmol as described
7373 cells were incubated at 4°C in serum-free medium containing
125
52
. GM
I-FGF2 (0.55 nM), 0.15%
gelatin, 20 mM Hepes buffer (pH 7.5), and increasing concentrations of PTX3. After 2 h, 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 h 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 of
other antagonists. After 24 h, cells were trypsinized and counted in a Burker chamber. HUVE cells
(2,500 cells/well in 96 well plates) were incubated for 24 h in complete EGM-2 medium. After
washing, cells were incubated for further 72 h 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 8,000 cells/cm2 in
1.0% gelatin-coated 48 well plates and incubated for 24 h 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 of PTX3.
Microphotograps were taken under an inverted microscope 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
s.c. in the dorsal scapular region with 1x106 parental, EGFP-infected, or PTX3-infected FGF2-TMAE cells (8 animals per group). The tumor volum was measured daily with calipers. At sacrifice,
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 µl of calcium-free medium containing fibrinogen (2.5 mg/ml) and thrombin (250 mU/ml) were
7
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added to each well and allowed to gel for 5 min at 37°C. Then, 500 µl of culture medium were
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 1-2 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, USA) adsorbed with FGF2
alone (0.028 nmoles/embryo) or in the presence of PTX3 (0.11 nmoles/embryo) dissolved in 5 µl of
PBS were implanted on the top of growing CAMs
55
. Sponges containing vehicle alone or PTX3
alone were used as controls. The angiogenic response was graded at day 12 under a
stereomicroscope on an arbitrary scale of 0-4+, with 0 representing no response and 4+ representing
the strongest activity.
8
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Results
PTX3/FGF2 interaction
In an effort to explore new molecular targets of PTX3, we evaluated its capacity to interact
with a variety of extracellular 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 to the PTX3 ligand C1q
32
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 vascular endothelial growth
factor (VEGF), while 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 µg/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 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
125
I-FGF2 in a dose-dependent manner and the interaction is prevented by a monoclonal anti-PTX3
antibody. Under the same experimental conditions,
125
I-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
125
I-FGF2 for the binding to immobilized
PTX3 despite the fact 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,
two other members of the FGF family 56, were tested for their capacity to compete with
125
I-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 calculated for the interaction of free
bPTX3 with immobilized FGF2 (see above).
9
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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 (see 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 h 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 1-3 h after the beginning of FGF2 treatment. Delayed
administration of PTX3 ( 6 h) 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.
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
125
I-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 if 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
to unlabeled PTX3. Accordingly, CRP exerts only a limited inhibition on the mitogenic activity
triggered by FGF2 in GM 7373 cells (Figure 3).
10
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PTX3 hampers the autocrine activity of endogenous FGF2 in ECs
ECs express FGF2 in vitro and in vivo (see
18
and references therein). Endogenous FGF2
plays an autocrine role in ECs following its release via 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, that 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 (Fig. 4A).
To confirm these observations, we took advantage of a FGF2-overexpressing mouse aortic
endothelial cell line (FGF2-T-MAE 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 as
similar inhibitory effect whereas CRP was ineffective (Figure 4B).
Taken together the 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
Since 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/µg of protein) were screened for PTX3 secretion
by ELISA. Four PTX3-producing clones, named B4, B8, B9, and B20 cells, and two 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 of PTX3/5x106
cells/48 h, respectively, whereas parental cells, BVV6, and BVV10 cells do not release detectable
amounts of PTX3. Mock and PTX3 transfectants were then compared to 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, PTX3producing cells showed a rate of growth significantly slower than that of FGF2-T-MAE cells and
11
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inversely related to the amount of released PTX3 (inset to Figure 5A). Similar results were observed
when PTX3-overexpression (45 ng of PTX3/5x106 cells/48 h) 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 s.c. injection in nude mice
of human PTX3-infected FGF2-T-MAE cells showed a reduced rate of growth when compared to
tumors originated by parental or EGFP-infected cells (Figure 5B). All xenografts expressed
significant amounts of FGF2 (6.8 ± 0.3 ng/mg of protein) whereas human PTX3 expression was
detectable only in PTX3-transduced lesions (15.0 ± 2.6 ng/mg of 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 1-2 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 Fig 7, PTX3 caused a significant inhibition of FGF2-induced angiogenesis whereas it did
not affect basal physiological 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 (Tab. 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.
12
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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
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 over mentioned 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 nor 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. Rusnati, unpublished observations), suggest that
PTX3 prevents the interaction of FGF2 to 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 via 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 biological 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 blood stream, 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 s.c. injection
of FGF2-activated 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 oxide
14,61
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 the inhibitory effect exerted by PTX3 cDNA
13
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overexpression in FGF2-T-MAE cells indicate 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
via 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 physiopathological 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 atherosclerosis
10,63
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 PTX3 36,42
may modulate the activity exerted by FGF2 on these cells. Indeed, preliminary experiments showed
the capacity of PTX3 to inhibit FGF2-dependent proliferation in smooth muscle cells (M. Camozzi,
unpublished data).
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
physiopathological settings.
14
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18
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Tables
Tab. 1: Binding of bPTX3 to immobilized cytokines, chemokines and growth factors.
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 ± S.E.M. of 2
determinations in duplicate.
Immobilized protein
Bound bPTX3
(O.D. 405 nm)
none
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
0.036 ± 0.020
Macrophage colony stimulating factor
-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
0.016 ± 0.002
IL-10
-0.025 ± 0.008
IL-12
0.004 ± 0.004
Monocyte chemotactic protein 1
0.014 ± 0.005
Lymphotactin
-0.015 ± 0.004
19
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Figure legends
Fig. 1. Binding of 125I-FGF2 to immobilized PTX3. (A) PTX3-coated wells were incubated
with
125
I-FGF2 in the absence ( ) or in the presence ( ) of anti-PTX3 antibody (5 µg/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., heat denatured). (D) Increasing
concentrations of
125
I-FGF2 were incubated onto wells coated with PTX3 (100 nM). For all the
experiments, bound radioactivity was measured after 2 h at 37°C. In panels A-C, each point is the
mean ± S.E.M of 3-4 determinations in duplicate whereas the data shown in panel D represent one
experiment (similar results were obtained in a second independent experiment). Inset to panel D:
Scatchard plot regression analysis of the 125I-FGF2 binding data to immobilized PTX3.
Fig. 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 ( ) or in the presence of
anti-PTX3 ( ) or irrelevant ( ) antibodies (both at 99 µg/ml). (B) GM 7373 cells were treated
with 0.4% FCS with no addition (control), FGF2 (1.66 nM), 10% FCS, DAG (15 µM), TPA (8.0
nM), EGF (0.6 nM), VEGF (0.7 nM), FGF4 (1.66 nM), or FGF8 (1.66 nM) in the absence (black
bars) or in the presence (grey bars) of PTX3 (66 nM). (C) GM 7373 cells, E1G11 cells, or HUVE
cells were incubated with FGF2 (0,55 nM) in the absence (open bars) or in the presence (black bars)
of PTX3 (222 nM). All mitogens induced a statistically significant increase in the proliferation rate
(Student’s t test, P< 0.05). Each point is the mean ± S.E.M. of 3-7 determinations in duplicate. (D)
PTX3 (66 nM) was added to GM 7373 cells at the indicated periods of time before/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 h after the beginning of
FGF2 treatment. Two additional independent experiments gave similar results. In panels A, C, and
D, data are expressed as percent of the increase in cell number in respect 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 ( ) and FGFRs ( ) was evaluated and
expressed as percent of the radioactivity measured in the absence of PTX3. Arrow points to the
inhibition measured in the presence of a 100-fold molar excess of unlabelled FGF2. Each point is
the mean ± S.E.M. of 3 determinations in duplicate.
20
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Fig. 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 (closed circles) or CRP (open circles). Then, the amount of bPTX3 bound to
immobilized FGF2 was measured and data were expressed as percent 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 (closed squares) or CRP (open squares). Then,
cells were counted and data were expressed as percent of the increase in cell proliferation in respect
to cells treated with FGF2 alone. Each point is the mean ± S.E.M. of 3 determinations in duplicate.
Fig. 4. Effect of PTX3 on the autocrine activity exerted by endogenous FGF2 in ECs. (A)
Wounded BAE cell monolayers were grown in the absence (a) or in the presence of neutralizing
anti-FGF2 antibodies (200 µg/ml) (b) or PTX3 (666 nM) (c). Microphotographs (10 x
magnification) were taken 16 h 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 h, the medium was replaced without (ctrl) or with the addition of neutralizing antiFGF2 antibody ( FGF2-Ab) (10 µg/ml), suramin (300 µM), PTX3 (222 nM), or CRP (222 nM).
After 3 days cells were counted and data were expressed as percent of cell proliferation in respect to
untreated controls. Each point is the mean ± S.E.M. of 4 determinations in duplicate. Student’s t
test: **, P< 0.01.
Fig. 5. Effect of PTX3 overexpression on endothelial FGF2-T-MAE cell growth in vitro and in
vivo. (A) Parental FGF2-T-MAE cells ( ), PTX3-overexpressing clones B4 ( ), B8 ( ), B9 ( ),
and B20 ( ), and mock transfected clones BVV6 ( ) and BVV10 ( ) 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 of PTX3/5x106 cells/48 h) (r = 0,88; p < 0.01). (B)
Parental ( ), EGFP-infected ( ), and PTX3-infected FGF2-T-MAE cells ( ) were injected s.c. in
nude mice and the size of the growing tumors was measured. Each point is the mean + S.D. of 8
animals. *, statistically different from parental plus EGFP-infected FGF2-T-MAE lesions (Student’s
t test, p<0.05).
Fig. 6. Effect of PTX3 overexpression on FGF2-T-MAE cells 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 h, aggregates were photographed and
21
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sprouts quantified by computerized image analysis. Each point is the mean ± S.E.M. of 4
determinations in duplicate. Student’s t-test: **, P< 0.01. (B) Representative images of aggregates
of FGF2-T-MAE (a), B8 (b) and B9 (c) cells. Original magnification: 40x.
Fig. 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 ± S.E.M. of 10-15 embryos per group. (B)Representative CAM s implanted with
gelatin sponges adsorbed with FGF2 alone (a) or FGF2 plus PTX3 (b). Original magnification: 5x.
22
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A
3
3
2
2
1
1
20
40
240
PT
X3
0
immobilized PTX3 (nM)
5
12
C
BS
fib
A
ro
ne
ct
in
ge
la
tin
vi
tro
ne
ct
in
0
0
6
B
4
D
9
4
125
I-FGF2 bound (pM)
0
2
bound/free
6
3
3
1
0
-
0
F
h. GF
d.
2
FG
F2
FG
F4
FG
cy
F8
to
ch
ro
m
e
hi C
st
on
e
125
3
I-FGF2 bound (cpm x10 )
4
0
2,8
150
300
0,2
0,1
5,6
8,4
11,2
125
I-FGF2 (nM)
M. Rusnati et al.
PTX3/FGF2 interaction - Fig. 1
23
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
100
16
A
B
12
50
10
25
4
8
10
100
PTX3 (nM)
0
C 100
75
75
50
50
25
25
0
0
D
E
100
75
50
10
00
10
0
0
100
T0
+
1
+ h
3h
+
6
+ h
12
h
as
h
h/
w
-3
G
M
73
73
E1
G
11
HU
VE
25
I-FGF2 bound (% of control)
100
DA
G
TP
A
EG
F
VE
G
F
FG
F4
FG
F8
0
co
nt
ro
l
FG
F2
FC
S
0
125
mitogenic activity (% of control)
2
cells/cm (x10 )
14
75
PTX3 (nM)
M. Rusnati et al.
PTX3/FGF2 interaction - Fig. 2
24
100
100
75
75
50
50
25
25
0
0
22
66
220
0
FGF2 mitogenic activity (% of control)
bPTX3 bound (% of control)
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
PTX3, CRP (nM)
M. Rusnati et al.
PTX3/FGF2 interaction - Fig. 3
25
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
100
a
75
** ** **
50
b
c
25
b
3
in
rl
ct 2-A am
TX
P
r
F
su
FG
P
CR
cell proliferation (% of control)
B
A
0
M. Rusnati et al.
PTX3/FGF2 interaction - Fig. 4
26
4
5
( x10 )
8
6
5
8
( x10 )
10
A
cell number
cell number
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
0
0
5
10 15
[PTX3]
4
20
2
0
0
(mm3)
tumor volume
600
2
4
days
6
8
B
400
*
200
0
*
* 9 * 10 11
01 8
12
13
14
days
M. Rusnati et al.
PTX3/FGF2 interaction - Fig. 5
27
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
A
sprouting activity
(relative units)
1,5
1,0
0,5
**
**
B8
B9
0,0
ctrl
BVV6
B
a
b
c
M. Rusnati et al.
PTX3/FGF2 interaction - Fig. 6
28
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
FGF2
B
PTX3
-
-
+
-
+
+
A
a
+
0
1
2
3
angiogenesis score
4
b
M. Rusnati et al.
PTX3/FGF2 interaction - Fig. 7
29
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
Prepublished online March 18, 2004;
doi:10.1182/blood-2003-10-3433
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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