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. Author manuscript; available in PMC: 2013 Jan 22.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2012 Jan 19;32(3):696–703. doi: 10.1161/ATVBAHA.111.243998

Long Pentraxin 3/TSG-6 Interaction: a Biological Rheostat for Fibroblast Growth Factor 2-Mediated Angiogenesis

Daria Leali 1,*, Antonio Inforzato 1,*, Roberto Ronca 1, Roberta Bianchi 1, Mirella Belleri 1, Daniela Coltrini 1, Emanuela Di Salle 1, Marina Sironi 1, Giuseppe Danilo Norata 1, Barbara Bottazzi 1, Cecilia Garlanda 1, Anthony J Day 1, Marco Presta 1
PMCID: PMC3551298  EMSID: EMS50901  PMID: 22267482

Abstract

Objective

Angiogenesis is regulated by the balance between pro- and anti-angiogenic factors and by extracellular matrix protein interactions. Fibroblast growth factor-2 (FGF2) is a major pro-angiogenic inducer inhibited by the interaction with the soluble pattern recognition receptor long pentraxin 3 (PTX3). PTX3 is locally co-expressed with its ligand TSG-6, a secreted glycoprotein that co-operates with PTX3 in extracellular matrix assembly. Here, we characterized the effect of TSG-6 on PTX3/FGF2 interaction and FGF2-mediated angiogenesis.

Methods and Results

Solid phase binding and surface plasmon resonance assays show that TSG-6 and FGF2 bind the PTX3 N-terminal domain with similar affinity. Accordingly, TSG-6 prevents FGF2/PTX3 interaction and suppresses the inhibition exerted by PTX3 on heparan sulfate proteoglycan/FGF2/FGF receptor complex formation and on FGF2-dependent angiogenesis in vitro and in vivo. Also, endogenous PTX3 exerts an inhibitory effect on vascularization induced by FGF2 in a murine s.c. Matrigel plug assay, the inhibition being abolished in Ptx3-null mice or by TSG-6 treatment in wild-type animals.

Conclusions

TSG-6 reverts the inhibitory effects exerted by PTX3 on FGF2-mediated angiogenesis through competition of FGF2/PTX3 interaction. This may provide a novel mechanism to control angiogenesis in those pathological settings characterized by the co-expression of TSG-6 and PTX3, in which the relative levels of these proteins may fine-tune the angiogenic activity of FGF2.

Keywords: angiogenesis, endothelium, FGF2, pentraxin, TSG-6

Angiogenesis, the process of new blood vessel formation from pre-existing ones, plays a key role in various physiological and pathological conditions, including wound healing, inflammation and cancer 1. The local, uncontrolled release of angiogenic growth factors and/or changes in the production of natural angiogenic inhibitors lead to disturbance of the angiogenic balance 2, which is responsible for the uncontrolled neovascularization that takes place during tumor growth and angiogenesis-dependent diseases 3.

Fibroblast Growth Factor 2 (FGF2) is a major heparin-binding angiogenic inducer 4. Growing evidence suggests that there is a tight cross talk between inflammatory and angiogenic responses during FGF2-mediated neovascularization (reviewed in 4). Indeed, elevated levels of FGF2 have been implicated in the pathogenesis of several diseases characterized by a deregulated angiogenic/inflammatory response, including cancer 4. FGF2 exerts its angiogenic activity by interacting with tyrosine-kinase FGF receptors (FGFRs) 5 and heparan sulfate proteoglycans (HSPGs) on the surface of endothelial cells (EC) and within the extracellular matrix (ECM) 6, 7, leading to the formation of productive HSPG/FGF2/FGFR ternary complexes that provide pro-angiogenic signals 8. FGF2-dependent angiogenesis is further modulated by a complex network of interactions involving serum and ECM proteins and their degradation products 9.

The soluble pattern recognition receptor pentraxin 3 (PTX3), also called TSG-14 10, is the prototypic member of the long pentraxin family 11. The mature PTX3 protein is composed of eight identical subunits held together by a disulfide bond network, where each protomer is comprised of a C-terminal pentraxin domain (as found in the short pentraxins) and a unique N-terminal region 12, 13. PTX3 is an ECM-associated protein produced at sites of inflammation by monocytes, ECs and smooth muscle cells (SMC) in response to inflammatory cytokines and bacterial components 10, 11, 14, 15, where all of these cell types are also major sources of FGF2 in vivo. PTX3 is believed to be an inflammatory mediator with unique and non-redundant functional roles at the crossroads between innate immunity (e.g. mediating complement activation and providing protection against opportunistic pathogens), fertility and angiogenesis 11, 16. This broad spectrum of biological activity is likely due to the structural complexity of the octameric PTX3 protein, shown recently to correspond to an elongated and asymetric molecule composed of two differently sized globular lobes connected by a short stalk 13, in which the N- and C-terminal regions of PTX3 mediate the binding to multiple ligands 13, 17, 18. In this regard, PTX3 binds FGF2 with high affinity and specificity 19 via its N-terminal region 13, 20. Recent work indicates that each PTX3 octamer can bind to two FGF2 molecules where these binding sites are composed of tetrameric assemblies of the N-terminal domain 13. Importantly, PTX3 inhibits FGF2-dependent EC proliferation in vitro and angiogenesis in vivo 19, 21, 22. Also, PTX3 inhibits FGF2-dependent SMC activation and intimal thickening after arterial injury 23. Thus, PTX3 may contribute to the modulation of FGF2 activity in different pathological settings characterized by the co-expression of the two proteins, such as inflammation, atherosclerosis, and neoplasia (see 19, 23 for further discussion).

TSG-6, the secreted product of tumor necrosis factor-stimulated gene-6 (also known as TNFAIP6), is an ~35-kDa protein, comprised mainly of a Link module 24 and a CUB_C domain 25, that is expressed by a wide variety of cell types, including leukocytes, SMCs and ECs in response to inflammatory stimuli 26-30. For example, TSG-6 is expressed by vascular SMCs following blood vessel injury and mechanical strain 31, 32 and has been detected at sites of neovascularization in the synovium of patients with rheumatoid arthritis 33. TSG-6 has been implicated in ECM assembly and remodeling where it binds to a wide range of ECM components 29, 30, including the glycosaminoglycan hyaluronan (HA) 24, 34, the heavy chains (HC) of inter-α-inhibitor (IαI) 35, 36, and PTX3 37. Interestingly, TSG-6, HA, IαI and PTX3 all co-operate in the formation of an ECM around the pre-ovulatory oocyte 35, 37, 38, where the production of this matrix is required for successful ovulation and fertilization in vivo, and the interaction of TSG-6 with PTX3 may contribute directly to matrix stabilisation via the formation of PTX3/TSG-6/HA complexes 37. Notably, the coordinated expression of TSG-6 and PTX3 by leukocytes (in inflammatory infiltrates) and ECs has been recently described in inflamed tissues 26. However, the effect of TSG-6 on the interaction of PTX3 with FGF2 and its anti-angiogenic activity has not yet been investigated.

In this study we demonstrate that TSG-6 reverts the inhibitory effects exerted by PTX3 on FGF2-mediated angiogenesis through competition of the FGF2/PTX3 interaction, pointing to a novel mechanism of modulation of the angiogenic process where the relative levels of TSG-6 and PTX3 dictate the biological activity of FGF2.

Methods

The detailed descriptions of the methods that were used in this study are provided in the supplemental materials (available online at http://atvb.ahajournals.org).

Solid phase binding assays

96 well microtiter plates were coated with PTX3, NtermPTX3 or CtermPTX3, TSG-6, Link_TSG6 or CUB_C_TSG6 and incubated with the proteins under test for 1 hour at 37 °C. Bound proteins were detected using the corresponding primary antibody. In competition experiments, bound biotinylated PTX3 (bPTX3) was revealed by incubation with alkaline phosphatase-conjugated streptavidin and absorbance was read at 405 nm.

Surface plasmon resonance

A BIAcore X system (BIAcore Inc., Piscataway, NJ) was used to analyze the binding of FGF2, wild-type and TSG-6 mutants, Link_TSG6 and CUB_C_TSG6 to PTX3 immobilized on CM4 sensor chips 20.

Cross-linking assay

FGF2 (11 pmoles) was incubated for 1 hour at room temperature with PTX3 (55 pmoles) in a 30 μL volume of PBS containing 1.25 mmol/L bis[sulfosuccinimidyl]suberate in the absence or presence of TSG-6 (110 pmoles). Reaction products were separated by SDS-PAGE and revealed by Western blotting with either anti-FGF2 or anti-PTX3 antibodies.

FGF2-mediated cell-cell adhesion assay

FGFR1-overexpressing, HSPG-deficient chinese hamster ovary cells (A745 CHO flg-1A; 50,000 cells/cm2) were incubated on glutaraldehyde-fixed wild type CHO-K1 cell monolayers with or without 1.66 nmol/L of FGF2 in the absence or presence of PTX3 or NtermPTX3 (220 nmol/L) and increasing doses of TSG-6. After 2 hours at 37 °C, A745 CHO flg-1A cells bound to the CHO-K1 cell monolayers were counted 39.

EC proliferation assay

Subconfluent cultures of ECs were incubated in medium containing 0.4% (v/v) FCS plus FGF2 (0.55 nmol/L) in the absence or presence of PTX3 (220 nmol/L) and increasing doses of wildtype or TSG-6 mutants or Link_TSG6. Following 24 hours incubation, cells were trypsinized and counted 20.

Chicken embryo chorioallantoic membrane (CAM) assay

Alginate beads containing vehicle or FGF2 (8 pmoles) with or without PTX3 (33 pmoles) and TSG-6 (83 pmoles) were placed on top of the CAM at day 11 of incubation. After 72 hours, blood vessels converging toward the implant were counted under a stereomicroscope by two observers in a double-blind fashion 40.

Matrigel plug angiogenesis assay

Six week old female C57BL/6 wild type and Ptx3-/- 41 mice were injected s.c. with 400 μL of Matrigel containing PBS or 4.0 pmoles of FGF2 and/or 33 pmoles of TSG-6. After 7 days, pellets were processed for total RNA extraction and the levels of expression of PTX3, FGF2, TSG-6 and VEGF-A were assayed by semi-quantitative RT-PCR in representative samples. The vascular response was quantified by evaluation of the levels of expression of the endothelial marker CD31 by quantitative RT-PCR.

Immunohistochemistry

Sections of human atherosclerotic carotid artery specimens, human gastric carcinoma and human pleomorphic parotid adenoma biopsies were immunostained with anti-human PTX3, anti-human TSG-6, and anti-human FGF2 polyclonal antibodies.

Throughout the whole paper, PTX3 concentrations are expressed as concentrations of PTX3 protomer.

Results

TSG-6 binds the N-terminal domain of PTX3 via the Link module

Previous observations suggest that TSG-6 interacts with PTX3 through its Link module at a distinct site from the HA-binding surface 37. As shown in Figure 1A, PTX3 bound to microtiter plates coated with full-length human TSG-6 or with its Link module domain (Link_TSG6) whereas little or no interaction was observed when a recombinant preparation of the CUB_C domain (CUB_C_TSG6) was applied. Accordingly, real time surface plasmon resonance analysis shows that both full length TSG-6 and Link_TSG6, but not CUB_C_TSG6, bind PTX3 immobilized to the sensorchip (Figure 1B). Kinetic fitting of sensorgrams gave equilibrium dissociation constants (kd) of 314 nmol/L and 648 nmol/L for the binding of full length TSG-6 and Link_TSG6 to PTX3, respectively (Supplemental Figure I). Similar results were obtained when Scatchard plot regression was performed on binding data acquired under state steady conditions (data not shown).

Figure 1.

Figure 1

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The Link module of TSG-6 binds PTX3. A) Microtiter plates were coated with 25 pmoles/well of TSG-6 (■),Link_TSG6 (△) or CUB_C_TSG6 (▲) and incubated with PTX3. Bound protein was detected using the anti-human PTX3 polyclonal antibody αPTX3pb. Data are expressed as mean ± SD (n = 12). B) TSG-6, Link_TSG6 and CUB_C_TSG6 were injected onto a PTX3-coated sensorchip and protein binding was monitored by surface plasmon resonance (BIAcore). Sensorgrams are representative of the binding response at analyte concentration of 300 nmol/L.

Consistent with the observation that PTX3 and HA recognize distinct surfaces on TSG-6 37, the TSG-6Y94F mutant, which has greatly reduced (~100-fold) HA-binding activity (42 and A.J Day, unpublished data, 2010), has a similar affinity as wildtype TSG-6 for PTX3 (supplemental Figure I). Conversely, the TSG-6Y47F mutant, that also has greatly impaired HA-binding activity (A.J Day, unpublished data, 2010), binds PTX3 with somewhat lower affinity than wildtype TSG-6 (~6-fold) suggesting that this amino acid may participate to some extent in PTX3 binding. Mutation of Glu-183, located within the CUB module, to Ser (TSG-6E183S) (A.J Day, unpublished data, 2010), had no affect on PTX3 binding, consistent with the finding that the CUB_C domain does not play a part in the interaction (supplemental Figure I). Taken together, these results demonstrate that the Link module, rather than the CUB_C domain, mediates the binding of TSG-6 to PTX3 and that this interaction occurs with an affinity similar to that reported for FGF2/PTX3 interaction (kd = 300 nmol/L 20).

To identify which region of the PTX3 molecule is involved in the interaction with TSG-6, microtiter plates coated with recombinant forms of the N-terminal and C-terminal domains of PTX3 (NtermPTX3 and CtermPTX3, respectively) were incubated with TSG-6. As shown in Figure 2A, TSG-6 bound to immobilized NtermPTX3 in a concentration-dependent manner, similar to that seen with the full length PTX3 protein, whereas no interaction of TSG-6 with CtermPTX3 was observed. Accordingly, Link_TSG6 bound immobilized full-length PTX3 and NtermPTX3 but not CtermPTX3, whereas CUB_C_TSG6 did not interact with any of the PTX3 domains (Figure 2B). Therefore, it can be concluded that PTX3 interacts with the Link module of TSG-6 via its N-terminal domain, which is also the site where FGF2 binds 13, 20, 43.

Figure 2.

Figure 2

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TSG-6 binds the N-terminal domain of PTX3. A) Full length PTX3 (■) or recombinant domains NtermPTX3 (△) and CtermPTX3 (▲) were immobilized on microtiter plates (15 pmoles/well) and incubated with TSG-6. Bound TSG-6 was detected using the anti-human TSG-6 polyclonal antibody RAH1. B) Microtiter plates were coated with 15 pmoles/well of full length PTX3 (black bars), NtermPTX3 (open bars) or CtermPTX3 (dashed bars). Following incubation with full length TSG-6, Link_TSG6 or CUB_C_TSG6 (all at 1 μmol/L), bound proteins were detected with RAH1 (for TSG-6 and CUB_C_TSG6) or Q75 (for Link_TSG6) antibodies. Data are expressed as mean ± SD (n = 12).

TSG-6 inhibits the PTX3/FGF2 interaction

As described above TSG-6 and FGF2 both bind PTX3 with similar affinities where these interactions are mediated via the N-terminal region of PTX3. This suggests that TSG-6 might be able to modulate the interaction of PTX3 with FGF2. Indeed, TSG-6 competed for the binding of biotinylated PTX3 (bPTX3) to microtiter plates coated with FGF2 with a potency (IC50 ~ 100 nmol/L) similar to that of non-biotinylated PTX3, here used as a positive control (Figure 3A). Also, TSG-6 inhibits the binding of NtermPTX3 fragment to immobilized FGF2 (Figure 3B), whereas the short pentraxin serum amyloid P component (SAP), which is homologous to the C-terminal domain of PTX3 but does not bind FGF2 and TSG-6 17, had no affect on bPTX3/FGF2 interaction. Furthermore, TSG-6 inhibited the chemical cross-linking of the FGF2/PTX3 complex in solution (supplemental Figure II). Thus, TSG-6 acts as an inhibitor of the FGF2 interaction with PTX3.

Figure 3.

Figure 3

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TSG-6 inhibits PTX3/FGF2 interaction. A) Microtiter plates were coated with FGF2 (25 pmoles/well) and incubated with 12 nmol/L biotinylated PTX3 (bPTX3) and various concentrations of TSG-6 (■). Bound bPTX3 was detected by incubation with ExtrAvidin™ and absorbance was read at 405 nm. Non-biotinylated PTX3 (△) and SAP (▲) were used as positive and negative controls, respectively. B) FGF2-coated microtiter plates were incubated with 50 nmol/L recombinant NtermPTX3 and the indicated amounts of TSG-6 (■). Bound NtermPTX3 was detected with the αPTX3pb antibody. SAP was used as a negative control (▲). In both panels, data are the mean ± SD of three independent experiments in quadruplicate and are expressed as percent of ligand binding in the absence of competitors.

TSG-6 restores HSPG/FGF2/FGFR ternary complex formation inhibited by PTX3

FGF2 exerts its biological activity by leading to the formation of a pro-angiogenic HSPG/FGF2/FGFR ternary complex in ECs 8. In keeping with its FGF2-antagonist activity, PTX3 inhibits the formation of this ternary complex in a cell-cell adhesion assay 39, 44 in which FGF2 mediates the interaction of HSPG-deficient CHO cells stably transfected with FGFR1 to a monolayer of CHO cells expressing HSPGs but not FGFRs (Figure 4A). To investigate the effect of TSG-6 on the inhibitory activity exerted by PTX3 on HSPG/FGF2/FGFR ternary complex formation, cells were incubated with varying concentrations of TSG-6 in the presence of constant amounts of FGF2 and PTX3. As shown in Figure 4A, TSG-6 restored FGF2-mediated cell-cell adhesion (i.e. the formation of the intercellular HSPG/FGF2/FGFR complex) in a dose-dependent fashion. It must be pointed out that TSG-6 alone (i.e. in the absence of FGF2 and PTX3) did not induce cell-cell interaction nor did it affect the FGF2-mediated formation of the HSPG/FGF2/FGFR complex when PTX3 was omitted. Similarly, TSG-6 also reversed the inhibitory effect of the NtermPTX3 fragment on HSPG/FGF2/FGFR complex formation (supplemental Figure III). These data indicate that TSG-6 can rescue the PTX3-mediated inhibition of FGF2 engagement with its receptors, presumably via its competition for the PTX-FGF2 interaction.

Figure 4.

Figure 4

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TSG-6 suppresses the inhibitory effect exerted by PTX3 on FGF2 activity. A) FGFR1-overexpressing, HSPG-deficient CHO cells were incubated on a monolayer of CHO-K1 cells with FGF2 (1.66 nmol/L) and the indicated amounts of TSG-6 in absence (○) or presence (●) of PTX3 (220 nmol/L). After 2 hours, bound cells were counted under an inverted microscope. Data are expressed as percentage of the cell-cell adhesion induced by FGF2 alone. Binding of TSG-6-treated cells in the absence of FGF2 and PTX3 is shown as control (□). B) GM7373 cells were incubated with 0.4% FCS containing 0.56 nmol/L FGF2 and the reported concentrations of TSG-6 in the absence (○) or presence (●) of PTX3 (220 nmol/L). After 24 hours cells were trypsinized and counted. Data are expressed as percentage of GM7373 cells stimulated with FGF2 only. The proliferation of GM7373 cells treated with TSG-6 only (i.e. in the absence of FGF2 and PTX3) is shown as a control (□). C, D) Retrovirus-infected PTX3 cells overexpressing PTX3 (PTX3_MAECs, in C) or NtermPTX3 (NtermPTX3_MAECs, in D) were incubated with 0.4% FCS plus 0.56 nmol/L FGF2 and the indicated amounts of TSG-6 (●). Following 24 hours incubation, cells were trypsinized and counted. Data are expressed as percentage of the proliferation measured in mock-infected cells stimulated with FGF2 only. The proliferation of cells treated with TSG-6 alone is shown as a control (○). In all graphs, intermediate markings on the x axis represent the actual concentrations of TSG-6 used in the different assays. Data are the mean ± SD of three experiments performed in triplicate.

TSG-6 suppresses the inhibitory effect of PTX3 on FGF2-dependent angiogenesis

PTX3 inhibits the mitogenic activity exerted by FGF2 on ECs, without affecting the activity of unrelated mitogens 19. On this basis, TSG-6 was assessed for its capacity to reverse the inhibition caused by PTX3 on FGF2-dependent EC proliferation. To this aim, fetal bovine aortic GM7373 ECs were incubated with FGF2 (0.56 nmol/L), PTX3 (220 nmol/L) and a range of TSG-6 concentrations. Consistent with its ability to restore HSPG/FGF2/FGFR ternary complex formation inhibited by PTX3, increasing doses of TSG-6 progressively restored FGF2-induced EC proliferation in the presence of PTX3 (Figure 4B). A similar effect was also seen with the TSG-6 mutants TSG-6Y47F, TSG-6Y94F, and TSG-6E183S (supplemental Figure IV) that all retain binding to PTX3 (see supplemental Figure I). It should be noted that TSG-6 alone did not affect EC proliferation when tested in the absence or in the presence of FGF2 (Figure 4B).

To further confirm the capacity of TSG-6 to restore EC proliferation by preventing FGF2/PTX3 interaction, we took advantage of an experimental model in which human PTX3 or the NtermPTX3 fragment are endogenously expressed by retrovirus-infected murine aortic ECs (PTX3_MAECs and NtermPTX3_MAECs, respectively) and inhibit EC proliferation in response to exogenous FGF2 20. As shown in Figure 4C,D, increasing doses of TSG-6 progressively restored the capacity of PTX3_MAEC and NtermPTX3_MAEC transfectants to proliferate in response to FGF2. Again, similar activities were seen with the TSG-6 mutant proteins (supplemental Figure IV). These and the data above demonstrate that neither the HA-binding function of TSG-6 24 nor its ability to form covalent complexes with the HCs of IαI 36 (i.e. properties impaired in these mutants) are necessary for TSG-6 to show activity in these angiogenesis assay systems.

The capacity of TSG-6 and PTX3 to affect FGF2-induced neovascularization in vivo was then investigated in a chicken embryo CAM angiogenesis assay 45 and in a murine s.c. Matrigel plug assay 46. In the CAM assay, alginate beads adsorbed with FGF2 (8.0 pmoles/embryo) exert a potent angiogenic response when applied on the top of the CAM as compared to beads adsorbed with vehicle. Consistent with the in vitro observations, the angiogenic response elicited by FGF2 was significantly reduced by the addition of 33 pmoles of PTX3 to the FGF2 implants (p < 0.001) and this inhibition was fully abolished by co-administration of 83 pmoles of TSG-6. No effect on CAM vascularization was instead exerted by TSG-6 alone (Figure 5A).

Figure 5.

Figure 5

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TSG-6/PTX3 cross-talk modulates FGF2-mediated angiogenesis in vivo. A) Chicken embryo CAMs were implanted at day 11 with alginate beads containing FGF2 (8 pmoles), PTX3 (33 pmoles) and TSG-6 (83 pmoles) or combinations of these proteins. At day 14, newly-formed thin blood microvessels converging toward the implant in a spoke-wheel pattern (readily distinguishable from preexisting, larger vessels with no directionality) 45 were counted. CAM implants that contained vehicle (PBS) or TSG-6 only were used as controls. Data are the mean ± SD (n = 7-11). *, p<0.01, Student’s t test. B) Wild-type (open bars) and Ptx3-null mice (black bars) where injected s.c. with Matrigel plugs containing PBS or 4 pmoles of FGF2 and/or 33 pmoles of TSG-6. After 7 days, pellets were processed for total RNA extraction and the vascular response was quantified by RT-PCR analysis of the expression of the endothelial marker CD31. Data are the mean ± SEM (n = 8-10). *, p<0.05 or better, n.s., not statistically significant, Student’s t test.

Next, the effect of TSG-6 on FGF2-induced angiogenesis was assessed in wild-type and Ptx3-null mice using a s.c. Matrigel plug assay (Figure 5B). It must be pointed out that the s.c. injection of Matrigel induces per sè a mild pro-inflammatory reaction 47, leading to the co-expression within the plug of PTX3, FGF2, TSG-6, and VEGF-A transcripts (supplemental Figure V). Also, no significant difference in the levels of FGF2, TSG-6, and VEGF-A expression occurred in PBS-treated Matrigel plugs injected in Ptx3-null mice when compared to plugs implanted in wild-type animals (supplemental Figure V). Consistent with the FGF2-antagonist activity of PTX3, we observed a significant increase of vascularization in both PBS-treated and FGF2-treated plugs implanted in Ptx3-deficient mice when compared to wild-type animals, as assessed by quantitative RT-PCR analysis of the levels of expression of the endothelial marker CD31 in the Matrigel plugs 48. A similar increase was observed when TSG-6 was added to PBS-treated and FGF2-treated Matrigel plugs implanted in wild-type animals, no further significant increase in vascularization being exerted by TSG-6 in Ptx3-null mice (Figure 5B).

Discussion

Taken together, the above results indicate that TSG-6 suppresses the inhibition exerted by PTX3 on FGF2-dependent angiogenesis both in vitro and in vivo. Thus, this provides the first direct evidence of a role for TSG-6 as a modulator of neovascularization. Our observations clearly indicate that TSG-6 exerts its ‘pro-angiogenic’ functions via inhibition of the PTX3/FGF2 interaction, where this may be mediated by the binding of TSG-6 to PTX3. Preliminary solid phase binding and surface plasmon resonance assays suggest that TSG-6 may also interact directly with FGF2 (A. Inforzato and A. J. Day, unpublished data, 2009). Thus, TSG-6 might prevent FGF2/PTX3 interaction through a combination of mechanisms that together lead to suppression of the inhibition exerted by PTX3 on the angiogenic activity of FGF2. Further work is required to fully dissect the molecular interplay between PTX3, FGF2 and TSG-6 so as to understand the mechanism of TSG-6 action.

As described above, PTX3 may contribute to the modulation of FGF2-driven angiogenesis in different pathological settings characterized by the co-expression of the two proteins, including inflammation, wound healing, atherosclerosis, and neoplasia [see 44, 49 for a further discussion]. The coordinated expression of TSG-6 and PTX3 has been described in inflamed tissues 26, 50 and in the cumulus oophorus 37, 50. Accordingly, FGF2, TSG-6 and PTX3 are co-expressed following s.c. injection of a Matrigel plug in mice (see supplemental Figure V), a mild pro-inflammatory experimental condition 47. Moreover, immunohistochemical analysis performed on a limited series of human specimens shows that FGF2, TSG-6 and PTX3 are co-expressed in human atherosclerotic carotid artery and in biopsies of human benign tumors (pleomorphic parotid adenoma) and cancer (gastric carcinoma)51, 52 (supplemental Figure VI). Further studies on larger cohorts of patients will be required to assess the relationship between the expression of these modulators of the angiogenic process and tissue vascularization in different inflammatory/cancerous conditions.

Our study indicates that the relative levels of TSG-6 and PTX3 likely dictate the biological activity of FGF2; a low TSG-6:PTX3 ratio exerting an inhibitory effect on FGF2-mediated angiogenesis whereas an high TSG-6:PTX3 ratio represents a permissive condition for the angiogenic activity of the growth factor. Thus, the interaction of TSG-6 and PTX3 might act as a biological rheostat for FGF2-dependent neovascularization, contributing to the complex extracellular protein interactome 9 that mediates the angiogenic process. These conclusions are supported by the observation that endogenous PTX3 exerts a significant inhibitory effect on vascularization induced by endogenous FGF2 or by the exogenously added growth factor in a murine Matrigel plug assay, this effect being abolished in Ptx3-null mice or by TSG-6 treatment in wild-type animals. As anticipated, no effect is exerted by TSG-6 in the absence of endogenous PTX3, i.e. in Ptx3-null mice.

Our studies indicate that TSG-6 can act as a novel indirect ‘pro-angiogenic’ cofactor by releasing FGF2 from the PTX3 constraints, no direct angiogenic activity being exerted by this protein when administered alone in the different assays. On the other hand, recent data have shown that intra-ocular TSG-6 treatment in a rat model of corneal wound healing can inhibit neovascularization 53, possibly as a consequence of its anti-inflammatory activity (e.g. inhibition of neutrophil infiltration) 30. Notably, the anti-angiogenic and anti-inflammatory effects were observed only when TSG-6 was administered within 4 hours after injury, no effect being observed when the inflammatory cell infiltrate was already established 30. Previous observations had shown that some anti-inflammatory cytokines, including erythropoietin 54 and interleukin-10 55 may promote angiogenesis, whereas tumor necrosis factor-α may exert pro- or anti-angiogenic effects depending upon the dose of the cytokine 56. Taken together, these data suggest that TSG-6 may have both pro-angiogenic and anti-angiogenic properties, such that the balance between promotion and inhibition of neovascularization may depend on context (e.g. the microenvironment). Further research is needed to investigate the precise role of TSG-6 as a modulator of angiogenesis and how this is regulated, for example, by components of the ECM.

Supplementary Material

1

Acknowledgements

We thank Patrizia Dell’Era, Ragnar Lindstedt and Giovanni Salvatori for reagents, Michela Corsini for having performed the CAM assays and Manuela Nebuloni and Fabio Pasqualini for assistance with immunohistochemistry.

Sources of Funding Supported by Arthritis Research UK (grant nos. 16539 and 18472), the Medical Research Council (grant no. G0701180) and Sigma-Tau to A.J.D. and by 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, and Fondazione Cariplo (grant 2008-2264 and NOBEL Project) to M.P. The contribution of the European Commission (“TOLERAGE” 2008-202156) and European Research Council (project HIIS) to A.I., M.S., B.B. and C.G. is gratefully acknowledged. A.I. was the recipient of fellowships from Fondazione Italiana per la Ricerca sul Cancro, Fondazione “Humanitas” per la Ricerca, and Scuola Europea di Medicina Molecolare (SEMM).

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