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Circulation Research. 2004;94:735-742
Published online before print February 12, 2004, doi: 10.1161/01.RES.0000122043.11286.57
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(Circulation Research. 2004;94:735.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Thromboxane A2 Receptor Agonists Antagonize the Proangiogenic Effects of Fibroblast Growth Factor-2

Role of Receptor Internalization, Thrombospondin-1, and {alpha}vß3

Anthony W. Ashton, Yan Cheng, Armin Helisch, J. Anthony Ware

From the Departments of Medicine (Cardiology) and Molecular Pharmacology (A.W.A, A.H., J.A.W.), the Albert Einstein College of Medicine, Yeshiva University, Bronx, NY; and Center for Experimental Therapeutics (Y.C.), University of Pennsylvania, Philadelphia, Pa.

Correspondence to Anthony Ashton, Room G01, Golding Building, Department of Cardiology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, NY 10461. E-mail ashton{at}aecom.yu.edu


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of the pathogenesis of many vascular events, including angiogenesis. Endothelial cells express TXA2 receptors (TP) but the effects of TP stimulation on angiogenesis remain controversial. In this study, we show that stimulation of endothelial cell TP impairs ligand-induced FGF receptor internalization and consequently abrogates FGF-2-induced endothelial cell migration in vitro and angiogenesis in vivo. Prevention of FGF-2-induced angiogenesis was associated with expression of the TPß isoform. The deficit in FGFR1 internalization was mediated through activation of TPß preventing the FGF-2-mediated decrease in p53 expression, thus enhancing thrombospondin-1 (TSP-1) release from EC and reducing FGFR1 internalization. Once released TSP-1 interacted with the {alpha}vß3 integrin on the EC surface. On stimulation, FGFR1 and {alpha}vß3 were found to associate in a complex. We determined that complex formation was important for receptor internalization as conditions that inhibit FGFR1 internalization, such as inappropriate ligation of {alpha}vß3 by either TSP-1 or a neutralizing antibody, disrupted the complex. These results establish a novel role for isoform specific regulation of angiogenesis by TP, provide the first functional significance for the existence of two TP isoforms in humans, and clarify the mechanism by which TP signaling regulates FGFR1 kinetics and signaling.


Key Words: thromboxane • angiogenesis • FGFR1 internalization • integrin {alpha}vß3


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Angiogenesis is central to the pathogenesis of, and recovery from, multiple disease processes, including ischemia and cancer. Elevated levels of one angiogenic factor, fibroblast growth factor-2 (FGF-2), have been documented in acute ischemic coronary syndromes.1,2 Serum FGF-2 levels are first detectable in the acute phase of AMI and are maintained for at least 14 days after the onset of ischemia.3 In vivo studies suggest that the cardioprotective effects of FGF-2 are significantly correlated with its ability to stimulate angiogenesis.4,5 Interestingly, the highest FGF-2 levels occur at the time of the most intense angiogenic response after ischemia in the myocardium. These data suggest that FGF-2 may modulate the angiogenic response to ischemic episodes3 in which new vessels are formed in the coronary collateral circulation and (partially) revascularize the heart.6

Integrin-mediated outside-in signals cooperate with growth factor receptors to promote cell proliferation and motility. Ligation of the integrin {alpha}vß3 is crucial for endothelial cell adhesion to matrix, and for migration and proliferation during angiogenesis.7,8 Indeed, {alpha}vß3 has become a prominent target for antiangiogenic therapy because of its importance to the angiogenic response. The angiogenic activity of FGF-2 is mediated, in part, through activation of {alpha}vß3.9,10 The integrin {alpha}vß3 contributes to distinct pathways of FGF-induced angiogenesis, including activation of the Ras/ERK11 and Src/FAK12 signaling pathways, and has been proposed to augment the signaling of FGF to strengthen angiogenic responses.10,13 Thrombospondin-1 (TSP-1) is a natural antagonist of {alpha}vß3. TSP-1 can interact with multiple cell surface receptors, such as CD47 and integrins, to inhibit the angiogenic response in ECs. Indeed, TSP-1 uses multiple pathways to modulate basic processes that inhibit endothelial cell migration. TSP-1 has been shown to negatively regulate focal adhesion formation on fibronectin,14 suggesting that integrin function may be impaired. Further, TSP-1 release is decreased or ablated in response to angiogenic stimuli that signal through {alpha}vß3, such as FGF-2.15

FGF-2-induced angiogenesis in vivo usually occurs in the setting of inflammation and ischemia.16,17 Such pathological environments contain multiple factors capable of modifying an angiogenic response. Thus, any pathophysiological effect of FGFs must take place in the context of crosstalk with modifiers present during disease. Thromboxane (TX) A2 receptor (TP) expression and activation are increased in multiple disease states, including ischemia and inflammation,18 and are potent stimuli moderating vascular responses. TP exists as two isoforms in humans, TP{alpha}19 and TPß20; however, the physiological significance of these two isoforms is currently unknown. Because TP activation occurs in the same settings as FGF-2 release, it seems likely that TP could modify angiogenic responses to multiple stimuli.

To examine the hypothesis that TP stimulation may act as an angiogenic modulator, we tested whether TP ligands interfere with the mitogenic, chemokinetic, and angiogenic properties of FGF-2. In this study, we report a novel mechanism by which TPß stimulation abrogates the proangiogenic properties of FGF-2. We also establish distinct (patho)physiological consequence of the activity of the two TP isoforms.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
An expanded Materials and Methods section can be found in the online data supplement available at http://circres.ahajournals.org.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
TP Stimulation Abrogates Endothelial Cell Migration and Angiogenesis in Response to FGF-2
To determine the effect of TP stimulation on the angiogenic potential of endothelial cells, we investigated the potential for migration in response to FGF-2 in the presence of the TXA2 mimetic IBOP. FGF-2 treatment increased the migration rate of human umbilical vein endothelial cells (HUVECs) to 18.3 µm/h (compared with 8.3 µm/h of control cells; P<=0.0001; Figure 1A). The accelerated migration in response to FGF-2 was concentration dependent with maximal effects at or above 20 ng/mL (data not shown). Costimulation with the TXA2 mimetic IBOP (100 nmol/L) abrogated the enhanced migration (9.6 µm/h; P<=0.005 versus FGF-2 alone; Figure 1A) in FGF-2-treated HUVECs. These results demonstrate that TP stimulation is an effective antagonist of FGF-2-induced migration with as little as 50 nmol/L IBOP abrogating the response (IC50 {approx}25 nmol/L, data not shown).



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  		Figure 1. IBOP prevents migration and angiogenesis in response to FGF-2 through the TPß isoform. HUVECs stimulated with the TXA2 mimetic IBOP and FGF-2 ({bullet}) showed slower migration (A) compared to those stimulated with FGF-2 alone ({blacksquare}). IBOP ({circ}) alone slowed migration (A) compared to HUVECs treated with media alone ({square}). B, Inhibition of FGF-2-induced migration by IBOP required the expression of the TPß isoform. Results are measured as micrometers traveled. All data are presented as mean±SD. C, Visualization of the angiogenic response in Matrigel plugs to FGF in the presence and absence of IBOP in normal (TP{alpha}) and TPß transgenic mice. Arrows highlight the location of vessels. D, Quantitation of vessel number in cross sections of matrigel plugs from WT ({square}) and TPß transgenic ({blacksquare}) mice reveals that the expression of TPß is required for the inhibition of angiogenesis by TXA2 mimetics in vivo. Results represent 6 mice per group and are mean±SD. Significance (P<=0.05) from *control and #FGF2 alone, respectively.

Next, we assessed the role of each TP isoform in modulating angiogenesis. HUVECs express both TP{alpha} and TPß by Western and Northern blot analysis (data not shown). TP-null ECs were transfected with vectors encoding TP{alpha} or TPß to examine the isoform specific modulation of EC migration. In the absence of IBOP, migration was similar to vector transfected (control) cells (Figure 1B) both in the presence and absence of FGF-2. TP{alpha} transfection did not alter EC migration in response to FGF-2 when treated with 50 nmol/L IBOP; however, the migration of TPß ECs was reduced to unstimulated levels (Figure 1B). Thus, TPß is necessary and sufficient for the reduction in FGF-2-induced migration mediated by IBOP, suggesting a divergence in the regulation of angiogenesis by the two receptor subtypes.

The matrigel plug model of angiogenesis was used to define the regulation of angiogenesis by individual TP isoforms in vivo using wild-type and TPß transgenic mice.21 Murine TP receptors closely resemble human TP{alpha} receptors (75% homology), with no murine homologue of TPß reported to date. Matrigel (500 µL) was subcutaneously injected, the plugs excised and vessel counts determined after 14 days (Figures 1C and 1D). Matrigel plugs containing saline (control), IBOP (500 nmol/L), or the TP receptor blocker SQ29548 (30 µmol/L) had low levels of angiogenesis (0.35 vessels/field) in both wild-type (WT) and TPß-expressing mice. FGF-2 induced equivalent angiogenesis in both strains with an average vessel count of 9.3±0.7 and 11.4±1.4 per field for WT (TP{alpha}) and TPß transgenics, respectively (P=NS between groups). Inclusion of IBOP in FGF-2 containing plugs modestly reduced vessel number by 20% in WT mice. In contrast, IBOP completely eliminated FGF-2-induced angiogenesis in the TPß transgenic (Figures 1D), which was prevented by inclusion of SQ29548. This result indicates that TPß expression correlates with the antiangiogenic activity of TXA2 receptor ligands in vivo and suggests strongly that TPß expression mediates the inhibition of FGF-2-induced migration and angiogenesis by TP ligands.

TPß Signaling Prevents HUVEC Migration and Angiogenesis by Inhibiting FGFR1 Internalization
We next investigated the mechanism by which TPß stimulation could potentially interfere with FGF-2 receptor signaling. In initial experiments, we found that FGFR1 expression and the subsequent activation of downstream effectors such as ERK1/2and PLC-{gamma} were not altered by IBOP in FGF-2-treated HUVECs (online Figure 1A). However, the surface expression of FGFR1 was greatly different on HUVECs treated with FGF-2 and IBOP versus FGF-2 alone. FGF-2 treatment reduced FGFR1 surface expression by up to 75%, as determined by ELISA using an antibody against FGFR1 ({square}) and ligand binding assays ({blacksquare}) (Figure 2A), whereas IBOP alone was without effect. Costimulation with IBOP dramatically increased the extent of FGFR1 binding compared with FGF-2 alone. The effect was reversed by SQ29548, indicating that it was mediated by TP.



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  		Figure 2. IBOP limits FGFR-1 internalization in response to FGF-2. A, FGFR-1 cell surface expression was elevated in cells treated with both FGF2 and IBOP compared to FGF2 alone as determined by [125I] FGF-2 binding ({blacksquare}) and ELISA ({square}). Results are presented as mean±SD (n=4). B, Enhanced cell surface expression of FGFR1 was due to a failure of FGFR1 to internalize. Release of [125I] FGF-2 fragments into the media of cells treated with FGF and IBOP ({square}) was significantly less than control cells (gray circles). Deficit was reversed with the TXA2 receptor blocker SQ29548 ({circ}). IBOP in the absence ({bullet}) or presence of SQ29548 ({diamondsuit}) was without effect. Significance (P<=0.05) from *control and #FGF2 alone, respectively (n=5).

In the absence of altered overall FGFR1 expression, the increase in FGFR1 surface expression might result from altered rates of internalization. We performed ligand internalization/degradation assays (Figure 2B) to determine FGFR1 internalization rates in HUVECs exposed to FGF-2 with and without IBOP. FGF-2 and IBOP costimulation decreased release of FGF-2 fragments, which corresponds to the rate of FGFR1 internalization, from HUVECs by 4.5-fold compared with controls, IBOP, SQ29548, or FGF-2 alone. Simultaneous addition of SQ29548 again reversed these effects, showing that the reduction in FGFR1 internalization was TP mediated. In contrast, the internalization of KDR and Tie receptors in response to ligand was not inhibited by TP costimulation (data not shown). Consistent with previous findings,22 the inhibition of FGFR1 internalization resulted in a 3-fold increase in receptor tyrosine phosphorylation (online Figure 1A). Together these results indicate that the IBOP-induced signaling deficit is associated with failure of FGFR1 to internalize in response to FGF-2.

The main consequence of FGFR1 internalization is the onset of a transcriptional program that initiates the cell cycle.23,24 We also found that costimulation with FGF-2 and IBOP produced a deficit in the proliferation of treated HUVECs (online Figure 1B). HUVECs treated with FGF-2 and IBOP display a delayed transition into S-phase with decreased magnitude of the response compared with HUVECs treated with FGF-2 alone. This deficit was correlated with decreased cyclin A induction by FGF-2 in the presence of IBOP (online Figure 1C). Thus, the inhibition of FGFR1 internalization by IBOP produces clear defects in FGF-2 signaling that may be responsible for the inhibition of the angiogenesis.

Deficit in FGFR1 Internalization Is Associated With Maintenance of TSP-1 Release From ECs
Next, we investigated the potential mechanism for the defective internalization of FGFR1. Tyrosine phosphorylation of FGFR1 and subsequent activation of PLC{gamma} and ERK coincide with increased mitogenic activity and receptor internalization.25 No decrease in FGFR1 phosphorylation, ERK, or PLC{gamma} activation by FGF-2 was observed in the presence of IBOP (online Figure 1A), indicating that this mechanism is unlikely. The second possibility considered involved a direct association of TSP-1 with FGF-2 in the extracellular compartment.26 Previous work has shown that FGF-2 downregulates TSP-1 release from HUVECs.15 A sandwich ELISA was established to measure TSP-1 release by FGF-2-stimulated HUVECs with and without IBOP. FGF-2 abrogated TSP-1 release from HUVECs at 24 hours, whereas IBOP alone was without effect (Figure 3A). IBOP costimulation reversed the downregulation of TSP-1 by FGF-2, returning the release to control levels. Thus, enhanced endogenous release of TSP-1 may be linked to impaired FGFR-1 internalization.



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  		Figure 3. TP stimulation prevents the FGF-2-induced downregulation of p53 and TSP-1. A, FGF stimulation of HUVECs potently downregulates TSP-1 release, as measured by ELISA, with the TXA2 mimetic IBOP abrogating that decrease. B, IBOP prevents the downregulation of p53 expression by FGF-2 in HUVECs. Data are from 5 experiments (mean±SD). *Significance (P<=0.05) from control. #Significance from FGF-2 treatment.

Active levels of p53 are a major determinant of TSP-1 transcription, synthesis, and release.27,28 Immunoblotting determined that FGF-2 treatment reduced p53 expression in HUVECs by 85% (Figure 3B), which was negated by costimulation with IBOP. We next confirmed the role of soluble TSP-1 as an inhibitor of FGF-2-induced migration (Figure 4A). Addition of exogenous, purified platelet TSP-1 to HUVECs abrogated FGF-2-induced, but not basal, migration in a concentration-dependent manner with an IC50 of 20 ng/mL and maximal effects at 60 ng/mL. Further, the inhibition of FGF-2-induced migration by exogenous TSP-1 occurs at concentrations (60 ng/mL) far lower than the difference in the level of TSP-1 release between FGF-2-treated HUVECs in the presence and absence of TXA2 mimetics (Figure 4A). These results indicate that the addition of exogenous TSP-1 can mimic the effects of TP costimulation and suggests that even small perturbations in the prevention of FGF-2-mediated TSP-1 downregulation by TXA2 may be sufficient to inhibit HUVEC migration in response to FGF-2.



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  		Figure 4. Antagonism of TSP function restores the chemokinetic activity of FGF-2 and FGFR1 internalization. A, FGF-2-induced, but not basal, migration is prevented by the addition of exogenous TSP-1, mimicking the changes induced by IBOP treatment. B, Transfection of HUVECs with TSP-1 antisense (T{alpha}s), but not random oligonucleotides (Tscr), prevented the inhibition of FGF-2-induced FGFR1 internalization ({square}) and migration ({blacksquare}) by IBOP. Results are measured as OD 415 nm and micrometers traveled for receptor ELISA and migration assay, respectively. Both data sets are presented as mean±SD (n=5). # and * denote significance (P>=0.01) from control and FGF-2, respectively.

To investigate further the relationship between increased TSP-1 release and altered FGF-2 signaling imposed by TP stimulation, we interfered with the synthesis, release, and signaling of TSP-1 using antisense oligonucleotides and blocking antibodies. Antisense oligonucleotides were used to prevent the reinduction of TSP-1 by TPß costimulation in FGF-2-treated HUVECs (Figure 4B). ELISA confirmed that TSP-1 antisense, but not scrambled, oligonucleotides abrogated the release of TSP-1 and decreased TSP-1 mRNA levels (online Figures 2A and 2B). Moreover, TSP-1 antisense completely counteracted the IBOP-induced increase in TSP-1 release in FGF-2-stimulated HUVECs. Neither oligonucleotide affected EC migration or FGFR1 surface expression in unstimulated HUVECs. Chemokinesis ({blacksquare}) and FGFR1 internalization ({square}, as determined by surface expression) in response to FGF-2 remained inhibited by IBOP in the presence of scrambled oligonucleotides (Figure 4B). However, transfection of TSP-1 antisense oligonucleotides prevented the blockade of FGF-2-mediated migration by IBOP. Similarly, FGFR1 internalization was restored in FGF-2- and IBOP-treated HUVECs using TSP-1 antisense oligonucleotides. These data indicate that enhancing TSP-1 release from HUVECs was essential for the antagonism of FGF-2 signaling by TPß stimulation.

Interaction Between the {alpha}vß3 Integrin and TSP-1 Is Correlated With a Deficit in FGFR1 Internalization and EC Migration
The final set of experiments were directed at determining the mechanism by which TSP-1 might prevent FGFR1 internalization. TSP-1 has multiple and complex interactions with cell surface moieties including heparan sulfate proteoglycans,29 CD36,30 CD47,31 and LRP.32 We found that inclusion of neutralizing antibodies that antagonize the interaction of TSP-1 with proteoglycans, CD36 and CD47, did not reverse the inhibition of migration by IBOP in FGF-2-stimulated HUVECs (Figure 5A). However, the use of an anti-TSP-1 polyclonal antibody restored the ability of HUVECs to migrate in response to FGF-2 in the presence of IBOP. These data indicated that interaction of TSP-1 with a cell surface receptor other than CD36, CD47, and heparan sulfate was responsible for the inhibition of migration.



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  		Figure 5. TSP-1 interacts with {alpha}vß3 to inhibit receptor internalization. A, Polyclonal antibody against TSP-1, but not antibodies that inhibit the interaction of TSP-1 with heparin (hep), CD36, or CD47, reversed the inhibition of FGF-2-induced migration due to TPß signaling. None of the antibodies used (Ab) nor preimmune IgG (IgG) inhibited the migration of HUVECs. B and C, IBOP induces the association of TSP-1 with the integrin {alpha}vß3 in FGF-2-stimulated HUVECs. To ensure specificity preimmune (–ve) IgG and an irrelevant antibody (I-IgG, anti-MHC class 1) were used as controls. D, Antagonism of the integrin dimer {alpha}vß3 antagonizes FGFR1 signaling in response to FGF-2. Selective antagonism of {alpha}vß3 function with a neutralizing antibody prevents both the migration of HUVECs ({square}) and the ability of FGFR1 to internalize ({blacksquare}) in response to FGF-2. Non-neutralizing IgG against {alpha}vß3 ({alpha}vß3 NN) was used as a negative control. Results are measured as OD 415 nm and micrometers traveled for receptor ELISA and migration assay, respectively. Both data sets are presented as mean±SD and represent 5 experiments. # and * denote significance (P>=0.01) from control and FGF-2, respectively.

A primary family of TSP-1 receptors not targeted by these neutralizing antibodies are the integrin family of proteins. HUVECs contain a distinct complement of integrins, including {alpha}2,3,5,6,V and ß1,3,5.33 TSP-1 has previously been shown to ligate the integrin heterodimers {alpha}IIbß3, {alpha}vß3, {alpha}3ß1, {alpha}4ß1, {alpha}5ß1, and {alpha}6ß4.34–36 In HUVECs treated with FGF-2 and IBOP, the greatest association of TSP-1 was with the integrins {alpha}v and ß3 versus FGF-2 alone, with lesser amounts of TSP-1 associated with the integrins {alpha}3 and ß1 (Figure 5B). Further, immunoprecipitation of {alpha}vß3, but not {alpha}3ß1, using complex specific antibodies found TSP-1 present (Figures 5B and 5C). This increase in TSP-1 association with {alpha}vß3 was only detectable in HUVECs treated with both FGF-2 and IBOP and did not increase beyond the association observed in control treated cells (Figure 5C).

FGF-2-mediated endothelial cell migration is most sensitive to perturbations in {alpha}vß3 function (online Figure 3). Thus, the ligation of {alpha}vß3 by TSP-1 may explain the deficits in migration and FGFR1 internalization in cells treated with IBOP and FGF-2. If so, the use of a neutralizing antibody against {alpha}vß3 should mimic the effects of IBOP stimulation on migration and FGFR1 trafficking in FGF-2-stimulated ECs. HUVEC migration (Figure 5D, {square}) and FGFR1 internalization in response to FGF-2 (Figure 5D, {blacksquare}) were both inhibited by a {alpha}vß3 neutralizing antibody. Preimmune IgG and a non-neutralizing antibody against {alpha}vß3 had no effect on FGF-2-induced migration (Figure 5D, online Figure 3). Antibodies against integrins {alpha}1–6 or ß1,2,4,5 were all ineffective in inhibiting FGF-2-induced migration (online Figure 3).

To understand the mechanism of how inhibition of {alpha}vß3 function could affect FGFR1 internalization and subsequently migration and angiogenesis, we examined the possibility that FGFR1 may associate with {alpha}vß3 as a part of the internalization process. Indeed, under conditions of FGF-2 stimulation, complex formation between {alpha}vß3 and FGFR1 was enhanced 10-fold (Figure 6A). Interaction of FGFR1 and {alpha}vß3, at baseline and under FGF-2 stimulation, was ablated by addition of IBOP (Figure 6A). Moreover, the association of FGFR1 and {alpha}vß3 in FGF-2- and IBOP-treated HUVECs was restored by antagonizing TSP-1 release with antisense oligonucleotides (Figure 6B). In addition, neutralizing antibodies against {alpha}vß3, but not non-neutralizing antibodies, mimicked the effects of IBOP and inhibited complex formation. These results indicated that normal {alpha}vß3 function and signaling are required to maintain FGFR1 signaling, subcellular redistribution, and potentially internalization after ligand binding. Collectively, these data demonstrate that the interaction with, and possibly the inappropriate ligation of, {alpha}vß3 by TSP-1 forms an essential part of the inhibitory pathway used by TPß stimulation to inhibit the angiogenic and chemokinetic effects of FGF-2.



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  		Figure 6. Antagonism of {alpha}vß3 function inhibits complex formation and internalization of FGFR1. A, FGF-2 induces complex formation between {alpha}vß3 and FGFR1, which is ablated by IBOP costimulation. Complex formation was detected by blotting for FGFR1 in immunocomplexes containing {alpha}vß3. B, Antagonism of {alpha}vß3 function antagonizes complex formation in response to FGF-2 and antagonism of TSP-1 release restores function. Use of a neutralizing antibody against {alpha}vß3, but not a non-neutralizing (Non) antibody or preimmune IgG (–ve), prevents {alpha}vß3-FGFR1 complex formation, consistent with the effects on FGFR1 internalization. Similarly, IBOP costimulation ablates complex formation, which is restored by TSP-1 antisense oligonucleotides.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study, we found that TP ligands abrogated the enhanced migration of endothelial cells in response to FGF-2 and suppressed angiogenesis in vivo. Further, the inhibition was dependent on the expression of the TPß isoform. The mechanism of inhibition was specific for the internalization of the FGF receptors. These data have important potential implications in clinical settings in which release of FGF and TP ligands occur simultaneously, such as during ischemia and after angioplasty. TXA2 may inhibit FGF-2-mediated endothelial cell migration during this process, thus prolonging the time required for reendothelialization and increasing the risk of SMC hypertrophy and neointimal formation due to plasma-derived mitogenic factors. Moreover, TXA2 may inhibit the migration of endothelial cells into infarcted tissue, and thus discourage revascularization, by antagonizing the signaling of FGF-2. Currently, the interplay between GPRC and tyrosine kinase receptors has only been described for the EGF receptor.37 The antagonism of the signaling pathways of FGF by TXA2 enriches our perception of the role for TP activation and expands the notion of "cross talk" between receptor tyrosine kinases and G protein-linked receptors.

Inhibition of the angiogenic response to FGF-2 was linked with the expression of the TPß isoform on endothelial cells. The original report of the TPß transgenic mice showed a decrease in placental size and microscopic evidence of ischemia.21 These observations concur with our present findings and suggest that vessel formation in the developing placenta was most likely suppressed by the ligation of TPß. Additionally, these observations may explain the dichotomy in the literature over the role of TP ligands in the regulation of angiogenesis. Although both TP isoforms are expressed in human ECs, little is known about their relative importance in EC biology. In small animal models of disease, inhibition of COX-1 or –2 results in the abrogation of TXA2 release and decreased neovascularization.38 Further, TXA2 synthase overexpression in tumor cells correlates with enhanced angiogenesis and growth rate39,40 and blockade of TP with SQ29548 has been shown to abrogate FGF and VEGF migration in vitro and in vivo.41,42 However, all of these findings have taken place in models that lack TPß expression. Our data suggest that TPß expression and subsequent signaling can overcome that from TP{alpha}, resulting in a phenotype that inhibits migration and angiogenesis in ECs expressing both isoforms. Thus, the existence of two TP isoforms in humans may have implications for its role in vascular disease.

We have also shown that ligation of {alpha}vß3 is necessary for the internalization of FGFR1 in response to ligand. Additionally, the defective internalization was specific for FGF receptors, as TPß costimulation did not alter KDR and Tie receptor internalization in response to ligand (data not shown). The integrin {alpha}vß3 contributes to distinct pathways of FGF-induced angiogenesis and has been proposed to augment the signaling of FGF to produce stronger angiogenic responses.10 However, the level of synergy between the FGF and {alpha}vß3 signaling cascades has previously been reported to influence only downstream mediators of angiogenesis. The dynamics of FGFR1 trafficking are complex but biologically important. Once formed, the FGF-2/FGFR1 complex is internalized and adopts a perinuclear location, at which it is hypothesized to alter transcriptional patterns.23,24 Sustained internalization of FGF-2/FGFR1 complexes has been shown to be crucial for the mitogenic and chemotactic effects of FGF-2.43 Little is known about the mechanism(s) that regulate FGF-2/FGFR1 complex internalization, although it is thought to be phosphorylation dependent44 and mediated through caveolae.45 Whereas such a significant role for {alpha}vß3 in FGFR1 internalization was unexpected, it is a logical regulatory step, because {alpha}vß3 also localizes to caveolae.46 ECM components have been shown to influence the internalization of other growth factor receptors, such as the insulin receptor.47 The present report additionally describes the integrin-mediated regulation of receptor tyrosine kinase internalization in response to ligand and that perturbation of this mechanism can alter the angiogenic properties of the agent. Indeed, FGFR1 and {alpha}vß3 appear to form a complex on FGF-2 stimulation as assessed by coimmunoprecipitation experiments, which is abrogated by TPß costimulation (Figure 6). TPß stimulation does not influence PLC{gamma} activation, caveolin-1 content, or {alpha}vß3 content in HUVECs (data not shown), indicating that these parameters were not the cause of the failure of FGFR1 to internalize.

The downregulation of p53 appears to be essential for the internalization of FGFR1 and the functioning of the FGF-{alpha}vß3 axis at multiple levels. Indeed, p53 expression and optimal function of FGFR1 signaling appear to be mutually exclusive.48 In addition, the loss of p53 due to FGF-2 signaling would enhance the angiogenic response through multiple mechanisms. p53 is not just an ontogeny suppressor, which is at odds with the mitogenic activity of FGF-2, but is also a major regulator of TSP-1 transcription.27,28 Because TSP-1 can negatively regulate the angiogenic response through {alpha}vß3 ligation, decreasing its release may be critical for the angiogenic activity of FGF-2. Loss of p53 itself also augments the signaling from {alpha}v integrins and suppression of p53 levels is a necessary part of the angiogenic response to FGF.49 Further, inappropriate ligation of {alpha}v integrins also induces p53 and abrogates levels of neovascularization. Thus, p53 may be a central target for down regulation by FGF-2.

In conclusion, the abrogation of FGF-2-induced downregulation of p53 by TPß induces release of cell-derived TSP1, resulting in the absence of EC migration and impaired angiogenesis. These findings suggest the hypothesis that inhibition of TP receptors, perhaps selective for the TPß isoform, could enhance myocardial revascularization following infarction.


*    Acknowledgments
 
This work was supported by NIH grants (HL47032, HL51043, and HL55552) and a postdoctoral fellowship (0020186T) and scientist development grant (0335416T) to A.W.A. from the American Heart Association. Transgenic mice with targeted overexpression of TPß to the endothelium (driven by the preproendothelin promotor) were obtained from Dr Garret Fitzgerald.


*    Footnotes
 
Original received September 22, 2003; revision received January 30, 2004; accepted February 3, 2004.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

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