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Circulation Research. 2004;95:372-379
Published online before print July 8, 2004, doi: 10.1161/01.RES.0000138300.41642.15
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(Circulation Research. 2004;95:372.)
© 2004 American Heart Association, Inc.

Molecular Medicine

Thromboxane A2 Receptor Signaling Inhibits Vascular Endothelial Growth Factor–Induced Endothelial Cell Differentiation and Migration

Anthony W. Ashton, J. Anthony Ware

From the Departments of Medicine (Cardiology) and Molecular Pharmacology, the Albert Einstein College of Medicine, Yeshiva University, Bronx, NY.

Correspondence to Anthony Ashton, Room G01, Golding Bldg, 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
 
Vascular endothelial growth factor (VEGF) is an important patho-physiological mediator of angiogenesis. VEGF-induced endothelial cell (EC) migration and angiogenesis often occur in complicated environments containing multiple agents capable of modifying the response. Thromboxane (TX) A2 is released from multiple cell types and is a prime mediator of pathogenesis of many vascular diseases. Human EC express both TXA2 receptor (TP) isoforms; however, the effects of individual TP isoforms on VEGF-induced EC migration and angogenesis are unknown. We report here that the TXA2 mimetic [1S-(1{alpha}, 2ß(5Z), 3{alpha}(1E, 3R), 4{alpha}]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5'-heptenoic acid (IBOP) (100 nmol/L) is a potent antagonist (IC50 30 nmol/L) of VEGF-induced EC migration and differentiation. TPß, but not TP{alpha}, expression is required for the inhibition of VEGF-induced migration and angiogenesis. IBOP costimulation suppressed nitric oxide (NO) release from VEGF-treated EC through decreased activation of Akt, eNOS, and PDK1. TPß costimulation also ablated the increase in focal adhesion formation in response to VEGF. This mechanism was characterized by decreased recruitment of focal adhesion kinase (FAK) and vinculin to the {alpha}vß3 integrin and reduced FAK and Src activation in response to VEGF. Addition of NO donors together with transfection of a constitutively active Src construct could circumvent the blockade of VEGF-induced migration by TP; however, neither intervention alone was sufficient. Thus, TP stimulation appears to limit angiogenesis, at least in part, by inhibiting the pro-angiogenic cytokine VEGF. These data further support a role for antagonism of TP activation in enhancing the angiogenic response in tissues exposed to elevated TXA2 levels in which revascularization is important.


Key Words: thromboxane • vascular endothelial growth factor • migration • focal adhesions


*    Introduction
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 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Angiogenesis, the formation of new blood capillaries, is central to normal physiological processes such as wound healing and development, as well as the pathophysiology of multiple diseases, including ischemia and cancer.1 The magnitude of neovascularization is thought to be determined by regulation of the balance between pro- and anti-angiogenic stimuli. Vascular endothelial growth factor (VEGF) has received attention as a key regulator of endothelial cell (EC) function and angiogenesis.2 VEGF is elevated in the serum of patients with acute ischemia from day 3 to 28 postinfarction (peak day 10 to 14) and stimulates neovascularization of the myocardium and development of collateral vessels.3,4 VEGF expression correlates both temporally and spatially with the onset and peak of the angiogenic response in several physiological situations,5,6 and VEGF elicits a strong angiogenic response in a variety of in vivo models.7,8 Antagonism of VEGF signaling inhibits angiogenesis and disease progression, indicating the essential role played by VEGF in ischemia-related and tumor angiogenesis.9–11

Key processes, modulated by VEGF, that contribute to angiogenesis include EC migration, proliferation, differentiation, and apoptosis.12 The angiogenic activity of VEGF is mediated through interaction with VEGFR2/KDR, a member of the Ig-like family of receptor tyrosine kinases.2 The signaling targets used by VEGF to elicit an angiogenic response from EC remain incompletely understood. The acute steps in VEGF-mediated signal transduction include receptor dimerization and subsequent autophosphorylation.2,12,13 The more distal elements in the VEGF signaling cascade include activation of PLC{gamma}, PI-3-kinase, Akt, eNOS, MAPK, focal adhesion kinase (FAK), paxillin, Src, and NCK.12,13 In EC, these pathways culminate in the expression of matrix-degrading enzymes,14 inhibition of apoptosis,15 and regulation of nitric oxide (NO) synthase expression.16 Another major component of the angiogenic response is the alteration of EC–extracellular matrix interactions. Experimental evidence indicates a role for VEGF in regulating cell–matrix interactions as VEGF enhances the expression of {alpha}1ß1 and {alpha}2ß2 integrins.17 Moreover, neutralizing antibodies to multiple integrins, including {alpha}vß3, {alpha}5ß1, and {alpha}vß5, antagonize VEGF-induced neovascularization.18,19 In addition, the integrin {alpha}vß3 has been shown to bind and augment the activity of VEGFR2.20

VEGF-induced angiogenesis in vivo always occurs in a (patho)physiological setting, such as inflammation or ischemia.1 The complex environments of these conditions present to EC multiple factors capable of modifying an angiogenic response. The effects of VEGF on angiogenesis, therefore, must take place in the context of "cross-talk" with modifiers present during disease, yet most studies have only explored the effects of VEGF in isolation. Thromboxane (TXA2) receptors (TP) are G-protein–coupled receptors expressed on many cell types. TXA2 acting through TP is a potent modulator of vascular responses.21 Both TXA2 levels and TP expression are increased in multiple disease states, including ischemia. TP exists as 2 isoforms in humans, TP{alpha}22 and TPß,23 which arise from alternate splicing of a single transcript with TP{alpha}, the product of a retained intron.23 The 2 receptor isoforms have equal affinity for TP ligands as the ligand-binding domains are shared, and the alternatively spliced tail is a poor discriminator of G-protein coupling.24 Thus, the physiologic significance of 2 isoforms in humans is poorly understood, because all other species have only 1 TP isoform.

To determine whether TP stimulation is a modulating factor for VEGF-induced migration and angiogenesis, we tested whether TP ligands interfere with the VEGF signaling, a potent ubiquitous factor involved in the promotion of angiogenesis in multiple (patho)physiological states. We report here that TP stimulation abrogates the angiogenic and chemotactic properties of VEGF on EC. The mechanism of inhibition is highly novel and involves the suppression of NO and FAK/Src activation by TPß. These pathways culminate in reduced focal adhesion formation on {alpha}vß3, which inhibits VEGF-induced angiogenesis. These data establish a (patho)physiological consequence of the existence of 2 TP isoforms in humans and increases our understanding of the paradoxical nature between TXA2 and the regulation of angiogenesis.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The methods used to obtain the results in this article were diverse. Many of the methods used, such as the methods for immunofluorescence and the chemotaxis chambers,25 as well as the cell culture, in vitro differentiation, and immunoblot,26 have all been previously published.

For an expanded Methods section, please see the online data supplement available at http://circres.ahajournals.org.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
TPß Stimulation Abrogates EC Migration and Angiogenesis in Response to VEGF
To assess the effects of TP stimulation on angiogenic potential, we investigated the migration of EC in response to VEGF in the presence and absence of the TXA2 mimetic [1S-(1{alpha}, 2ß(5Z), 3{alpha}(1E, 3R), 4{alpha}]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxab icyclo-[2.2.1]heptan-2yl]-5'-heptenoic acid (IBOP). VEGF-induced robust chemotaxis of HUVEC in the Boyden chamber assay (Figure 1A) in a concentration-dependant manner with maximal effects at ≥20 ng/mL. Costimulation with the TXA2 mimetic IBOP (100 nmol/L) abrogated the VEGF-induced migration (Figure 1A). In vitro, the angiogenic response to VEGF was similarly blunted by the presence of IBOP (Figure 1B). IBOP, at concentrations as low as 50 nmol/L, abrogated both the chemotactic and angiogenic responses to VEGF, with the IC50 for both processes {approx}30 nmol/L. The angiogenic and chemotactic responses to VEGF were also ablated by another TP agonist, U46619, at concentrations ≥200 nmol/L (data not shown). These results demonstrate that TP stimulation is an effective antagonist of VEGF-induced migration and angiogenesis.



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  		Figure 1. TP inhibits the chemotactic and angiogenic effects of VEGF in vitro. The chemotactic response of HUVEC (A) to VEGF (20 ng/mL) was ablated in the presence of IBOP (100 nmol/L) as was the potential to form tubes on matrigel (B). Determination of the IC50 for the inhibition of VEGF-induced migration (C) and angiogenesis (D) by IBOP. Migration and angiogenesis were quantitated after 5 hours and 8 hours, respectively. The data are expressed as mean±SD. # and * Indicate significance (P≤ 0.01) from control (Ctrl) and VEGF alone, respectively.

Human umbilical vein endothelial cells (HUVEC) express both TP{alpha} and TPß by Western and Northern blot analysis (data not shown), yet the role of each TP isoform in modulating angiogenesis is undefined. TP-null EC 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 2A) both in the presence and absence of VEGF. TP{alpha} transfection did not alter EC migration in response to VEGF when treated with 100 nmol/L IBOP; however, the migration of TPß EC was reduced to unstimulated levels (Figure 2A). This reduction was reversed by the thromboxane receptor blocker SQ29548 (Figure 2A). Similar data were observed on matrigel; whereas TPß expression was required for the inhibition of VEGF-induced tube formation (Figure 2B). Stimulation of TP{alpha} receptors produced no inhibition of VEGF-induced angiogenesis. Currently, the only functional segregation reported between TP{alpha} and TPß are differences in the proximal receptor kinetics and the relative preference for G{alpha}h. The anti-angiogenic properties of TPß do not correlate with either the coupling to G{alpha}h or the differences in receptor kinetics (see online Figure I), indicating the antagonism of angiogenesis is independent of both responses. Thus, TPß expression is necessary and sufficient to reduce the angiogenic potential of VEGF-treated EC in the presence of TP ligands through a currently unidentified mechanism. This suggests a divergence in the regulation of angiogenesis by the 2 receptor subtypes.



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  		Figure 2. TPß expression is required for the anti-angiogenic effects of thromboxane receptor ligands. Inhibition of VEGF-induced migration (A) and angiogenesis (B) by the TP ligand IBOP in TXA2 receptor-null EC reconstituted with empty vector ({square}), TP{alpha} ({OV0071}), or TPß ({blacksquare}). Cells were stimulated with IBOP (100 nmol/L), VEGF (20 ng/mL), or both. Control (Ctrl) cells were stimulated with media alone. Results are presented as mean±SD (n=4). # and * Indicate significance (P≤0.01) from control (Ctrl) and VEGF alone, respectively.

The Changes in VEGF Signaling Induced by IBOP Are not an Acute Event in Receptor Signaling
We have previously observed that TPß costimulation can perturb acute events in receptor tyrosine kinase signaling in other systems, such as fibroblast growth factor (FGF)-R1 internalization in response to FGF-2.27 To determine the deficit in VEGF signaling that results in the abrogation of its pro-angiogenic properties, we first examined whether TPß altered signaling cascades that are proximal to VEGFR2. Autophosphorylation of VEGFR2 (Figure 3B) and its subsequent internalization from the membrane (Figure 3A) were only observed in the presence of VEGF and were not altered by costimulation by IBOP. Another signaling event critical to the angiogenic response to VEGF is ERK activation.12 The VEGF-induced increase in ERK phophorylation was also unaltered by IBOP stimulation (Figure 3C). These data indicated that the cross-talk between the 2 receptor systems was specific to a few key pathways and not a general inhibition of VEGF signaling. This point of cross-talk must also be in a downstream pathway because the earliest events in VEGF signaling were preserved in the presence of TXA2 mimetics.



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  		Figure 3. Stimulation of HUVEC with TP does not abrogate acute aspects of VEGFR2 signaling. A, Surface expression of VEGFR2 was used to document receptor internalization by enzyme-linked immunosorbent assay. VEGFR2 internalized in response to VEGF and this was not ablated by IBOP. Results are presented as mean±SD (n=4). #Significance (P≤0.01) from control (Ctrl). B, Similarly, VEGFR2 tyrosine phosphorylation on VEGF stimulation was not altered by the presence of IBOP as determined by immunoprecipitation–Western blotting. C, Downstream activation of ERK by VEGF was not decreased by costimulation with IBOP, indicating that the inhibition of VEGF signaling was not universal. The blots shown are representative of 4 individual experiments.

VEGF-Induced Focal Adhesion Formation Is Abrogated by TPß Costimulation in Human EC
The activation of integrins and their respective signaling pathways is a well-described system used by VEGF to stimulate angiogenesis. We found that VEGF-stimulated significant changes in focal adhesion formation (Figure 4A). Compared with controls, in which focal adhesions were few and small, VEGF increased the average number and the average size of focal adhesions per cell by 4-fold and 3-fold, respectively (Figure 4B). Both of these parameters were reduced to the levels found in unstimulated HUVEC by IBOP costimulation (Figure 4A and 4B).



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  		Figure 4. TP abrogates VEGF-induced focal adhesion formation. A, Focal adhesions were visualized by immunofluorescence staining of HUVEC with an anti-vinculin antibody. Photographs (x1000) are representative of 3 independent experiments. B, The size ({blacksquare}) and number ({square}) of focal adhesions in HUVEC were estimated from photomicrographs using image analysis software. Results are presented as mean±SD (n=4). # and * Indicate significance (P≤0.01) from control (Ctrl) and VEGF alone, respectively. C, Changes in FAK and vinculin association with various integrin subunits were visualized by immunoprecipitation–Western blotting. Autoradiographs are representative of 3 independent experiments.

To determine which integrins were affected by IBOP, we examined the extent to which they participated in the forming of focal adhesions by immunoprecipitating the various integrin subunits and probing the resulting complexes for the presence of FAK and vinculin (Figure 4C). These 2 components of the focal adhesion complex were chosen as they are recruited at different stages of the process of focal adhesion formation. VEGF stimulation increased association of FAK and vinculin to the integrins {alpha}v and ß3, which was subsequently confirmed using a complex-specific antibody against {alpha}vß3 (Figure 4C). No other integrin appeared to be a target for VEGF-mediated focal adhesion formation (Figure 4C). This is consistent with our previously published data.25 However, in the presence of IBOP, the VEGF-mediated increase of the association of FAK and vinculin with {alpha}vß3 did not occur. These data were consistent with the focal adhesion data and further demonstrate that TPß potently regulates multiple VEGF signaling pathways important for angiogenesis.

TPß Stimulation Potently Inhibits the Activation of the 2 Most Critical Pathways for VEGF-Mediated Chemotaxis and Focal Adhesion Formation
The downstream pathways used by VEGF to induce EC chemotaxis and angiogenesis are complex but increasingly well-understood. Two signaling cascades, the PI 3-kinase/NO and Src/FAK pathways, have been described as essential for VEGF-induced chemotaxis and focal adhesion formation. NO acts as an autocrine factor on the EC to regulate many basal processes, including focal adhesion formation28 and permeability,29 but is also essential for VEGF-induced survival, chemotaxis, and angiogenesis.30 Thus, we examined NO production by VEGF-treated EC in the presence and absence of TP stimulation. Figure 5A shows the linear accumulation of NO in conditioned media from VEGF-treated EC. The enhanced accumulation of NO was ablated by costimulation with the TP agonist IBOP (100 nmol/L), resulting in NO levels obtained from unstimulated cells. NO production by VEGF results from a cascade of phosphorylation events resulting in the sequential activation of PI3 kinase, PDK1, Akt, and eNOS. As acute events in VEGFR2 signaling appeared unaltered by IBOP costimulation (Figure 3), we began analysis of this cascade from the point closest to the generation of NO. VEGF stimulation of HUVEC resulted in increased phosphorylation of eNOS (Ser1177) and Akt (Ser473) (Figure 5B), both required for NO production. Akt is activated by PDK-1 after recruitment of both to the membrane by inositol-3,4,5-phosphate (IP3) through their pleckstrin homology domains. PDK1 translocated to the membrane in response to VEGF; however, this was abrogated by TP stimulation (Figure 5B). This result indicated that the generation of IP3 in response to VEGF was abrogated, and subsequently TPß inhibited all downstream signaling (Figure 5B). We determined if PI3 kinase was activated in response to VEGF as it generates the IP3 required for initiating the NO cascade. As can be seen in Figure 5B, VEGF stimulation recruited the p85 subunit of PI3 kinase into a complex with VEGFR2 and caused it to become tyrosine-phosphorylated (a marker of activation). IBOP costimulation did not abrogate PI3 kinase activation, thus the decrease in IP3 levels was not the result of inhibition of IP3 generation.



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  		Figure 5. TP stimulation inhibits the activation of PI-3k/NO and Src/FAK in response to VEGF. A, HUVEC were incubated with media alone, IBOP (100 nmol/L, {circ}), VEGF (20 ng/mL, {square}), or both ({blacksquare}), and NO release (measured as NO2/NO3 content) was quantitated by enzyme-linked immunosorbent assay. VEGF increased the rate of NO accumulation 12-fold more than unstimulated cells. Results are presented as mean±SD (n=4). B, Activation of signaling intermediates in the NO synthesis pathway by VEGF was abrogated by TP costimulation. The proteins assayed are presented in sequential order from most distal to most proximal to VEGFR2. C and D, Changes in overall Tyr phosphorylation (p-Y; C) or at specific Tyr residues (D) in FAK by VEGF with and without IBOP were visualized by immunoblotting. E and F, Similar experiments were also performed to assess site specific phosphorylation of Src tyrosine residues. Loading was determined by reprobing the membranes for nonphosphorylated FAK and Src, respectively. G, Src kinase activity was measured by immune–kinase assay in lysates from HUVEC treated with BOP (100 nmol/L), VEGF (20 ng/mL), or both for 4 hours. H, The VEGF-induced association of Src with VEGFR2 was inhibited by IBOP as determined by immunoprecipitation–Western blotting. Autoradiographs are representative of 3 independent experiments. # and * Indicate significance (P≤0.01) from control (Ctrl) and VEGF alone, respectively.

In addition to this striking inhibition of VEGF-induced NO production, we also found that TPß costimulation ablated signaling through the FAK/Src pathway. Total Tyr phosphorylation of FAK was stimulated by VEGF and abrogated by IBOP costimulation (Figure 5C). This was not the case for FGF-2, indicating the pathway was specific to VEGFR2 signaling (Figure 5C). FAK can be phosphorylated on multiple Tyr residues, each with a distinct function.31 Using site-specific antibodies (Figure 5D) we observed significant increases in phosphorylation at residues Tyr397, the autophosphorylation site, Tyr 576, Tyr861, and Tyr925 in response to VEGF. Costimulation with IBOP (100 nmol/L) ablated the phosphorylation of all the residues in response to VEGF, indicating a universal inhibition of FAK activation.

Src enhances FAK activation and focal adhesion formation in response to VEGF12 by phosphorylation of residue Y576/Y577, which enhances autophosphorylation. Interestingly, IBOP stimulation inhibited FAK phosphorylation at residue Y576/Y577 in response to VEGF. We therefore hypothesized that the deficit in focal adhesion formation lay not at the level of FAK activation but rather 1 level up at the point of Src activation. To assess Src activation, we used phospho-specific antibodies. Src is activated through a mechanism involving phosphorylation on Y416 and derepression of Y527 through a dephosphorylation event.32 VEGF treatment increased Y416 phosphorylation {approx}10-fold, with a concomitant loss of Y527 phosphorylation (Figure 5E). IBOP costimulation ablated the VEGF-induced increase in Y416 phosphorylation and enhanced phosphorylation at residue Y527, lost when VEGF was used alone. This finding was specific to VEGF because the FGF-2 induced phosphorylation of Y416 and dephosphorylation of Y527 still occur in the presence of IBOP (Figure 5F). The Src kinase activity contained in lysates from VEGF-treated HUVEC (Figure 5G) displayed an 11-fold increase over control lysates, consistent with the increase in Y416 phosphorylation observed in Figure 5E. IBOP, however, abrogated the enhanced Src kinase activity (Figure 5G). Thus, in the presence of IBOP, the inability to dephosphorylate Y527 keeps Src in an inactive conformation, the active site occluded by the C-terminus, which prevents FAK activation and focal adhesion formation in response to VEGF.

Src activation by VEGF results from a direct interaction with VEGFR2.12 VEGF treatment, but not media or IBOP alone, resulted in the association of Src with VEGFR2 when VEGFR2 was immunoprecipitated from HUVEC lysates (Figure 5H). This was ablated in the presence of IBOP costimulation. Conversely, the association of PI3 kinase and VEGFR2 is not compromised by IBOP (Figure 5B), indicating the effect is specific to Src. These data suggest that Src recruitment to VEGFR2, the most proximal step in activation, was the source of cross-talk that abrogated FAK-mediated signaling in response to VEGF.

The Combination of NO Donors and a CA–Src Relieve the Blockade of VEGF-Induced Migration Induced by TPß Costimulation in Human EC
We have now identified 2 potential mechanisms by which TPß stimulation could inhibit VEGF-induced migration and angiogenesis. However, which of these pathways was causal in this relationship was not determined. To examine the relationships between the 2 pathways and the inhibition of VEGF’s angiogenic properties by TPß, we circumvented the block of both pathways through either replacing the lost metabolite (through the use of NO donors) or preventing the Src inactivation through transfection of a constitutively active point mutant. Levels of overexpression achieved were {approx}5-fold (Figure 6A). We then used HUVEC overexpressing CA–Src, along with S-nitroso-N-acetylpenicillamine, in the Boyden chamber ({square} in Figure 6B) and tube-forming ({blacksquare} in Figure 6B) assays to examine if the combination could relieve the blockage of VEGF-induced angiogenesis by IBOP. HUVEC chemotaxis was minimally stimulated by CA–Src and not at all by SNAP (200 µmol/L). Use of the NO donor alone did not reverse the inhibition of VEGF-induced migration by IBOP (Figure 6B). The CA–Src construct was equally ineffective at reversing the blockade (Figure 6B). However, when SNAP was added to HUVEC expressing the CA—Src, IBOP-mediated inhibition of VEGF-induced migration was alleviated. This was also true for the tube-formation assay in which only restoration of both pathways simultaneously resulted in the restoration of VEGF angiogenic potential in the presence of IBOP. These results indicated that these 2 pathways are both important for VEGF-induced migration and both need to be active for the process of angiogenesis to occur. Finally, TPß stimulation appears to regulate VEGF-mediated angiogenesis through attacking multiple key signaling pathways, which results in a complex mechanism of checks and balances that ensure the demise of angiogenic potential in VEGF-treated EC.



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  		Figure 6. The angiogenic potential of VEGF-treated EC is restored by circumventing the inhibition of both Src activation and NO production by IBOP. A, Blot indicating the level of Src in HUVEC transfected with the Src-Y527F constitutively active point mutant. {alpha}-Tubulin was used as the loading control. Autoradiographs are representative of 3 independent experiments. B, HUVEC migration ({square}) and angiogenesis ({blacksquare}) in response to VEGF, in the presence and absence of IBOP, were examined in combination with either a NO donor (SNAP, 200 µmol/L) or transfection of a CA–Src construct. Migration and angiogenesis in media alone (Ctrl) were the sources of comparison. Data are expressed as cells or tubes per field (mean±SD, n=3). # and * Indicate significance (P≤0.01) from control (Ctrl) and VEGF.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study, we found that TP ligands abrogated the chemotactic and angiogenic properties of EC in response to VEGF. Further, the inhibition was dependent on the expression of the TPß isoform. These data have important potential implications in settings where release of VEGF and TP ligands coincide, such as tumor growth and ischemia. Our data indicate that in EC expressing the TPß isoform, TXA2 may inhibit the migration of EC toward a source of VEGF, such as an infarcted tissue or tumor, and thus discourage neovascularization by antagonizing VEGFR2 signaling. Currently, the interplay between GPRC and tyrosine kinase receptors has primarily been restricted to the EGF receptor.33 The antagonism of the signaling pathways of VEGF 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 VEGF was linked with the expression of the TPß isoform on EC. The primary deficit in the original report of the TPß transgenic mice was a decrease in placental size with microscopic evidence of ischemia.34 Our present observations concur with these findings and suggest that vessel formation in the developing placenta was most likely suppressed by TPß ligation. Additionally, these observations may explain the dichotomy in the literature over the role of TP ligands in the regulation of angiogenesis. Little is known about the relative importance of both TP isoforms to EC biology in humans. Inhibition of COX-1 or COX-2 results in the abrogation of TXA2 release and decreased neovascularization in small animal models of disease.35 Further, TXA2 synthase overexpression in tumor cells correlates with enhanced angiogenesis and growth rate,36 and blockade of TP with SQ29548 has been shown to abrogate FGF and VEGF migration in vitro and in vivo.37,38 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 EC expressing both isoforms. Thus, the existence of 2 TP isoforms in humans may have implications for its role in vascular disease.

The mechanism by which TPß inhibits VEGF-induced migration and angiogenesis is complex. Focal adhesion re-organization is essential to the angiogenic response to VEGF.39 The relationship between focal adhesion size and migration is biphasic in nature. Small focal adhesions transmit strong propulsive tractions and are highly adhesive whereas large, bright, mature focal adhesions exert weaker forces.40 Thus, the increased FA size in VEGF-treated cells is most likely an attempt to modulate cell–matrix interactions and allow for a chemotactic response. The reduction in FA size correlates with the ablation of migration in cells treated with VEGF and IBOP. The reversion to small highly adhesive complexes in the presence of IBOP probably shifts the balance away from the moderate levels of adhesion required for migration to occur toward a highly adherent cell with such strong cell–matrix interactions that migration cannot occur. In addition, the mechanism of FAK and Src inactivation by TPß is not ubiquitous. In similar experiments, TPß activation did not perturb Src activation in response to FGF-2 (Figure 5), which may reflect fundamental differences in the basic biology of the chemotactic versus chemokinetic migration.

Our data have shown that the activation of both the NO and Src/FAK pathways by VEGF is essential for modulation of focal adhesions and migration. The inability of NO donors or a CA–Src alone to relieve the blockade of VEGF-induced migration raises the question as to whether these pathways function in series or parallel, with both required to sustain a chemotactic response. Reciprocal regulation is an integral aspect of the relationship between the PI3-k/NO and FAK/Src pathways. NO donors and ectopic expression of eNOS stimulate tyrosine phosphorylation of FAK and Src and increase their association through a cGMP-dependent process.41,42 c-Src activation is also an early step in the cGMP-dependent anti-apoptotic actions of NO.43 FAK activity is sensitive to redox conditions and can be activated by changes in redox state.44 NO generation provides a highly reducing environment, which would change the oxidative conditions in the cell and allow for FAK activation. Conversely, Src controls the transcription and stability of eNOS mRNA.45 Src activation by VEGF is essential for generating IP3 formation in response to VEGF,46 and inhibitors of Src kinase activity have been shown to ablate NO production,47 PI3-kinase, and Akt activation48 under basal and VEGF-stimulated conditions. Thus, these 2 pathways, essential for the angiogenic properties of VEGF, appear to function in an integrated manner, with both required for migration.

To conclude, the activation of TPß abrogates the activation of 2 critical pathways, PI3-k/NO and FAK/Src, by VEGF resulting in the absence of migration and impaired angiogenesis. These findings suggest the hypothesis that inhibition of TP receptors, perhaps selective inhibition of the TPß isoform, could enhance myocardial revascularization after infarction.


*    Acknowledgments
 
This work was supported by National Institutes of Health grants HL47032, HL51043, and HL55552 (to J.A.W.) and by a postdoctoral fellowship and scientist development grant from the Heritage affiliate of the American Heart Association (postdoctoral fellowship 0020186T, scientist development grant 0335416T; to A.W.A.). The constitutively active point mutant of Src was a gift from R.G. Pestell.


*    Footnotes
 
Original received January 5, 2004; revision received June 25, 2004; accepted June 25, 2004.


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

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