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(Circulation Research. 2000;87:739.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Reversal of Angiogenesis In Vitro, Induction of Apoptosis, and Inhibition of Akt Phosphorylation in Endothelial Cells by Thromboxane A₂

Yunling Gao¹, Ryoji Yokota¹, Shaoqing Tang, Anthony W. Ashton, J. Anthony Ware

From the Departments of Medicine (Cardiovascular Division) (Y.G., R.Y., S.T., A.W.A., J.A.W.) and Molecular Pharmacology (J.A.W.), Albert Einstein College of Medicine of Yeshiva University, Bronx, NY. Current address for R.Y. is Department of Cardiology, Nara Hospital, Kinki University School of Medicine, Ikoma City, Nara, Japan.

Correspondence to J. Anthony Ware, MD, Cardiovascular Division, Department of Medicine, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Ave, Bronx, NY 10461. E-mail jaware{at}aecom.yu.edu\\ © 2000 American Heart Association, Inc.

	Abstract

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Abstract—Thromboxane A₂ (TxA₂) causes platelet aggregation, vasoconstriction, and inhibition of endothelial cell (EC) migration and prevents vascular tube formation via its specific receptors (TP), of which there are two isoforms (TP and TPß), both expressed in human ECs. In this study, we demonstrate that the TxA₂ mimetic IBOP increases apoptosis of human ECs and inhibits the phosphorylation of Akt kinase, an intracellular mediator required for cell survival. Treatment with IBOP destroyed EC networks formed on a basement membrane matrix in vitro. To distinguish the role of the TP isoforms, each isoform was expressed in TP-null ECs to create TP and TPß ECs. IBOP induced apoptosis and inhibited phosphorylation of Akt kinase in both TP and TPß. IBOP increased cAMP levels in TP but not in TPß. Apoptosis induced by IBOP in TP was not affected by either the adenylyl cyclase activator forskolin or the protein kinase A inhibitor 14-22 amide or H-89, whereas that in TPß was suppressed by forskolin and enhanced by the protein kinase A inhibitor 14-22 amide or H-89, suggesting that the TP isoforms differ in their signal pathways in mediating apoptosis. In conclusion, apoptosis may be the mechanism by which TxA₂-mediated destruction of vascular structures in ECs occurs; although both TP isoforms induce apoptosis, possibly via inhibiting Akt phosphorylation, the signaling differs in each isoform, in that activation of the adenylyl cyclase pathway prevents apoptosis caused by TPß, but not by TP, stimulation.

Key Words: thromboxane A₂ • endothelial cells • apoptosis • Akt kinase • cAMP

	Introduction

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The eicosanoid thromboxane A₂ (TxA₂) is released from activated platelets, monocytes, and damaged vessel wall and causes platelet aggregation, vasoconstriction, and hypertrophy of vascular smooth muscle.^{1 2} TxA₂ and its receptor (TP) are known mediators of unstable coronary disease,³ acute myocardial infarction,⁴ reocclusion after coronary thrombolysis,⁵ pregnancy-induced hypertension,^{6 7} and ischemia of multiple organs.¹ The expression of TPs^{8 9} in the vasculature is widespread, but whether TxA₂ affects the growth or survival of endothelial cells (ECs) is unknown.

TxA₂-induced functions are mediated by TPs that are members of the G protein–coupled receptor family. Thus far, two TP isoforms have been cloned, one from placenta (TP)¹⁰ and one from ECs (TPß).¹¹ These isoforms differ in their alternately spliced cytoplasmic C-terminal tails. These tails have an important role in regulating TP signaling and can be phosphorylated (eg, by the cGMP-dependent kinase).¹² The differences in the cytoplasmic domains have been shown to confer association with different G proteins. Although both receptors are linked to G_q/G₁₁,¹³ TP also interacts with G_s to stimulate a concomitant increase in cAMP after ligand stimulation, whereas TPß causes a ligand-induced decrease in the elevated cAMP level produced by an adenylyl cyclase activator.¹⁴ Recent reports show that the mechanisms and kinetics for desensitization and internalization differ between these two TP isoforms,^{15 16} suggesting that each receptor is linked to a different signal transduction pathway. Whether each receptor triggers a discrete pathway in ECs or mediates distinct EC events is unknown. Recently, we reported that activation of the TP by the TxA₂ mimetics IBOP and U46619 inhibits EC migration in a denudation-injury model and prevents the formation of vascular networks in human umbilical vein ECs (HECs) in vitro,¹⁷ both of which are fundamental events in angiogenesis and in remodeling. Because the balance between positive and negative regulators of EC survival determines vascular formation, we hypothesized that TP stimulation might be involved in the regulation of EC growth or survival. It has been established that activation of the serine/threonine protein kinase Akt, also known as protein kinase B or Rac kinase, is involved in embryonic vascular development and neoangiogenesis, and the phosphatidylinositol 3'-kinase (PI3-kinase)/Akt pathways are the targets of the antiapoptotic effects of many cytokines and angiogenic growth factors.^{18 19}

In this study, we demonstrate that TP stimulation degrades capillary formation in ECs in vitro, and that TP stimulation induces EC apoptosis and inhibits phosphorylation of the survival protein kinase Akt. Furthermore, TP isoforms differ in the signal transduction pathway used to cause apoptosis.

	Materials and Methods

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Materials, Cell Culture, and Stable Lines of Rat Capillary ECs (RECs)
The TxA₂ mimetic IBOP and antagonist SQ29548 were purchased from Cayman Chemical. [³H]SQ29548 was from NEN Life Science. Forskolin, protein kinase A (PKA) inhibitor 14-22 amide, and H-89 were obtained from Calbiochem. HECs were isolated and cultured until the third passage in M199 containing 20% newborn calf serum and 5% human serum.¹⁷ RECs were cultured in M199 with 15% FBS.²⁰ Differential expression plasmids (the GFP/pRc plasmid²¹ ) containing TP or TPß cDNA were constructed. RECs were transfected with vector, TP, or TPß construct using lipofectin (Gibco BRL). Stable clones were selected by neomycin and sorted by GFP fluorescence using a FACScan flow cytometer. Expression of TP was determined by reverse transcription–polymerase chain reaction (RT-PCR) using SuperScript One-Step RT-PCR kit (Gibco BRL). A sense primer, 3AF, AGACAGTGCTGCGAAACCCG (nucleotides 800 to 819), corresponding to the conserved region, and three antisense primers were used, 3BR, TCTTCCAATGTCTGCATGCCC (nucleotides 1315 to 1295) in the TP-specific region, and 3CR, TGTAATCCCAGCTGCTCGGGA (nucleotides 1764 to 1745), and 4AS, GGAGTCTCACTCTGTGGCCCA (nucleotides 1006 to 987), representing the portion of the cytoplasmic tail common to both isoforms after TP-specific region. TP binding sites were determined as described.¹⁵ [Ca²⁺]_i responding to IBOP stimulation was measured using fura 2–based digital imaging microscopy as described.²²

In Vitro Tube Formation Assay
Cells (1.5x10⁵/well) were seeded on Matrigel in 12-well plates for 13 hours to allow tube formation, then stimulated with different concentrations of IBOP alone or plus SQ29548 for an additional 24 hours. The number of enclosed networks of tubes was determined as described.¹⁷ Incomplete networks were not counted.

Analysis of EC Apoptosis
Cells (1x10⁶) were harvested and stained by either fluorescein isothiocyanate–conjugated annexin V (for HECs) or annexin V-biotin/streptavidin-allophycocyanin (for RECs) and propidium iodide dye following the manufacturer’s protocol (PharMingen). Apoptotic cells were determined by flow cytometry. For morphology, HECs on Matrigel were stained in situ with Hoechst 33342 for 10 minutes and fixed with 3.7% formaldehyde in PBS for 15 minutes. Images were captured using a Nikon Eclipse 600 microscope with a Nikon Coolpix 950 digital camera attached. For quantification of apoptotic cells, tubular networks formed on Matrigel were liberated by dispase (1 mL/well) digestion for 2 hours. The dispase was then inactivated by 10 mmol/L EDTA. The dispersed cells were collected by centrifuging at 200g for 5 minutes. To determine DNA content, liberated cells were incubated with 100 µL Hoechst 33342 (1 mmol/L in PBS) for 30 minutes on ice and analyzed by flow cytometry. The number of apoptotic cells was estimated as the percentage of total cells to the left of the G₀/G₁ peak.

Akt Phosphorylation Assay
Akt phosphorylation was analyzed with the phosphospecific Akt (Ser473) antibody (New England BioLabs) by Western blotting.²¹

cAMP Enzyme Immunoassay (EIA) Measurement
cAMP measurements of the cell lysates were performed by an enzyme immunoassay (EIA) according to the manufacturer’s instruction (Amersham).

Statistical Analysis
One- or two-way ANOVA was performed. Post hoc test was performed by Fisher’s least significant difference method. Differences were considered significant at values of P<0.01.

	Results

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Expression of TP Isoforms in HEC and REC Clones
Using RT-PCR, we have determined that HECs express both TP isoforms (Figure 1A, left), consistent with a previous report.⁹ To determine whether TP isoforms mediate different aspects of TP function, each TP isoform was expressed in RECs (which are TP-null) to create separate clones of TP and TPß (Figure 1A, right). Scatchard analysis (Figure 1B) of each TP isoform using [³H]SQ29548 binding revealed that both TP and TPß expressed a similar number of receptors (B_max=125.2±7.8 and 135.1±9.2 fmol/million cells, respectively). There was no significant difference in the K_d between TP and TPß (11.2±1.3 and 10.2±1.7 nmol/L, respectively). Endogenous TP expression in HECs was also confirmed by [³H]SQ29548 binding (B_max=14.6±2.5 fmol/million cells, K_d=9.8±1.2 nmol/L). As a functional index, [Ca²⁺]_i levels after IBOP stimulation were measured in Figure 1C. HEC and REC TP and TPß showed significant increase in [Ca²⁺]_i on IBOP stimulation. Thus, we demonstrated that both TP isoforms were expressed in HECs, and we created ECs that separately express each TP isoform to study what differences, if any, existed in their ability to modulate EC function.

View larger version (27K): [in this window] [in a new window]   Figure 1. Figure 1. Characterization of TP isoforms in HEC and REC clones. A, RT-PCR detection of the TP in HEC and REC clones. TP: the products (516 bp) amplified with 3AF and 3BR in the TP-specific region. TPß: the products amplified with 3AF and 3CR (305 bp) or 3AF and 4AS (206 bp) in the consensus tail region after the TP-specific region. Experiments were repeated 4 times. B, Scatchard analysis of TP and TPß ECs. Both TP and TPß expressed a similar number of receptors and exhibited comparable affinity for [3H]SQ29548. Independent experiments were performed 4 times, and 2 different clones of each isoform were used. C, Effects of IBOP on [Ca2+]i in HEC and REC clones. [Ca2+]i was measured in fura 2–loaded cells stimulated with 100 nmol/L IBOP. *P<0.001 vs vector.

Reversal of Angiogenesis In Vitro and Apoptosis of HECs by TxA₂
HECs were incubated with IBOP on the Matrigel as described in Materials and Methods. Loss of preformed EC tubes after IBOP treatment was observed in a concentration-dependent manner. This degradation of the tubes was specifically prevented by the TP antagonist SQ29548 (Figure 2A), indicating that this effect resulted from stimulation of the TP. A representative experiment of degradation of EC tubes on the Matrigel is shown in Figure 2B. To determine whether IBOP-induced apoptosis might be the cause, we examined whether IBOP-treated ECs demonstrated evidence of apoptosis. IBOP increased HEC apoptosis 2-fold after 24 hours (Figure 3A, left) and >3-fold after 48 hours (Figure 3A, right), as determined by flow cytometry. The TP antagonist SQ29548 (Figure 3) also blocked these effects. Apoptosis of HECs from Matrigel was determined morphologically by Hoechst 33342 stain and also by flow cytometry. IBOP-treated HECs from Matrigel did not form tubes and exhibited a random arrangement of cells with either condensed or fragmented nuclei (Figure 3B), which were considered apoptotic, and the number of apoptotic cells was increased 55% compared with untreated cells (Figure 3C). Thus, IBOP mediated the degradation of preformed capillary tubes in vitro and induced apoptosis in HECs.

View larger version (48K): [in this window] [in a new window]   Figure 2. Figure 2. IBOP-induced degradation of HEC tubes formed in vitro. A, HECs were seeded for 13 hours to allow tube formation, then treated with IBOP alone or IBOP with SQ29548 (10 µmol/L) for 24 hours. The number of networks was counted 24 hours after stimulation. Results shown are mean±SE. Five independent experiments were performed. P<0.01 vs 0 nmol/L; *P<0.01 vs +SQ29548. B, Top, Decrease in networks corresponding to the increase in concentration of IBOP. Bottom, Suppression of the IBOP effect by SQ29548. Representative of 5 different experiments.

View larger version (22K): [in this window] [in a new window]   Figure 3. Figure 3. Induction of HEC apoptosis by IBOP. A, HECs were cultured in the presence or absence of serum to provide the negative or positive control for apoptosis. HECs were cultured in 2% BSA with or without IBOP (100 nmol/L) for 24 hours (left) or 48 hours (right). After stimulation, cells both in the media and on the bottom of the dishes were collected. Apoptotic cells were determined by flow cytometry for annexin V and propidium iodide staining. Results shown are mean±SE of 3 independent experiments with 3 different HEC preparations. #P<0.01 vs with serum; *P<0.01 vs control. B, HECs were incubated on Matrigel for 13 hours to form tubes, then stimulated with IBOP for 24 hours and stained with Hoechst 33342 in situ. Control cultures (left) formed tubes between islands of monolayered HECs. Arrows denote proliferating cells at ends of the tube. IBOP-treated HECs (right) exhibited the absence of tube formation and random arrangement of cells. Arrows denote cells with either condensed (C) or fragmented nuclei (F). Results are representative of 3 individual experiments. C, Cells in the Matrigel were stained with Hoechst 33342 and collected after dispase digestion and analyzed by flow cytometry. The sub–G1 peak on the generated histograms (left) was used to quantify the percentage of apoptotic cells present on Matrigel (right). The data are shown as the mean±SE from 5 experiments. *P<0.001 vs control.

Reversal of Angiogenesis In Vitro and Apoptosis in REC TP and TPß by TxA₂
To determine whether the TP isoforms had different effects in IBOP-induced tube degradation and apoptosis, we tested whether IBOP causes tube degradation and apoptosis in only TP ECs or TPß ECs or both. IBOP degraded formed tubes and induced apoptosis in both TP and TPß ECs, which was specifically blocked by SQ29548 (Figures 4 and 5). Vector-transfected cells did not show tube degradation or increased apoptosis with IBOP. Thus, these results suggest that either TP isoform can mediate IBOP-induced reversal of angiogenesis in vitro and EC apoptosis.

View larger version (73K): [in this window] [in a new window]   Figure 4. Figure 4. IBOP-induced degradation of tubes formed in vitro via TP or TPß. A, RECs were seeded for 13 hours to form tubes, then treated with IBOP (100 nmol/L) alone or IBOP with SQ29548 (10 µmol/L) for 24 hours. The number of networks was counted 24 hours after stimulation. Results shown are mean±SE. *P<0.01 vs IBOP or IBOP+SQ29548. Three independent experiments were performed. B, Representative pictures show the decreases in networks in TP and TPß ECs treated with IBOP, and the effects of IBOP were suppressed by SQ29548.

View larger version (23K): [in this window] [in a new window]   Figure 5. Figure 5. Induction of apoptosis by IBOP in TP or TPß ECs. RECs were serum-deprived for 2 days, then stimulated with or without IBOP (100 nmol/L) or IBOP with SQ29548 (5 µmol/L) for 24 hours in serum-free medium. Tumor necrosis factor- (TNF-) (100 U/mL) and cycloheximide (CHX) (2.5 µg/mL) were added as a positive control for apoptosis. After stimulation, cells both in the media and on the bottom of the dishes were collected. Apoptotic cells were determined as described in Materials and Methods. Results shown are mean±SE of 3 independent experiments. #P<0.001 vs control or IBOP in vector; *P<0.01 vs control or IBOP with SQ29548 in TP and TPß.

TxA₂ Inhibits Phosphorylation of Akt Kinase
Given that the Akt signal pathway is an important determinant of EC survival, we determined whether IBOP induces apoptosis by counteracting serum-induced Akt activation. Akt phosphorylation was analyzed by Western blotting with a specific phospho-Akt antibody, which has been shown to correlate with its enzyme activity.¹⁹ In the presence of serum, apoptosis is inhibited and Akt is phosphorylated and activated. Stimulation with IBOP in HECs (Figure 6A) and in RECs expressing either TP isoform (Figure 6B) markedly decreased the amount of phosphorylated Akt. TP antagonist SQ29548 specifically blocked the dephosphorylation of Akt by IBOP. These results suggest that IBOP induces apoptosis in ECs, probably via inhibiting the Akt signal transduction pathway.

View larger version (42K): [in this window] [in a new window]   Figure 6. Figure 6. Inhibition of Akt phosphorylation by IBOP in HECs and in TP or TPß RECs. HECs (A) and RECs (B) were cultured in the presence of serum to activate Akt maximally, then treated with or without IBOP (100 nmol/L) in serum-free medium for 20 minutes. SQ29548 (5 µmol/L) was added 20 minutes before IBOP. Western blotting was performed with the anti–phospho-Akt antibody (top). The blots were then reprobed with anti–Akt antibody as a loading control (bottom). Representative of 3 different experiments.

Effect of Adenylyl Cyclase Stimulation or Inhibition on Apoptosis Induced by TxA₂
Previous reports suggested that the TP isoforms have distinct effects on cAMP metabolism,¹⁴ and changes in cAMP levels are associated with apoptosis in different kinds of cells.^{23 24 25 26} Thus, we determined whether the cAMP-dependent pathway contributes to IBOP-induced apoptosis by each TP isoform. Figure 7 demonstrates a differential response in cAMP levels after IBOP in each of the ECs. TP ECs showed a concentration-dependent increase in cAMP levels, whereas neither TPß ECs nor vector ECs showed a significant change in cAMP levels. To test whether modification of the cAMP pathway affects apoptosis in either TP or TPß ECs, cells were treated with either the adenylyl cyclase activator forskolin or the cell-permeable myristoylated PKA inhibitor 14-22 amide and H-89. Neither forskolin nor the two PKA inhibitors affected IBOP-induced apoptosis in TP cells (Figure 8A). On the other hand, forskolin suppressed IBOP-induced apoptosis, and both of the PKA inhibitors enhanced IBOP-induced apoptosis in TPß cells (Figure 8B). These results suggest that the inhibition of adenylyl cyclase by TPß stimulation may have an important role in inducing EC apoptosis, but that TP stimulation, although it affects cAMP metabolism, may induce apoptosis by a separate mechanism.

View larger version (14K): [in this window] [in a new window]   Figure 7. Figure 7. Differential response in cAMP levels to IBOP in TP and TPß ECs. Cells were incubated with IBMX (0.1 mmol/L) for 10 minutes and with various concentrations of IBOP for an additional 10 minutes, and the cAMP levels were measured by EIA as described in Materials and Methods. Data points are mean±SE from 4 replicate wells of a single experiment. The results were confirmed in 2 additional experiments. *P<0.01, TP vs 0 nmol/L, TPß, and Vector.

View larger version (20K): [in this window] [in a new window]   Figure 8. Figure 8. Effect of forskolin, PKA inhibitor (PKAI), and H-89 on IBOP-induced apoptosis in TP or TPß ECs. Cells were serum-deprived for 2 days, then stimulated with different reagents as indicated for 24 hours in serum-free medium. TNF- (100 U/mL) and CHX (2.5 µg/mL) were added as a positive control for apoptosis. After stimulation, cells both in the media and on the bottom of the dishes were collected. Apoptotic cells were determined by flow cytometry for annexin V and propidium iodide staining. A, Forskolin (FSK) (10 µmol/L), PKAI (1 µmol/L), and H-89 (1 µmol/L) had no effect on IBOP (100 µmol/L)-induced apoptosis in TP ECs. B, FSK decreased and PKAI and H-89 increased IBOP-induced apoptosis in TPß ECs. Three experiments were performed using 2 different clones from each isoform. #P<0.001 vs vector control; *P<0.01 vs TP control; P<0.01 vs TPß control and IBOP+FSK.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
TxA₂ contributes to thrombosis and ischemia by causing aggregation of platelets and vasoconstriction,¹ and TxA₂ levels are elevated during those states.^{3 6} Thus, any effects that TxA₂ has on endothelial function or angiogenesis would be relevant in patients with ischemia. In this study, we demonstrated that in vitro tube formation in HECs was reversed with the TxA₂ mimetic, an effect that resulted from its interaction with TP and may reflect EC apoptosis. These results suggest that TxA₂ may prevent and reverse angiogenesis via EC apoptosis during conditions such as myocardial infarction or myocarditis in which TxA₂ is formed and released from platelets or macrophages.²⁷ By suppressing angiogenesis, TxA₂ might also affect the vascular remodeling process.

What is the mechanism(s) by which TP stimulation induces apoptosis and reverses vascular tube formation? One possibility is that EC tubes on Matrigel require growth factors, such as basic fibroblast growth factor (FGF), for their maintenance and survival. Reversal of angiogenesis was recently noted in HECs after deprivation of basic FGF, which was followed by apoptosis.²⁸ Increasing evidence of crosstalk between G protein and tyrosine kinase receptors²⁹ suggests that one of the possible mechanisms for our observations is that TxA₂-derived signals may interact with and inhibit such a growth factor survival signaling pathway.

A second possibility, not necessarily mutually exclusive, is based on the effect of TxA₂ on Akt. Akt is a growth factor–regulated serine/threonine kinase that contains a pleckstrin homology (PH) domain and is activated by a mechanism involving PI3-kinase. Binding of PI3-kinase products to the PH domain results in translocation of Akt to the plasma membrane where it is activated by phosphorylation by upstream kinases including PI3-kinase. Activated Akt provides a universal survival signal to protect cells from apoptosis induced by various stresses.^{19 30} In this study, we have demonstrated that IBOP markedly reduces the phosphorylation of Akt in ECs. Phosphorylation of Akt correlates with its kinase activity, which, in turn, is proportional to its ability to inhibit apoptosis and promote survival.^{19 31} Thus, these results suggest that TxA₂ may induce apoptosis by inhibiting the Akt signal pathway.

As noted above, human TP exists as at least two isoforms (TP and TPß). Both isoforms are expressed in a number of tissues and cells including platelets, placenta, and ECs. To test the differential role of TP isoforms in mediating EC apoptosis, we expressed each isoform in RECs, which have no native functional TP. Both TP and TPß ECs showed enhanced apoptosis and decreased phospho-Akt level after IBOP stimulation, suggesting that both TP isoforms mediate EC apoptosis, although it is not clear how these isoforms are regulated in vivo.

The next hypothesis that we tested is that the TP isoforms induced apoptosis using different intracellular mediators. Each TP isoform uses a signaling pathway with some features that are shared, and at least some that differ. Hirata et al¹⁴ reported that in human platelets TP was functionally coupled to G_s whereas TPß was coupled to G_i. Thus, in addition to differences in desensitization¹⁵ and internalization,¹⁶ the TP isoforms have contrasting effects on cAMP mobilization.

There are data to suggest that these changes in cAMP may be related to the ability of TxA₂ to induce apoptosis; in tissues other than ECs, such changes in cAMP levels have been linked to this process. No consistent direction for the association of cAMP changes with apoptosis has been established. In some cases, increased cAMP promotes,^{23 24} and in others prevents,^{25 26} apoptosis. In vascular ECs, TxA₂ causes vascular EC damage (as estimated by adenine release), and a phosphodiesterase inhibitor, which increases cAMP levels, prevents this TxA₂-induced damage, although whether the effects of TxA₂ resulted from apoptosis in that study is unknown.³² In the present study, we sought to determine whether PKA inhibition could be responsible for the apoptosis in TPß ECs, or whether PKA activation could mediate IBOP-induced apoptosis in TP ECs. The adenylyl cyclase activator forskolin suppressed and the PKA inhibitors increased TxA₂-induced apoptosis in TPß, suggesting that the inhibition of PKA (a decrease of the cAMP level) may promote apoptosis in these cells. On the other hand, the PKA inhibitors did not block TxA₂-induced apoptosis in TP, nor was such apoptosis enhanced by forskolin. These results indicate that PKA inhibition induced by the TPß isoform might mediate the proapoptotic effect of TP, but that PKA activation induced by the TP might not be related to apoptosis in ECs. These data suggest that stimulation of each TP isoform may induce apoptosis via a different downstream pathway, only one of which (TPß) appears to be associated with cAMP.

In disease states such as myocardial infarction and pregnancy-induced hypertension, an elevated number of TPs has been found in platelets,^{4 7} and there are also increased levels of circulating TxA₂.^{3 6} Our finding that TxA₂ damages ECs suggests that the eicosanoid might further aggravate these diseases by lessening the thromboresistant and vasodilatory properties of ECs. In addition, TxA₂ might prevent and reverse angiogenesis or collateral development in ischemic hearts, a process that protects hearts from further ischemia and promotes recovery.³³ Thus, we speculate that a TxA₂ receptor blocker and/or TxA₂ synthase inhibitor might promote angiogenesis and improve the process of remodeling of the tissue over several weeks.

In conclusion, we demonstrate TP-mediated reversal of tube formation in vitro and also a possible mechanism for this phenomenon: TP-mediated apoptosis of HECs at least partially through dephosphorylation of Akt kinase. TP isoforms mediate apoptosis through at least two separate signal transduction pathways. These observations suggest that TP stimulation of ECs may have an important effect on pathophysiological conditions marked by TxA₂ release that requires vascular growth or repair.

	Acknowledgments

 

This work was supported by HL47032 and HL51043 from the National Institutes of Health. Dr Yokota was partly supported by the program JSPS Fellowship for Research at Centers of Excellence Abroad under the sponsorship of the Japan Society for the Promotion of Science. We thank Drs Weixin Zhao and George J. Christ for measuring the intracellular calcium concentrations.

	Footnotes

 
¹ Both authors contributed equally to this study.

Received May 23, 2000; revision received August 14, 2000; accepted August 29, 2000.

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