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(Circulation Research. 2000;86:967.)
© 2000 American Heart Association, Inc.

Cellular Biology

Fluid Shear Stress Induces Lipocalin-Type Prostaglandin D₂ Synthase Expression in Vascular Endothelial Cells

Yoji Taba, Toshiyuki Sasaguri, Megumi Miyagi, Takeo Abumiya, Yoshikazu Miwa, Toshiko Ikeda, Masako Mitsumata

From the Departments of Bioscience (Y.T., T.S., M. Miyagi, Y.M.) and Epidemiology (T.A.), National Cardiovascular Center Research Institute, Osaka; Third Department of Internal Medicine (Y.T., M. Miyagi), University of the Ryukyus School of Medicine, Okinawa; and Department of Pathology (T.I., M. Mitsumata), School of Medicine, Yamanashi Medical University, Yamanashi, Japan.

Correspondence to Toshiyuki Sasaguri, MD, PhD, Department of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan. E-mail sasaguri{at}ri.ncvc.go.jp

	Abstract

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Abstract—Ligands for peroxisome proliferator–activated receptor , such as the thiazolidinedione class of antidiabetic drugs and 15-deoxy-^12,14-prostaglandin J₂ (15d-PGJ₂), modulate various processes in atherogenesis. In search of cells that generate prostaglandin D₂ (PGD₂), the metabolic precursor of 15d-PGJ₂, we identified PGD₂ from culture medium of endothelial cells. To study how PGD₂ production is regulated in endothelial cells, we investigated the role of fluid shear stress in the metabolism of PGD₂. Endothelial cells expressed the mRNA for the lipocalin-type PGD₂ synthase (L-PGDS) both in vitro and in vivo. Loading laminar shear stress using a parallel-plate flow chamber markedly enhanced the gene expression of L-PGDS, with the maximal effect being obtained at 15 to 30 dyne/cm². The expression began to increase within 6 hours after loading shear stress and reached the maximal level at 18 to 24 hours. In contrast, shear stress did not alter the expression levels of PGI₂ synthase and thromboxane A₂ synthase. In parallel with the increase in the expression level of L-PGDS, endothelial cells released PGD₂ and 15d-PGJ₂ into culture medium. These results demonstrate that shear stress promotes PGD₂ production by stimulating L-PGDS expression and suggest the possibility that a peroxisome proliferator–activated receptor ligand is produced in vascular wall in response to blood flow.

Key Words: shear stress • vascular endothelial cells • prostaglandins

	Introduction

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Prostaglandins (PGs) of the J₂ family, including PGJ₂, ¹²-PGJ₂, and 15-deoxy-^12,14-PGJ₂ (15d-PGJ₂), are the metabolites of PGD₂.^{1 2} These cyclopentenone PGs were known as antitumor and antiviral PGs,³ before they were revealed to be naturally occurring ligands for peroxisome proliferator–activated receptor (PPAR), one of the ligand-activated nuclear receptor transcription factors.^{4 5} Among these PGs, 15d-PGJ₂ is the most effective activator for PPAR.

PPAR ligands have been shown to modulate multiple processes in atherogenesis since they were found to promote the differentiation of macrophages, increasing oxidized LDL uptake by stimulating the expression of a scavenger receptor CD36.^{6 7} They also inhibit inflammatory cytokine production in monocytes.⁸ In vascular smooth muscle cells (VSMCs), they prevent migration by inhibiting the expression of matrix metalloproteinase-9.⁹ We reported that PGJ₂ and ¹²-PGJ₂ strongly inhibit proliferation of VSMCs,¹⁰ and we recently found that 15d-PGJ₂ not only arrests the cell cycle but also promotes the differentiation of VSMCs, inducing smooth muscle–specific myosin heavy chains.¹¹ PPAR ligands also have been shown to inhibit angiogenesis in vitro and in vivo.¹²

PGs of the J₂ family can be naturally generated from PGD₂ in the presence of albumin.^{1 2} Although we have detected PGD₂ from the culture medium of vascular endothelial cells in a preliminary study,¹³ it is uncertain that PGD₂ is physiologically produced in the vascular wall. We therefore investigated whether endothelial cells express PGD₂ synthase (PGDS) to produce PGD₂ and, as a consequence, 15d-PGJ₂. In this study, we particularly focused on the role of fluid shear stress, because blood flow is one of the crucial factors that regulate endothelial cell function, and its turbulence may promote atherosclerosis by disturbing homeostasis in vascular wall maintained by endothelial cells.¹⁴

Here we report for the first time that steady laminar shear stress stimulates endothelial cells to produce PGD₂ and 15d-PGJ₂ by upregulating the expression of PGDS.

	Materials and Methods

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Chemicals
PGs were purchased from Cayman Chemical Co. Phorbol 12-myristate 13-acetate (PMA) was from Sigma Chemical Co.

Cell Culture
Vascular endothelial cells, VSMCs, and blood monocytes were isolated and cultured as described.^{15 16 17}

Shear Stress Apparatus
Steady laminar shear stress was loaded on endothelial cells grown on a gelatin-coated polyester sheet (Plastic Suppliers) in a parallel-plate flow chamber as described.¹⁸

Chromatography of Arachidonic Acid Metabolites
Confluent cells were incubated in medium containing 9.25 kBq/mL [¹⁴C]arachidonic acid (Amersham Pharmacia Biotech) at 37°C. After 3 hours, the conditioned medium was collected and acidified to pH 3.0 by adding HCl. Four times the volume of ethyl acetate was then added, and the mixture was centrifuged at 1000g for 10 minutes at 4°C. The upper phase was collected and evaporated under nitrogen. After dissolving with methyl acetate, samples were applied to a silica-gel thin-layer chromatography (TLC) plate (silica gel 60 HPTLC, Merck), which was developed with chloroform/ethyl acetate/ethanol/acetic acid (20:20:4:1, vol/vol). The plate was analyzed for radioactivity with a bioimage analyzer (BAS-2500, Fuji Photo Film Co) or exposed to an x-ray film at -80°C. Radioactive spots were scraped off and re-extracted with ethanol. The extracts mixed with standard PGs were applied to a Cosmosil 5C18 column (Nacalai Tesque) using an HPLC system (Waters) and were eluted with 17 mmol/L H₃PO₄/CH₃CN (7:3, vol/vol). The elution fractions were counted for radioactivity with a scintillation counter (LS5801, Beckman Instruments). Standard PGs were detected at a wavelength of 192.5 nm.

Enzyme Immunoassay (EIA)
One milliliter of culture medium was acidified to pH 3.0 by adding HCl and applied to an Amprep C2 minicolumn (Amersham Pharmacia Biotech). After a wash with water, 10% ethanol, and hexane, PGs were eluted with methyl formate. Next, the eluent was evaporated, and residues were dissolved in the assay buffer included in the EIA kits for PGD₂ and 15d-PGJ₂ (Cayman Chemical Co). The concentrations of PGs were determined using these kits according to the manufacturer’s protocol and using a microplate reader (ImmunoMini NJ-2300, Nalge Nunc International).

Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
RT-PCR was performed as described,¹⁹ using PCR primers synthesized on the basis of the GenBank database.

Northern Blotting
Northern blotting was performed as described,¹⁶ using cDNAs cloned by RT-PCR.

In Situ Hybridization
The riboprobe was synthesized from a template cDNA fragment in the presence of digoxigenin-UTP (DIG) and T3 RNA polymerase (Promega) for antisense or T7 RNA polymerase (Promega) for sense, using a DIG RNA labeling kit (Roche Diagnostics). Fresh autopsy specimens were fixed with 4% paraformaldehyde in PBS overnight at 4°C. Hybridization was carried out by incubating the sections with a denatured DIG-labeled antisense or sense riboprobe (1 µg/mL) in a moist chamber for 16 hours at 42°C. After RNase treatment, the sections were incubated with an anti-DIG antibody conjugated with alkaline phosphatase. Hybridized probes were detected by revealing phosphatase activity with 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate.

Immunohistochemistry
Immunostaining was performed as previously described.²⁰

Statistics
Results are expressed as mean±SD. Statistical significance was assessed by Student t test.

	Results

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Detection of PGD₂ in Endothelial Cell Culture Medium
In search of vascular cells that are able to produce PGD₂, we analyzed arachidonic acid metabolites of VSMCs, endothelial cells, and monocytes. The cells were incubated with [¹⁴C]arachidonic acid for 3 hours in the absence or presence of PMA, and then the metabolites contained in the culture media were analyzed by TLC (Figure 1A). The most abundant product in VSMCs seemed to be PGE₂, but there was no band that corresponded with the authentic standard of PGD₂. In endothelial cells, a weak band corresponding with PGD₂ was found, and its level was elevated in the presence of PMA. Metabolites re-extracted from this band were analyzed by HPLC (Figure 1B). A radioactive peak consistent with authentic PGD₂ was found in the extract. In monocytes, relatively strong bands were found close to authentic PGD₂ (Figure 1A). However, HPLC analysis revealed that they were not PGD₂, given that no radioactivity was found in the fractions in which authentic PGD₂ was eluted (not shown).

View larger version (50K): [in this window] [in a new window]   Figure 1. Endothelial cells produce PGD2. A, Human umbilical artery smooth muscle cells, umbilical vein endothelial cells, and blood monocytes were incubated with [14C]arachidonic acid for 3 hours in the absence or presence of PMA (10 nmol/L). The metabolites in the medium were analyzed by TLC. Positions of authentic standards are also shown. RPMI indicates RPMI 1640; HS, human serum obtained from a healthy volunteer; EC, endothelial cells; and Mo/M, monocytes/macrophages. B, TLC band corresponding with PGD2 (arrowhead) was re-extracted and analyzed by HPLC. Arrow indicates position where an authentic standard PGD2 was eluted.

Shear Stress Stimulates the Production of PGD₂ and 15d-PGJ₂ in Endothelial Cells
Although we detected PGD₂ in culture medium of endothelial cells, the amount of the PG produced under static conditions seemed to be small. Considering that the synthesis of PGI₂ is upregulated by fluid shear stress,²¹ we wondered whether shear stress also stimulates PGD₂ production. After exposing endothelial cells to shear stress (15 dyne/cm²) for 24 hours, we incubated the cells in fresh medium with [¹⁴C]arachidonic acid under static conditions for a further 3 hours and analyzed the PGs released into the medium by TLC (Figure 2). The amount of PGD₂ released from the cells pre-exposed to shear stress was 3 times that generated by the control cells.

View larger version (52K): [in this window] [in a new window]   Figure 2. Shear stress simulates PGD2 production in endothelial cells. After being exposed to shear stress (15 dyne/cm2) for 24 hours, endothelial cells were further incubated in fresh medium containing [14C]arachidonic acid under static conditions for 3 hours. A, Arachidonic acid metabolites in the medium were analyzed by TLC. B, Degrees of intensity of the TLC bands corresponding with PGD2 were quantified using an image analyzer. Values were standardized to those of static controls and are shown as fold increase (n=3). **P<0.01. SC indicates static control; SS, shear stress.

PGD₂ released into culture medium was quantified by EIA. After exposing endothelial cells to shear stress (15 dyne/cm²) for 24 hours, the cells were cultured in fresh medium under static conditions for various times, and the concentrations of PGD₂ were determined (Figure 3A). In cells pre-exposed to shear stress, the concentration of PGD₂ increased linearly until 3 hours had passed. Therefore, to evaluate the activity of cells to synthesize PGD₂, we exposed cells to shear stress (15 dyne/cm²) for various periods and measured the amount of PGD₂ synthesized during the next 3 hours under static conditions (Figure 3B). In sheared cells, the level of PGD₂ began to increase within 6 hours and achieved a plateau in 18 to 24 hours. In contrast, the level was not significantly changed in the static control cells.

View larger version (16K): [in this window] [in a new window]   Figure 3. EIA for PGD2 and 15d-PGJ2. A, After being exposed to shear stress (15 dyne/cm2) for 24 hours, endothelial cells were further incubated in fresh medium under static conditions for the period indicated. Concentrations of PGD2 in medium were measured by EIA. B and C, After being exposed to shear stress (15 dyne/cm2) for the period indicated, endothelial cells were further incubated in fresh medium under static conditions for 3 hours. Concentrations of PGD2 (B) and 15d-PGJ2 (C) were determined by EIA. Values obtained at time 0 were subtracted. Data are mean±SD (n=4). *P<0.05, **P<0.01. , Static control (SC); •, shear stress (SS).

We then measured 15d-PGJ₂ by EIA using the same protocol as in Figure 3B (Figure 3C). In sheared cells, the level of 15d-PGJ₂ also began to increase within 6 hours, but it continued to increase until 24 hours without reaching a plateau, whereas in controls, the increase was small and had reached a plateau by 18 hours.

Shear Stress Induces L-PGDS Expression
To determine the mechanism for the accelerated production of PGD₂ in sheared cells, we examined the effect of shear stress on the expression of PGDS, which mediates the isomeric conversion of PGH₂ to PGD₂.²² RNAs extracted from endothelial cells were analyzed by RT-PCR (Figure 4A). In static cells, the expression level of the mRNA for the lipocalin-type PGDS (L-PGDS) seemed to be very low. However, it was elevated by shear stress depending on the time of loading of the stress. In parallel to the increase in the amount of synthesized PGD₂, the level of L-PGDS mRNA began to increase within 6 hours and reached a maximal level at 18 to 24 hours. However, the mRNA for the hematopoietic-type PGDS (H-PGDS) was not detected in endothelial cells even after exposure to shear stress.

View larger version (38K): [in this window] [in a new window]   Figure 4. Shear stress induces L-PGDS mRNA. After exposing endothelial cells to shear stress (15 dyne/cm2), we extracted total cellular RNAs at the times indicated. A, RNAs (1 µg) were analyzed by RT-PCR for L-PGDS (top) and H-PGDS (bottom). PCR primers used were as follows: human L-PGDS, 5'-AGGTCTCCGTGCAGCCCAACTT-3' and 5'-GTTCCGTCATGCACTTATCGGTTTGG-3'; human H-PGDS, 5'-CCTTGGGCAGAGAAAAAGCAAGA-3' and 5'-AACATGGATCAGCTAGAGTTTGG-3'; and GAPDH, 5'-TCCACCACCCTGTTGCTGTA-3' and 5'-ACCACAGTCCATGCCATCAC-3'. SC indicates static control; SS, shear stress; and PC, positive control (RNAs from human placenta). B, RNAs (10 µg/lane) were analyzed by Northern blotting for L-PGDS and ß-actin. C, Data obtained in panel B were quantified. Expression levels of L-PGDS mRNA normalized to those of ß-actin mRNA were standardized to the value obtained at 0 hours and are shown as fold increase. One representative result of 3 independent experiments is shown.

To quantify the expression levels of L-PGDS, we performed Northern blotting (Figures 4B and 4C). A 0.9-kb band, which has been reported to be human L-PGDS mRNA,²³ was clearly detected, and an additional weak band was detected at 5 to 6 kb. The alterations in their levels were in parallel. There was no increase in the mRNA level when cells were cultured under static conditions. However, the expression level in sheared cells was elevated from 6 hours, and it was 13 times as high as that in the static control cells at 24 hours.

The mRNAs for PGI₂ synthase (PGIS) and thromboxane A₂ synthase (TXS) also were expressed in endothelial cells (Figure 5). However, in contrast to L-PGDS expression, their levels were not influenced by shear stress, although PGIS expression tended to slightly increase 18 to 24 hours after a change of medium.

View larger version (85K): [in this window] [in a new window]   Figure 5. Effect of shear stress on expression of PGIS and TXS. Total cellular RNAs extracted at the times indicated were analyzed for expression of PGIS, TXS, GAPDH, and ß-actin by RT-PCR (A) and Northern blotting (B). PCR primers used were as follows: human PGIS, 5'-TCTCCTCGACTTCTCCTACAGC-3' and 5'-TTGACCGCATAACTCCTCCCCA-3', and human TXS, 5'-AAGATGGGAAGAGGTCAGAGGTG-3' and 5'-CACCCAGTAGAGGAGAAAACGTC-3'. SC indicates static control; SS, shear stress.

The shear stress–induced L-PGDS expression depended on the strength of the stress (Figure 6). Shear stress of relatively high strength (10 to 30 dyne/cm²) markedly stimulated L-PGDS mRNA expression, with the maximal effect being obtained at 15 to 30 dyne/cm². However, the increase in the level of the mRNA was small in cells cultured under shear stress of lower strength (<5 dyne/cm²).

View larger version (44K): [in this window] [in a new window]   Figure 6. Dependence of L-PGDS mRNA expression on shear strength. A, Endothelial cells were exposed to various strengths of shear stress (0 to 30 dyne/cm2) for 24 hours. Total cellular RNAs (10 µg) were analyzed by Northern blotting for L-PGDS and ß-actin. B, Data obtained in panel A were quantified. Expression levels of PGDS normalized to those of ß-actin are shown as fold increase against the value obtained at time 0 (n=3). Statistical analysis was made by comparing the expression levels with that in cells incubated under static conditions for 24 hours. *P<0.05, **P<0.01.

L-PGDS Expression in Arterial Endothelium In Vivo
Finally, we examined whether L-PGDS is expressed in endothelial cells in vivo by in situ hybridization (Figure 7). L-PGDS mRNA was clearly detected in human aortic endothelial cells in all of the autopsy cases studied. Cells in the intima, probably migrating VSMCs, also expressed L-PGDS mRNA, whereas VSMCs in the media were not stained.

View larger version (152K): [in this window] [in a new window]   Figure 7. Expression of L-PGDS mRNA in human aorta. Sections of the aortas obtained from autopsy cases were hybridized with an L-PGDS antisense (a) or its sense (b) riboprobe and stained by incubating with an anti-DIG antibody conjugated with alkaline phosphatase. Arrows indicate L-PGDS–positive endothelial cells; arrowheads, L-PGDS–positive intimal cells. The sections also were immunostained with monoclonal antibodies to human CD34 (QBEND 10, Chemicon International Inc) (c) and human muscle-specific actin (HHF-35, Enzo Diagnostics, Inc) (d) as markers for endothelial cells and VSMCs, respectively. The examinations were performed in 4 cases, and representative samples from a 69-year-old female are shown.

	Discussion

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We found that vascular endothelial cells express L-PGDS mRNA in vitro and in vivo and that shear stress stimulates its expression in vitro, thereby stimulating the production of PGD₂ and 15d-PGJ₂. These results indicated the possibility that a PPAR ligand is produced in vascular wall in response to mechanical stimuli generated by blood flow.

Enzymes that catalyze the isomeric conversion of PGH₂ to PGD₂ are classified into 2 species, namely L-PGDS and H-PGDS.²² Although these 2 enzymes catalyze the same reaction, they have no homology in amino acid sequence.^{23 24} L-PGDS found to be expressed in endothelial cells had been known as ß-trace, a 26-kDa protein that is a major soluble component of cerebrospinal fluid, before it was revealed to function as a PGDS.²⁵ In the meantime, L-PGDS belongs to the lipocalin superfamily that includes various secretory lipid-transporter proteins,²² and therefore, it could operate as a carrier protein for small lipophilic molecules. However, the expression of H-PGDS, which is a glutathione-requiring enzyme mainly expressed in antigen-presenting cells, mast cells, and megakalyoblasts,²² was not detected in endothelial cells.

In the central nervous system, PGD₂ produced by L-PGDS has been suggested to regulate the sleep-awakening cycle.²² Recently, L-PGDS was found in human serum at a concentration of 0.2 to 0.4 µg/mL, and its level in coronary circulation was elevated in patients with ischemic heart disease.²⁶ L-PGDS was expressed in cardiomyocytes, atrial endocardial cells, and VSMCs in the atherosclerotic intima.²⁶ However, our study suggests that endothelial cells may be a major source of L-PGDS and PGD₂ contained in serum.

The factors controlling L-PGDS gene expression are poorly identified except for thyroid hormone receptor. The expression level of L-PGDS mRNA decreases in hypothyroid rat brain.²⁷ Recently it has been revealed that the complex of T₃ and thyroid hormone receptor-ß elevates the L-PGDS promoter activity by binding to a thyroid hormone response element.^{28 29} Further studies are needed to identify the transcription factors involved in the L-PGDS expression in response to shear stress.

The manner in which shear stress regulates PGD₂ synthesis seemed to be different from that for PGI₂. Shear stress markedly upregulated L-PGDS expression, whereas there was no difference in the expression levels of PGIS between the sheared and static cells, despite the fact that shear stress stimulates the release of PGI₂ from endothelial cells.²¹ Shear stress activates enzymes involved in arachidonic acid metabolism, such as phospholipase C,^{30 31} diacylglycerol lipase,³⁰ and cytosolic phospholipase A₂,³² and it also stimulates the expression of cyclooxygenase-2.³³ Therefore, shear stress may stimulate the common pathway for PG synthesis beginning with the liberation of arachidonic acid and ending with the synthesis of PGH₂; however, its downstream may be diversely regulated. L-PGDS activity is upregulated by increasing its expression, whereas PGIS is activated by post-translational modification but not by elevating its expression. The possibility remains that L-PGDS activity also is regulated by protein modification.

It is of clinical interest that L-PGDS is accumulated in coronary circulation of angina patients.²⁶ At this stage, however, it is not clear whether this is a protective response against ischemia or an undesirable factor for patients. PGD₂ reportedly prevents platelet aggregation through activation of adenylate cyclase³⁴ and induces endothelium-dependent arterial relaxation.³⁵ Its metabolites, the J₂ family PGs, display various effects on the process of atherogenesis, as mentioned previously. Most of their effects appear to be preventive for the formation of atherosclerotic lesions and to be beneficial for stabilizing the plaques. However, these PGs could be proatherogenic, first because it is undetermined whether the PPAR ligand–induced differentiation of macrophages is antiatherogenic or proatherogenic.^{6 7} Secondly, 15d-PGJ₂ has been reported to induce endothelial cell apoptosis.³⁶ However, a completely opposite result has been obtained in our study, namely that 15d-PGJ₂ inhibited endothelial cell apoptosis (Y. Taba et al, unpublished data, 1999).

Considering that the formation of atherosclerotic lesions is less frequent in the areas exposed to laminar and high shear stress,¹⁸ it is important to study whether PGs produced by L-PGDS contribute to the shear stress–mediated prevention of atherosclerosis. As far as our in vitro experiments are concerned, laminar shear stress–induced L-PGDS mRNA expression depends on the strength of the stress. Under high shear stress (15 to 30 dyne/cm²) corresponding to the stress in arteries, L-PGDS expression was markedly stimulated, whereas under low shear stress (<5 dyne/cm²) corresponding to that in veins, the stimulation was weak. We examined whether the in vivo expression levels of L-PGDS are different among the areas of human aortas and their major branches where the strength of shear stress is supposed to be different. However, endothelial cells ubiquitously expressed L-PGDS mRNA without significant difference in the expression levels among the areas (not shown). It should be further investigated whether changes in the pattern of blood flow, such as turbulence, influence the expression level and activity of this enzyme.

Intimal cells, which were probably VSMCs, exhibited L-PGDS signals in all of the autopsy cases studied, despite the fact that we did not detect PGD₂ in the culture medium of VSMCs. RT-PCR revealed that cultured VSMCs expressed only a trace of L-PGDS mRNA (not shown). Therefore, the amount of PGD₂ produced in cultured cells may have been too small to be detected by TLC, which is not a very sensitive method. The reason why intimal cells clearly expressed L-PGDS mRNA is unclear. They may be activated under in vivo conditions by unknown stimuli derived from surrounding cells to induce L-PGDS expression.

We hypothesize that laminar shear stress stimulates the production of PGD₂ and 15d-PGJ₂ by upregulating the expression of L-PGDS in endothelial cells and consequently prevents the development of atherosclerotic lesions.

	Acknowledgments

 
This study was supported in part by grants from the Ministry of Health and Welfare (Research Grants for Cardiovascular Diseases 9A-4 and 11C-1); Ministry of Education, Science, and Culture (a Grant-in-Aid for Scientific Research); Science and Technology Agency (Special Coordination Funds for Promoting Science and Technology [Encouragement System of Center of Excellence]); Japan Cardiovascular Research Foundation; and Sankyo Foundation of Life Science.

Received November 29, 1999; accepted March 20, 2000.

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