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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:1764.)
© 2001 American Heart Association, Inc.

Vascular Biology

cAMP Response Element-Binding Protein Mediates Thrombin-Induced Proliferation of Vascular Smooth Muscle Cells

Tomotake Tokunou; Toshihiro Ichiki; Kotaro Takeda; Yuko Funakoshi; Naoko Iino; Akira Takeshita

From the Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.

Correspondence to Toshihiro Ichiki, MD, Department of Cardiovascular Medicine, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, 812-8582 Fukuoka, Japan. E-mail ichiki{at}cardiol.med.kyushu-u.ac.jp

	Abstract

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Abstract—— Thrombin is a potent mitogen for vascular smooth muscle cells (VSMCs) and plays an important role in the progression of atherosclerosis. Although recent reports have suggested that cAMP response element-binding protein (CREB) is necessary for the survival of neuronal cells, the role of CREB in VSMC proliferation is not determined. We examined the role of CREB in thrombin-induced VSMC proliferation and the effect of thrombin on phosphorylation of CREB at Ser133, which is a critical marker for activation by Western blot analysis. Thrombin induced phosphorylation of CREB in a dose-dependent manner. An oligopeptide, SFLLRN, which activates the thrombin receptor, also induced the phosphorylation of CREB. Inhibition of extracellular signal-regulated protein kinase or inhibition of p38 mitogen-activated protein kinase suppressed the thrombin-induced CREB phosphorylation. Inhibition of the epidermal growth factor receptor by AG1478 also inhibited the thrombin-induced CREB phosphorylation. Overexpression of the dominant-negative form of CREB inhibited thrombin-induced c-fos mRNA expression and incorporation of [³H]thymidine and [³H]leucine. These results suggest that CREB-dependent gene transcription plays a critical role in thrombin-induced proliferation and hypertrophy of VSMCs. Transactivation of the epidermal growth factor receptor and 2 mitogen-activated protein kinase pathways are involved in this process. CREB may be a novel transcription factor mediating the vascular remodeling process induced by thrombin.

Key Words: thrombin • cAMP response element-binding protein • epidermal growth factor receptor • mitogen-activated protein kinase

	Introduction

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Thrombin belongs to the multifunctional serine protease family¹ and plays an important role in the blood coagulation cascade through the cleavage of fibrinogen to fibrin. Thrombin also activates intracellular signaling pathways through the thrombin receptor (protease-activated receptor-1 [PAR-1]). Thrombin activates PAR-1 by cleaving its amino-terminal exodomain to unmask a new receptor amino-terminus beginning with the sequence of SFLLRN, which functions as a tethered ligand.² The synthetic oligopeptide SFLLRN activates PAR-1 independently of proteolysis. PAR-1 is a member of the 7-transmembrane domain receptor family. PAR-1 couples with G_q, which activates phospholipase Cß,³ and G_i, which inhibits adenylate cyclase.⁴ Recently, it has been reported that transactivation of the epidermal growth factor receptor (EGF-R) played an important role in thrombin signaling.⁵

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMCs),⁶ and PAR-1 is widely expressed in the atherosclerotic lesion,⁷ suggesting that thrombin may contribute to inflammatory and proliferative changes of the vascular wall, which are believed to be crucial for atherogenesis. Indeed, inhibition of thrombin by heparin⁸ or hirudin⁹ prevented neointimal formation after balloon angioplasty.

cAMP response element (CRE)-binding protein (CREB) is a 43-kDa nuclear transcription factor¹⁰ that was originally found to be activated by cAMP-dependent protein kinase (protein kinase A).¹¹ Phosphorylation of serine residue at 133 (Ser133) is necessary for transcriptional activation. Recent studies have shown that phosphorylation of Ser133 is also mediated by extracellular signal-regulated protein kinase (ERK),¹² p38 mitogen-activated protein kinase (MAPK),¹³ calmodulin-dependent kinase (CaMK),¹⁴ and Akt protein kinase¹⁵ pathways. Phosphorylation of CREB at Ser133 permits an access of the transcriptional coactivator, designated the CREB-binding protein.¹⁶

Overexpression of the dominant-negative CREB transgene induced apoptosis in T cells¹⁷ in response to activation signals, and inhibition of CREB function induced neuronal cell death.¹⁸ These results suggest that CREB is critical for the survival of these cell types. However, these changes were not observed in mice with targeted deletion of the CREB gene.¹⁹ This discrepancy is explained by upregulation of other CREB family genes in CREB knockout mice. Therefore, overexpression of the dominant-negative form of CREB has an advantage over gene deletion for the functional analysis of CREB. In the present study, we examined the role of CREB in thrombin-stimulated VSMCs by adenovirus-mediated overexpression of dominant-negative CREB.

	Methods

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Reagents
DMEM and FBS were purchased from GIBCO-BRL. Thrombin was purchased from Ito Ham Co. One unit per milliliter of thrombin is approximately equivalent to 20 nmol/L. 6-Amino acid peptide (SFLLRN), a PAR-1 agonist, was purchased from Bachem. PD98059 and U0126, inhibitors of ERK kinase, were purchased from Research Biochemicals International and Promega Co, respectively. SB203580 and FR167653, inhibitors of p38 MAPK, were generous gifts from SmithKline Beecham Pharmaceuticals and Fujisawa Pharmaceutical Co, Ltd, respectively. AG1478, an inhibitor of EGF-R, and [Tyr(SO₃H)⁶³]-hirudin fragment (54-65) were obtained from Sigma Chemical Co. All antibodies used in the experiments were obtained from New England Biolabs except for the horseradish peroxidase-conjugated second antibodies (anti-rabbit or anti-mouse IgG, Vector Laboratories Inc). A recombinant adenovirus vector expressing a mutant of CREB (AdCREBM1)²⁰ in which the phosphorylation site at Ser133 was changed to alanine was a gift from Dr Anthony J. Zeleznik (University of Pittsburgh, Pittsburgh, Pa). Unless mentioned otherwise, other chemical reagents were purchased from Wako Pure Chemicals.

Cell Culture
VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats and maintained as described previously.²¹ Passages between 5 and 15 were used. VSMCs were grown to confluence, growth-arrested in DMEM with 0.1% BSA for 2 days, and used for the experiments.

Western Blot Analysis
VSMCs were lysed in sample buffer (5 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 7.6, 1% Triton X-100, 50 mmol/L NaCl, 30 mmol/L sodium phosphate, 50 mmol/L NaF, 1% aprotinin, 0.5% pepstatin A, 2 mmol/L phenylmethylsulfonyl fluoride, and 5 mmol/L leupeptin). Protein concentrations were determined with the bicinchoninic acid protein assay kit (Pierce Chemical Co). Cell lysates (20 µg) were heated at 95°C for 5 minutes, electrophoresed on 12% SDS-PAGE, and transferred to polyvinylidene difluoride membrane (Millipore). The blots were blocked with TBS-T (20 mmol/L Tris-HCl, pH 7.6, 137 mmol/L NaCl, and 0.1% Tween 20) containing 10% nonfat dry milk at room temperature for 1 hour. Phosphorylated CREB at Ser133 was detected by a phospho-CREB antibody (recognizes only phosphorylated form) by enhanced chemiluminescence (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. The membranes were exposed to x-ray film. The membranes were stripped by incubation in buffer containing 62.5 mmol/L Tris-HCl, 2% SDS, and 100 mmol/L 2-mercaptoethanol at 50°C for 1 hour and reprobed with an antibody against CREB (recognizes phosphorylated and nonphosphorylated forms) by the same procedure. The intensity of the bands was quantified with a MacBAS bioimaging analyzer (Fujifilm). Activation of ERK and p38 MAPK was examined with the same method.

Transfection of CRE/Luciferase Fusion DNA Construct to VSMCs
VSMCs (5x10⁵ cells) were prepared in a 6-cm tissue culture dish. After 48 hours, 5 µg of CRE/luciferase fusion DNA (3 copies of the CRE site are located upstream of a TATA-like promoter from the herpes simplex virus thymidine kinase promoter [Clontec Laboratories Inc]) and 2 µg of the LacZ gene driven by the simian virus 40 promoter-enhancer sequence were introduced to VSMCs via the DEAE dextran method according to the manufacturer’s instructions (Promega Corp). After transfection, the cells were cultured in DMEM with 10% FBS for 24 hours, washed twice with PBS, and stimulated with 1 U/mL thrombin for 9 hours in DMEM with 0.1% BSA. Then luciferase activity was measured and normalized by ß-galactosidase activity as described previously.²¹

Infection of AdCREBM1 and AdLacZ
Confluent VSMCs were washed 2 times with PBS and incubated with AdCREBM1 or adenovirus vector expressing LacZ (AdLacZ) for 2 hours at room temperature in PBS under gentle agitation. Then the cells were washed 3 times with PBS, cultured in DMEM with 0.1% BSA for 2 days, and used for the experiments. Multiplicity of infection (MOI) indicates the number of virus per cell added to culture dish. Infection efficiency of adenovirus is almost 100%, as determined by the staining of the ß-galactosidase expressed by AdLacZ (data not shown).

Northern Blot Analysis
Total RNA was prepared according to an acid guanidinium-phenol-chloroform extraction method.²² Northern blot analysis of c-fos mRNA was performed as described previously.²¹ The hybridized membrane was stripped by boiling in 0.5% SDS solution and hybridized to a ³²P-labeled 18S rRNA probe to obtain a reference for the amount of applied RNA. The radioactivity of hybridized bands of c-fos or 18S rRNA was quantified with a MacBAS bioimaging analyzer (Fujifilm).

DNA and Protein Synthesis
After infection with AdCREBM1 or AdLacZ, VSMCs were cultured in DMEM with 0.1% BSA for 2 days. Then the cells were stimulated with thrombin for an additional 48 hours. VSMCs were labeled with [³H]thymidine or [³H]leucine during the last 24 hours of stimulation. The cells were washed with PBS, fixed in 10% trichloroacetic acid, and then washed with a mixture of ethanol and ether (2:1). The cells were lysed in 0.5N NaOH, and the radioactivity of incorporated [³H]thymidine or [³H]leucine was measured with a liquid scintillation counter.

Statistical Analysis
Statistical analyses were performed by 1-way ANOVA and multiple comparison (Fisher) tests if appropriate. A value of P<0.05 was considered significant. Data were expressed as mean±SE.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Phosphorylation of CREB at Ser133 by Thrombin
To investigate whether CREB is activated by thrombin, we performed Western blot analysis by using an antibody that recognizes the phosphorylated form of CREB at Ser133. Phosphorylation of CREB was significantly increased in thrombin-stimulated VSMCs compared with unstimulated cells (Figure 1A). Figure 1B indicates that thrombin dose-dependently increased CREB phosphorylation. Figure 1C shows that an oligopeptide, SFLLRN (10 µmol/L), also induced phosphorylation of CREB at 10 minutes after stimulation, suggesting that the thrombin-induced phosphorylation of CREB is mediated by PAR-1. Hirudin binds to thrombin and inhibits thrombin function. Thrombin preincubated with hirudin for 5 minutes failed to induce CREB phosphorylation (Figure 1D).

View larger version (34K): [in this window] [in a new window]   Figure 1. Phosphorylation of CREB by thrombin. Phosphorylation of CREB was detected by Western blot analysis with use of a phospho-specific CREB antibody. The density of the specific band was scanned and quantified with an imaging analyzer. The ratio of phosphorylated CREB (phospho CREB [p-CREB]) to total CREB is shown. The ratio of untreated cells was designated as 1.0. Results are expressed as mean±SE. *P<0.01 vs control (C [unstimulated cells]). A, VSMCs were stimulated with thrombin (1 U/mL) for the indicated periods (n=4). B, VSMCs were stimulated for 10 minutes with varying concentrations of thrombin (n=5). C, VSMCs were stimulated for 10 minutes with thrombin (10-2 U/mL) or SFLLRN (10 µmol/L). The same results were obtained in other independent experiments (n=3). D, VSMCs were stimulated for 10 minutes with thrombin (10-2 U/mL) that was preincubated with or without hirudin (1 µmol/L) for 5 minutes. The same results were obtained in other independent experiments (n=3).

MAPKs Are Important for Thrombin-Induced CREB Phosphorylation
We examined the pathways responsible for thrombin-induced phosphorylation of CREB. Preincubation with PD98059 (30 µmol/L) partially inhibited the thrombin-induced CREB phosphorylation (Figure 2A). SB203580 (10 µmol/L) also partially suppressed the thrombin-induced CREB phosphorylation (Figure 2A). A combination of PD98059 and SB203580 additively suppressed the thrombin-induced CREB phosphorylation. Figure 2C and 2D indicates that PD98059 and SB203580 almost completely suppressed thrombin-induced the activation of ERK and p38 MAPK, respectively, suggesting that the concentrations of these inhibitors were sufficient. To confirm the role of MAPKs, we used other MAPK inhibitors. U0126 (50 µmol/L) and FR167653 (1 µmol/L) also inhibited the thrombin-induced CREB phosphorylation (Figure 2B). Inhibition of EGF-R by AG1478 (2.5 µmol/L) also inhibited the thrombin-induced CREB phosphorylation (Figure 2E). AG1478 almost completely inhibited thrombin-induced activation of ERK and p38 MAPK (Figure 2E).

View larger version (37K): [in this window] [in a new window]   Figure 2. Effects of inhibitors of MAPKs and EGF-R on thrombin-induced CREB phosphorylation. VSMCs were preincubated with 30 µmol/L PD98059 and/or 10 µmol/L SB203580 (A) or 50 µmol/L U0126 and/or 1 µmol/L FR167653 (B) for 30 minutes and stimulated with thrombin (10-2 U/mL) for 10 minutes. Phosphorylation of CREB (p-CREB, A and B), ERK (p-ERK, C), and p38 MAPK (p-p38, D) was detected by Western blot analysis. VSMCs were preincubated with 2.5 µmol/L AG1478 for 30 minutes and stimulated with thrombin (10-2 U/mL) for 10 minutes (E). Phosphorylation of CREB, ERK, and p38 MAPK was detected by Western blot analysis. The same results were obtained in other independent experiments (n=3).

Activation of CRE-Dependent Transcription by Thrombin
We investigated whether thrombin activated CRE-dependent gene transcription by using a CRE/luciferase reporter construct. As shown in Figure 3, thrombin (1 U/mL) increased luciferase activity by 2.1-fold compared with that of unstimulated cells (P<0.01). The enhancement of luciferase activity by thrombin was significantly (P<0.01) suppressed by PD98059 and/or SB203580.

View larger version (20K): [in this window] [in a new window]   Figure 3. Activation of CRE-dependent transcription by thrombin. A CRE/luciferase fusion DNA (5 µg) was introduced to VSMCs. Forty-eight hours after transfection, the VSMCs were preincubated with PD98059 (30 µmol/L) and/or SB203580 (10 µmol/L) for 30 minutes and stimulated with thrombin (1 U/mL) for 9 hours. Stimulation with forskolin (0.1 and 0.5 µmol/L) for 9 hours was used as a positive control. Then luciferase and ß-galactosidase assays were performed. The luciferase activity was normalized by the ß-galactosidase activity. The fold induction of normalized luciferase activity by thrombin is indicated. Results are expressed as mean±SE (n=4). *P<0.01 vs thrombin-treated cells.

Inhibition of Thrombin-Induced c-fos mRNA Expression by Overexpression of Dominant-Negative Form of CREB
To clarify the role of CREB in thrombin signaling, we overexpressed the dominant-negative form of CREB by an adenovirus vector (AdCREBM1),²⁰ which inhibits CREB function by replacing endogenous CREB with the overexpressed mutant CREB rather than by inhibition of phosphorylation. Although immunoreactivity of CREB was increased in an MOI-dependent manner by AdCREBM1 (Figure 4A), thrombin-induced CREB phosphorylation was not increased. CRE is one of the important cis DNA elements in the c-fos gene promoter.²³ Infection of AdCREBM1 strongly inhibited thrombin-induced c-fos mRNA expression in VSMCs (Figure 4B).

View larger version (33K): [in this window] [in a new window]   Figure 4. AdCREBM1 suppressed thrombin-induced c-fos mRNA expression. A, VSMCs were infected with 10 MOI and 30 MOI of AdCREBM1, which expresses unphosphorylatable CREB, and stimulated with thrombin (10-2 U/mL) for 10 minutes. Phosphorylation of CREB and total CREB expression level were detected by Western blot analysis. The same results were obtained in other independent experiments (n=3). B, VSMCs were infected with AdCREBM1 (30 and 100 MOI) or AdLacZ (100 MOI) and stimulated with or without thrombin (10-2 U/mL) for 30 minutes (n=3). Thrombin-induced c-fos mRNA expression was detected by Northern blot analysis, and the radioactivities of the bands were measured with an imaging analyzer. The radioactivity of c-fos mRNA was normalized with that of rRNA. Results are expressed as mean±SE. NS indicates not significant. *P<0.05; *P<0.01.

Inhibition of Thrombin-Induced DNA and Protein Synthesis by AdCREBM1
We examined the role of CREB for thrombin-induced proliferation and hypertrophy of VSMCs. VSMCs were infected with AdCREBM1, and [³H]thymidine and [³H]leucine incorporation were measured. Incorporation of [³H]thymidine and [³H]leucine in thrombin-stimulated cells was increased significantly by 3.0-fold (P<0.01) and 2.3-fold (P<0.01), respectively, compared with the incorporation in unstimulated cells (Figure 5A and 5B). Infection of AdLacZ (30 MOI) did not affect the thrombin-induced incorporation of thymidine or leucine. However, infection of AdCREBM1 almost completely inhibited the thrombin-induced [³H]thymidine and [³H]leucine incorporation (Figure 5A and 5B). PD98059 and/or SB203580, which inhibited thrombin-induced phosphorylation of CREB, also inhibited the thrombin-induced [³H]thymidine and [³H]leucine incorporation (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 5. Inhibition of thrombin-induced DNA and protein synthesis by AdCREBM1. VSMCs were infected with AdCREBM1 (30 MOI) or AdLacZ (30 MOI) and incubated with or without thrombin (2 U/mL) for 48 hours. [3H]Thymidine incorporation (A) and [3H]leucine incorporation (B) were measured. Results are expressed as mean±SE (n=3). [3H]Thymidine and [3H]leucine incorporation of unstimulated cells was designated as 100%. *P<0.01 vs unstimulated cells.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We have found in the present study that (1) thrombin stimulated CREB phosphorylation and CRE-dependent gene transcription, (2) thrombin-induced phosphorylation of CREB was dependent on EGF-R transactivation and 2MAPK pathways, and (3) overexpression of the dominant-negative form of CREB inhibited thrombin-induced proliferation and hypertrophy of VSMCs. The present study, to the best of our knowledge, is the first report showing that CREB is a critical transcription factor for thrombin-induced proliferation of VSMCs.

Various protein kinases are reported to phosphorylate CREB at Ser133.^11–15 We showed that ERK and p38 MAPK were involved in thrombin-induced CREB activation. We confirmed the role of ERK and p38 MAPK for thrombin-induced CREB phosphorylation by using second inhibitors for ERK (U0126) and p38 MAPK (FR167653). We also examined the effect of H89 (a protein kinase A inhibitor), KN93 (a CaMKII inhibitor), and wortmannin (an inhibitor of phosphatidylinositol 3-kinase that activates Akt/PKB) on thrombin-induced CREB phosphorylation. However, none of these inhibitors affected thrombin-induced CREB phosphorylation. The effect of these inhibitors on thrombin-induced CREB phosphorylation is summarized in the Table.

View this table: [in this window] [in a new window]   Table 1. Effect of Protein Kinase Inhibitors on Thrombin-Induced CREB Phosphorylation

p90RSK-2,¹² downstream from ERK, was reported to phosphorylate CREB, and MAPK-activated protein kinase-2,²⁴ downstream from p38 MAPK, also phosphorylates CREB. Our data suggest that p90RSK-2 and MAPK-activated protein kinase-2 may phosphorylate CREB in response to thrombin. Furthermore, another protein kinase designated mitogen- and stress-activated protein kinase-1 (MSK1),²⁵ which is activated by ERK and p38 MAPK, was reported. Activated MSK1 phosphorylates CREB at Ser133. Suppression of ERK and p38 MAPK is required to suppress the activation of MSK1. Therefore, it may be possible that MSK1 mediates thrombin-induced phosphorylation of CREB. Further study is necessary to determine the kinase that directly phosphorylates CREB downstream from ERK and p38 MAPK.

Transactivation of EGF-R is indispensable for thrombin-induced ERK activation.⁵ Because AG1478 suppressed thrombin-induced ERK and p38 MAPK activation, inhibition of thrombin-induced CREB phosphorylation by AG1478 may be ascribed to the suppression of these MAPK pathways.

Although thrombin induced CREB phosphorylation by severalfold, CRE promoter activity was upregulated by 2.1-fold. The reason for this discrepancy is not clear. However, competition of phosphorylated CREB between CRE sites of endogenous genes and CRE-luciferase plasmid may occur. Most of the phosphorylated CREB may bind to and activate endogenous genes. Therefore, CRE-luciferase activity may not be upregulated to the same extent as the level of CREB phosphorylation.

Thrombin rapidly induced c-fos mRNA expression as previously reported.²³ CRE in the promoter region plays a critical role in the induction of c-fos gene expression in response to mitogens. We showed that overexpression of the dominant-negative form of CREB strongly suppressed thrombin-induced c-fos mRNA expression, confirming the previous results. Also, thrombin-induced incorporation of thymidine and leucine was almost completely blocked by AdCREBM1. Because a number of genes are reported to have a CRE site in the promoter region, it is difficult to identify the target gene(s) that is critically involved in the suppression of VSMC growth by AdCREBM1. CRE may play a critical role in the expression of 1 gene and may be important in the thrombin-induced growth of VSMCs. Alternatively, accumulation of partial inhibition of CRE-dependent gene expression, such as the effect of AdCREBM1 that we observed on thrombin-induced c-fos mRNA expression, may cause strong growth inhibition. In addition to c-fos, 1 of the candidate genes is proliferating cell nuclear antigen (PCNA), an auxiliary factor of DNA polymerase . PCNA is necessary for DNA replication, and the promoter of the PCNA gene contains a CRE site.²⁶ Further study is necessary to identify the critical gene(s) that is inhibited by the dominant-negative form of CREB.

Overexpression of wild-type CREB in VSMCs did not affect basal and thrombin-induced incorporation of thymidine or leucine (data not shown). It was previously reported that the concentration of CREB in the nucleus of PC12 cells was so high that the high-affinity CREs were expected to be nearly saturated.²⁷ CREB was readily detected by Western blot analysis in our VSMCs. Therefore, we assume that our VSMCs may also express a sufficient amount of CREB, and overexpression of CREB did not show any additional effect on basal and thrombin-induced incorporation of thymidine or leucine.

Apart from the central role of the blood coagulation cascade, thrombin is a potent mitogen for VSMCs. Proliferation of VSMCs by thrombin²⁸ requires reactive oxygen species (ROS), as recently reported in platelet-derived growth factor-induced²⁹ and angiotensin II-induced³⁰ mitogenesis. Diphenyleneiodonium, an inhibitor of NAD(P)H oxidase, inhibited thrombin-induced VSMC proliferation and ROS production,²⁸ suggesting that PAR-1 activates NAD(P)H oxidase. We have not examined whether ROS is involved in CREB activation. Rao et al³¹ reported that N-acetylcysteine, an antioxidant, inhibited thrombin-induced ERK and p38 MAPK activation. Therefore, thrombin-induced ROS may regulate CREB activation through MAPKs.

In normal arteries, the thrombin receptor is mainly expressed in the endothelial layer.⁷ In human atheroma, PAR-1 was widely expressed in the areas rich in macrophages and in VSMCs.⁷ PAR-1 expression was induced as early as 6 hours after balloon injury of the carotid artery,³² and the upregulation of PAR-1 expression continued throughout vascular lesion formation for up to 2 weeks. Gallo et al⁹ reported that prolonged and continuous treatment with hirudin suppressed neointimal formation in the balloon injury model of porcine coronary arteries. It has also been reported that mice lacking PAR-1 suffer from less neointimal formation in response to balloon injury of the artery.³³ These results suggest that thrombin and its receptor, PAR-1, are critically involved in the progression of atherosclerosis. We have shown in the present study that CREB is a crucial transcription factor for thrombin-induced mitogenesis and hypertrophy of VSMCs. CREB may be a novel target in the prevention of atherogenesis.

	Acknowledgments

 
This study was supported in part by a grant from the Kaibara Morikazu Memorial Foundation, Fukuoka, Japan; by the Welfide Medicinal Research Foundation, Osaka, Japan; and by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture (Nos. 1177035 and 12877113).

	Footnotes

 
Consulting Editor for this article was Alan M. Fogelman, MD, Professor of Medicine and Executive Chair, Departments of Medicine and Cardiology, UCLA School of Medicine, Los Angeles, Calif.

Received May 7, 2001; accepted August 30, 2001.

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