This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, S. Articles by Iwao, H. Search for Related Content PubMed PubMed Citation Articles by Kim, S. Articles by Iwao, H. Pubmed/NCBI databases Compound via MeSH Substance via MeSH

(Circulation. 1998;97:1731-1737.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Angiotensin Blockade Inhibits Activation of Mitogen-Activated Protein Kinases in Rat Balloon-Injured Artery

Shokei Kim, MD; Yasukatsu Izumi, MD; Masahiko Yano; Akinori Hamaguchi; Katsuyuki Miura, MD; Shinya Yamanaka, MD; Hitoshi Miyazaki, PhD; ; Hiroshi Iwao, MD

From the Department of Pharmacology (S.K., Y.I., M.Y., A.H., K.M., S.Y., H.I.), Osaka City University Medical School, Osaka, Japan, and the Gene Experiment Center and Center for Tsukuba Advanced Research Alliance (H.M.), University of Tsukuba, Ibaraki, Japan.

Correspondence to Shokei Kim, MD, Department of Pharmacology, Osaka City University Medical School, 1–4-54 Asahimachi, Abeno, Osaka 545, Japan. E-mail kims{at}msic.med.osaka-cu.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—The effect of balloon injury on the arterial signal transduction pathway has not been examined. In vitro studies show that extracellular signal-regulated kinases (ERKs) and c-Jun NH₂-terminal kinases (JNKs), belonging to the mitogen-activated protein kinase (MAPK) family, play a critical role in the activation of transcription factor activator protein-1 (AP-1) and cell proliferation or apoptosis. However, the activation and role of MAPKs in vascular diseases in vivo remain to be determined. Therefore, we examined the effect of balloon injury on arterial MAPKs and the possible role of angiotensin II.

Methods and Results—Arterial JNK and ERK activities were measured by in-gel kinase assay. AP-1 DNA binding activity was determined by gel mobility shift analysis. After balloon injury of rat carotid artery, JNK (p46JNK and p55JNK) and ERK (p44ERK and p42ERK) activities were increased as early as 2 minutes, reached their peak (6- to 18-fold) at 5 minutes, and thereafter rapidly declined to control levels. JNK and ERK activations were followed by a 3.9-fold increase in arterial AP-1 DNA binding activity, which contained c-Jun and c-Fos proteins. Arterial JNK activation at 2 or 5 minutes was remarkably suppressed by E4177 (an angiotensin AT1 receptor antagonist) and cilazapril (an ACE inhibitor). E4177 also prevented activation of ERKs by suppressing their tyrosine phosphorylation, whereas cilazapril failed to prevent such activation. The increased AP-1 DNA binding activity was significantly inhibited by both E4177 and cilazapril.

Conclusions—Arterial JNKs and ERKs are dramatically activated by balloon injury associated with the activation of the AP-1 complex. These MAPK activations, followed by AP-1 activation, are mediated at least in part by the AT1 receptor. Thus, activation of JNKs and ERKs may be responsible for balloon injury–induced neointima formation.

Key Words: angiotensin • balloon • signal transduction • muscle, smooth • remodeling

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Arterial balloon injury, which causes endothelial denudation and stretching of the medial smooth muscle cells, leads to progressive neointimal thickening.^{1 2 3} This arterial repair process, characterized by vascular smooth muscle cell proliferation,^{1 2} apoptosis,^{4 5} or migration,³ has been shown to be associated with significant changes in numerous gene expressions, such as immediate-early genes, growth factors, and extracellular matrix components.^{6 7 8} Generally, the gene expression is controlled by the intracellular signal transduction pathway (protein kinase cascade), indicating that the characterization of the cellular signal transduction pathway activated by balloon injury is essential to elucidate the molecular mechanism of neointima formation. However, in contrast to detailed studies on gene expression, the effects of balloon injury on cellular protein kinases have not been examined.

ERKs^{9 10} and JNKs^{11 12 13} are protein serine/threonine kinases and belong to the MAPK family. ERKs and JNKs are regulated by different upstream activators and play a central role in cell proliferation or apoptosis¹⁴ and the regulation of various transcription factors such as AP-1^{15 16 17} and numerous gene expressions. However, previous reports on the regulation and function of MAPKs have largely come from in vitro studies using cultured cells, and the in vivo role of MAPKs remains unclear.¹⁸

In rat balloon-injured artery, both an angiotensin AT1 receptor antagonist and an ACE inhibitor are well known to prevent neointima formation by suppressing the proliferation of vascular smooth muscle cells.^{19 20 21} In the present study, we examined MAPK activity in rat balloon-injured artery and obtained the first evidence that JNKs and ERKs are dramatically activated by balloon injury mediated by the AT1 receptor.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Drugs
Cilazapril, an ACE inhibitor, was donated by Nippon Roche, Ltd (Tokyo, Japan). E4177, a specific and potent nonpeptide AT1 receptor antagonist,^{22 23} was provided by Eisai Co, Ltd (Tokyo, Japan).

Balloon Injury
All procedures were in accordance with institutional guidelines for animal research. Ten- to 11-week-old male Sprague-Dawley rats (Clea Japan, Tokyo) weighing 350 to 400 g were used in the present study. Rats were fed standard laboratory chow (MF, Oriental Kobo) and given tap water ad libitum. The present study was performed on 479 rats.

The first set of experiments (n=217 rats) were performed to examine the effects of balloon injury on arterial JNK and ERK activity and arterial AP-1 DNA binding activity. For balloon injury, rats were anesthetized with sodium pentobarbital (40 mg/kg IP), and endothelial denudation of the left common carotid artery was carried out by three passages of a Fogarty 2F balloon catheter (Baxter Healthcare), as previously described.⁶ For examination of ERK and JNK activities at 2, 5, 15, and 60 minutes and 3 and 24 hours after balloon injury, the carotid arteries were perfused via the left ventricle with PBS (pH 7.4) containing 2.5 mmol/L EDTA, 2 mmol/L ß-glycerophosphate, 10 mmol/L NaF, 1 mmol/L sodium orthovanadate (Na₃VO₄), and 100 µg/mL PMSF at 4°C at a flow rate of 40 mL/min for 1 minute. After perfusion, injured left common carotid artery and noninjured right common carotid artery (control) were immediately excised, placed on PBS precooled at 4°C, dissected from adherent fat and connective tissues on ice, then frozen in liquid nitrogen and stored at -80°C until use. Extreme care was taken to be certain that the arteries were not stretched on dissection. For examination of AP-1 DNA binding activity at 1, 3, 6, and 24 hours after balloon injury, bilateral common carotid arteries were collected in the same manner as for examination of ERK and JNK activity and stored at -80°C.

The second set of experiments (n=262 rats) were undertaken to examine the effects of an ACE inhibitor and an angiotensin AT1 receptor antagonist on ERK and JNK activity and AP-1 DNA binding activity in balloon-injured artery. Rats were separated into three groups, including (1) vehicle-treated group (control group; 88 rats), (2) E4177-treated group (20 mg · kg^-1 · d^-1; 87 rats), and (3) cilazapril-treated group (10 mg · kg^-1 · d^-1; 87 rats). Preliminary experiments showed that E4177 (20 mg · kg^-1 · d^-1) and cilazapril (10 mg · kg^-1 · d^-1) prevented neointima formation after balloon injury to a similar extent. Therefore, in the present experiments, we used these drugs at the above-mentioned doses. Cilazapril and E4177, suspended with 5% gum arabic solution, were given to rats by gastric gavage once a day from 3 days before balloon injury until the end of the experiments. The control group of rats (vehicle-treated rats) were given an equal volume of gum arabic solution in the same manner. For balloon injury, rats were anesthetized with sodium pentobarbital (40 mg/kg IP), and balloon injury of the left common carotid artery was carried out as described above. The injured left carotid artery and noninjured right carotid artery were collected as described above at 2 and 5 minutes after balloon injury for the measurement of JNKs and ERKs and at 3 hours for the measurement of AP-1 DNA binding activity. Arterial tissues were stored at -80°C until use.

Preparation of Arterial Protein Extracts for Protein Kinase Assay
Arterial protein extracts were prepared from the pooled tissue from five to six rat carotid arteries in each group to minimize animal-to-animal and procedural variability. Arteries, pooled from five to six rats, were homogenized on ice with polytron homogenizer (PCU-11, Kinematica AG) in lysis buffer (20 mmol/L HEPES) (pH 7.2), 25 mmol/L NaCl, 2 mmol/L EGTA, 50 mmol/L NaF, 1 mmol/L Na₃VO₄, 25 mmol/L ß-glycerophosphate, 0.2 mmol/L DTT, 1 mmol/L PMSF, 60 µg/mL aprotinin, 2 µg/mL leupeptin, and 0.1% Triton X-100). After incubation at 4°C for 30 minutes, the homogenates were sonicated (Sonifier 250, Branson Ultrasonics Co) on ice for 1 minute and centrifuged at 10 000g at 4°C for 30 minutes. The protein concentrations of the supernatants were measured with a protein assay kit (Pierce) and stored at -80°C until protein kinase assay.

Measurement of Arterial JNK and ERK Activity
JNK and ERK activities were measured by use of the in-gel kinase method, as previously described in detail.²⁴ GST-c-Jun(1–79) and MBP were used as the substrate of JNKs and ERKs, respectively. In brief, samples of arterial protein extracts (10 µg), denatured in Laemmli sample buffer, were electrophoresed on SDS-polyacrylamide (12%) gel containing 0.1 mg/mL of GST-c-Jun(1–79) for JNK assay or 0.5 mg/mL of MBP for ERK assay. After electrophoresis, protein kinases in the gels were denatured by guanidine-HCl and renatured in Tris-HCl (pH 8.0), and the gels were incubated with (-³² P)ATP, washed extensively, dried, and subjected to autoradiography, as described in detail elsewhere.²⁴ The densities of autoradiograms were measured by use of a bioimaging analyzer (BAS-2000, Fuji Photo Film Co).

Identification of JNKs and ERKs by Immunoprecipitation With Specific Antibodies
To confirm that arterial JNK and ERK activities can be specifically measured by in-gel kinase assay, we performed in-gel kinase assay of arterial extracts immunoprecipitated with specific antibodies. Specific antibodies used were as follows: polyclonal rabbit IgG(c-17) specifically recognizing both p46JNK and p55JNK; polyclonal rabbit anti-p44ERK IgG(c-16); and polyclonal rabbit anti-p42ERK IgG(c-14). All antibodies were purchased from Santa Cruz Biotechnology, Inc. Normal rabbit IgG (control) was purchased from Vector Laboratories, Inc. Arterial protein extract (50 µg of protein) was preabsorbed with 10 µL of recombinant protein A-agarose (50% vol/vol) (Upstate Biotechnology) at 4°C for 2 hours. After centrifugation at 10 000g at 4°C for 15 minutes, the supernatants were incubated with each specific antibody (1 µg each) or normal rabbit IgG (1 µg) at 4°C for 2 hours, and 20 µL of recombinant protein A-agarose (50% vol/vol) was added, followed by incubation at 4°C for 12 hours. After centrifugation at 800g for 10 minutes, the pellets were washed four times with lysis buffer containing 0.5 mol/L NaCl. Finally, the pellets were suspended with 25 µL of lysis buffer, boiled for 5 minutes in Laemmli sample buffer, and centrifuged, and the resulting supernatants were electrophoresed on 12% SDS–polyacrylamide gel containing 0.2 mg/mL of GST-c-Jun or 0.5 mg/mL of MBP and subjected to in-gel kinase assay for JNKs or ERKs, as described above.

Western Blot Analysis
By using rabbit polyclonal phospho-specific ERK antibody (New England Biolabs, Inc) recognizing tyrosine-phosphorylated forms (active forms) of p44ERK and p42ERK, we measured arterial phosphorylated ERK proteins with Western blot analysis. Arterial protein extracts (10 µg protein), prepared as described above, were boiled for 5 minutes in Laemmli sample buffer, then electrophoresed on an SDS–polyacrylamide gel (12%), and the separated proteins were electrophoretically transferred to Hybond-PVDF membranes (Amersham Life Sciences). Complete protein transfer to the membrane was ensured by staining the gels with Coomassie blue. Nonspecific background was blocked by incubating the membrane with 5% bovine serum albumin in TBS-T at 4°C overnight. The membrane was then incubated with phospho-specific ERK antibody (1:1000 dilution) for 1 hour at room temperature, washed 4 times with TBS-T, and then incubated with horseradish peroxidase–conjugated donkey anti-rabbit immunoglobulin (Amersham) at a dilution of 1:5000 in TBS-T. After a further washing with TBS-T, the membrane was treated with ECL reagent (Amersham), and chemiluminescence was detected by exposure to Hyperfilm-ECL. The intensity of the bands was measured by use of a Macintosh LC-III computer with an optical scanner (EPSON GT-8000, Seoko), using the public domain NIH Image program.

Gel Mobility Shift Assay
For gel mobility shift assay, seven to eight rat carotid arteries were pooled to obtain one sample. Arteries were homogenized in 0.4 mL of 20 mmol/L HEPES (pH 7.9) containing 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1.5 mmol/L MgCl₂, 20% glycerol, 10 mmol/L NaF, 1 mmol/L Na₃VO₄, 0.2 mmol/L DTT, 20 mmol/L ß-glycerophosphate, 0.5 mmol/L PMSF, 60 µg/mL aprotinin, and 2 µg/mL leupeptin, incubated on ice for 15 minutes, and centrifuged at 15 000 rpm at 4°C for 10 minutes. The resulting supernatant was assayed for protein concentrations and stored at -80°C until use. The detailed procedure of the gel mobility shift assay has been described previously.²⁴ In brief, the samples of arterial protein extracts (10 µg protein) were incubated with 10 fmol of a ³² P-labeled oligonucleotide probe containing the consensus AP-1 binding sequence (5'-CGCTTGATGACTCAGCCGGAA-3') at room temperature for 20 minutes in 20 µL of the binding buffer, consisting of 20 mmol/L HEPES (pH 7.9), 0.2 mmol/L EDTA, 0.2 mmol/L EGTA, 80 mmol/L NaCl, 0.3 mmol/L MgCl₂, 1 mmol/L DTT, 0.2 mmol/L PMSF, 6% glycerol, and 2 µg of polydeoxyinosinic-deoxycytidylic acid (poly[dI-dC]; Pharmacia) as a nonspecific competitor. For competition experiments, a mutant AP-1 oligonucleotide competitor (5'-CGCTTGATGACTTGGCCGGAA-3') was also used. The DNA-protein complexes were electrophoresed on 4% nondenaturing polyacrylamide gels, and the gels were then dried, subjected to autoradiography, and analyzed with the use of a bioimaging analyzer (BAS-2000), as described previously.²⁴ As a positive control sample of AP-1, we used nuclear extracts from raf-1–transformed rat fibroblasts stimulated with phorbol ester (3611-RF-phorbol), known to be rich in AP-1 (Santa Cruz Biotechnology, Inc).

Supershift assays were performed with rabbit polyclonal anti–c-Fos IgG raised against the amino acids 128 to 152 portion of c-Fos and rabbit anti–c-Jun IgG raised against the amino acids 247 to 263 portion of c-Jun (Santa Cruz Biotechnology, Inc). Each antibody (1 µg each) was added to the samples after the initial binding reaction between the arterial protein extracts and ³² P-labeled consensus AP-1 oligonucleotide; the reaction was allowed to occur at room temperature for 1 hour and subjected to electrophoresis, as described above.

Statistical Analysis
Data are expressed as mean±SEM. Statistical significance was determined with one-way ANOVA, followed by Duncan multiple-range test (SuperANOVA, Abacus Concepts). Differences were considered statistically significant at a value of P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
JNK and ERK Activity in Arterial Extracts
As shown by in-gel assay with GST-c-Jun (Figure 1A) used as a substrate, it was apparent that the 46-kD and 55-kD kinase bands in crude arterial extracts were due to p46JNK and p55JNK, respectively. In-gel assay with MBP (Figure 1B) showed that the 42-kD and 44-kD kinase bands corresponded to p42ERK and p44ERK, respectively. Thus, in this study, JNK and ERK activities in crude arterial extracts were successfully measured by using an in-gel kinase assay.

View larger version (18K): [in this window] [in a new window]   Figure 1. Identification of JNK and ERK activity in crude arterial extracts. A, In-gel kinase assay with GST-c-Jun as a substrate was carried out on crude arterial extracts (lane 1), the immunoprecipitates with anti-JNK IgG recognizing both p46JNK and p55JNK (lane 2), and the precipitates with normal rabbit IgG (lane 3). B, In-gel kinase assay with MBP as a substrate was performed on crude arterial extracts (lane 1), the immunoprecipitates with both anti-p44ERK IgG and anti-p42ERK IgG (lane 2), and the precipitates with normal rabbit IgG (lane 3). In both A and B, there was no band of protein kinase activity in the sample treated with normal rabbit IgG (lane 3). The positions of molecular mass markers are indicated by 97.4 kD, 66 kD, 46 kD, and 30 kD. K indicates kilodalton.

Time Course of Arterial JNK and ERK Activities After Balloon Injury
As shown by autoradiograms in Figure 2A, arterial JNKs consisted of two isoforms, p46JNK and p55JNK, and the majority of arterial JNK activity was due to p46JNK. Compared with noninjured artery, arterial p46JNK and p55JNK activities were increased by 3.6- and 2.2-fold, respectively, as early as 2 minutes after balloon injury and reached peak levels (17.9- and 6.0-fold, respectively; P<.01) at 5 minutes. Although p46JNK and p55JNK activities remained increased by 3.1-fold (P<.01) and 2.4-fold (P<.05), respectively, at 15 minutes, their activities rapidly decreased and returned almost to control levels at 60 minutes. At 24 hours after balloon injury, conversely, arterial p46JNK activity was decreased to 49.1% of control value (P<.05).

View larger version (27K): [in this window] [in a new window]   Figure 2. JNK (A) and ERK (B) activity at 0, 2, 5, 15, and 60 minutes and 3 and 24 hours after balloon injury of rat carotid artery. Upper panels indicate representative autoradiograms showing the activities of p46JNK and p55JNK (A) and p44ERK and p42ERK (B) from two different samples at 0, 2, 5, and 15 minutes after balloon injury, determined by in-gel kinase assay. The mean value of each JNK or ERK activity from noninjured artery (0 minute) is defined as 1. Each bar represents mean±SEM (n=3 to 4). P<.05, *P<.01 vs control (0 minute).

As indicated by autoradiograms in Figure 2B, arterial ERKs were composed of two isoforms, p44ERK and p42ERK. Like the time course of JNK activity, p44ERK and p42ERK activities were increased by 8.4- and 10.7-fold (P<.01), respectively, at 2 minutes after balloon injury and peaked (10.3- and 14.0-fold, respectively; P<.01) at 5 minutes. Thereafter, activity of both ERKs rapidly declined but remained higher (P<.01) than control values at 3 hours after injury. At 24 hours after injury, conversely, p44ERK activity was significantly lower than control (48% of control value; P<.01), and p42ERK also tended to be lower than control (although not statistically significant).

Effects of E4177 and Cilazapril on Arterial JNK Activity
Figure 3 illustrates the effects of E4177 (AT1 receptor antagonist) and cilazapril (ACE inhibitor) on arterial JNK activity at 2 and 5 minutes after balloon injury. Both drugs prevented the increase in p46JNK and p55JNK activity by >86% and 80% at 2 and 5 minutes (P<.01), respectively, after injury. There was no significant difference between E4177 and cilazapril in the inhibitory effects of JNK activation at either time point.

View larger version (32K): [in this window] [in a new window]   Figure 3. Effects of E4177 and cilazapril on arterial JNK activity at 2 and 5 minutes after balloon injury. Upper panels show representative autoradiograms of JNK activity from two different samples from each group. Veh, E4177, and Cilaza indicate balloon-injured artery from rats treated with vehicle, E4177, and cilazapril, respectively; non-inj indicates noninjured artery. Each bar represents mean±SEM (n=5). The mean value of p46JNK and p55JNK activity in noninjured artery is expressed as 1. *P<.01 vs vehicle.

Effects of E4177 and Cilazapril on ERK Activity and Tyrosine Phosphorylation of ERKs
Figure 4 indicates the effects of E4177 and cilazapril on arterial ERK activity at 2 and 5 minutes after balloon injury. At 2 minutes, E4177 tended to suppress p44ERK or p42ERK activation (although not statistically significant). At 5 minutes (peak point), E4177 significantly prevented the activation of p44ERK and p42ERK by 42% and 47% (P<.01), respectively. On the other hand, cilazapril did not significantly inhibit the activation of p44ERK or p42ERK at 2 or 5 minutes after balloon injury.

View larger version (28K): [in this window] [in a new window]   Figure 4. Effects of E4177 and cilazapril on arterial ERK activity at 2 and 5 minutes after balloon injury. Upper panels show representative autoradiograms of ERK activity from two different samples from each group. Abbreviations as in Figure 3. Each bar represents mean±SEM (n=5). The mean value of p44ERK and p42ERK activity in noninjured artery is expressed as 1. *P<.01 vs vehicle.

To examine whether the inhibition of arterial ERK activation by E4177 was due to the inhibition of tyrosine phosphorylation of ERK, we specifically determined tyrosine-phosphorylated p44ERK and P42ERK contents in arterial extracts at 5 minutes after balloon injury with Western blot analysis using specific antibody recognizing only tyrosine-phosphorylated p44ERK and p42ERK. Figure 5 shows that tyrosine phosphorylation of p44ERK and p42ERK was significantly increased in injured artery and that E4177 but not cilazapril significantly prevented tyrosine phosphorylation of both ERK isoforms.

View larger version (31K): [in this window] [in a new window]   Figure 5. Tyrosine-phosphorylated p44ERK and p42ERK contents in arterial extracts at 5 minutes after balloon injury. Tyrosine-phosphorylated p44ERK and p42ERK were specifically measured by Western blot analysis with specific antibody recognizing only tyrosine-phosphorylated p44ERK and p42ERK. Abbreviations as in Figure 3. Each bar represents mean±SEM (n=5). The mean value of tyrosine-phosphorylated p44ERK and p42ERK in noninjured artery is expressed as 1. P<.05, *P<.01 vs vehicle.

Time Course of Arterial AP-1 DNA Binding Activity After Balloon Injury and Effects of E4177 and Cilazapril
As shown in Figure 6, the results obtained with the gel mobility shift assay of 3611-RF, used as a positive control for AP-1, confirmed that specific AP-1 DNA binding complex could be successfully detected by our present method. Furthermore, the major band (band A) in rat carotid arterial extracts (lanes 1 to 3 in Figure 6) had nearly the same mobility as the AP-1 DNA complex in the positive control (3611-RF). In both carotid arterial extracts (lanes 1 to 3) and 3611-RF (lanes 4 to 6), the use of 10 fmol of a labeled AP-1 oligonucleotide probe gave less nonspecific binding (bands B and C) than the use of 90 or 40 fmol of an AP-1 probe. Therefore, all gel mobility shift assays examining carotid arterial AP-1 DNA binding were performed with the use of 10 fmol of a labeled AP-1 probe.

View larger version (82K): [in this window] [in a new window]   Figure 6. Validity of gel mobility shift assay using a labeled AP-1 oligonucleotide probe. In lanes 1 to 3, carotid arterial extracts (10 µg protein), collected at 3 hours after balloon injury, were subjected to gel mobility shift assay using different concentrations of a labeled AP-1 probe (90, 40, and 10 fmol in lanes 1, 2, and 3, respectively). In lanes 4 to 14, nuclear extracts (10 µg protein) from raf-1–transformed rat fibroblasts (3611-RF), which are known to be rich in AP-1, were subjected to gel mobility shift assay. The detailed method is described in "Methods." The major band (corresponding to position A) in 3611-RF was effectively competed for by addition of 10-, 40-, 100-, and 200-fold excess of a cold AP-1 oligonucleotide (lanes 7, 8, 9, and 10, respectively) but not by addition of 200-fold excess of a cold mutant AP-1 oligonucleotide (lane 11). Furthermore, as shown by arrows, this major band in 3611-RF supershifted with the addition of anti–c-Fos IgG (lane 12) or anti–c-Jun IgG (lane 13) but not with control IgG (lane 14). These results showed that the major band (corresponding to position A) in 3611-RF indeed represented specific AP-1 complex containing both c-Fos and c-Jun, confirming the validity of our assay. Gel mobility shift assay of carotid arterial extracts (lanes 1 to 3) showed one major band (band A) and two minor bands (bands B and C). The major band (band A) in arterial extracts had the same mobility as specific AP-1 DNA complex in 3611-RF (positive control).

We further characterized band A in arterial extracts in Figure 6. As shown in Figure 7A, the band designated with a half bracket (corresponding to band A in Figure 6) was efficiently competed for by increasing concentrations of a cold AP-1 oligonucleotide but not by a mutant AP-1. Furthermore, the addition of anti–c-Fos or anti–c-Jun antibody to the binding reaction produced supershifted complexes. Thus, the band designated with a half bracket (Figure 7A) had nearly the same characteristics as the specific AP-1 band in 3611-RF (a positive control) with regard to electrophoretic mobility, competition with cold competitors, and supershift with specific antibodies, thereby confirming that this band indeed represented specific AP-1 DNA binding activity.

View larger version (60K): [in this window] [in a new window]   Figure 7. Specificity of AP-1 DNA binding activity from carotid artery (A) and time course of arterial AP-1 DNA binding activity after balloon injury (B). A, Arterial protein extracts, collected at 3 hours after balloon injury, were incubated with a 32P-labeled AP-1 consensus oligonucleotide probe in the absence of unlabeled AP-1 oligonucleotide probe (-), and in the presence of 10-, 40-, 100-, and 200-fold molar excess of unlabeled AP-1 probe (X 10, X 40, X 100, and X 200, respectively) and 200-fold molar excess of unlabeled mutant AP-1 probe (X 200 mutant AP-1). Furthermore, supershift assays were performed with anti–c-Fos IgG (Anti-c-Fos), anti–c-Jun IgG (Anti-c-Jun), or nonimmunized rabbit IgG (Normal IgG). The half bracket indicates specific AP-1 DNA binding complexes. NS indicates nonspecific binding; F, free probe; and unlabeled AP-1 oligo., unlabeled AP-1 oligonucleotide competitor. B, Representative autoradiogram of gel mobility shift assay of arterial AP-1 binding activity (top) and the value of arterial AP-1 DNA binding activity (bottom) at 0, 1, 3, 6, and 24 hours after balloon injury. Each bar represents mean±SEM (each, n=3). The mean value of AP-1 binding activity in noninjured artery (time 0) is expressed as 1. P<.05, *P<.01 vs time 0.

As shown in Figure 7B, arterial AP-1 DNA binding activity was increased by 3.9-fold (P<.01) at 3 hours after injury and gradually decreased thereafter.

As shown in Figure 8, treatment with E4177 and cilazapril prevented the increase in arterial AP-1 DNA binding activity at 3 hours after balloon injury by 47% (P<.01) and 36% (P<.01), respectively.

View larger version (42K): [in this window] [in a new window]   Figure 8. Effect of cilazapril and E4177 on arterial AP-1 DNA binding activity at 3 hours after balloon injury. Arterial AP-1 DNA binding activity at 3 hours after balloon injury was compared among rats treated with vehicle (Veh), cilazapril (Cilaza), and E4177. Left, Representative autoradiogram of gel mobility shift assay of two different samples from each group. For gel mobility shift assay, seven to eight rat carotid arteries were pooled and extracted to obtain one sample. The half bracket indicates specific AP-1 binding complexes. NS indicates nonspecific binding; F, free probe. Right, Each bar represents mean±SEM (each, n=4). The mean value in vehicle is defined as 100.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Cells respond to extracellular stress or stimuli by activating intracellular signal transduction pathways, which cause the changes in various gene expressions. These molecular changes finally lead to the modification of cell function, including cellular phenotypic changes, or cell growth or apoptosis. Previous studies demonstrate that numerous growth-associated genes responsible for vascular remodeling, such as proto-oncogenes, growth factors, or extracellular matrices, are activated in balloon-injured rat artery,^{6 7 8} indicating the contribution of the altered gene expressions to neointima formation after balloon injury. However, the signal transduction pathway underlying the changes in gene expression in balloon-injured artery has not been examined, which encouraged us to examine the activation of MAPKs in vivo. In the present study, using in-gel kinase assay, we successfully measured arterial MAPK activity and obtained the first evidence that both JNKs and ERKs are rapidly and dramatically activated in balloon-injured artery.

Accumulating evidence on the in vivo effects of AT1 receptor antagonists and ACE inhibitors indicates that angiotensin II, via the AT1 receptor, plays a critical role in the development of various vascular diseases induced by balloon injury,^{19 20 21} hypertension,^{21 25} or diabetes.²³ However, the signal transduction pathway associated with the vascular protective effects of angiotensin II blockade in vivo remains to be determined. In vitro studies of cultured vascular smooth muscle cells show that ERKs are activated by angiotensin II.^{26 27} Furthermore, very recently, angiotensin II has been shown to activate JNKs in cultured hepatocytes²⁸ and neonatal rat cardiac myocytes,²⁹ although the effect of angiotensin II on JNKs in cultured vascular smooth muscle cells has not been reported. Therefore, to examine the possible contribution of the AT1 receptor to the activation of ERKs and JNKs in balloon-injured artery, we examined the effects of an AT1 receptor antagonist and an ACE inhibitor. In the present study, both the AT1 receptor antagonist and the ACE inhibitor significantly inhibited the activation of JNKs in injured artery, demonstrating that angiotensin II, via the AT1 receptor, is responsible for balloon injury–induced arterial JNK activation. Furthermore, in-gel kinase assay and Western blot analysis showed that the AT1 receptor antagonist also significantly inhibited arterial ERK activation at its peak time point (5 minutes) after balloon injury by suppressing the tyrosine phosphorylation of ERKs. Thus, our present work provided the first evidence that balloon injury–induced activation of JNKs and ERKs is at least in part mediated by the AT1 receptor. Furthermore, in light of the fact that JNKs and ERKs have different upstream signaling cascades and substrate specificities,^{9 18 30} the AT1 receptor seems to be responsible for the activation of multiple signaling cascades in the balloon-injured artery.

It has been believed that JNKs or ERKs play a central role in the formation of transcription factor AP-1 complex.¹⁵ JNKs, which are the only potent activator of c-Jun protein, increase c-Jun transactivational activity by their phosphorylation or induce c-jun mRNA expression.^{11 12 13} Furthermore, JNKs can also induce c-fos mRNA expression.¹⁷ On the other hand, unlike JNKs, ERKs cannot activate c-Jun protein or induce c-jun mRNA expression,¹⁶ although they can induce c-fos mRNA expression by phosphorylating Elk-1/TCF transcription factors.¹⁵ Previously, we and other groups of investigators reported that arterial mRNA expression of c-jun and c-fos is significantly enhanced 30 to 60 minutes after balloon injury. However, it remains to be determined whether or not the increased arterial mRNAs for c-jun and c-fos induced by balloon injury can lead to the increase in DNA binding activity of the AP-1 complex. Our present study, using gel shift analysis, provided the first evidence that AP-1 DNA binding activity, which contained c-Jun and c-Fos proteins, is significantly increased in balloon-injured artery. AP-1 is involved in the expression of numerous genes responsible for cell proliferation³¹ and tissue remodeling, such as collagenase,¹⁸ endothelin-1,³² or transforming growth factor-ß,³³ by binding the AP-1 consensus sequence present in their promoter region. Therefore, it is likely that the activation of AP-1 is implicated in intimal thickening after balloon injury.

Our present data on gel shift analysis of AP-1 activity, taken together with our previous findings that the AT1 receptor antagonist prevents the induction of c-fos and c-jun mRNAs in balloon-injured artery,⁶ demonstrate that the inhibition of c-fos and c-jun gene expression by the AT1 receptor antagonist is associated with suppression of the activation of the AP-1 complex. Of note are the observations that the inhibitory effect of the ACE inhibitor on arterial AP-1 DNA binding activity was comparable to that of the AT1 receptor antagonist, although the ACE inhibitor suppressed JNKs but not ERKs. Homodimers of c-Jun proteins or heterodimers of c-Jun and c-Fos proteins can form a stable AP-1 complex.^{15 31} On the other hand, unlike c-Jun, c-Fos cannot form homodimers and therefore needs c-Jun to form the AP-1 complex.³¹ Furthermore, the expression and activation of c-Jun protein are significantly induced by JNKs but not by ERKs.^{15 16} These findings suggest that the suppression of arterial AP-1 activity by the AT1 receptor antagonist and ACE inhibitor in balloon injury might be mediated, at least in part, by the suppression of JNK activation and that JNKs might be responsible for AP-1 activation, although our present work provided no direct evidence of this.

The present study did not enable us to elucidate the reason for the differential effects of the AT1 receptor antagonist and ACE inhibitor on ERKs. A previous report³⁴ clearly showed that the inhibition of neointima formation in balloon-injured artery by an ACE inhibitor was at least partly mediated by increased bradykinin accumulation. Furthermore, in vitro investigations indicate that bradykinin causes the activation of ERKs.^{35 36} Therefore, our present work suggests that the lack of a decrease in arterial ERK activity by an ACE inhibitor might be partly due to the increased bradykinin accumulation and that both the ACE inhibitor and the AT1 receptor antagonist prevent neointima formation with different effects on the intracellular signal transduction cascades. However, further study is needed to elucidate our proposal.

In conclusion, we obtained the first evidence that JNKs and ERKs, the two main subgroups of the MAPK family, are rapidly and dramatically activated in balloon-injured artery, and that this is associated with activation of the transcription factor AP-1 complex. The activation of JNKs and ERKs in balloon-injured artery is at least partially mediated by the angiotensin AT1 receptor. We propose that the activation of MAPKs may be involved in vascular remodeling after balloon injury. However, further work is needed to determine whether our present observations apply to human atherosclerosis, because the balloon-injury model has limited relevance for human atherosclerosis.

	Selected Abbreviations and Acronyms

 

AP-1 = activator protein-1 AT1 = angiotensin II type 1 DTT = dithiothreitol ERK = extracellular signal-regulated kinase GST = glutathione S-transferase JNK = c-Jun NH2-terminal kinase MAPK = mitogen-activated protein kinase MBP = myelin basic protein TBS-T = Tris-buffered saline (pH 7.6) containing 0.1% Tween 20

	Acknowledgments

 
This work was supported in part by a grant-in-aid for scientific research (09670101 and 09470527) from the Ministry of Education, Science, and Culture. We are grateful to Dr Masahiko Hibi (Osaka University Medical School) for providing GST-c-Jun(1–79) plasmid, and Eriko Gomi and Kazuko Tsukahara for in-gel kinase assay and Western blot analysis.

Received September 19, 1997; revision received November 12, 1997; accepted November 23, 1997.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Clowes AW, Reidy MA, Clowes MM. Kinetics of cellular proliferation after arterial injury, I: smooth muscle cell growth in the absence of endothelium. Lab Invest. 1983;49:327–333.[Medline] [Order article via Infotrieve]

2. Clowes AW, Reidy MA, Clowes MM. Mechanisms of stenosis after arterial injury. Lab Invest. 1983;49:208–215.[Medline] [Order article via Infotrieve]

3. Clowes AW, Schwartz SM. Significance of quiescent smooth muscle migration in the injured rat carotid artery. Circ Res. 1985;56:139–145.[Abstract/Free Full Text]

4. Isner JM, Kearney M, Bortman S, Passeri J. Apoptosis in human atherosclerosis and restenosis. Circulation. 1995;91:2703–2711.[Abstract/Free Full Text]

5. Perlman H, Maillard L, Krasinski K, Walsh K. Evidence for the rapid onset of apoptosis in medial smooth muscle cells after balloon injury. Circulation. 1997;95:981–987.[Abstract/Free Full Text]

6. Kim S, Kawamura M, Wanibuchi H, Ohta K, Hamaguchi A, Omura T, Yukimura T, Miura K, Iwao H. Angiotensin II type 1 receptor blockade inhibits the expression of immediate-early genes and fibronectin in rat injured artery. Circulation. 1995;92:88–95.[Abstract/Free Full Text]

7. Miano JM, Tota RR, Vlasic N, Danishefsky KJ, Stemerman MB. Early proto-oncogene expression in rat aortic smooth muscle cells following endothelial removal. Am J Pathol. 1990;137:761–765.[Abstract]

8. Majesky MW, Reidy MA, Bowen-Pope DF, Hart CE, Wilcox JN, Schwartz SM. PDGF ligand and receptor gene expression during repair of arterial injury. J Cell Biol. 1990;111:2149–2158.[Abstract/Free Full Text]

9. Davis RJ. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993;268:14553–14556.[Free Full Text]

10. Nishida E, Gotoh Y. The MAP kinase cascade is essential for diverse signaling pathways. Trends Biochem Sci. 1993;18:128–131.[Medline] [Order article via Infotrieve]

11. Derijard B, Hibi M, Wu IH, Barrett T, Su B, Deng T, Karin M, Davis RJ. JNK1: a protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell. 1994;76:1025–1037.[Medline] [Order article via Infotrieve]

12. Hibi M, Lin A, Smeal T, Minden A, Karin M. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 1993;7:2135–2148.[Abstract/Free Full Text]

13. Kyriakis JM, Banerjee P, Nikolakaki E, Dai T, Rubie EA, Ahmad MF, Avruch J, Woodgett JR. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 1994;369:156–160.[Medline] [Order article via Infotrieve]

14. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 1995;270:1326–1331.[Abstract/Free Full Text]

15. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem. 1995;270:16483–16486.[Free Full Text]

16. Minden A, Lin A, Smeal T, Derijard B, Cobb M, Davis R, Karin M. c-Jun N-terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen-activated protein kinases. Mol Cell Biol. 1994;14:6683–6688.[Abstract/Free Full Text]

17. Cavigelli M, Dolfi F, Claret FX, Karin M. Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation. EMBO J. 1995;14:5957–5964.[Medline] [Order article via Infotrieve]

18. Force T, Pombo CM, Avruch JA, Bonventre JV, Kyriakis JM. Stress-activated protein kinases in cardiovascular disease. Circ Res. 1996;78:947–953.[Free Full Text]

19. Powell JS, Clozel JP, Muller RKM, Kuhn H, Hefti F, Hosang M, Baumgartner HR. Inhibitors of angiotensin-converting enzyme prevent myointimal proliferation after vascular injury. Science. 1989;245:186–188.[Abstract/Free Full Text]

20. Dzau VJ, Gibbons GH, Pratt RE. Molecular mechanism of vascular renin-angiotensin system in myointimal hyperplasia. Hypertension. 1991;18(suppl II):II-100–II-105.

21. Timmermans PBMWM, Wong PC, Chiu AT, Herblin WF, Benfield P, Carini DJ, Lee RJ, Wexler R, Saye J, Smith R. Angiotensin II receptors and angiotensin II receptor antagonists. Pharmacol Rev. 1993;45:205–251.[Medline] [Order article via Infotrieve]

22. Okunishi H, Song K, Oka Y, Kobayashi T, Kawamoto T, Ishihara H, Mori N, Miyazaki M. In vitro pharmacology of a novel non-peptide angiotensin II-receptor antagonist, E4177. Jpn J Pharmacol. 1993;62:239–244.[Medline] [Order article via Infotrieve]

23. Kim S, Wanibuchi H, Hamaguchi A, Miura K, Yamanaka S, Iwao H. Angiotensin blockade improves cardiac and renal complications of type II diabetic rats. Hypertension. 1997;30:1054–1061.[Abstract/Free Full Text]

24. Hamaguchi A, Kim S, Yano M, Yamanaka S, Iwao H. Activation of glomerular mitogen-activated protein kinases in angiotensin II-mediated hypertension. J Am Soc Nephrol. 1998;9:372–380.[Abstract]

25. Kim S, Ohta K, Hamaguchi A, Omura T, Yukimura T, Miura K, Inada Y, Ishimura Y, Chatani F, Iwao H. Angiotensin II type 1 receptor antagonist inhibits the gene expression of transforming growth factor-ß1 and extracellular matrix in cardiac and vascular tissues of hypertensive rats. J Pharmacol Exp Ther. 1994;273:509–515.[Abstract/Free Full Text]

26. Duff JL, Berk BC, Corson MA. Angiotensin II stimulates the pp44 and pp42 mitogen-activated protein kinases in cultured rat aortic smooth muscle cells. Biochem Biophys Res Commun. 1992;188:257–264.[Medline] [Order article via Infotrieve]

27. Tsuda T, Kawahara Y, Ishida Y, Koide M, Shii K, Yokoyama M. Angiotensin II stimulates two myelin basic protein/microtubule-associated protein 2 kinases in cultured vascular smooth muscle cells. Circ Res. 1992;71:620–630.[Abstract/Free Full Text]

28. Zohn IE, Yu H, Li X, Cox AD, Earp HS. Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase. Mol Cell Biol. 1995;15:6160–6168.[Abstract]

29. Kudo S, Komuro I, Mizuno T, Yamazaki T, Zou Y, Shiojima I, Takekoshi N, Yazaki Y. Angiotensin II stimulates c-Jun NH₂-terminal kinase in cultured cardiac myocytes of neonatal rats. Circ Res. 1997;80:139–146.[Abstract/Free Full Text]

30. Cano E, Mahadevan LC. Parallel signal processing among mammalian MAPKs. Trends Biochem Sci. 1995;20:117–122.[Medline] [Order article via Infotrieve]

31. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta. 1991;1072:129–157.[Medline] [Order article via Infotrieve]

32. Lee ME, Dhadly MS, Temizer DH, Clifford JA, Yoshizumi M, Quertermous T. Regulation of endothelin-1 gene expression by fos and jun. J Biol Chem. 1991;266:19034–19039.[Abstract/Free Full Text]

33. Kim SJ, Angel P, Lafyatis R, Hattori K, Kim KY, Sporn MB, Karin M, Roberts AB. Autoinduction of transforming growth factor-ß1 is mediated by the AP-1 complex. Mol Cell Biol. 1990;10:1492–1497.[Abstract/Free Full Text]

34. Farhy RD, Carretero OA, Ho KL, Scicli AG. Role of kinins and nitric oxide in the effects of angiotensin converting enzyme inhibitors on neointima formation. Circ Res. 1993;72:1202–1210.[Abstract/Free Full Text]

35. Malarkey K, Chilvers ER, Lawson MF, Plevin R. Stimulation by endothelin-1 of mitogen-activated protein kinases and DNA synthesis in bovine tracheal smooth muscle cells. Br J Pharmacol. 1995;116:2267–2273.[Medline] [Order article via Infotrieve]

36. Fleming I, Fisslthaler B, Busse R. Calcium signaling in endothelial cells involves activation of tyrosine kinases and leads to activation of mitogen-activated protein kinases. Circ Res. 1995;76:522–529.[Abstract/Free Full Text]

This article has been cited by other articles:

				L. J. Sommerville, C. Xing, S. E. Kelemen, S. Eguchi, and M. V. Autieri Inhibition of allograft inflammatory factor-1 expression reduces development of neointimal hyperplasia and p38 kinase activity Cardiovasc Res, January 1, 2009; 81(1): 206 - 215. [Abstract] [Full Text] [PDF]

				M. Matsumoto, P. C. Dimayuga, C. Wang, J. Kirzner, M. Cercek, J. Yano, K.-Y. Chyu, P. K. Shah, and B. Cercek Exogenous heat shock protein-70 inhibits cigarette smoke-induced intimal thickening Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2008; 295(4): R1320 - R1327. [Abstract] [Full Text] [PDF]

				Y. Tian, L. J. Sommerville, A. Cuneo, S. E. Kelemen, and M. V. Autieri Expression and Suppressive Effects of Interleukin-19 on Vascular Smooth Muscle Cell Pathophysiology and Development of Intimal Hyperplasia Am. J. Pathol., September 1, 2008; 173(3): 901 - 909. [Abstract] [Full Text] [PDF]

				Y. Han, J. Deng, L. Guo, C. Yan, M. Liang, J. Kang, H. Liu, A. M. Graham, and S. Li CREG promotes a mature smooth muscle cell phenotype and reduces neointimal formation in balloon-injured rat carotid artery Cardiovasc Res, June 1, 2008; 78(3): 597 - 604. [Abstract] [Full Text] [PDF]

				E. Laaksamo, R. Tulamo, M. Baumann, R. Dashti, J. Hernesniemi, S. Juvela, M. Niemela, and A. Laakso Involvement of Mitogen-Activated Protein Kinase Signaling in Growth and Rupture of Human Intracranial Aneurysms Stroke, March 1, 2008; 39(3): 886 - 892. [Abstract] [Full Text] [PDF]

				R. A. Deaton, C. Su, T. G. Valencia, and S. R. Grant Transforming Growth Factor-{beta}1-induced Expression of Smooth Muscle Marker Genes Involves Activation of PKN and p38 MAPK J. Biol. Chem., September 2, 2005; 280(35): 31172 - 31181. [Abstract] [Full Text] [PDF]

				C. G. Egan, C. L. Wainwright, R. M. Wadsworth, and G. F. Nixon PDGF-induced signaling in proliferating and differentiated vascular smooth muscle: Effects of altered intracellular Ca2+ regulation Cardiovasc Res, August 1, 2005; 67(2): 308 - 316. [Abstract] [Full Text] [PDF]

				Q. Zhao, M. Ishibashi, K.-i. Hiasa, C. Tan, A. Takeshita, and K. Egashira Essential Role of Vascular Endothelial Growth Factor in Angiotensin II-Induced Vascular Inflammation and Remodeling Hypertension, September 1, 2004; 44(3): 264 - 270. [Abstract] [Full Text] [PDF]

				W. Ni, S. Kitamoto, M. Ishibashi, M. Usui, S. Inoue, K.-i. Hiasa, Q. Zhao, K.-i. Nishida, A. Takeshita, and K. Egashira Monocyte Chemoattractant Protein-1 Is an Essential Inflammatory Mediator in Angiotensin II-Induced Progression of Established Atherosclerosis in Hypercholesterolemic Mice Arterioscler Thromb Vasc Biol, March 1, 2004; 24(3): 534 - 539. [Abstract] [Full Text]

				Y. Izumi, S. Kim, M. Yoshiyama, Y. Izumiya, K. Yoshida, A. Matsuzawa, H. Koyama, Y. Nishizawa, H. Ichijo, J. Yoshikawa, et al. Activation of Apoptosis Signal-Regulating Kinase 1 in Injured Artery and Its Critical Role in Neointimal Hyperplasia Circulation, December 2, 2003; 108(22): 2812 - 2818. [Abstract] [Full Text] [PDF]

				R. Kishore, C. Luedemann, E. Bord, D. Goukassian, and D. W. Losordo Tumor Necrosis Factor-Mediated E2F1 Suppression in Endothelial Cells: Differential Requirement of c-Jun N-Terminal Kinase and p38 Mitogen-Activated Protein Kinase Signal Transduction Pathways Circ. Res., November 14, 2003; 93(10): 932 - 940. [Abstract] [Full Text] [PDF]

				H. Kawano, S. Kim, K. Ohta, T. Nakao, H. Miyazaki, T. Nakatani, and H. Iwao Differential Contribution of Three Mitogen-Activated Protein Kinases to PDGF-BB-Induced Mesangial Cell Proliferation and Gene Expression J. Am. Soc. Nephrol., March 1, 2003; 14(3): 584 - 592. [Abstract] [Full Text] [PDF]

				S. Kim, Y. Izumi, Y. Izumiya, Y. Zhan, M. Taniguchi, and H. Iwao Beneficial Effects of Combined Blockade of ACE and AT1 Receptor on Intimal Hyperplasia in Balloon-Injured Rat Artery Arterioscler Thromb Vasc Biol, August 1, 2002; 22(8): 1299 - 1304. [Abstract] [Full Text] [PDF]

				Y. Zhan, S. Kim, H. Yasumoto, M. Namba, H. Miyazaki, and H. Iwao Effects of Dominant-Negative c-Jun on Platelet-Derived Growth Factor-Induced Vascular Smooth Muscle Cell Proliferation Arterioscler Thromb Vasc Biol, January 1, 2002; 22(1): 82 - 88. [Abstract] [Full Text] [PDF]

				A. Otani, H. Takagi, H. Oh, S. Koyama, and Y. Honda Angiotensin II Induces Expression of the Tie2 Receptor Ligand, Angiopoietin-2, in Bovine Retinal Endothelial Cells Diabetes, April 1, 2001; 50(4): 867 - 875. [Abstract] [Full Text]

				A. Parenti, L. Brogelli, S. Donnini, M. Ziche, and F. Ledda ANG II potentiates mitogenic effect of norepinephrine in vascular muscle cells: role of FGF-2 Am J Physiol Heart Circ Physiol, January 1, 2001; 280(1): H99 - H107. [Abstract] [Full Text] [PDF]

				R. M. Touyz and E. L. Schiffrin Signal Transduction Mechanisms Mediating the Physiological and Pathophysiological Actions of Angiotensin II in Vascular Smooth Muscle Cells Pharmacol. Rev., December 1, 2000; 52(4): 639 - 672. [Abstract] [Full Text] [PDF]

				S. Kim, Y. Zhan, Y. Izumi, H. Yasumoto, M. Yano, and H. Iwao In Vivo Activation of Rat Aortic Platelet-Derived Growth Factor and Epidermal Growth Factor Receptors by Angiotensin II and Hypertension Arterioscler Thromb Vasc Biol, December 1, 2000; 20(12): 2539 - 2545. [Abstract] [Full Text] [PDF]

				Y. Izumi, S. Kim, Y. Zhan, M. Namba, H. Yasumoto, and H. Iwao Important Role of Angiotensin II-Mediated c-Jun NH2-Terminal Kinase Activation in Cardiac Hypertrophy in Hypertensive Rats Hypertension, October 1, 2000; 36(4): 511 - 516. [Abstract] [Full Text] [PDF]

				F. Jung, J. Haendeler, C. Goebel, A. M. Zeiher, and S. Dimmeler Growth factor-induced phosphoinositide 3-OH kinase/Akt phosphorylation in smooth muscle cells: induction of cell proliferation and inhibition of cell death Cardiovasc Res, October 1, 2000; 48(1): 148 - 157. [Abstract] [Full Text] [PDF]

				J. Haendeler, M. Ishida, L. Hunyady, and B. C. Berk The Third Cytoplasmic Loop of the Angiotensin II Type 1 Receptor Exerts Differential Effects on Extracellular Signal-Regulated Kinase (ERK1/ERK2) and Apoptosis via Ras- and Rap1-Dependent Pathways Circ. Res., April 14, 2000; 86(7): 729 - 736. [Abstract] [Full Text] [PDF]

				S. Kim and H. Iwao Molecular and Cellular Mechanisms of Angiotensin II-Mediated Cardiovascular and Renal Diseases Pharmacol. Rev., March 1, 2000; 52(1): 11 - 34. [Abstract] [Full Text] [PDF]

				S. Kim, Y. Zhan, Y. Izumi, and H. Iwao Cardiovascular Effects of Combination of Perindopril, Candesartan, and Amlodipine in Hypertensive Rats Hypertension, March 1, 2000; 35(3): 769 - 774. [Abstract] [Full Text] [PDF]

				K. Sekiguchi, T. Yokoyama, M. Kurabayashi, F. Okajima, and R. Nagai Sphingosylphosphorylcholine Induces a Hypertrophic Growth Response Through the Mitogen-Activated Protein Kinase Signaling Cascade in Rat Neonatal Cardiac Myocytes Circ. Res., November 26, 1999; 85(11): 1000 - 1008. [Abstract] [Full Text] [PDF]

				F. L. Day, L. A. Rafty, C. N. Chesterman, and L. M. Khachigian Angiotensin II (ATII)-inducible Platelet-derived Growth Factor A-chain Gene Expression Is p42/44 Extracellular Signal-regulated Kinase-1/2 and Egr-1-dependent and Mediated via the ATII Type 1 but Not Type 2 Receptor. INDUCTION BY ATII ANTAGONIZED BY NITRIC OXIDE J. Biol. Chem., August 20, 1999; 274(34): 23726 - 23733. [Abstract] [Full Text] [PDF]

				A. Hamaguchi, S. Kim, Y. Izumi, Y. Zhan, S. Yamanaka, and H. Iwao Contribution of Extracellular Signal-Regulated Kinase to Angiotensin II–Induced Transforming Growth Factor-{beta}1 Expression in Vascular Smooth Muscle Cells Hypertension, July 1, 1999; 34(1): 126 - 131. [Abstract] [Full Text] [PDF]

				S. K. Sharma, D. Chapman, R. Temsah, T. Netticadan, D. P. Brasil, and N. S. Dhalla Prevention of Vascular Apoptosis in Myocardial Infarction by Losartan Journal of Cardiovascular Pharmacology and Therapeutics, January 1, 1999; 4(2): 77 - 84. [Abstract] [PDF]

				M. Yano, S. Kim, Y. Izumi, S. Yamanaka, and H. Iwao Differential Activation of Cardiac c-Jun Amino-Terminal Kinase and Extracellular Signal-Regulated Kinase in Angiotensin II–Mediated Hypertension Circ. Res., October 5, 1998; 83(7): 752 - 760. [Abstract] [Full Text] [PDF]

				S. Eguchi, P. J. Dempsey, G. D. Frank, E. D. Motley, and T. Inagami Activation of MAPKs by Angiotensin II in Vascular Smooth Muscle Cells. METALLOPROTEASE-DEPENDENT EGF RECEPTOR ACTIVATION IS REQUIRED FOR ACTIVATION OF ERK AND p38 MAPK BUT NOT FOR JNK J. Biol. Chem., March 9, 2001; 276(11): 7957 - 7962. [Abstract] [Full Text] [PDF]

				U. Schmitz, K. Thommes, I. Beier, W. Wagner, A. Sachinidis, R. Dusing, and H. Vetter Angiotensin II-induced Stimulation of p21-activated Kinase and c-Jun NH2-terminal Kinase Is Mediated by Rac1 and Nck J. Biol. Chem., June 15, 2001; 276(25): 22003 - 22010. [Abstract] [Full Text] [PDF]

				Y. Izumi, S. Kim, M. Namba, H. Yasumoto, H. Miyazaki, M. Hoshiga, Y. Kaneda, R. Morishita, Y. Zhan, and H. Iwao Gene Transfer of Dominant-Negative Mutants of Extracellular Signal-Regulated Kinase and c-Jun NH2-Terminal Kinase Prevents Neointimal Formation in Balloon-Injured Rat Artery Circ. Res., June 8, 2001; 88(11): 1120 - 1126. [Abstract] [Full Text] [PDF]

				M. Kume, K. Komori, T. Matsumoto, T. Onohara, K. Takeuchi, Y. Yonemitsu, and K. Sugimachi Administration of a Decoy Against the Activator Protein-1 Binding Site Suppresses Neointimal Thickening in Rabbit Balloon-Injured Arteries Circulation, March 12, 2002; 105(10): 1226 - 1232. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kim, S. Articles by Iwao, H. Search for Related Content PubMed PubMed Citation Articles by Kim, S. Articles by Iwao, H. Pubmed/NCBI databases Compound via MeSH Substance via MeSH