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 Takeda, K. Articles by Takeshita, A. Search for Related Content PubMed PubMed Citation Articles by Takeda, K. Articles by Takeshita, A. Related Collections Gene expression

(Circulation. 2000;102:1834.)
© 2000 American Heart Association, Inc.

Basic Science Reports

Peroxisome Proliferator-Activated Receptor Activators Downregulate Angiotensin II Type 1 Receptor in Vascular Smooth Muscle Cells

Kotaro Takeda, MD; Toshihiro Ichiki, MD; Tomotake Tokunou, MD; Yuko Funakoshi, MD; Naoko Iino, MS; Katsuya Hirano, MD; Hideo Kanaide, MD; Akira Takeshita, MD

From the Departments of Cardiovascular Medicine (K.T., T.I., T.T., Y.F., N.I., A.T.) and Molecular Cardiology (K.H., H.K.), 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

Top Abstract Introduction Methods Results Discussion References

 
Background—Peroxisome proliferator-activated receptor (PPAR) activators, such as troglitazone (Tro), not only improve insulin resistance but also suppress the neointimal formation after balloon injury. However, the precise mechanisms have not been determined. Angiotensin II (Ang II) plays crucial roles in the pathogenesis of atherosclerosis, hypertension, and neointimal formation after angioplasty. We examined the effect of PPAR activators on the expression of Ang II type 1 receptor (AT₁-R) in cultured vascular smooth muscle cells (VSMCs).

Methods and Results—AT₁-R mRNA and AT₁-R protein levels were determined by Northern blot analysis and radioligand binding assay, respectively. Natural PPAR ligand 15-deoxy-^12,14-prostaglandin J₂, as well as Tro, reduced the AT₁-R mRNA expression and the AT₁-R protein level. The PPAR activators also reduced the calcium response of VSMCs to Ang II. PPAR activators suppressed the AT₁-R promoter activity measured by luciferase assay but did not affect the AT₁-R mRNA stability, suggesting that the suppression occurs at the transcriptional level.

Conclusions—PPAR activators reduced the AT₁-R expression and calcium response to Ang II in VSMCs. Downregulation of AT₁-R may contribute to the inhibition of neointimal formation by PPAR activators.

Key Words: receptors • prostaglandins • troglitazone • angiotensin • muscle, smooth • cells

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Peroxisome proliferator-activated receptor (PPAR) belongs to the family of steroid/thyroid hormone nuclear receptor transcription factors, and 3 isoforms (designated , , and ) have been identified.¹ Ligand-activated PPAR forms heterodimer with retinoid X receptors, binds to specific DNA sequence [PPAR response element (PPRE)], and activates target gene transcription.¹ PPAR is highly expressed in adipocytes and activated macrophages and is involved in fatty acid metabolism, adipocyte differentiation,² and inhibition of macrophage activation.³ Both PPAR and PPAR are expressed in vascular smooth muscle cells (VSMCs).⁴

PPAR is activated by natural ligand 15-deoxy-^12,14-prostaglandin J₂ (15-d-PGJ₂)⁵ and synthetic ligands (thiazolidinediones),⁶ including troglitazone (Tro) and pioglitazone (Pio). The thiazolidinediones decrease plasma glucose and insulin levels and improve insulin resistance.⁷ The thiazolidinediones are also reported to decrease blood pressure in a hypertensive rat model⁸ and to inhibit neointimal formation of balloon-injured vessels in rats.⁹ The suppression of the mitogen-activated protein (MAP) kinase pathway¹⁰ and the inhibition of migration^{9 11} and proliferation^{8 9} of VSMCs by PPAR activators are considered to be responsible for the inhibition of neointimal formation. However, precise mechanisms have not been clearly determined. On the other hand, the lipid-lowering fibrates, such as bezafibrate and fenofibrate, activate PPAR and are reported to inhibit the cytokine production in VSMCs.⁴

Angiotensin II (Ang II) plays crucial roles in the pathogenesis of atherosclerosis and hypertension.¹² Ang II causes VSMC hypertrophy, extracellular matrix production, and the expression of various growth factors.¹³ Although 2 Ang II receptor isoforms, designated type 1 receptor (AT₁-R)¹⁴ and type 2 receptor (AT₂-R),¹⁵ have been cloned, most of the cardiovascular effects are mediated by AT₁-R. AT₁-R of VSMCs is increased in atherosclerotic lesion and neointima after balloon injury,¹⁶ and ACE inhibitors and AT₁-R antagonists suppress neointimal formation.¹⁷ These results suggest that upregulation of AT₁-R and enhancement of Ang II actions in vessel wall contribute to the progression of atherosclerosis and neointimal formation after angioplasty.

The aim of the present study was to determine whether PPAR activators affect the AT₁-R gene expression in VSMCs. We demonstrated that PPAR activator, but not PPAR activator, was one of the negative regulators of AT₁-R gene expression. Because Ang II is reported to inhibit insulin signaling,¹⁸ PPAR activator-induced AT₁-R downregulation, at least in part, may contribute to not only the inhibition of neointimal formation but also the improvement in insulin resistance.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Reagents
Tro, Pio, and bezafibrate were provided by Sankyo Pharmaceutical Co, Takeda Chemical Industries, and Kissei Pharmaceutical Co, respectively. BSA and ionomycin were purchased from Sigma Chemical Co. DMEM and FBS were purchased from GIBCO BRL. [-³²P]dCTP and [¹²⁵I]Sar¹,Ile⁸-Ang II were purchased from DuPont-New England Nuclear. 15-d-PGJ₂ was purchased from Cayman Chemical Co. Tro, 15-d-PGJ₂, and Pio were dissolved in dimethyl sulfoxide (DMSO), and bezafibrate was dissolved in water. Fura-2/AM (an acetoxymethyl ester form of Fura-2) was purchased from Dojido. Other chemical reagents were purchased from Wako Pure Chemicals unless mentioned specifically.

Cell Culture
VSMCs were isolated from the thoracic aorta of Sprague-Dawley rats and maintained as described previously.¹⁹ Passages between 6 and 12 were used for the experiments.

Northern Blot Analysis
Total RNA was prepared according to an acid guanidinium thiocyanate-phenol-chloroform extraction method, and Northern blot analysis of AT₁-R and 18S ribosomal (r)RNA was performed as described previously.¹⁹ The radioactivity of hybridized bands of AT₁-R mRNA and 18S rRNA was quantified with a MacBAS Bioimage Analyzer (Fuji Photo Film Co).

Measurement of Cell Viability
Confluent VSMCs were serum deprived for 48 hours and then treated with 15-d-PGJ₂, Tro, or Pio. After 24 hours of incubation, these cells were harvested with trypsin-EDTA and stained with 0.4% trypan blue. The total and dead cells were counted with an hemocytometer.

Estimation of Number of AT₁-R Binding Sites
Confluent VSMCs in 24-well dishes were cultured in DMEM supplemented with 0.1% BSA for 48 hours and incubated with vehicle or 15-d-PGJ₂ (10 µmol/L) for 12 hours. The number of AT₁-R binding sites was estimated through the binding of [¹²⁵I]Sar¹,Ile⁸-Ang II as described previously.¹⁹ Protein concentrations were determined with the bicinchoninic acid protein assay kit (Pierce Chemical Co).

Measurement of AT₁-R Gene Promoter Activity
The AT₁-R promoter-luciferase fusion DNA construct (-980 bp) was described previously.¹⁹ VSMCs (4x10⁵) were prepared in a 6-cm tissue culture dish. After 48 hours, 5 µg AT₁-R promoter-luciferase fusion DNA construct and 2 µg LacZ gene driven by simian virus 40 (SV40) promoter-enhancer sequence were introduced to VSMCs via the DEAE-dextran method as previously described.¹⁹ These cells were cultured in DMEM supplemented with 10% FBS for 24 hours and stimulated with 15-d-PGJ₂, Tro, or bezafibrate in DMEM containing 0.1% BSA for 24 hours. The luciferase activity was measured and normalized by ß-galactosidase activity as described previously.¹⁹

Measurement of Intracellular Calcium Response
VSMCs were incubated in DMEM containing 5 µmol/L Fura-2/AM for 1 hour and then pretreated with vehicle, 15-d-PGJ₂, or Tro for 10 minutes (short-term treatment). Alternately, VSMCs were pretreated with vehicle or these PPAR activators for the indicated periods (6 to 12 hours) before Fura-2/AM loading (long-term treatment). Then, VSMCs were washed with buffer containing 5 mmol/L KCl, 10 mmol/L HEPES, 5.5 mmol/L D-glucose, 1 mmol/L MgCl₂, 135 mmol/L NaCl, and 1 mmol/L CaCl₂ and stimulated with 100 nmol/L Ang II. Intracellular calcium concentration ([Ca²⁺]_i) was measured with a fluorescence spectrophotometer (CAM-230; Japan Spectroscopie) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 500 nm. The fluorescence data were expressed as percentages, with the values at rest and at the peak response obtained with 25 µmol/L ionomycin assigned to be 0% and 100%, respectively.

Statistical Analysis
Statistical analyses of the relative AT₁-R mRNA expression were performed with 1-way ANOVA and Fisher’s test if appropriate. The difference of dissociation constant (K_d) and AT₁-R binding site (B_max) were compared by Mann-Whitney U test. Degradation of AT₁-R mRNA was analyzed by 2-way ANOVA. Data are shown as mean±SEM. P<0.05 was considered to be statistically significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
15-d-PGJ₂ Suppresses AT₁-R mRNA Expression
VSMCs were incubated with 10 µmol/L 15-d-PGJ₂, and an mRNA level of AT₁-R was determined. Figure 1A shows that the expression level of AT₁-R mRNA was significantly reduced by 15-d-PGJ₂ compared with the control level at 6 hours, and the reduction was reached a maximum at 12 hours of incubation. Figure 1B shows that incubation with varying concentrations of 15-d-PGJ₂ for 12 hours resulted in a dose-dependent suppression of AT₁-R mRNA level.

View larger version (31K): [in this window] [in a new window]   Figure 1. Suppression of AT1-R mRNA expression by 15-d-PGJ2 in VSMCs. VSMCs were incubated with 15-d-PGJ2 (10 µmol/L) for various periods as indicated (A). VSMCs were incubated with 15-d-PGJ2 (B) at concentrations varying from 1 to 10 µmol/L for 12 hours. Total RNA was isolated, and expression of AT1-R mRNA and 18S rRNA was determined by Northern blot analysis. Left, Representative autoradiography. Right, Radioactivity of AT1-R mRNA bands was counted with a Bioimage Analyzer and was normalized with radioactivity of 18S rRNA. Values (mean±SEM) are expressed as a percent of control culture (100%) (n=3). **P<0.01 vs control.

Suppression of AT₁-R mRNA Expression Was Mediated by PPAR
Both PPAR and PPAR are expressed in VSMCs.⁴ To examine whether the downregulation of AT₁-R mRNA by 15-d-PGJ₂ is mediated by PPAR, we determined the effect of Tro or a PPAR activator, bezafibrate, on AT₁-R mRNA expression. Tro downregulated the AT₁-R mRNA expression in a time- (Figure 2A) and dose- (Figure 2B) dependent manner, whereas bezafibrate did not affect the expression of AT₁-R mRNA (Figure 2C). Tro was reported to have an antioxidant effect.²⁰ To exclude the possibility that an antioxidant effect of Tro is responsible for the suppression of AT₁-R mRNA expression, we examined the effect of Pio, which did not have an antioxidant effect.²⁰ Pio also suppressed the AT₁-R mRNA expression, as did Tro (Figure 2D).

View larger version (39K): [in this window] [in a new window]   Figure 2. Suppression of AT1-R mRNA expression by Tro in VSMCs. VSMCs were incubated with 20 µmol/L Tro (A), bezafibrate (C), or Pio (D) for varying time periods as indicated. VSMCs were incubated with Tro at varying concentrations as indicated for 6 hours (B). Expression of AT1-R mRNA and 18S rRNA was determined with method described in legend to Figure 1. Values (mean±SEM) are expressed as a percent of control culture (100%) (n=3). *P<0.05 vs control. **P<0.01 vs control.

Because PPAR activators were reported to have a proapoptotic effect in several cell lines,²¹ we measured the viability of VSMCs with trypan blue exclusion assay. Treatments of VSMCs with 15-d-PGJ₂ (10 µmol/L), Tro (20 µmol/L), or Pio (20 µmol/L) for 24 hours showed statistically unchanged differences in cell viability compared with control (in percent of viable cells: control 96.6±0.4%, 15-d-PGJ₂ 97.8±0.6%, Tro 97.9±0.4%, Pio 97.8%±0.9%; n=4).

PPAR Activators Downregulate the AT₁-R Number in VSMCs
Figure 3 shows saturation curve (A) and Scatchard plot analysis (B) of the binding of [¹²⁵I]Sar¹,Ile⁸-Ang II to vehicle (0.1% DMSO)- and 15-d-PGJ₂ (10 µmol/L)–treated VSMCs for 12 hours. Binding to vehicle-treated cells revealed a B_max value of 0.89 pmol/mg protein and a K_d value of 7.14 nmol/L. On the other hand, 15-d-PGJ₂–treated cells showed significantly reduced B_max (0.46 pmol/mg protein) and statistically unchanged K_d (7.23 nmol/L) values. Tro (20 µmol/L) also significantly reduced the B_max value without changing the K_d value of AT₁-R in VSMCs (data not shown). These data indicate that PPAR activators significantly reduced the AT₁-R number without changing the affinity.

View larger version (20K): [in this window] [in a new window]   Figure 3. Effects of 15-d-PGJ2 on AT1-R number in VSMCs. VSMCs were incubated with vehicle or 15-d-PGJ2 (10 µmol/L) for 12 hours. Binding assays with [125I]Sar1,Ile8-Ang II were performed. Unlabeled Sar1,Ile8-Ang II (10 µmol/L) was used to determine nonspecific binding. Specific binding was calculated by subtracting nonspecific binding from total binding. Saturation curve (A) and Scatchard plot analysis (B) are shown. Bmax and Kd values are described in text. 15-d-PGJ2 significantly reduced AT1-R number (P<0.01) without affecting its affinity (n=3).

Effect of PPAR Activators on AT₁-R mRNA Stability
We examined whether PPAR activators affected the AT₁-R mRNA stability. VSMCs were stimulated with vehicle, 15-d-PGJ₂ (10 µmol/L), or Tro (20 µmol/L) for 6 hours and then treated with actinomycin D (5 µg/mL). Figure 4A shows that the degradation rate of AT₁-R mRNA did not differ significantly among the 3 groups. Two-hour treatment of these PPAR activators also did not affect the AT₁-R mRNA stability (data not shown). To clarify the early phase of destabilization process, VSMCs were pretreated with actinomycin D for 30 minutes and then stimulated with vehicle, 15-d-PGJ₂, or Tro. The half-life of AT₁-R mRNA was unchanged among the 3 groups (Figure 4B). These data indicate that PPAR activators do not change AT₁-R mRNA stability.

View larger version (20K): [in this window] [in a new window]   Figure 4. Effects of PPAR activators on AT1-R mRNA stability. VSMCs were incubated with vehicle (), 15-d-PGJ2 (10 µmol/L, ), and Tro (20 µmol/L, ) for 6 hours, and then actinomycin D (5 µg/mL) was added (A). VSMCs were preincubated with actinomycin D for 30 minutes and then incubated with vehicle, 15-d-PGJ2, and Tro (B). Total RNA was isolated at indicated time points, and expression of AT1-R mRNA and 18S rRNA was determined with method described in legend to Figure 1. Expression levels of AT1-R mRNA in VSMCs before (A) and after (B) addition of actinomycin D in each group were set as 100% (n=3).

PPAR Activators Suppress AT₁-R Promoter Activity
To examine whether PPAR activators suppress AT₁-R promoter activity, AT₁-R promoter-luciferase fusion DNA construct was introduced into VSMCs. Then, the VSMCs were stimulated with 15-d-PGJ₂, Tro, or bezafibrate at varying concentrations (as indicated in the figure) for 24 hours. Consistent with the results of Northern blot analysis, 15-d-PGJ₂ and Tro significantly suppressed AT₁-R promoter activity in a dose-dependent manner (Figures 5A and 5B) but bezafibrate did not (Figure 5C).

View larger version (23K): [in this window] [in a new window]   Figure 5. Effect of PPAR activators on AT1-R promoter activity. AT1-R promoter-luciferase DNA construct and LacZ gene were introduced to VSMCs with DEAE-dextran method. Then, VSMCs were stimulated with 15-d-PGJ2 (A), Tro (B), or bezafibrate (C) for 24 hours at concentrations indicated. Luciferase activity was normalized by ß-galactosidase activity. Relative luciferase activity of unstimulated VSMC (control) was set as 100%. Relative luciferase activities are shown as mean±SEM (n=3). **P<0.01 vs control. *P<0.05 vs control.

De Novo Protein Synthesis Is Not Required for PPAR Activator–Induced Downregulation of AT₁-R Expression
To examine whether PPAR activator–induced downregulation of AT₁-R mRNA requires de novo protein synthesis, we examined the effect of cycloheximide (10 µg/mL). Although incubation with cycloheximide alone for 12 hours upregulated the AT₁-R mRNA expression, 15-d-PGJ₂ (Figure 6) and Tro (data not shown) significantly suppressed the AT₁-R mRNA level in the presence of cycloheximide. These data suggest that PPAR activator–induced AT₁-R downregulation does not require de novo protein synthesis.

View larger version (21K): [in this window] [in a new window]   Figure 6. Effect of cycloheximide (CHX) on AT1-R downregulation. VSMCs were pretreated with or without cycloheximide (10 µg/mL) for 30 minutes and then incubated in presence or absence of 15-d-PGJ2 (10 µmol/L) for 12 hours. Expression of AT1-R mRNA and 18S rRNA was determined with method described in legend to Figure 1. Values (mean±SEM) are expressed as a percent of control culture (100%) (n=3). **P<0.01 vs control. *P<0.01 vs control.

PPAR Activators Decrease Calcium Response to Ang II
We next examined whether PPAR activator–induced AT₁-R downregulation decreased the response of VSMCs to Ang II stimulation. VSMCs were pretreated with vehicle, 15-d-PGJ₂ (10 µmol/L), or Tro (20 µmol/L) for the indicated periods. Then, the VSMCs were stimulated with 100 nmol/L Ang II, and [Ca²⁺]_i was measured. A brief pretreatment (10 minutes) with these compounds did not affect Ang II–induced calcium response. Ang II–induced maximal [Ca²⁺]_i increases were 69.5±2.7%, 70.6±5.5%, and 66.4±6.2% (in percent of maximum fluorescence induced by ionomycin treatment) in vehicle-, 15-d-PGJ₂–, and Tro-treated VSMCs, respectively (Figure 7A). However, long-term pretreatment with 15-d-PGJ₂ or Tro significantly decreased the calcium response to Ang II (Figure 7B). Ang II–induced maximal [Ca²⁺]_i increase in vehicle-treated VSMCs (control) was 61.4±4.2%, but those in 15-d-PGJ₂–treated VSMCs (for 12 hours) and Tro-treated VSMCs (for 6 hours) were 28.9±4.0% (P<0.01 versus control) and 44.0±4.5% (P<0.05 versus control), respectively.

View larger version (27K): [in this window] [in a new window]   Figure 7. Effect of PPAR activators on intracellular calcium response to Ang II. A, VSMCs were pretreated with vehicle, 15-d-PGJ2 (10 µmol/L), or Tro (20 µmol/L) for 10 minutes. B, VSMCs were also pretreated with vehicle or 15-d-PGJ2 for 12 hours or Tro for 6 hours, respectively. Then, these VSMCs were stimulated with 100 nmol/L Ang II, and [Ca2+]i was measured. A representative record is shown. Ten-minute treatment with 15-d-PGJ2 or Tro did not affect Ang II–induced calcium response (vehicle 69.5±2.7%, 15-d-PGJ2 70.6±5.5%, Tro 66.4±6.2% given in percent of maximum fluorescence induced by ionomycin treatment). However, long-term treatment with 15-d-PGJ2 or Tro significantly decreased calcium response to Ang II (vehicle 61.4±4.2%, 15-d-PGJ2 28.9±4.0% [P<0.01 vs vehicle], and Tro 44.0±4.5% [P<0.05 vs vehicle]).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we demonstrated that PPAR activators reduced the expression of AT₁-R in cultured VSMCs. PPAR activators reduced AT₁-R promoter activity without affecting AT₁-R mRNA stability, suggesting that PPAR activators suppress AT₁-R gene expression at the transcriptional level rather than at the posttranscriptional level. AT₁-R expression was specifically suppressed by PPAR activators in VSMCs, because PPAR activators did not affect ACE mRNA (data not shown) or rRNA expression. In addition, Ang II–induced biological response, an increase in [Ca²⁺]_i, also was significantly suppressed by 15-d-PGJ₂ and Tro. Although Tro is reported to inhibit voltage-dependent calcium current after brief pretreatment,²² the Ang II–induced calcium response was not affected by a brief incubation with these PPAR activators. Therefore, the decreased response of [Ca²⁺]_i to Ang II after long-term treatment with PPAR activators probably reflects the reduction in AT₁-R number.

The synthetic PPAR ligands thiazolidinediones, including Tro, have been shown to improve insulin resistance.⁷ Insulin resistance is related not only to the pathogenesis of diabetes mellitus but also to the progression of atherosclerosis.²³ Although Tro was reported to suppress the neointimal formation after balloon injury,⁹ the precise mechanisms have not been clearly determined. Our data suggest that downregulation of AT₁-R expression and decreased Ang II action by PPAR activators may be involved, at least in part, in the inhibition of neointimal formation by Tro. In addition, Goetze et al¹⁰ reported that Tro inhibited Ang II–induced extracellular signal–regulated kinase (ERK)1/2 activation in VSMCs. Because ERK1/2 activity is important for VSMC proliferation, inhibition of the ERK pathway may be another mechanism for the suppression of neointimal formation by Tro.^{9 10}

It was previously reported that Tro regulated various gene expressions.^{3 11 24} Although Tro is a ligand of PPAR, 2 different mechanisms were reported: PPAR-dependent and PPAR-independent signaling mechanisms. Matrix metalloproteinase-9 mRNA gene expression was decreased by both Tro and 15-d-PGJ₂ treatment, indicating the process was mediated by the PPAR-dependent pathway.¹¹ On the other hand, Tro upregulated inducible nitric oxide (NO) synthase mRNA expression in VSMCs, whereas 15-d-PGJ₂ did not affect it, suggesting that the action of Tro was independent of PPAR.²⁴ In PPAR-dependent pathway, ligand-activated PPAR positively regulated some gene expression via binding to specific DNA sequence PPRE¹ or inhibited other gene expression in part by through antagonism of the activities of the transcriptional factor, such as activator protein-1 and nuclear factor-B.³ The PPAR-independent pathway has not been clearly determined. We demonstrated here that both Tro and 15-d-PGJ₂ suppressed AT₁-R gene expression, suggesting that the suppression of AT₁-R expression in VSMCs was mediated by PPAR-dependent mechanism rather than PPAR-independent pathway. There is no consensus of PPRE in AT₁-R gene promoter up to -980 bp. It may be possible that the PPAR activator–induced AT₁-R downregulation is due to an interference with other transcriptional factors by ligand-activated PPAR. However, the mechanism of PPAR activator–induced AT₁-R downregulation is not clearly determined at this point. Although an antioxidant effect of Tro may play a role in the downregulation of AT₁-R, this explanation is unlikely because 15-d-PGJ₂ and Pio, which are without an antioxidant effect, also suppressed the AT₁-R expression.

Because it was previously reported that Tro affected the stability of inducible NO synthase mRNA,²⁴ we investigated whether PPAR activators affected the AT₁-R mRNA stability in VSMCs. PPAR activators did not affect AT₁-R mRNA stability but suppressed AT₁-R promoter activity. These data suggest that PPAR activators negatively regulate AT₁-R gene transcription.

In an insulin-resistant state, the plasma insulin level was highly elevated. Insulin is a positive regulator of AT₁-R gene expression and enhances the Ang II signaling.²⁵ Furthermore, Ang II inhibits the insulin signaling at multiple steps in VSMCs.¹⁸ These may lead to a further increase in the plasma insulin level. These sequences (an increase in insulin level, upregulation of AT₁-R, an inhibition of insulin signaling by Ang II, and a further increase in the insulin level) may form a vicious circle. These may cause further progression of atherosclerosis and insulin resistance. Thiazolidinediones, synthetic PPAR ligands, restore insulin responsiveness and significantly decrease plasma insulin level in a diabetic model.²⁶ The decrease in plasma insulin level may result in a reduction in AT₁-R expression. In addition, we described here that Tro and Pio directly decreased AT₁-R expression in VSMCs independent of insulin concentration. It is expected that the activation of PPAR by Tro and Pio downregulates AT₁-R gene expression through both direct and indirect mechanisms in an insulin-resistant state in vivo.

In conclusion, we demonstrated that activation of PPAR significantly downregulated AT₁-R expression. The suppression of AT₁-R expression may contribute not only to an improvement in insulin resistance but also to inhibition of the progression of neointima formation, atherosclerosis, and high blood pressure.

	Acknowledgments

 
This work was supported in part by Kaibara Morikazu Science Promotion Foundation (Fukuoka, Japan) and Uehara Memorial Foundation (Tokyo, Japan).

Received March 2, 2000; revision received April 25, 2000; accepted May 15, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Schoonjans K, Martin G, Staels B, et al. Peroxisome proliferator-activated receptors: orphans with ligands and functions. Curr Opin Lipidol. 1997;8:159–166.[Medline] [Order article via Infotrieve]
   
2. Tontonoz P, Hu E, Spiegelman BM. Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor. Cell. 1994;79:1147–1156.[Medline] [Order article via Infotrieve]
   
3. Ricote M, Li AC, Willson TM, et al. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature. 1998;391:79–82.[Medline] [Order article via Infotrieve]
   
4. Staels B, Koenig W, Habib A, et al. Activation of human aortic smooth-muscle cells is inhibited by PPAR but not by PPAR activators. Nature. 1998;393:790–793.[Medline] [Order article via Infotrieve]
   
5. Forman BM, Tontonoz P, Chen J, et al. 15-Deoxy-delta 12,14-prostaglandin J₂ is a ligand for the adipocyte determination factor PPAR gamma. Cell. 1995;83:803–812.[Medline] [Order article via Infotrieve]
   
6. Spiegelman BM. PPAR-gamma: adipogenic regulator and thiazolidinedione receptor. Diabetes. 1998;47:507–514.[Abstract]
   
7. Nolan JJ, Ludvik B, Beerdsen P, et al. Improvement in glucose tolerance and insulin resistance in obese subjects treated with troglitazone. N Engl J Med. 1994;331:1188–1193.[Abstract/Free Full Text]
   
8. Dubey RK, Zhang HY, Reddy SR, et al. Pioglitazone attenuates hypertension and inhibits growth of renal arteriolar smooth muscle in rats. Am J Physiol. 1993;265:R726–R732.[Abstract/Free Full Text]
   
9. Law RE, Meehan WP, Xi XP, et al. Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia. J Clin Invest. 1996;98:1897–1905.[Medline] [Order article via Infotrieve]
   
10. Goetze S, Xi XP, Graf K, et al. Troglitazone inhibits angiotensin II-induced extracellular signal-regulated kinase 1/2 nuclear translocation and activation in vascular smooth muscle cells. FEBS Lett. 1999;452:277–282.[Medline] [Order article via Infotrieve]
    
11. Marx N, Schonbeck U, Lazar MA, et al. Peroxisome proliferator-activated receptor activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ Res. 1998;83:1097–1103.[Abstract/Free Full Text]
    
12. Goodfriend TL, Elliott ME, Catt KJ. Drug therapy: angiotensin receptors and their antagonists. N Engl J Med. 1996;334:1649–1654.[Free Full Text]
    
13. Gibbons GH, Pratt RE, Dzau VJ. Vascular smooth muscle cell hypertrophy vs. hyperplasia: autocrine transforming growth factor-beta 1 expression determines growth response to angiotensin II. J Clin Invest. 1992;90:456–461.
    
14. Sasaki K, Yamano Y, Bardhan S, et al. Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor. Nature. 1991;351:230–233.[Medline] [Order article via Infotrieve]
    
15. Kambayashi Y, Bardhan S, Takahashi K, et al. Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. J Biol Chem. 1993;268:24543–24546.[Abstract/Free Full Text]
    
16. Viswanathan M, Stromberg C, Seltzer A, et al. Balloon angioplasty enhances the expression of angiotensin II AT₁ receptors in neointima of rat aorta. J Clin Invest. 1992;90:1707–1712.
    
17. Osterrieder W, Muller RK, Powell JS, et al. Role of angiotensin II in injury-induced neointima formation in rats. Hypertension. 1991;18(suppl II):II-60–II-64.
    
18. Folli F, Kahn CR, Hansen H, et al. Angiotensin II inhibits insulin signaling in aortic smooth muscle cells at multiple levels: a potential role for serine phosphorylation in insulin/angiotensin II crosstalk. J Clin Invest. 1997;100:2158–2169.[Medline] [Order article via Infotrieve]
    
19. Ichiki T, Usui M, Kato M, et al. Downregulation of angiotensin II type 1 receptor gene transcription by nitric oxide. Hypertension. 1998;31:342–348.[Abstract/Free Full Text]
    
20. Inoue I, Katayama S, Takahashi K, et al. Troglitazone has a scavenging effect on reactive oxygen species. Biochem Biophys Res Commun. 1997;235:113–116.[Medline] [Order article via Infotrieve]
    
21. Elstner E, Muller C, Koshizuka K, et al. Ligands for peroxisome proliferator-activated receptor gamma and retinoic acid receptor inhibit growth and induce apoptosis of human breast cancer cells in vitro and in BNX mice. Proc Natl Acad Sci U S A. 1998;95:8806–8811.[Abstract/Free Full Text]
    
22. Nakamura Y, Ohya Y, Onaka U, et al. Inhibitory action of insulin-sensitizing agents on calcium channels in smooth muscle cells from resistance arteries of guinea-pig. Br J Pharmacol. 1998;123:675–682.[Medline] [Order article via Infotrieve]
    
23. Howard G, O’Leary DH, Zaccaro D, et al. Insulin sensitivity and atherosclerosis: the Insulin Resistance Atherosclerosis Study (IRAS) Investigators. Circulation. 1996;93:1809–1817.[Abstract/Free Full Text]
    
24. Hattori Y, Hattori S, Kasai K. Troglitazone upregulates nitric oxide synthesis in vascular smooth muscle cells. Hypertension. 1999;33:943–948.[Abstract/Free Full Text]
    
25. Nickenig G, Roling J, Strehlow K, et al. Insulin induces upregulation of vascular AT₁ receptor gene expression by posttranscriptional mechanisms. Circulation. 1998;98:2453–2460.[Abstract/Free Full Text]
    
26. Shinohara E, Kihara S, Ouchi N, et al. Troglitazone suppresses intimal formation following balloon injury in insulin-resistant Zucker fatty rats. Atherosclerosis. 1998;136:275–279.[Medline] [Order article via Infotrieve]
    

This article has been cited by other articles:

				X. Ruan, F. Zheng, and Y. Guan PPARs and the kidney in metabolic syndrome Am J Physiol Renal Physiol, May 1, 2008; 294(5): F1032 - F1047. [Abstract] [Full Text] [PDF]

				Q. Zhang, F. He, R. Kuruba, X. Gao, A. Wilson, J. Li, T. R. Billiar, B. R. Pitt, W. Xie, and S. Li FXR-mediated regulation of angiotensin type 2 receptor expression in vascular smooth muscle cells Cardiovasc Res, February 1, 2008; 77(3): 560 - 569. [Abstract] [Full Text] [PDF]

				Z. Yousefipour, H. Hercule, L. Truong, A. Oyekan, and M. Newaz Ciglitazone, a Peroxisome Proliferator-Activated Receptor {gamma} Inducer, Ameliorates Renal Preglomerular Production and Activity of Angiotensin II and Thromboxane A2 in Glycerol-Induced Acute Renal Failure J. Pharmacol. Exp. Ther., August 1, 2007; 322(2): 461 - 468. [Abstract] [Full Text] [PDF]

				D. R. Cha, X. Zhang, Y. Zhang, J. Wu, D. Su, J. Y. Han, X. Fang, B. Yu, M. D. Breyer, and Y. Guan Peroxisome Proliferator Activated Receptor {alpha}/{gamma} Dual Agonist Tesaglitazar Attenuates Diabetic Nephropathy in db/db Mice Diabetes, August 1, 2007; 56(8): 2036 - 2045. [Abstract] [Full Text] [PDF]

				T. S. Elton and M. M. Martin Angiotensin II Type 1 Receptor Gene Regulation: Transcriptional and Posttranscriptional Mechanisms Hypertension, May 1, 2007; 49(5): 953 - 961. [Full Text] [PDF]

				M. Pravenec and T. W. Kurtz Molecular Genetics of Experimental Hypertension and the Metabolic Syndrome: From Gene Pathways to New Therapies Hypertension, May 1, 2007; 49(5): 941 - 952. [Abstract] [Full Text] [PDF]

				J. T. Crossno Jr., C. V. Garat, J. E. B. Reusch, K. G. Morris, E. C. Dempsey, I. F. McMurtry, K. R. Stenmark, and D. J. Klemm Rosiglitazone attenuates hypoxia-induced pulmonary arterial remodeling Am J Physiol Lung Cell Mol Physiol, April 1, 2007; 292(4): L885 - L897. [Abstract] [Full Text] [PDF]

				M. A. Potenza, F. L. Marasciulo, M. Tarquinio, M. J. Quon, and M. Montagnani Treatment of Spontaneously Hypertensive Rats With Rosiglitazone and/or Enalapril Restores Balance Between Vasodilator and Vasoconstrictor Actions of Insulin With Simultaneous Improvement in Hypertension and Insulin Resistance Diabetes, December 1, 2006; 55(12): 3594 - 3603. [Abstract] [Full Text] [PDF]

				A. Zanchi, C. Perregaux, M. Maillard, D. Cefai, J. Nussberger, and M. Burnier The PPAR{gamma} agonist pioglitazone modifies the vascular sodium-angiotensin II relationship in insulin-resistant rats Am J Physiol Endocrinol Metab, December 1, 2006; 291(6): E1228 - E1234. [Abstract] [Full Text] [PDF]

				I. Imayama, T. Ichiki, K. Inanaga, H. Ohtsubo, K. Fukuyama, H. Ono, Y. Hashiguchi, and K. Sunagawa Telmisartan downregulates angiotensin II type 1 receptor through activation of peroxisome proliferator-activated receptor {gamma} Cardiovasc Res, October 1, 2006; 72(1): 184 - 190. [Abstract] [Full Text] [PDF]

				O. A. Gudbrandsen, M. Hultstrom, S. Leh, L. Monica Bivol, O. Vagnes, R. K. Berge, and B. M. Iversen Prevention of Hypertension and Organ Damage in 2-Kidney, 1-Clip Rats by Tetradecylthioacetic Acid Hypertension, September 1, 2006; 48(3): 460 - 466. [Abstract] [Full Text] [PDF]

				U. Campia, L. A. Matuskey, and J. A. Panza Peroxisome Proliferator-Activated Receptor-{gamma} Activation With Pioglitazone Improves Endothelium-Dependent Dilation in Nondiabetic Patients With Major Cardiovascular Risk Factors Circulation, February 14, 2006; 113(6): 867 - 875. [Abstract] [Full Text] [PDF]

				F. Blaschke, Y. Takata, E. Caglayan, R. E. Law, and W. A. Hsueh Obesity, Peroxisome Proliferator-Activated Receptor, and Atherosclerosis in Type 2 Diabetes Arterioscler. Thromb. Vasc. Biol., January 1, 2006; 26(1): 28 - 40. [Abstract] [Full Text] [PDF]

				K. Benkirane, F. Amiri, Q. N. Diep, M. El Mabrouk, and E. L. Schiffrin PPAR-{gamma} inhibits ANG II-induced cell growth via SHIP2 and 4E-BP1 Am J Physiol Heart Circ Physiol, January 1, 2006; 290(1): H390 - H397. [Abstract] [Full Text] [PDF]

				K. Benkirane, E. C. Viel, F. Amiri, and E. L. Schiffrin Peroxisome Proliferator-Activated Receptor {gamma} Regulates Angiotensin II-Stimulated Phosphatidylinositol 3-Kinase and Mitogen-Activated Protein Kinase in Blood Vessels In Vivo Hypertension, January 1, 2006; 47(1): 102 - 108. [Abstract] [Full Text] [PDF]

				S Soumian, R Gibbs, A Davies, and C Albrecht mRNA expression of genes involved in lipid efflux and matrix degradation in occlusive and ectatic atherosclerotic disease J. Clin. Pathol., December 1, 2005; 58(12): 1255 - 1260. [Abstract] [Full Text] [PDF]

				A. C. Calkin, J. M. Forbes, C. M. Smith, M. Lassila, M. E. Cooper, K. A. Jandeleit-Dahm, and T. J. Allen Rosiglitazone Attenuates Atherosclerosis in a Model of Insulin Insufficiency Independent of Its Metabolic Effects Arterioscler. Thromb. Vasc. Biol., September 1, 2005; 25(9): 1903 - 1909. [Abstract] [Full Text] [PDF]

				Y. Guan Peroxisome Proliferator-Activated Receptor Family and Its Relationship to Renal Complications of the Metabolic Syndrome J. Am. Soc. Nephrol., November 1, 2004; 15(11): 2801 - 2815. [Abstract] [Full Text] [PDF]

				S. Wakino, K. Hayashi, T. Kanda, S. Tatematsu, K. Homma, K. Yoshioka, I. Takamatsu, and T. Saruta Peroxisome Proliferator-Activated Receptor {gamma} Ligands Inhibit Rho/Rho Kinase Pathway by Inducing Protein Tyrosine Phosphatase SHP-2 Circ. Res., September 3, 2004; 95(5): e45 - e55. [Abstract] [Full Text] [PDF]

				H. L. Keen, M. J. Ryan, A. Beyer, S. Mathur, T. E. Scheetz, B. D. Gackle, F. M. Faraci, T. L. Casavant, and C. D. Sigmund Gene expression profiling of potential PPAR{gamma} target genes in mouse aorta Physiol Genomics, June 17, 2004; 18(1): 33 - 42. [Abstract] [Full Text] [PDF]

				N. Marx, H. Duez, J.-C. Fruchart, and B. Staels Peroxisome Proliferator-Activated Receptors and Atherogenesis: Regulators of Gene Expression in Vascular Cells Circ. Res., May 14, 2004; 94(9): 1168 - 1178. [Abstract] [Full Text] [PDF]

				A. Zanchi, A. Chiolero, M. Maillard, J. Nussberger, H.-R. Brunner, and M. Burnier Effects of the Peroxisomal Proliferator-Activated Receptor-{gamma} Agonist Pioglitazone on Renal and Hormonal Responses to Salt in Healthy Men J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1140 - 1145. [Abstract] [Full Text] [PDF]