Peroxisome Proliferator-Activated Receptor γ Activators Downregulate Angiotensin II Type 1 Receptor in Vascular Smooth Muscle Cells
Background—Peroxisome proliferator-activated receptor γ (PPARγ) 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 PPARγ 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 PPARγ ligand 15-deoxy-Δ12,14-prostaglandin J2, as well as Tro, reduced the AT1-R mRNA expression and the AT1-R protein level. The PPARγ activators also reduced the calcium response of VSMCs to Ang II. PPARγ 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—PPARγ 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 PPARγ activators.
Peroxisome proliferator-activated receptor (PPAR) belongs to the family of steroid/thyroid hormone nuclear receptor transcription factors, and 3 isoforms (designated α, γ, and δ) 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 PPARγ 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 PPARγ and PPARα are expressed in vascular smooth muscle cells (VSMCs).4
PPARγ is activated by natural ligand 15-deoxy-Δ12,14-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 migration9 11 and proliferation8 9 of VSMCs by PPARγ 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 PPARα 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 PPARγ activators affect the AT1-R gene expression in VSMCs. We demonstrated that PPARγ activator, but not PPARα activator, was one of the negative regulators of AT1-R gene expression. Because Ang II is reported to inhibit insulin signaling,18 PPARγ 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.
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. [α-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.
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 (4×105) 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 β-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 PPARγ 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 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 (Kd) and AT1-R binding site (Bmax) 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.
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.
Suppression of AT1-R mRNA Expression Was Mediated by PPARγ
Both PPARγ and PPARα are expressed in VSMCs.4 To examine whether the downregulation of AT1-R mRNA by 15-d-PGJ2 is mediated by PPARγ, we determined the effect of Tro or a PPARα 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⇓).
Because PPARγ 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).
PPARγ 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 Bmax value of 0.89 pmol/mg protein and a Kd value of 7.14 nmol/L. On the other hand, 15-d-PGJ2–treated cells showed significantly reduced Bmax (0.46 pmol/mg protein) and statistically unchanged Kd (7.23 nmol/L) values. Tro (20 μmol/L) also significantly reduced the Bmax value without changing the Kd value of AT1-R in VSMCs (data not shown). These data indicate that PPARγ activators significantly reduced the AT1-R number without changing the affinity.
Effect of PPARγ Activators on AT1-R mRNA Stability
We examined whether PPARγ 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 PPARγ 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 PPARγ activators do not change AT1-R mRNA stability.
PPARγ Activators Suppress AT1-R Promoter Activity
To examine whether PPARγ 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⇓).
De Novo Protein Synthesis Is Not Required for PPARγ Activator–Induced Downregulation of AT1-R Expression
To examine whether PPARγ 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 PPARγ activator–induced AT1-R downregulation does not require de novo protein synthesis.
PPARγ Activators Decrease Calcium Response to Ang II
We next examined whether PPARγ 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.
In the present study, we demonstrated that PPARγ activators reduced the expression of AT1-R in cultured VSMCs. PPARγ activators reduced AT1-R promoter activity without affecting AT1-R mRNA stability, suggesting that PPARγ activators suppress AT1-R gene expression at the transcriptional level rather than at the posttranscriptional level. AT1-R expression was specifically suppressed by PPARγ activators in VSMCs, because PPARγ 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 PPARγ activators. Therefore, the decreased response of [Ca2+]i to Ang II after long-term treatment with PPARγ activators probably reflects the reduction in AT1-R number.
The synthetic PPARγ 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 PPARγ 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 PPARγ, 2 different mechanisms were reported: PPARγ-dependent and PPARγ-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 PPARγ-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 PPARγ.24 In PPARγ-dependent pathway, ligand-activated PPARγ 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-κB.3 The PPARγ-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 PPARγ-dependent mechanism rather than PPARγ-independent pathway. There is no consensus of PPRE in AT1-R gene promoter up to −980 bp. It may be possible that the PPARγ activator–induced AT1-R downregulation is due to an interference with other transcriptional factors by ligand-activated PPARγ. However, the mechanism of PPARγ 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 PPARγ activators affected the AT1-R mRNA stability in VSMCs. PPARγ activators did not affect AT1-R mRNA stability but suppressed AT1-R promoter activity. These data suggest that PPARγ 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 PPARγ 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 PPARγ 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 PPARγ 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.
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.
- Copyright © 2000 by American Heart Association
Spiegelman BM. PPAR-gamma: adipogenic regulator and thiazolidinedione receptor. Diabetes. 1998;47:507–514.
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.
Marx N, Schonbeck U, Lazar MA, et al. Peroxisome proliferator-activated receptor γ activators inhibit gene expression and migration in human vascular smooth muscle cells. Circ Res. 1998;83:1097–1103.
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.
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.
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.
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.
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.
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.
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.
Hattori Y, Hattori S, Kasai K. Troglitazone upregulates nitric oxide synthesis in vascular smooth muscle cells. Hypertension. 1999;33:943–948.
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.