Rosiglitazone Attenuates Endothelin-1–Induced Vasoconstriction by Upregulating Endothelial Expression of Endothelin B Receptor
Thiazolidinediones improve insulin resistance and endothelial dysfunction. However, the mechanisms underlying the vasoprotective effects of thiazolidinediones remain to be fully elucidated. The present study aimed to examine the molecular mechanism for the anti-vasoconstrictive effects of rosiglitazone in response to endothelin (ET) 1. Mouse aortas were treated with rosiglitazone for 24 hours, and ET-1–induced vasoconstriction was assessed by wire myography. The results showed that rosiglitazone attenuated ET-1–induced contraction in mouse aortas; this effect was abolished by ET-B receptor (ETBR) antagonist, NO synthase inhibitor, and by the removal of endothelium. By using Northern blotting, real-time RT-PCR, Western blotting, and immunohistochemical techniques, we found that rosiglitazone upregulated expression of ETBR at both mRNA and protein levels in mouse aortas and human vascular endothelial cells. The induction of ETBR was prevented by peroxisome proliferator-activated receptor-γ antagonism. Luciferase reporter assay showed that rosiglitazone enhanced ETBR gene promoter activity. Furthermore, chromatin immunoprecipitation assays demonstrated that peroxisome proliferator-activated receptor-γ can directly bind to ETBR gene promoter. Furthermore, in vivo treatment with rosiglitazone also attenuated the ET-1–induced vasoconstrictions and increased the ETBR expression in mouse aortas and mesenteric arteries. In conclusion, these results demonstrate that rosiglitazone attenuated ET-1–induced vasoconstriction through the upregulation of endothelial ETBR, which is a peroxisome proliferator-activated receptor-γ direct target.
Endothelial dysfunction is associated with insulin resistance and type 2 diabetes mellitus. Thiazolidinediones (TZDs), like rosiglitazone and pioglitazone, are peroxisome proliferator-activated receptor (PPAR)-γ ligands and have been widely used for patients with type 2 diabetes mellitus. They exert cardiovascular benefit by improving glucose and lipid homeostasis and insulin resistance. In addition, TZDs also have direct protective effects on endothelial function independent of their insulin-sensitizing action.1–3⇓⇓
The endothelium is crucially involved in the maintenance of vascular homeostasis.4 Normal endothelial function mainly depends on the capacity of endothelial cells to produce NO, which is vasoprotective by inhibiting cytokine expression, smooth muscle cell migration, leukocyte adhesion, and platelet aggregation, as well as improving vasodilatory function.5 TZDs improve endothelial dysfunction. PPAR-γ activators inhibit thrombin-induced endothelin (ET) 1 production in human vascular endothelial cells.6 In vivo, treatment with rosiglitazone decreases blood pressure7 and partially restores the impaired relaxation of carotid arteries to acetylcholine without affecting the gene expression of major modulators of blood pressure, including endothelial NO synthase, angiotensin II type 1 receptors, and preproendothelin 1. PPAR-γ activators also modulate cardiac remodeling in mineralocorticoid-induced hypertension, which was associated with a decreased production of ET-1.8 In vitro experiments indicate that TZDs cause vasodilatation partially by decreasing the effect of ET-1.9,10⇓ The Prospective Pioglitazone Clinical Trial in Macrovascular Events indicates that pioglitazone reduced the composite of all-cause mortality, nonfatal myocardial infarction, and stroke in type 2 diabetics who have a high risk of macrovascular events.11 However, the mechanisms underlying the vasoprotective effect of TZDs remain to be fully elucidated. In the present study, we observed the effect of rosiglitazone on ET-1–induced vasoconstriction and the underlying molecular mechanism.
Materials and Methods
Reagents and Chemicals
Polyclonal rabbit anti-ET-B receptor (ETBR) antibody was from Abcam. Polyclonal rabbit anti-ETAR and anti–PPAR-γ were from Santa Cruz Biotechnology. ET-1, acetylcholine, U46619, NG-nitro-l-arginine methyl ester (l-NAME), GW9662, and bisphenol A diglycidyl ether (BADGE) were from Sigma-Aldrich Co, rosiglitazone and troglitazone were from GlaxoSmithKline, and ABT627 and A192621 were from Abbott Laboratories.
Adult male C57BL/6J mice (10 weeks old) were housed under a 12-hour light/12-hour dark cycle and fed ad libitum. Mice received daily oral administration of 10 mg/kg−1 of rosiglitazone or vehicle via gastric gavage for 2 weeks. The animal protocols were approved by the institutional animal care and use committee and were consistent with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Blood Vessel Preparation
The mice were euthanized by cervical dislocation. Thoracic aortas and mesenteric resistance arteries were dissected out, cleaned of adhering connective tissue, and cut into several ring segments of ≈2 mm in length each. Isolated mouse aortic rings were incubated in DMEM supplemented with 10% FBS, plus 100 IU/mL of penicillin and 100 μg/mL of streptomycin with rosiglitazone (1 or 10 μmol/L) or vehicle control for 24 hours, then transferred into Krebs solution and mounted in a myograph. Real-time changes in arterial tone were measured as described.12
Isometric Tension Measurement
Blood vessels were prepared as described previously.13 Briefly, each ring was suspended between 2 small tungsten wires in an organ chamber (Multi Myograph System) filled with Krebs-Henseleit solution and placed under a previously determined optimal resting tension of 3 mN for aortas and 1 mN for mesenteric resistance arteries and left for 90-minute equilibration. The concentration-dependent contractions to ET-1 (1 to 50 nmol/L) were compared in control or rosiglitazone-treated rings in the absence and presence of l-NAME (100 μmol/L). The effects of antagonists of both ETAR and ETBR were tested on ET-1–induced contractions. In some experiments, the endothelium was mechanically disrupted, which was confirmed by a complete loss of relaxation to acetylcholine.
Human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells were isolated and cultured as described.5
RNA Isolation and Northern Blotting
Total RNA was isolated from HUVECs using TRIzol reagent (Invitrogen), fractionated on formaldehyde-agarose gels, transferred to nylon membranes, and hybridized to cDNA probes for human ETBR and GAPDH cDNAs. The cDNA probes for ETBR and GAPDH were synthesized by RT-PCR, purified, and labeled with 32P-dCTP using the Prime-a-Gene Labeling System (Promega), as described.5
Real-Time Quantitative RT-PCR
Total RNA was reverse transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase and oligo-dT primer. Real-time PCR involved SYBR-green dye and Taq polymerase; the results were analyzed with the Opticon Monitor analysis software. The sequences of the primers were listed in Table S1 (available in the online Data Supplement at http://hyper.ahajournals.org).
Protein Extraction and Western Blotting
Aortas were isolated and frozen in liquid nitrogen after rosiglitazone treatment and homogenized in radioimmunoprecipitation assay lysis buffer. Proteins were extracted from the vessels and HUVECs as described.5 Western analyses were performed with appropriate primary antibodies and a horseradish peroxidase–conjugated secondary antibody followed by ECL detection (Amersham Biosciences).
HUVECs were coinfected with AdPPAR-γ that encodes a mouse PPAR-γ1 fused to a minimal VP16 transactivation domain and AdtTA that encodes a tetracycline-responsive transactivator and maintained in the presence or absence of tetracycline (0.1 μg/mL, a tet-off expression) for 48 hours as described.5
Cross-sections in 5-μm thickness were cut in paraffin-embedded aortic rings, treated with citrate buffer for antigen retrieval, incubated with 3% H2O2 to block endogenous peroxidase, and blocked in 5% normal goat serum. Anti-ETBR antibody was added and incubated overnight at 4°C, followed by Biotin-SP–conjugated secondary antibodies (Jackson Immunoresearch), then incubated with streptavidin–horseradish peroxidase conjugate (Zymed), and visualized by diaminobenzidine (Vector Labs).
Chromatin Immunoprecipitation Assay
Cells were cross-linked with 1% formaldehyde and quenched before harvest and sonication. The sheared chromatin was immunoprecipitated with 5 μg of anti–PPAR-γ antibody (or control IgG) and protein A Sepharose beads. The eluted immunoprecipitates were digested with proteinase K, and DNA was extracted and underwent PCR with the primers flanking the putative peroxisome proliferator response element binding motifs.
Plasmids, Transfection, and Reporter Assay
The pGL3/hETBR promoter-luciferase reporter was made by PCR cloning. Briefly, a 5′-flanking region (−1415 to +28) of the human ETBR gene was amplified from human genomic DNA using specific primers and subcloned into pGL3-basic. Plasmids were transfected into bovine aortic endothelial cells with the use of Lipofectamine 2000 (Invitrogen). The plasmid expressing β-galactosidase (pRSV-gal) was cotransfected to normalize the transfection efficiency. At 48 hours after transfection, cell lysates were harvested to measure luciferase and β-galactosidase activities.
Quantitative data are mean±SEM. Arterial contractions were expressed as active tension [tone developed/(2×ring length in millimeters)].13 Statistical analyses were performed with 1- or 2-way ANOVA or Student t test; Bonferroni post hoc tests were performed when >2 treatments were compared (GraphPad Prism, version 4.0, GraphPad), with statistical significance set at P<0.05. Nonquantitative results were representative of ≥3 independent experiments.
Rosiglitazone Attenuates Vasoconstrictive Effect of ET-1
To investigate the effect of rosiglitazone on vasoconstriction induced by ET-1, male C57BL/6J mouse thoracic aortic rings were treated with rosiglitazone (10 μmol/L) for 24 hours, and the constrictive responses to ET-1 were examined. Original recordings in Figure 1A show that, in aortas with endothelium, ET-1 produced concentration-dependent contractions, which were significantly reduced by 24-hour treatment with rosiglitazone (Figure 1B). By contrast, the acute (30-minute) exposure to 10 μmol/L of rosiglitazone did not modulate ET-1–induced contractions (data not shown). Rosiglitazone (24-hour) treatment did not affect U46619 (a thromboxane A2 agonist)-induced contraction (Figure 1C; n=5). Likewise, the 60 mmol/L of K+-induced contraction was comparable in control (2.48±0.10 mN/mm) and rosiglitazone-treated (2.41±0.13 mN/mm) rings (P>0.05; n=4).
Inhibitory Effect of Rosiglitazone Is Abolished by ETBR Receptor Antagonist, NO Synthase Inhibitor, and Endothelium Denudation
To examine which ET receptor subtype contributed to the effects of rosiglitazone on ET-1–induced vasoconstriction, endothelium-intact and endothelium-denuded aortic rings treated with rosiglitazone were examined in the presence or absence of ETAR and ETBR antagonists. The specificities of ETAR and ETBR antagonists were also tested on ET-1–induced contractions. Finally, the role of endothelium-derived NO was examined in rings with endothelium pretreated with l-NAME, an endothelial NO synthase inhibitor.
The aortic contraction in response to ET-1 was mediated through ETAR, because the selective ETAR antagonist ABT627 (10 nmol/L) abolished the contraction in control and rosiglitazone-treated rings (Figure 2A). The attenuated ET-1–induced contraction of rosiglitazone-treated rings was restored by the presence of a selective ETBR antagonist, A192621 (10 nmol/L), whereas this antagonist did not modulate the evoked contractions in control rings (Figure 2B). The difference in the amplitude of contractions between control and rosiglitazone-treated rings was lost in rings that had been previously exposed to 100 μmol/L of l-NAME for 30 minutes (Figure 2C) or in rings without endothelium (Figure 2D).
TZDs Increase mRNA and Protein Levels of ETBR in Aortas and Endothelial Cells
To investigate how rosiglitazone affected the expression of ETBR, male C57BL/6J mice thoracic aortas were treated with 1 and 10 μmol/L of rosiglitazone for 24 hours and examined for the ETBR protein level. Western blotting showed an upregulation of ETBR with rosiglitazone treatment, which was much greater in endothelium-intact than in endothelium-denude aortas (Figure 3A), whereas the ETAR expression was unchanged (Figure 3B). Immunohistochemical staining also showed that ETBR was expressed in both endothelial cells and vascular smooth muscle cells at low levels in normal mouse aortas (Figure 3C), and rosiglitazone treatment increased the ETBR expression, which was primarily confined to endothelial cells (Figure 3D).
To further investigate the effects of PPAR-γ activation on the expression of ETBR in endothelial cells, we first determined the ETBR mRNA level of HUVECs treated with troglitazone (10 μmol/L) or rosiglitazone (10 μmol/L) for 24 hours. The results showed that both PPAR-γ agonists upregulated the ETBR mRNA expression. ETBR mRNA was significantly increased in HUVECs with 24-hour treatment with troglitazone or rosiglitazone as detected by Northern blotting (Figure 4A). Moreover, rosiglitazone and troglitazone increased the ETBR mRNA expression in both a time- and concentration-dependent manner (Figure 4B to 4C and 4D to 4E). In addition, selective PPAR-γ antagonists GW9662 and BADGE abolished the stimulatory effects of TZDs on ETBR expression. In contrast, adenoviral overexpression of a constitutively active PPAR-γ increased ETBR mRNA and protein levels in HUVECs (Figure S1), indicating that the upregulation of ETBR by TZDs was PPAR-γ specific.
PPAR-γ Binds and Activates the ETBR Promoter
Sequence analysis using the transcription element search system (http://www.cbil.upenn.edu/tess) revealed 2 potential PPAR-responsive elements (PPRE) within the 2400-bp region upstream of the first exon of human ETBR gene. Chromatin immunoprecipitation assay indicated that PPAR-γ could directly bind to ETBR gene the PPRE located at −1301/−1289 (PPRE1) in the flanking region of human ETBR gene (Figure 5A), whereas the amplification with the primers flanking the −2146/−2134 site (PPRE2) or the −37/−23 site (RXRE) detected no binding for PPAR-γ. These results suggested that the PPRE1 could mediate the induction of ETBR gene by TZDs.
To further examine whether rosiglitazone activated the ETBR gene transcription activity, bovine aortic endothelial cells were transfected with pGL3/hETBR −1415-Luc plasmid containing human ETBR gene promoter region (ETBR gene 5′-flanking −1415 to +28 sequence that contains the PPRE1 motif) and then treated with rosiglitazone and/or pretreated with PPAR-γ–specific antagonists GW9662. Reporter gene luciferase assay shows that PPAR-γ agonist rosiglitazone enhanced ETBR gene promoter activity (P<0.05), and this effect was prevented by GW9662 (Figure 5B), indicating that ETBR is a direct target gene of PPAR-γ.
In Vivo Rosiglitazone Treatment Attenuates ET-1–Induced Vasoconstrictions
To further support the in vitro effects, mice were treated with rosiglitazone at 10 mg/kg−1 for 2 weeks, and vascular reactivities were examined by myograph. ET-1–induced vasoconstriction was attenuated after rosiglitazone treatment in both the aortas and mesenteric resistance arteries (Figure 6A and 6C). In the presence of NO synthase inhibitor, the inhibitory effects of rosiglitazone treatment on ET-1–induced vasoconstrictions were abolished in aortas and mesenteric resistance arteries (Figure 6B and 6D). Furthermore, in vivo rosiglitazone treatment also upregulated ETBR expression while leaving ETAR expression unaltered (Figure 6E and 6F) in mouse aortas. In addition, selective ETBR agonist sarafotoxin 6c induced vasodilatations in mesenteric resistance arteries only from rosiglitazone- but not vehicle-treated mice (Figure S2), suggesting a functional role of the TZD-induced ETBR in improving vascular response in vivo.
In the present study, we demonstrate for the first time that rosiglitazone upregulates the expression of ETBR in endothelial cells and attenuates ET-1–induced vasoconstriction induced through an endothelial ETBR- and NO-related pathway. In addition, we show that PPAR-γ activation induced ETBR expression in human endothelial cells.
ET-1 is a potent vasoconstrictive and growth-promoting factor and has been implicated in the pathogenesis of hypertension, atherosclerosis, restenosis after angioplasty, cardiac hypertrophy, and congestive heart failure.14,15⇓ The ET family consists of 3 isoforms, including ET-1, ET-2, and ET-3. ET-1 is the predominant member generated by vascular endothelial cells and exerts physiological and pathophysiological function through 2 different type receptors: ETAR and ETBR. Although ETAR is expressed mainly in vascular smooth muscle cells and mediates vasoconstriction and cell hypertrophy,14 ETBR is predominantly located on endothelial cells and involved in the release of NO and prostacyclin, causing vasorelaxation, inhibiting platelet aggregation, and promoting the clearance of ET-1.16,17⇓ On the other hand, ETBR is slightly expressed in vascular smooth muscle cells and contributes to vasoconstriction and ET-1 clearance.18,19⇓ Interestingly, it had been reported that PPAR-γ activation inhibited ET-1–induced cardiac hypertrophy20 and oxidized low-density lipoprotein-induced ET-1 secretion in endothelial cells. More recently, it was demonstrated that rosiglitazone treatment decreased blood pressure and was effective in preventing the progression of renal injury in deoxycorticosterone acetate-salt hypertension through reducing the overexpression of ET-1 in the kidney21 and that rosiglitazone decreased blood pressure and renal injury in a female mouse model of systemic lupus erythematosus accompanied by a reduction of urinary ET.22
The mechanisms underlying the vasoprotective effects of TZDs remain largely unknown. The present study clearly shows that 24-hour treatment with rosiglitazone suppressed ET-1–induced and ETAR-mediated contraction only in rings with endothelium. The attenuated contraction is most likely caused by the enhanced expression and function of ETBR in the endothelial cells based on the following observations. First, the effect of rosiglitazone on the aortic contraction was acutely prevented by a selective ETBR antagonist. Second, inhibition of NO production restored the reduced contraction in rosiglitazone-treated rings. Third, the reduced contraction disappeared in rings without a functional endothelium. The functional results are supported by an increased protein expression for ETBR but not ETAR in rosiglitazone-treated mouse aortas. By contrast, ETAR is expressed in both endothelium and underlying vascular smooth muscle cells. It is reported that TZD treatments reduced blood pressure and ameliorated vascular dysfunction in genetically hypertensive rats.23,24⇓ Our findings suggest additional benefit of the use of rosiglitazone in the preservation of endothelial function through the increased expression and activity of ETBR and enhanced NO availability in endothelial cells.
The present study shows that TZDs upregulated ETBR gene and protein expression through a PPAR-γ–dependent mechanism. Both agonists enhanced the ETBR expression in HUVECs, as confirmed by Northern blotting, real-time RT-PCR, and Western blotting. The upregulation of ETBR was prevented by cotreatment with PPAR-γ antagonists GW9662 and BADGE. Adenovirus-mediated overexpression of constitutively active PPAR-γ also increased the levels of ETBR mRNA and protein in HUVECs. Likewise, rosiglitazone increased the ETBR protein expression in mouse aortas, an effect prevented by the removal of endothelium. These findings indicate that the upregulation of ETBR by TZDs in endothelial cells is PPAR-γ specific. Further results with reporter assay suggest that rosiglitazone increased ETBR gene promoter activity in a PPAR-γ–dependent manner. The results with chromatin immunoprecipitation assays confirm that PPAR-γ directly bound to the PPRE site between −1301 and −1289 in the 5′-flanking region of human ETBR gene. These observations suggest that the ETBR gene is a direct target of transcriptional factor PPAR-γ.
Atherosclerosis and hypertension often coexist with and are common complications of diabetes mellitus.22,25⇓ The prevalence of atherosclerosis and hypertension in diabetic patients is approximately twice compared with that of nondiabetics.26 Conversely, the chance of developing type 2 diabetes mellitus in atherosclerosis and hypertensive individuals is also 2- to 4-fold higher than that in normotensive individuals. It is noted that rosiglitazone treatment improved endothelial function and inflammation in patients with type 2 diabetes mellitus27 and that rosiglitazone lowered blood pressure and protected endothelial function in Zucker fatty rats.28 TZDs could exert beneficial effects against the occurrence of cardiovascular disease by enhancing ETBR expression and NO production, which may be associated with a direct protection of endothelial function by inhibiting cytokine expression, smooth muscle cell migration, leukocyte adhesion, and platelet aggregation, as well as promoting endothelium-dependent relaxations. It was also suggested that TZDs improved blood flow by inhibiting neointimal formation after balloon injury in insulin-resistant rats.29 In light of the fact that the TZDs have both PPAR-γ–dependent and –independent effects, it is possible that the antihypertensive effect of TZDs may be mediated to the PPAR-γ–independent mechanisms. However, recent studies using tissue-specific PPAR-γ knockout or transgenic models have clearly demonstrated critical roles of vascular PPAR-γ in vascular relaxation and blood pressure regulation.30–33⇓⇓⇓ In this study, we also observed that in vivo treatment with rosiglitazone attenuated ET-1–induced vasoconstrictions in both conduit and resistance arteries, of which the effects were mediated through NO-related pathway accompanied with an upregulation of ETBR expression. Furthermore, selective ETBR agonist sarfarotoxin 6c caused vasodilatations in mesenteric resistance arteries of rosiglitazone-treated mice, which were sensitive to ETBR and NO synthase inhibition (Figure S1). Collectively, these data further support the notion that TZDs increased expression of ETBR and enhanced NO availability in endothelial cells, which offers an additional explanation of how TZDs improve endothelial function.
The present study demonstrates that ETBR gene is a direct target gene of PPAR-γ, and TZDs activate PPAR-γ and inhibit ET-1–induced vasoconstriction through upregulation of endothelial ETBR and promotion of NO function in blood vessels. These results may provide novel insight into our understanding of the molecular mechanisms underlying the vasoprotective effects of TZDs in treatment of the vascular complications associated with a metabolic syndrome, such as hypertension.
Sources of Funding
This work was supported by the National Natural Science Foundation of China/Research Grants Council Joint Research Scheme (30931160434, N_CUHK428/09) and grants from the National Natural Science Foundation of China (30890041 and 30821001).
J.T., W.T.W., and X.Y.T. contributed equally to this work.
- Received January 14, 2010.
- Revision received February 1, 2010.
- Accepted May 5, 2010.
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