Rosiglitazone Attenuated Endothelin-1-Induced Vasoconstriction of Pulmonary Arteries in the Rat Model of Pulmonary Arterial Hypertension via Differential Regulation of ET-1 Receptors

Pulmonary arterial hypertension (PAH) is a fatal disease characterized by a progressive increase in pulmonary arterial pressure leading to right ventricular failure and death. Activation of the endothelin (ET)-1 system has been demonstrated in plasma and lung tissue of PAH patients as well as in animal models of PAH. Recently, peroxisome proliferator-activated receptor γ (PPARγ) agonists have been shown to ameliorate PAH. The present study aimed to investigate the mechanism for the antivasoconstrictive effects of rosiglitazone in response to ET-1 in PAH. Sprague-Dawley rats were exposed to chronic hypoxia (10% oxygen) for 3 weeks. Pulmonary arteries from PAH rats showed an enhanced vasoconstriction in response to ET-1. Treatment with PPARγ agonist rosiglitazone (20 mg/kg per day) with oral gavage for 3 days attenuated the vasocontractive effect of ET-1. The effect of rosiglitazone was lost in the presence of L-NAME, indicating a nitric oxide-dependent mechanism. Western blotting revealed that rosiglitazone increased ETBR but decreased ETAR level in pulmonary arteries from PAH rats. ETBR antagonist A192621 diminished the effect of rosiglitazone on ET-1-induced contraction. These results demonstrated that rosiglitazone attenuated ET-1-induced pulmonary vasoconstriction in PAH through differential regulation of the subtypes of ET-1 receptors and, thus, provided a new mechanism for the therapeutic use of PPARγ agonists in PAH.


Introduction
Pulmonary arterial hypertension (PAH) is characterized by a progressive increase of pulmonary vascular resistance, leading to right ventricular failure and death [1]. ET-1 plasma level was elevated in the patients and experimental models for PAH [2,3]. Expression of ET-1 was increased in lung tissues of PAH patients, predominantly in pulmonary arteries [4,5]. ET-1 has 2 major subtypes of receptors: ET-A receptor (ET A R) is expressed on vascular smooth muscle cells (SMCs) and mediates vasoconstriction, whereas ET-B receptor (ET B R) is predominantly expressed in endothelial cells (ECs), where it primarily mediates vasodilatation and the clearance of ET-1. Expression of ET A R was upregulated in the lung tissues and pulmonary arteries from PAH patients with a wellestablished pathophysiological role [6][7][8]. However, a role of ET B R was rather controversial with the reports of unaltered, increased, or decreased expressions in the vessel tissues from various PAH conditions [9][10][11][12][13][14][15].
Emerging evidence suggests that peroxisome proliferatoractivated receptor-(PPAR ) agonists might have therapeutic role in treating PAH [16]. PPAR regulates the transcription of genes involved in glucose and lipid metabolism, inflammation, as well as vascular remodeling [17][18][19]. The Data were mean ± SEM from 5 to 7 rats. * < 0.05 CH + RSG versus CH group. expression of PPAR was reduced in the lungs from the PAH patients and the rat models [20,21]. Similarly, mice with deletion of PPAR in SMCs or ECs developed PAH. Pharmacological activation of PPAR ameliorated PAH. [21][22][23][24][25]. In ECs, PPAR activators inhibited thrombin-or oxidized low-density lipoproteins-(LDL-) induced ET-1 production [26,27]. In particular, we recently observed that PPAR agonist rosiglitazone attenuated ET-1-induced vasoconstriction through upregulation of ET B R in ECs [28]. However, whether the regulation of ET B R accounts for the ameliorative effect of PPAR agonists in PAH arteries remains to be elucidated. In the present study, we examined the role of rosiglitazone on ET-1-induced vasocontraction of pulmonary arteries in rat PAH models and the underlying mechanism.

Animals, Cell
Culture, and Reagents. Male Sprague-Dawley rats were used and the experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals with the approval by the institutional committee. Polyclonal rabbit anti-ET B R antibody was from Abcam. Polyclonal rabbit anti-ET A R was from Santa Cruz Biotechnology. ET-1 and Gnitro-L -arginine methyl ester ( L -NAME) were from Sigma-Aldrich Co., rosiglitazone was from GlaxoSmithKline, and A192621 was from Abbott Laboratories.

Chronic Hypoxia Induced PAH in
Rat. Rats were exposed to normobaric hypoxia (10% oxygen) or normoxia (21% oxygen) for 3 weeks and then treated with rosiglitazone (20 mg/kg per day) or water with oral gavage for 3 days.

Isometric Tension Measurement.
Left lungs were removed and placed in oxygenated Krebs-Henseleit solution. Pulmonary arteries were carefully dissected from adjacent connective tissue and cut into several ring segments of ≈2 mm long for measuring isometric force. Organ chambers (Multi Myograph System, Danish Myo Technology A/S) were filled with (37 ∘ C) Krebs solution containing (in mmol/L) 119.0 NaCl, 4.7 KCl, 2.5 CaCl 2 , 1.0 MgCl 2 , 25.0 NaHCO 3 , 1.2 KH 2 PO 4 , and 11.0 D -glucose. The Krebs solution in the organ bath was initially open to room air, being bubbled with mixed 95% O 2 and 5% CO 2 . Each ring was suspended between 2 tungsten wires (diameter, 40 m) in the chamber under optimal resting tension (2.5 mN as previously determined for the pulmonary arteries) and left for 90-minute equilibration. Vasoreactivity was measured to compare contractions in response to ET-1 (1 to 50 nmol/L) in the absence and presence of L -NAME (100 mol/L). The effects of antagonist of ET B R were tested on ET-1-induced contractions.

Western Blot Analysis.
Pulmonary arteries were dissected, frozen in liquid nitrogen, and homogenized in RIPA lysis buffer containing protease inhibitors. Protein lysates separated on 12.5% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred to PVDF membranes, which were blocked with 5% nonfat milk in Tris-buffered saline-Tween (0.2%) (TBS-T) for 1 h, incubated overnight with primary antibody and then horseradish peroxidase-(HRP-) conjugated secondary antibody, and visualized with ECL reagent.

Statistical Analysis.
Results represent mean ± SEM. Comparisons among groups involved ANOVA followed by unpaired Student's t-test. < 0.05 was considered statistically significant.

Rosiglitazone Ameliorated ET-1-Mediated Vasoconstriction in Rats with PAH.
To investigate the effect of rosiglitazone on vasoconstriction of pulmonary arteries induced by ET-1, pulmonary arteries from normoxia-, chronic hypoxia-(CH-), and rosiglitazone-treated CH-rats were dissected from groups of animals for isometric tension measurement responding to ET-1. The ET-1-induced contractions in pulmonary arteries were elevated in PAH rats compared to the normoxic rats. Treatment with PPAR agonist rosiglitazone (20 mg/kg per day) reversed the vasocontractive effect of ET-1 ( Figure 1). However, this effect of rosiglitazone was abolished by the treatment with the inhibitor of endothelial nitric oxide synthase (eNOS) L -NAME, indicating a NO-dependent mechanism (Figure 2).

Rosiglitazone Increased ET R Protein Levels in Pulmonary
Arteries from PAH Rats. To understand the mechanism for the effect of rosiglitazone on ET-1-induced vasocontraction in pulmonary arteries, we examined the protein level of ET B R with Western blotting. As shown in Figure

Inhibitory Effect of Rosiglitazone Is Abolished by ET R Antagonist.
To examine the functional role of ET B R in mediating the rosiglitazone effect on ET-1-induced vasoconstriction, pulmonary arteries were dissected from normoxia-, CH-, and rosiglitazone-treated CH-rats to measure the ET-1-responsive isometric tension in the presence or absence of A192621, a selective ET B R antagonist. In normoxic and PAH rats, A192621 (10 nmol/L) did not significantly alter the ET-1induced contraction (Figures 4(a) and 4(b)). However, in the rosiglitazone-treated pulmonary arteries, A192621 abolished the ameliorative effect on the ET-1-induced vasocontraction (Figure 4(c)).

Discussion
The vascular effects of ET-1 are mediated by 2 pharmacologically distinct G protein-coupled receptors, ET A R and ET B R [29]. ET A R is mostly expressed in SMCs and mediates the vasoconstrictive and proliferative effects of ET-1 [30]. However, ET B R expressed in ECs mediates endothelialdependent vasodilatation by stimulating the production of NO and prostacyclin, prevents apoptosis, and promotes the clearance of ET-1 [31,32]. ET B R is present in low densities on vascular smooth muscle cells where its activation induces vasoconstriction [33,34]. Since ET B R elicits vasodilation and vasoconstriction, its vascular functions in pulmonary arterial hypertension need to be further characterized. ET B Rdeficient rats developed exacerbated PAH after exposure to chronic hypoxia, characterized by elevated pulmonary arterial pressure, diminished cardiac output, increased right ventricular hypertrophy, and increased total pulmonary resistance. Plasma ET-1 level and mRNA of ET-converting enzyme-1 (ECE-1) were much higher in lungs from ET B Rdeficient rats compared with control rats. In ET B R-deficient rats, the pulmonary vessels showed less endothelial NO synthase (eNOS) and NO production, supporting a role of NO in ET B R-mediated vasodilation in the pulmonary vasculature [35]. Other studies in monocrotaline (MCT) induced PAH rats also showed that ET B R deficiency accelerated the progression of PAH and neointimal lesion [36,37]. Although both ET A R antagonist (ambrisentan) and dual ET A R/ET B R antagonist (bosentan) have been approved for treatment of PAH [38], selective antagonists for ET A R and ET B R appeared to have different effects on PAH. In a dog model for PAH, ET B R antagonist RES-701-1 was found to increase pulmonary arterial pressure whereas sarafotoxin S6c, an ET B R agonist, decreased pulmonary arterial resistance [39]. In addition, ET B R antagonist also elevated ET-1 concentrations in both in vivo and in vitro studies [40]. These findings suggest that activation of ET B R may play a protective role in the PAH. In addition to three categories of FDA-approved treatments including prostanoids, ET-1 receptor antagonists, and phosphodiesterase 5 (PDE5) inhibitors, PPAR agonists thiazolidinediones (TZDs) including rosiglitazone and PPAR Research 5 pioglitazone have shown beneficial effects in animal models of PAH. In rodent PAH models induced by MCT or hypoxia and those associated with insulin resistance, TZDs were found to effectively reduce pulmonary arterial pressure and right ventricular hypertrophy [21,22,24,25,41]. Recently, we showed that rosiglitazone reversed pulmonary arterial remodeling and inhibited vasoconstriction in response to serotonin in the rat PAH models induced by MCT and hypoxia. Although the molecular mechanisms underlying the TZD effects on PAH development remain unclear, a generally accepted hypothesis is that TZDs may act via their receptor PPAR to modulate the expression of key genes involved in the pathogenesis of PAH such as ET-1, eNOS, p27 KIP1 , adiponectin, apoE, MMP, and RhoA/ROCK. In this study, we provided in vivo evidence that rosiglitazone ameliorated ET-1-induced vasocontraction in the pulmonary arteries of PAH rats (Figure 1). The ameliorative effect of rosiglitazone was mediated via differential regulation of ET-1 receptors. In particular, the upregulation of ET B R might play a major role because rosiglitazone treatment increased the expression of ET B R in the pulmonary arteries ( Figure 3) and A192621, a selective antagonist of ET B R, abrogated the effect ( Figure 4). Conversely, rosiglitazone inhibited the induction of ET A R in the pulmonary arteries of PAH rats (Supplemental Figure 2). It is conceivable that rosiglitazone may have the vasoprotective effects by altering the ratio of ET A/B receptors. ET B R in ECs may increase Ca 2+ influx and the activation of eNOS, which leads to the production of NO and induction of vascular relaxation. This notion is corroborated with the result that the effect of rosiglitazone was abolished in the presence of L -NAME, an inhibitor of eNOS ( Figure 2). Importantly, the induction of endothelial ET B R is considered to be a PPAR -specific mechanism as we previously identified ET B R to be a direct target gene of PPAR [28].

Conclusions
In conclusion, we demonstrated that rosiglitazone upregulated the expression of ET B R, which mediated the decreased vasoconstriction in the rat models of PAH. This finding suggested a new mechanism for the protective role of PPAR in the development of PAH.