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Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells
LFS-14827; No of Pages 5 Life Sciences xxx (2016) xxx–xxx
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Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells Satoshi Sakai ⁎, Hidekazu Maruyama, Taizo Kimura, Kazuko Tajiri, Junya Honda, Satoshi Homma, Kazutaka Aonuma, Takashi Miyauchi Division of Cardiovascular Medicine, Department of Clinical Medicine, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
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Article history: Received 1 November 2015 Received in revised form 23 March 2016 Accepted 24 March 2016 Available online xxxx Keywords: Endothelin receptor antagonist Apoptosis Pulmonary arterial smooth muscle cell Pulmonary vascular remodeling
a b s t r a c t Aims: Vascular remodeling results from aberrations in the balance between cell proliferation and death, which is seen in the obstructive vasculature of pulmonary arterial hypertension (PAH). Endothelin (ET)-1 has a potent proliferative activity on vascular smooth muscle cells, and ET receptor inhibitors are used to treat PAH; however, it remains unclear whether ET receptor inhibition contributes to the apoptosis of pulmonary arterial smooth muscle cells (PASMCs), another cause of pulmonary vascular remodeling. Main methods: Cultured human PASMCs were treated with the ETA receptor antagonist BQ-123 (100 μM), or the ETB antagonist A-192621 (1 – 100 μM) or BQ-788 (1 – 100 μM) for 48 h. The cells were then incubated for another 24 h with or without doxorubicin (DOX, 1 μM), an anthracyclin antitumor antibiotic that promotes p53-mediated apoptosis. Cell viability and apoptosis were evaluated by MTT assays, caspase-3/7 activity assays, and Western blots for cleaved caspase-3 expression. Key findings: The viability of PASMCs was significantly decreased by A-192621 and BQ-788, in a dose-dependent manner. A-192621 and BQ-788 significantly increased the caspase-3/7 activity and cleaved caspase-3 expression in PASMCs. The PASMCs' susceptibility to DOX-induced apoptosis was significantly higher in the presence of A192621 and BQ-788 than with vehicle. However, BQ-123 did not affect these parameters. Significance: Blockade of the ETB receptor increases the extent of apoptosis and susceptibility to DOX-induced apoptosis in PASMCs. Apoptosis caused by ETB receptor blockade in PASMCs may be one of the mechanisms by which vascular remodeling is reduced in ET receptor inhibitor-based PAH treatments. © 2016 Elsevier Inc. All rights reserved.
1. Background Vascular remodeling results from aberrations in the balance between cell proliferation and death [10]. The enhanced proliferation of vascular smooth muscle cells is a major phenotype of obstructive vasculature diseases such as cessation-induced restenosis [21] and pulmonary arterial hypertension (PAH) [19]. Endothelin (ET)-1 [23] has a potent proliferative activity on vascular smooth muscle cells [13], through the receptor ETA [2]. Huang et al. showed that the expression of ET signaling and cell-proliferation pathway components are increased in the thickened vascular wall of the pulmonary arteries in PAH patients [8]. Thus, ET-1 is involved in the development of pulmonary arterial smooth muscle cell (PASMC) proliferation, and ET receptor antagonists have an ameliorating effect on PAH and are used therapeutically [4,6,14]. Meanwhile, some researchers have reported that ET-1 has a neuroprotective activity on olfactory epithelium [12] and that the blockade ⁎ Corresponding author. E-mail address: [email protected] (S. Sakai).
of ETB receptors induces apoptosis in glioma cells [15] and melanoma cells [11], suggesting that ET-1 signaling protects some kinds of cells from cell death. These findings led to the proposal that ET receptor antagonists suppress cell proliferation not only by inhibiting cell duplication but also by promoting cell death. However, it remains unclear whether ET receptor inhibition contributes to the apoptosis of PASMCs, another cause of pulmonary vascular remodeling. Apoptosis is a process of programmed cell death initiated through one of two pathways, the intrinsic and extrinsic pathways. Both pathways induce apoptosis by activating caspases [22], which are classified into two groups, initiator caspases and executioner caspases, and ultimately kill the cell by degrading proteins indiscriminately. The initiator for the intrinsic apoptosis pathway is caspase-9 and for the extrinsic pathway is caspase-8; caspase-3 is an executioner caspase that is activated upon its cleavage by one of the initiator caspases, caspase-8 or caspase-9. Therefore, the caspase-3 activity reflects the apoptosis level. We hypothesized that the inhibitory effect of ET receptor antagonists on pulmonary vascular remodeling is achieved through the promotion of cell death in addition to the blockade of ET-1′s proliferation-promoting activity in PASMCs. Here we investigated the effects of ETA and ETB
http://dx.doi.org/10.1016/j.lfs.2016.03.044 0024-3205/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: S. Sakai, et al., Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells, Life Sci (2016), http://dx.doi.org/10.1016/j.lfs.2016.03.044
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S. Sakai et al. / Life Sciences xxx (2016) xxx–xxx
receptor antagonists on apoptosis in cultured human PASMCs by measuring the capase-3 activity and activated caspase-3 expression.
luminometer (LB960, Berthold Technologies, Bad Wildbad, Germany) and corrected for the fluorogenetically determined cell viability.
2. Materials and methods
2.3.1. Western blots PASMCs at 2.5 × 106 cells/dish from each group were lysed on ice with RIPA buffer containing protease inhibitors (#11836153001, Roche), as described previously [16,17]. The protein concentration was determined with a bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Samples were separated by SDS-polyacrylamide gel electrophoresis in 10–20% polyacrylamide gradient gels, and transferred to a PVDF membrane. The membrane was blocked with 3% skim milk in trisbuffered saline (TBS) containing 0.1% Tween 20 (TBST), incubated with primary antibodies (1:1000) in a solution (W00H, DRC Co., Tokyo, Japan) overnight at 4 °C, and then reacted with a horseradish peroxidase-conjugated goat anti-rabbit antibody (#7074, Cell Signaling Technology, Boston, MA, USA) at 1:10,000 in a solution (W00B, DRC) at room temperature for 60 min. The blots were visualized with a chemiluminescent reagent (ImmunoStar, Wako) and CCD camera system (LightCapture II, Atto Co., Tokyo, Japan), and analyzed with software (CS Analyzer ver.3.0, Atto Co.). The primary antibodies were rabbit anti-cleaved caspase-3 (#9661, Cell Signaling Technology) and rabbit anti-tubulin (#2148, Cell Signaling Technology). The positive control for cleaved caspase-3 was DOX-treated PASMCs.
2.1. Study protocol Normal human pulmonary arterial smooth muscle cells (PASMCs) were purchased (CC-2581, Lonza Co., Walkersville, WD, USA). The PASMCs were cultured on plates in smooth muscle cell basal medium containing 5% fetal bovine serum (FBS) and other supplements (CC3128, Lonza Co.). After the cells reached 70–80% confluence, the medium was changed to DMEM (Wako, Osaka, Japan) containing 1% FBS and 1× insulin-transferrin-selenium supplement (Gibco, Grand Island, NY, USA) for 24 h, to reduce the possibility of apoptosis induction by serum-free conditions. The PASMCs were treated with the ETA receptor antagonist BQ-123 (100 μM), the ETB antagonist A-192621 (1 – 100 μM) or BQ-788 (1 – 100 μM), or ET-1 (0.1 μM) for 48 h. The PASMCs were then incubated for another 24 h with or without doxorubicin (DOX) (1 μM, Wako, Osaka, Japan), an anthracyclin antitumor antibiotic that is known to induce p53-mediated apoptosis [7]. DOX was added to evaluate whether the blockade of ET receptors increased the cells' susceptibility to apoptosis. Cell viability was evaluated by MTT assay (yellow tetrazole, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); apoptosis was evaluated by Western blots for cleaved caspase3, the active form, which is a critical executioner of apoptosis, and by the caspase-3/7 activity. We performed above assays in the PASMCs b10 passages. Because apoptosis is reported to be induced by ETB receptor antagonists in certain cell lines [11,15], we also assayed the caspase3/7 activity in GI-1 cells [9] (RBRC-RCB0763, RIKEN BioResource Center, Tsukuba, Japan) and MMAc cells [5] (RBRC-RCB0808), which are derived from human glioma and melanoma, respectively, as a positive control for apoptosis by ETB blockade. The GI-1 and MMAc cells (within 10 passages) were cultured in DMEM containing 10% FBS and treated with ET antagonists and DOX under the same conditions as the experiments using PASMCs. 2.2. MTT assay
2.3.2. Statistical analysis The values are shown as means ± SEM. Data were compared by a one-way ANOVA with Tukey-Kramer's HSD test using the JMP ver 11.0 statistical software (SAS Institute, Cary, NC, USA). Differences were considered significant at a P-value b 0.05. 3. Results 3.1. Cell viability MTT assays showed that the ETB antagonist A-192621 markedly reduced the cell viability of PASMCs in a dose-dependent manner (Fig. 1). Another ETB antagonist BQ-788 also decreased the viability, although to a lesser extent than with A-192621 (Fig. 1). However, the ETA antagonist BQ-123 or ET-1 did not affect the viability (Fig. 1). The cell viability
Cell viability was determined by cleavage of the yellow tetrazolium salt MTT (3-[4,5-di-methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to purple formazan crystals, which is seen in metabolically active cells (#1465007, Roche, Mannheim, Germany). Cells (7 × 104/well) were cultured in a 96-well microplate and treated with each ET receptor antagonist (1 – 100 μM) or ET-1 (0.1 μM), and DOX (1 μM) as described in the study protocol. The MTT labeling reagent was added to cells, which were incubated for 4 h. A solubilization solution was then added, and the cells were incubated overnight. The purple formazan crystals were then measured by a microplate reader (Varioskan, Thermo Scientific Inc., Waltham, MA, USA) at a spectrophotometric absorbance of 562 nm. 2.3. Caspase-3/7 activity Caspase-3/7 activity was measured by a luminescent assay kit (G8092, Promega, Madison, WI, USA). PASMCs, GI-1 cells, and MMAc cells (7 × 104 cells/well) were cultured in a 96-well microplate in 1% FBSDMEM, treated with BQ-123, A-192621, BQ-788, or ET-1 for 48 h, and then incubated with or without DOX for another 24 h. Before measuring the luminescence, the cell viability was measured to correct the caspase-3/7 activity for the intact viable cell number using a fluorogenic substrate (glycyl-phenylalanyl-aminofluorocoumarin) (G6081, Promega) by a microplate reader (Varioskan, Thermo Scientific Inc.). A luminogenic peptide substrate containing the DEVD sequence for caspase-3/7 activity was added to the cell lysate, and the mixture was incubated at 37 °C for 1 h. The total caspase-3/7 activity was luminometrically quantified by a
Fig. 1. ETB receptor blockade decreases the cell viability in PASMCs. Effect of an endothelin (ET)A receptor antagonist, BQ-123 (100 μM), ETB receptor antagonists (A-192621 and BQ788 [1 – 100 μM]), and ET-1 on the cell viability of pulmonary arterial smooth muscle cells (PASMCs) with or without doxorubicin (DOX) exposure, determined by MTT assay (n = 3 – 6 for each treatment combination). The medium was changed to DMEM containing 1% fetal bovine serum for 24 h, then the PASMCs were incubated with each ET receptor antagonist or ET-1 for 48 h. The PASMCs were then incubated with or without DOX (1 μM) for another 24 h. Values are expressed as the mean ± SEM. **P b 0.01 compared with the control (vehicle treatment without DOX). #P b 0.05, ##P b 0.01 compared to the same dose of each antagonist without DOX.
Please cite this article as: S. Sakai, et al., Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells, Life Sci (2016), http://dx.doi.org/10.1016/j.lfs.2016.03.044
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after DOX exposure tended to be lower than that without DOX in cells receiving the same amount of ETA or ETB antagonist; DOX significantly lowered the viability at some dosages of A-192621 and BQ-788 (Fig. 1). These results together suggest that blockade of the ETB receptor, but not ETA inhibition or ET-1, decreases the cell viability and increases the PASMCs' susceptibility to an apoptosis-promoting agent. 3.2. Caspase-3/7 activity The caspase-3/7 activity in PASMCs was significantly increased in a dose-dependent manner by the ETB antagonist A-192621 treatment (Fig. 2), although the other ETB antagonist BQ-788 did not affect the activity in the absence of DOX (Fig. 2). DOX exposure significantly augmented the caspase-3/7 activity in PASMCs treated with A-192621 or BQ-788 (Fig. 2). However, neither the ETA antagonist BQ-123 nor ET-1 altered the caspase-3/7 activity or the cells' susceptibility to DOX (Fig. 2). These results suggest that the ETB antagonists, but not the ETA antagonist, increase the caspase-3/7 activity in a dose-dependent manner and augment the cells' susceptibility to DOX-induced apoptosis. When the cells were treated with A-192621 at 100 μM, the caspase-3/ 7 activity was almost abolished, because high doses of A-192621 are cytotoxic to PASMCs. The caspase-3/7 activity of GI-1 cells, which were derived from human glioma, was similar to that of PASMCs (Fig. 4A), and A-192621 significantly increased this activity as well as the caspase-3/7 activity induced by DOX exposure (Fig. 4A). The Caspase-3/7 activity of MMAc cells, which were derived from human melanoma, in the absence of DOX was also similar to that in PASMCs (Fig. 4B); however, the increased activity in response to DOX exposure was markedly enhanced in MMAc cells (Fig. 4B), regardless of whether A-192621, BQ-788, BQ123, or ET-1 was present. These results suggest that the susceptibility to DOX is cell-dependent, with MMAc cells being very sensitive to DOX.
Fig. 3. Expression of an apoptosis executioner protein, cleaved caspase-3, in PASMCs determined by Western blot. (A) Representative Western blots of cleaved caspase-3. PASMCs were treated with various concentrations of the endothelin (ET)B receptor antagonist (A-192621 [1 – 100 μM]) for 48 h in DMEM containing 1% fetal bovine serum; the cells were then incubated with or without doxorubicin (DOX, 1 μM) for another 24 h. (B) Bar graph showing the relative expression of cleaved caspase-3 to tubulin, an internal control, analyzed by densitometry software. Values are expressed as the mean ± SEM (n = 3 – 4 in each treatment combination). *P b 0.01 compared with the control (vehicle treatment without DOX). ##P b 0.01 compared to the vehicle treatment with DOX. The values were compared between DOX (−) and DOX (+) at same dose of A-192621; P.C. indicates positive control.
3.3. Expression of cleaved caspase-3 The expression in PASMCs of cleaved caspase-3, the active form of caspase-3, which is a critical executioner of apoptosis, was significantly increased by A-192621 in a dose-dependent manner, regardless of DOX treatment (Fig. 3A and B). The dose-dependent increase in cleaved caspase-3 expression was significantly greater with DOX exposure
Fig. 2. ETB receptor blockade increases the caspase-3/7 activity in PASMCs. Effect of an endothelin (ET)A receptor antagonist, BQ-123 (100 μM), ETB receptor antagonists (A192621 and BQ-788 [1 – 100 μM]), and ET-1 on the caspase-3/7 activity in pulmonary arterial smooth muscle cells (PASMCs) with or without doxorubicin (DOX) exposure (n = 3 ~ 6 for each treatment combination). The medium was changed to DMEM containing 1% fetal bovine serum for 24 h, then the PASMCs were incubated with each ET receptor antagonist for 48 h. The PASMCs were then incubated with or without DOX (1 μM) for another 24 h. Values are expressed as the mean ± SEM. **P b 0.01 compared with the control (vehicle treatment without DOX). ##P b 0.01 compared to the same dose of each antagonist without DOX.
than without DOX. These results suggest that A-192621 induces apoptosis in a dose-dependent manner and increases the cells' susceptibility to apoptosis by DOX treatment. 4. Discussion In the present study, the blockade of ETB, but not of ETA, receptors increased the level of apoptosis in human PASMCs and enhanced the cells' susceptibility to DOX-induced apoptosis under our experimental conditions. That is, A-192621 and BQ-788, but not BQ-123, significantly reduced the viability of PASMCs, increased the cleaved caspase-3 protein level, and enhanced the caspase-3/7 activity in a dose-dependent manner; furthermore, DOX exposure significantly decreased the cell viability and increased the cleaved caspase-3 expression and caspase-3/7 activity, compared to DOX-untreated PASMCs that received an identical dose of A-192621 or BQ-788. ET-1 is reported to have an antiapoptotic action on endothelial cells [20], vascular smooth muscle cells [10], and melanoma cells [3] suffering from apoptosis-promoting stimuli such as serum deprivation, suggesting that ET-1 may act as a cell survival factor. In addition, the blockade of ETB receptors is reported to induce apoptosis in endothelial cells [20], glioblastoma cells [15], and melanoma cells [11]. In the present study, we revealed for the first time that ETB receptor antagonists dose-dependently induce apoptosis in PASMCs. The results were similar to previous reports using glioma [15] and melanoma cells [11], which we also confirmed in this study (Fig. 4A and B). We also demonstrated for the first time that ETB antagonists increase the PASMCs' susceptibility to apoptosis induced by DOX. These results suggest that ETB blockade induces apoptosis not only in glioma cells, melanoma cells, and endothelial cells, but also in PASMCs and possibly other cells. These findings also indicate that ETB receptor signaling is involved in cell survival.
Please cite this article as: S. Sakai, et al., Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells, Life Sci (2016), http://dx.doi.org/10.1016/j.lfs.2016.03.044
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Fig. 4. ETB receptor blockade increases the caspase-3/7 activity in GI-1 and MMAc cells. Effect of an endothelin (ET)A receptor antagonist, BQ-123 (100 μM) and ETB receptor antagonists (A192621 and BQ-788 [1 – 100 μM]) on the caspase-3/7 activity in GI-1 cells, which were derived from human glioma (A) and of MMAc cells, which were derived from human melanoma (B), with or without doxorubicin (DOX) exposure (n = 3 – 6 for each treatment combination). The medium was changed to DMEM containing 1% fetal bovine serum for 24 h, then the GI-1 or MMAc cells were incubated with each ET receptor antagonist for 48 h. The cells were then incubated with or without DOX (1 μM) for another 24 h. Bar graph values are expressed as the mean ± SEM. *P b 0.05, **P b 0.01 compared with the control (vehicle treatment without DOX). # P b 0.05, ##P b 0.01 compared to the same dose of each antagonist without DOX.
Further study is needed to elucidate whether ETB receptor inhibition leads to the apoptosis of PASMCs in vivo. The data obtained in this study also imply that the therapeutic inhibitory effect of ET receptor antagonists on pulmonary vascular remodeling would have two mechanisms of action. One is attributed to interference of the ETA-stimulated cell proliferative pathway, as shown in previous reports [2,13], and the other is blockade of the ETB-related cell survival pathway. In this model, ET receptor antagonists shift the cell division and cell death-balance of PASMCs toward the inhibition of cell proliferation by suppressing cell division via ETA blockade and by promoting cell death via ETB blockade, synergistically (Fig. 5). Here we used normal human PASMCs, in which the ETB receptor subtype is quantitatively and functionally less prominent than the ETA receptor; however, the ETB receptor is reported to become more highly expressed and functionally important [1] in the pulmonary vascular smooth muscle cells of patients with PAH. Moreover, the PASMCs from PAH patients are reported to be resistant to apoptosis. Sakao et al. reported that apoptosis-resistant endothelial cells and vascular smooth muscle cells proliferate in patients with PAH [18]. Therefore, it is possible that the ETB receptor pathway contributes to the proliferation of PASMCs in patients with PAH through the suppression of apoptosis. Furthermore, these findings also suggest that in the treatment of PAH, the blockade of both ETB and ETA receptors would attenuate the pulmonary vascular remodeling more efficiently than the blockade of ETA receptors alone. In the present study, the magnitude of the decrease in cell viability and of the increase in caspase-3/7 activity was greater in A-192621treated than in BQ-788-treated PASMCs as well as GI-1 and MMAc cells. Similar results were obtained in a glioblastoma cell line [15]. These findings suggest that A-192621 has a stronger apoptosispromoting effect than BQ-788. This difference might be explained by the molecular nature of these ETB antagonists; BQ-788 is a peptide, while A-192621 is not.
5. Conclusion The blockade of ETB receptors increases the extent of apoptosis and susceptibility to an apoptotic agent in PASMCs. The contribution of apoptosis by ETB receptor blockade in PASMCs may be a mechanism by which vascular remodeling is decreased in treating PAH.
Fig. 5. Schematic summary of the relationship between endothelin (ET) receptor subtypes (ETAR and ETBR) and the effect of ET receptor blockade on pulmonary vascular remodeling.
Please cite this article as: S. Sakai, et al., Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells, Life Sci (2016), http://dx.doi.org/10.1016/j.lfs.2016.03.044
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Conflict of interest There are no conflicts of interest. Acknowledgements This work was supported by JSPS KAKENHI, grant numbers 15K15318, 16H05220, 25293125, 15K19365, 15K19364, 25670757, and 24590654 and by a grant from the Miyauchi Project of the Tsukuba Advanced Research Alliance at the University of Tsukuba. We thank Naomi Koharazawa-Tojyo and Mari Nakagawa for their technical assistance. References [1] M. Bauer, H. Wilkens, F. Langer, S.O. Schneider, H. Lausberg, H.J. Schafers, Selective upregulation of endothelin B receptor gene expression in severe pulmonary hypertension, Circulation 105 (2002) 1034–1036. [2] M.R. Dashwood, P. Noertersheuser, M. Kirchengast, K. Muenter, Altered endothelin1 binding following balloon angioplasty of pig coronary arteries: effect of the ETA receptor antagonist, LU 135252, Cardiovasc. Res. 43 (1999) 445–456. [3] J. Eberie, L.F. Fecker, C.E. Orfanos, C.C. Geilen, Endothelin-1 decreases basic apoptotic rates in human melanoma cell lines, J. Investig. Dermatol. 119 (2002) 549–555. [4] S. Eddahibi, B. Raffestin, M. Clozel, M. Levame, S. Adnot, Protection from pulmonary hypertension with an orally active endothelin receptor antagonist in hypoxic rats, Am. J. Phys. 268 (1995) 828–835. [5] J. Furuta, A. Kaneda, Y. Umebayashi, F. Otuka, T. Sugimura, T. Ushijima, Silencing of the thrombomodulin gene in human malignant melanoma, Melanoma Res. 15 (2005) 15–20. [6] N. Galie, H. Olschewski, R.J. Oudiz, F. Torres, A. Frost, H.A. Ghofrani, D.B. Badesch, M.D. McGoon, V.V. McLaughlin, E.B. Roecker, M.J. Gerber, C. Dufton, B.L. Wiens, L.J. Rubin, Ambrisentan for the treatment of pulmonary arterial hypertension: results of the ambrisentan in pulmonary arterial hypertension, randomized, double-blind, placebo-controlled, multicenter, efficacy (ARIES) study 1 and 2, Circulation 117 (2008) 3010–3019. [7] J.W. Han, C.A. Dionne, N.L. Kedersha, V.S. Goldmacher, p53 status affects the rate of the onset but not the overall extent of doxorubicin-induced cell death in rat-1 fibroblasts constitutively expressing c-Myc, Cancer Res. 57 (1997) 176–182. [8] H. Huang, P. Zhang, Z. Wang, F. Tang, Z. Jiang, Activation of endothelin-1 receptor signaling is associated with neointima formation, neoangiogenesis and irreversible pulmonary artery hypertension in patients with congenital heart disease, Circ. J. 75 (2011) 1463–1471.
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Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells Satoshi Sakai ⁎, Hidekazu Maruyama, Taizo Kimura, Kazuko Tajiri, Junya Honda, Satoshi Homma, Kazutaka Aonuma, Takashi Miyauchi Division of Cardiovascular Medicine, Department of Clinical Medicine, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
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Article history: Received 1 November 2015 Received in revised form 23 March 2016 Accepted 24 March 2016 Available online xxxx Keywords: Endothelin receptor antagonist Apoptosis Pulmonary arterial smooth muscle cell Pulmonary vascular remodeling
a b s t r a c t Aims: Vascular remodeling results from aberrations in the balance between cell proliferation and death, which is seen in the obstructive vasculature of pulmonary arterial hypertension (PAH). Endothelin (ET)-1 has a potent proliferative activity on vascular smooth muscle cells, and ET receptor inhibitors are used to treat PAH; however, it remains unclear whether ET receptor inhibition contributes to the apoptosis of pulmonary arterial smooth muscle cells (PASMCs), another cause of pulmonary vascular remodeling. Main methods: Cultured human PASMCs were treated with the ETA receptor antagonist BQ-123 (100 μM), or the ETB antagonist A-192621 (1 – 100 μM) or BQ-788 (1 – 100 μM) for 48 h. The cells were then incubated for another 24 h with or without doxorubicin (DOX, 1 μM), an anthracyclin antitumor antibiotic that promotes p53-mediated apoptosis. Cell viability and apoptosis were evaluated by MTT assays, caspase-3/7 activity assays, and Western blots for cleaved caspase-3 expression. Key findings: The viability of PASMCs was significantly decreased by A-192621 and BQ-788, in a dose-dependent manner. A-192621 and BQ-788 significantly increased the caspase-3/7 activity and cleaved caspase-3 expression in PASMCs. The PASMCs' susceptibility to DOX-induced apoptosis was significantly higher in the presence of A192621 and BQ-788 than with vehicle. However, BQ-123 did not affect these parameters. Significance: Blockade of the ETB receptor increases the extent of apoptosis and susceptibility to DOX-induced apoptosis in PASMCs. Apoptosis caused by ETB receptor blockade in PASMCs may be one of the mechanisms by which vascular remodeling is reduced in ET receptor inhibitor-based PAH treatments. © 2016 Elsevier Inc. All rights reserved.
1. Background Vascular remodeling results from aberrations in the balance between cell proliferation and death [10]. The enhanced proliferation of vascular smooth muscle cells is a major phenotype of obstructive vasculature diseases such as cessation-induced restenosis [21] and pulmonary arterial hypertension (PAH) [19]. Endothelin (ET)-1 [23] has a potent proliferative activity on vascular smooth muscle cells [13], through the receptor ETA [2]. Huang et al. showed that the expression of ET signaling and cell-proliferation pathway components are increased in the thickened vascular wall of the pulmonary arteries in PAH patients [8]. Thus, ET-1 is involved in the development of pulmonary arterial smooth muscle cell (PASMC) proliferation, and ET receptor antagonists have an ameliorating effect on PAH and are used therapeutically [4,6,14]. Meanwhile, some researchers have reported that ET-1 has a neuroprotective activity on olfactory epithelium [12] and that the blockade ⁎ Corresponding author. E-mail address: [email protected] (S. Sakai).
of ETB receptors induces apoptosis in glioma cells [15] and melanoma cells [11], suggesting that ET-1 signaling protects some kinds of cells from cell death. These findings led to the proposal that ET receptor antagonists suppress cell proliferation not only by inhibiting cell duplication but also by promoting cell death. However, it remains unclear whether ET receptor inhibition contributes to the apoptosis of PASMCs, another cause of pulmonary vascular remodeling. Apoptosis is a process of programmed cell death initiated through one of two pathways, the intrinsic and extrinsic pathways. Both pathways induce apoptosis by activating caspases [22], which are classified into two groups, initiator caspases and executioner caspases, and ultimately kill the cell by degrading proteins indiscriminately. The initiator for the intrinsic apoptosis pathway is caspase-9 and for the extrinsic pathway is caspase-8; caspase-3 is an executioner caspase that is activated upon its cleavage by one of the initiator caspases, caspase-8 or caspase-9. Therefore, the caspase-3 activity reflects the apoptosis level. We hypothesized that the inhibitory effect of ET receptor antagonists on pulmonary vascular remodeling is achieved through the promotion of cell death in addition to the blockade of ET-1′s proliferation-promoting activity in PASMCs. Here we investigated the effects of ETA and ETB
http://dx.doi.org/10.1016/j.lfs.2016.03.044 0024-3205/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: S. Sakai, et al., Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells, Life Sci (2016), http://dx.doi.org/10.1016/j.lfs.2016.03.044
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receptor antagonists on apoptosis in cultured human PASMCs by measuring the capase-3 activity and activated caspase-3 expression.
luminometer (LB960, Berthold Technologies, Bad Wildbad, Germany) and corrected for the fluorogenetically determined cell viability.
2. Materials and methods
2.3.1. Western blots PASMCs at 2.5 × 106 cells/dish from each group were lysed on ice with RIPA buffer containing protease inhibitors (#11836153001, Roche), as described previously [16,17]. The protein concentration was determined with a bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Samples were separated by SDS-polyacrylamide gel electrophoresis in 10–20% polyacrylamide gradient gels, and transferred to a PVDF membrane. The membrane was blocked with 3% skim milk in trisbuffered saline (TBS) containing 0.1% Tween 20 (TBST), incubated with primary antibodies (1:1000) in a solution (W00H, DRC Co., Tokyo, Japan) overnight at 4 °C, and then reacted with a horseradish peroxidase-conjugated goat anti-rabbit antibody (#7074, Cell Signaling Technology, Boston, MA, USA) at 1:10,000 in a solution (W00B, DRC) at room temperature for 60 min. The blots were visualized with a chemiluminescent reagent (ImmunoStar, Wako) and CCD camera system (LightCapture II, Atto Co., Tokyo, Japan), and analyzed with software (CS Analyzer ver.3.0, Atto Co.). The primary antibodies were rabbit anti-cleaved caspase-3 (#9661, Cell Signaling Technology) and rabbit anti-tubulin (#2148, Cell Signaling Technology). The positive control for cleaved caspase-3 was DOX-treated PASMCs.
2.1. Study protocol Normal human pulmonary arterial smooth muscle cells (PASMCs) were purchased (CC-2581, Lonza Co., Walkersville, WD, USA). The PASMCs were cultured on plates in smooth muscle cell basal medium containing 5% fetal bovine serum (FBS) and other supplements (CC3128, Lonza Co.). After the cells reached 70–80% confluence, the medium was changed to DMEM (Wako, Osaka, Japan) containing 1% FBS and 1× insulin-transferrin-selenium supplement (Gibco, Grand Island, NY, USA) for 24 h, to reduce the possibility of apoptosis induction by serum-free conditions. The PASMCs were treated with the ETA receptor antagonist BQ-123 (100 μM), the ETB antagonist A-192621 (1 – 100 μM) or BQ-788 (1 – 100 μM), or ET-1 (0.1 μM) for 48 h. The PASMCs were then incubated for another 24 h with or without doxorubicin (DOX) (1 μM, Wako, Osaka, Japan), an anthracyclin antitumor antibiotic that is known to induce p53-mediated apoptosis [7]. DOX was added to evaluate whether the blockade of ET receptors increased the cells' susceptibility to apoptosis. Cell viability was evaluated by MTT assay (yellow tetrazole, 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); apoptosis was evaluated by Western blots for cleaved caspase3, the active form, which is a critical executioner of apoptosis, and by the caspase-3/7 activity. We performed above assays in the PASMCs b10 passages. Because apoptosis is reported to be induced by ETB receptor antagonists in certain cell lines [11,15], we also assayed the caspase3/7 activity in GI-1 cells [9] (RBRC-RCB0763, RIKEN BioResource Center, Tsukuba, Japan) and MMAc cells [5] (RBRC-RCB0808), which are derived from human glioma and melanoma, respectively, as a positive control for apoptosis by ETB blockade. The GI-1 and MMAc cells (within 10 passages) were cultured in DMEM containing 10% FBS and treated with ET antagonists and DOX under the same conditions as the experiments using PASMCs. 2.2. MTT assay
2.3.2. Statistical analysis The values are shown as means ± SEM. Data were compared by a one-way ANOVA with Tukey-Kramer's HSD test using the JMP ver 11.0 statistical software (SAS Institute, Cary, NC, USA). Differences were considered significant at a P-value b 0.05. 3. Results 3.1. Cell viability MTT assays showed that the ETB antagonist A-192621 markedly reduced the cell viability of PASMCs in a dose-dependent manner (Fig. 1). Another ETB antagonist BQ-788 also decreased the viability, although to a lesser extent than with A-192621 (Fig. 1). However, the ETA antagonist BQ-123 or ET-1 did not affect the viability (Fig. 1). The cell viability
Cell viability was determined by cleavage of the yellow tetrazolium salt MTT (3-[4,5-di-methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to purple formazan crystals, which is seen in metabolically active cells (#1465007, Roche, Mannheim, Germany). Cells (7 × 104/well) were cultured in a 96-well microplate and treated with each ET receptor antagonist (1 – 100 μM) or ET-1 (0.1 μM), and DOX (1 μM) as described in the study protocol. The MTT labeling reagent was added to cells, which were incubated for 4 h. A solubilization solution was then added, and the cells were incubated overnight. The purple formazan crystals were then measured by a microplate reader (Varioskan, Thermo Scientific Inc., Waltham, MA, USA) at a spectrophotometric absorbance of 562 nm. 2.3. Caspase-3/7 activity Caspase-3/7 activity was measured by a luminescent assay kit (G8092, Promega, Madison, WI, USA). PASMCs, GI-1 cells, and MMAc cells (7 × 104 cells/well) were cultured in a 96-well microplate in 1% FBSDMEM, treated with BQ-123, A-192621, BQ-788, or ET-1 for 48 h, and then incubated with or without DOX for another 24 h. Before measuring the luminescence, the cell viability was measured to correct the caspase-3/7 activity for the intact viable cell number using a fluorogenic substrate (glycyl-phenylalanyl-aminofluorocoumarin) (G6081, Promega) by a microplate reader (Varioskan, Thermo Scientific Inc.). A luminogenic peptide substrate containing the DEVD sequence for caspase-3/7 activity was added to the cell lysate, and the mixture was incubated at 37 °C for 1 h. The total caspase-3/7 activity was luminometrically quantified by a
Fig. 1. ETB receptor blockade decreases the cell viability in PASMCs. Effect of an endothelin (ET)A receptor antagonist, BQ-123 (100 μM), ETB receptor antagonists (A-192621 and BQ788 [1 – 100 μM]), and ET-1 on the cell viability of pulmonary arterial smooth muscle cells (PASMCs) with or without doxorubicin (DOX) exposure, determined by MTT assay (n = 3 – 6 for each treatment combination). The medium was changed to DMEM containing 1% fetal bovine serum for 24 h, then the PASMCs were incubated with each ET receptor antagonist or ET-1 for 48 h. The PASMCs were then incubated with or without DOX (1 μM) for another 24 h. Values are expressed as the mean ± SEM. **P b 0.01 compared with the control (vehicle treatment without DOX). #P b 0.05, ##P b 0.01 compared to the same dose of each antagonist without DOX.
Please cite this article as: S. Sakai, et al., Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells, Life Sci (2016), http://dx.doi.org/10.1016/j.lfs.2016.03.044
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after DOX exposure tended to be lower than that without DOX in cells receiving the same amount of ETA or ETB antagonist; DOX significantly lowered the viability at some dosages of A-192621 and BQ-788 (Fig. 1). These results together suggest that blockade of the ETB receptor, but not ETA inhibition or ET-1, decreases the cell viability and increases the PASMCs' susceptibility to an apoptosis-promoting agent. 3.2. Caspase-3/7 activity The caspase-3/7 activity in PASMCs was significantly increased in a dose-dependent manner by the ETB antagonist A-192621 treatment (Fig. 2), although the other ETB antagonist BQ-788 did not affect the activity in the absence of DOX (Fig. 2). DOX exposure significantly augmented the caspase-3/7 activity in PASMCs treated with A-192621 or BQ-788 (Fig. 2). However, neither the ETA antagonist BQ-123 nor ET-1 altered the caspase-3/7 activity or the cells' susceptibility to DOX (Fig. 2). These results suggest that the ETB antagonists, but not the ETA antagonist, increase the caspase-3/7 activity in a dose-dependent manner and augment the cells' susceptibility to DOX-induced apoptosis. When the cells were treated with A-192621 at 100 μM, the caspase-3/ 7 activity was almost abolished, because high doses of A-192621 are cytotoxic to PASMCs. The caspase-3/7 activity of GI-1 cells, which were derived from human glioma, was similar to that of PASMCs (Fig. 4A), and A-192621 significantly increased this activity as well as the caspase-3/7 activity induced by DOX exposure (Fig. 4A). The Caspase-3/7 activity of MMAc cells, which were derived from human melanoma, in the absence of DOX was also similar to that in PASMCs (Fig. 4B); however, the increased activity in response to DOX exposure was markedly enhanced in MMAc cells (Fig. 4B), regardless of whether A-192621, BQ-788, BQ123, or ET-1 was present. These results suggest that the susceptibility to DOX is cell-dependent, with MMAc cells being very sensitive to DOX.
Fig. 3. Expression of an apoptosis executioner protein, cleaved caspase-3, in PASMCs determined by Western blot. (A) Representative Western blots of cleaved caspase-3. PASMCs were treated with various concentrations of the endothelin (ET)B receptor antagonist (A-192621 [1 – 100 μM]) for 48 h in DMEM containing 1% fetal bovine serum; the cells were then incubated with or without doxorubicin (DOX, 1 μM) for another 24 h. (B) Bar graph showing the relative expression of cleaved caspase-3 to tubulin, an internal control, analyzed by densitometry software. Values are expressed as the mean ± SEM (n = 3 – 4 in each treatment combination). *P b 0.01 compared with the control (vehicle treatment without DOX). ##P b 0.01 compared to the vehicle treatment with DOX. The values were compared between DOX (−) and DOX (+) at same dose of A-192621; P.C. indicates positive control.
3.3. Expression of cleaved caspase-3 The expression in PASMCs of cleaved caspase-3, the active form of caspase-3, which is a critical executioner of apoptosis, was significantly increased by A-192621 in a dose-dependent manner, regardless of DOX treatment (Fig. 3A and B). The dose-dependent increase in cleaved caspase-3 expression was significantly greater with DOX exposure
Fig. 2. ETB receptor blockade increases the caspase-3/7 activity in PASMCs. Effect of an endothelin (ET)A receptor antagonist, BQ-123 (100 μM), ETB receptor antagonists (A192621 and BQ-788 [1 – 100 μM]), and ET-1 on the caspase-3/7 activity in pulmonary arterial smooth muscle cells (PASMCs) with or without doxorubicin (DOX) exposure (n = 3 ~ 6 for each treatment combination). The medium was changed to DMEM containing 1% fetal bovine serum for 24 h, then the PASMCs were incubated with each ET receptor antagonist for 48 h. The PASMCs were then incubated with or without DOX (1 μM) for another 24 h. Values are expressed as the mean ± SEM. **P b 0.01 compared with the control (vehicle treatment without DOX). ##P b 0.01 compared to the same dose of each antagonist without DOX.
than without DOX. These results suggest that A-192621 induces apoptosis in a dose-dependent manner and increases the cells' susceptibility to apoptosis by DOX treatment. 4. Discussion In the present study, the blockade of ETB, but not of ETA, receptors increased the level of apoptosis in human PASMCs and enhanced the cells' susceptibility to DOX-induced apoptosis under our experimental conditions. That is, A-192621 and BQ-788, but not BQ-123, significantly reduced the viability of PASMCs, increased the cleaved caspase-3 protein level, and enhanced the caspase-3/7 activity in a dose-dependent manner; furthermore, DOX exposure significantly decreased the cell viability and increased the cleaved caspase-3 expression and caspase-3/7 activity, compared to DOX-untreated PASMCs that received an identical dose of A-192621 or BQ-788. ET-1 is reported to have an antiapoptotic action on endothelial cells [20], vascular smooth muscle cells [10], and melanoma cells [3] suffering from apoptosis-promoting stimuli such as serum deprivation, suggesting that ET-1 may act as a cell survival factor. In addition, the blockade of ETB receptors is reported to induce apoptosis in endothelial cells [20], glioblastoma cells [15], and melanoma cells [11]. In the present study, we revealed for the first time that ETB receptor antagonists dose-dependently induce apoptosis in PASMCs. The results were similar to previous reports using glioma [15] and melanoma cells [11], which we also confirmed in this study (Fig. 4A and B). We also demonstrated for the first time that ETB antagonists increase the PASMCs' susceptibility to apoptosis induced by DOX. These results suggest that ETB blockade induces apoptosis not only in glioma cells, melanoma cells, and endothelial cells, but also in PASMCs and possibly other cells. These findings also indicate that ETB receptor signaling is involved in cell survival.
Please cite this article as: S. Sakai, et al., Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells, Life Sci (2016), http://dx.doi.org/10.1016/j.lfs.2016.03.044
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Fig. 4. ETB receptor blockade increases the caspase-3/7 activity in GI-1 and MMAc cells. Effect of an endothelin (ET)A receptor antagonist, BQ-123 (100 μM) and ETB receptor antagonists (A192621 and BQ-788 [1 – 100 μM]) on the caspase-3/7 activity in GI-1 cells, which were derived from human glioma (A) and of MMAc cells, which were derived from human melanoma (B), with or without doxorubicin (DOX) exposure (n = 3 – 6 for each treatment combination). The medium was changed to DMEM containing 1% fetal bovine serum for 24 h, then the GI-1 or MMAc cells were incubated with each ET receptor antagonist for 48 h. The cells were then incubated with or without DOX (1 μM) for another 24 h. Bar graph values are expressed as the mean ± SEM. *P b 0.05, **P b 0.01 compared with the control (vehicle treatment without DOX). # P b 0.05, ##P b 0.01 compared to the same dose of each antagonist without DOX.
Further study is needed to elucidate whether ETB receptor inhibition leads to the apoptosis of PASMCs in vivo. The data obtained in this study also imply that the therapeutic inhibitory effect of ET receptor antagonists on pulmonary vascular remodeling would have two mechanisms of action. One is attributed to interference of the ETA-stimulated cell proliferative pathway, as shown in previous reports [2,13], and the other is blockade of the ETB-related cell survival pathway. In this model, ET receptor antagonists shift the cell division and cell death-balance of PASMCs toward the inhibition of cell proliferation by suppressing cell division via ETA blockade and by promoting cell death via ETB blockade, synergistically (Fig. 5). Here we used normal human PASMCs, in which the ETB receptor subtype is quantitatively and functionally less prominent than the ETA receptor; however, the ETB receptor is reported to become more highly expressed and functionally important [1] in the pulmonary vascular smooth muscle cells of patients with PAH. Moreover, the PASMCs from PAH patients are reported to be resistant to apoptosis. Sakao et al. reported that apoptosis-resistant endothelial cells and vascular smooth muscle cells proliferate in patients with PAH [18]. Therefore, it is possible that the ETB receptor pathway contributes to the proliferation of PASMCs in patients with PAH through the suppression of apoptosis. Furthermore, these findings also suggest that in the treatment of PAH, the blockade of both ETB and ETA receptors would attenuate the pulmonary vascular remodeling more efficiently than the blockade of ETA receptors alone. In the present study, the magnitude of the decrease in cell viability and of the increase in caspase-3/7 activity was greater in A-192621treated than in BQ-788-treated PASMCs as well as GI-1 and MMAc cells. Similar results were obtained in a glioblastoma cell line [15]. These findings suggest that A-192621 has a stronger apoptosispromoting effect than BQ-788. This difference might be explained by the molecular nature of these ETB antagonists; BQ-788 is a peptide, while A-192621 is not.
5. Conclusion The blockade of ETB receptors increases the extent of apoptosis and susceptibility to an apoptotic agent in PASMCs. The contribution of apoptosis by ETB receptor blockade in PASMCs may be a mechanism by which vascular remodeling is decreased in treating PAH.
Fig. 5. Schematic summary of the relationship between endothelin (ET) receptor subtypes (ETAR and ETBR) and the effect of ET receptor blockade on pulmonary vascular remodeling.
Please cite this article as: S. Sakai, et al., Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells, Life Sci (2016), http://dx.doi.org/10.1016/j.lfs.2016.03.044
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Conflict of interest There are no conflicts of interest. Acknowledgements This work was supported by JSPS KAKENHI, grant numbers 15K15318, 16H05220, 25293125, 15K19365, 15K19364, 25670757, and 24590654 and by a grant from the Miyauchi Project of the Tsukuba Advanced Research Alliance at the University of Tsukuba. We thank Naomi Koharazawa-Tojyo and Mari Nakagawa for their technical assistance. References [1] M. Bauer, H. Wilkens, F. Langer, S.O. Schneider, H. Lausberg, H.J. Schafers, Selective upregulation of endothelin B receptor gene expression in severe pulmonary hypertension, Circulation 105 (2002) 1034–1036. [2] M.R. Dashwood, P. Noertersheuser, M. Kirchengast, K. Muenter, Altered endothelin1 binding following balloon angioplasty of pig coronary arteries: effect of the ETA receptor antagonist, LU 135252, Cardiovasc. Res. 43 (1999) 445–456. [3] J. Eberie, L.F. Fecker, C.E. Orfanos, C.C. Geilen, Endothelin-1 decreases basic apoptotic rates in human melanoma cell lines, J. Investig. Dermatol. 119 (2002) 549–555. [4] S. Eddahibi, B. Raffestin, M. Clozel, M. Levame, S. Adnot, Protection from pulmonary hypertension with an orally active endothelin receptor antagonist in hypoxic rats, Am. J. Phys. 268 (1995) 828–835. [5] J. Furuta, A. Kaneda, Y. Umebayashi, F. Otuka, T. Sugimura, T. Ushijima, Silencing of the thrombomodulin gene in human malignant melanoma, Melanoma Res. 15 (2005) 15–20. [6] N. Galie, H. Olschewski, R.J. Oudiz, F. Torres, A. Frost, H.A. Ghofrani, D.B. Badesch, M.D. McGoon, V.V. McLaughlin, E.B. Roecker, M.J. Gerber, C. Dufton, B.L. Wiens, L.J. Rubin, Ambrisentan for the treatment of pulmonary arterial hypertension: results of the ambrisentan in pulmonary arterial hypertension, randomized, double-blind, placebo-controlled, multicenter, efficacy (ARIES) study 1 and 2, Circulation 117 (2008) 3010–3019. [7] J.W. Han, C.A. Dionne, N.L. Kedersha, V.S. Goldmacher, p53 status affects the rate of the onset but not the overall extent of doxorubicin-induced cell death in rat-1 fibroblasts constitutively expressing c-Myc, Cancer Res. 57 (1997) 176–182. [8] H. Huang, P. Zhang, Z. Wang, F. Tang, Z. Jiang, Activation of endothelin-1 receptor signaling is associated with neointima formation, neoangiogenesis and irreversible pulmonary artery hypertension in patients with congenital heart disease, Circ. J. 75 (2011) 1463–1471.
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Please cite this article as: S. Sakai, et al., Antagonists to endothelin receptor type B promote apoptosis in human pulmonary arterial smooth muscle cells, Life Sci (2016), http://dx.doi.org/10.1016/j.lfs.2016.03.044