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Endothelin-Bone morphogenetic protein type 2 receptor interaction induces pulmonary artery smooth muscle cell hyperplasia in pulmonary arterial hypertension Hidekazu Maruyama, MD, PhD,a Céline Dewachter, MD, PhD,a Asmae Belhaj, MD,a,b,c Benoit Rondelet, MD, PhD,a,b,c Satoshi Sakai, MD, PhD,d Myriam Remmelink, MD, PhD,e Jean-Luc Vachiery, MD,f Robert Naeije, MD, PhD,a and Laurence Dewachter, PharmD, PhDa From the aLaboratory of Physiology and Physiopathology, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium; bDepartment of Thoracic Surgery, Erasmus University Hospital, Brussels, Belgium; cDepartment of CardioVascular and Thoracic Surgery and Lung Transplantation, Mont-Godinne University Hospital, Dinant, Belgium; d Cardiovascular Division, Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan; and the Departments of eAnatomopathology and fCardiology, Erasmus University Hospital, Brussels, Belgium.
KEYWORDS: Pulmonary arterial hypertension; endothelin; BMPR2; gremlin; pulmonary artery smooth muscle cell; proliferation
BACKGROUND: Endothelin receptor antagonists improve pulmonary arterial hypertension (PAH). Mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2) predispose to PAH. Here, we sought to determine whether there might exist interactions between these 2 signaling pathways and their effect on the acquisition of the altered phenotype of pulmonary artery smooth muscle cells (PA-SMCs) observed in PAH. METHODS: Expression of BMPR2, of the BMP agonist BMP4, and of the BMP antagonists gremlin1 and gremlin2 was evaluated in lungs and in PA-SMCs from 6 PAH patients and 14 controls treated with endothelin-1. Endothelin-1 pre-treated PA-SMCs were assessed for proliferation, apoptosis, and downstream signaling activation of Smad1/5/8 and p38 mitogen-activated protein kinase (p38MAPK) after BMP2 treatment. RESULTS: In PA-SMCs from PAH patients, expression of BMPR2 and BMP4 decreased, whereas expression of gremlin1 and gremlin2 increased compared with controls. Treatment of control PA-SMCs with endothelin-1 induced a dose-dependent increase in gremlin1 and gremlin2, whereas BMPR2 and BMP4 expression decreased, reaching similar levels as those observed in PAH cells. In control PA-SMCs, endothelin-1 pre-treatment reduced inhibitor of DNA binding 1 (Id1) expression and Smad1/5/8 activation induced by BMP2, whereas it enhanced p38MAPK activation. Moreover, BMP2 decreased serum-induced proliferation and increased the pro-apoptotic Bax/Bcl-2 ratio. These effects were attenuated by endothelin-1 pre-treatment. Endothelin-1 did not alter BMPR2 signaling in PA-SMCs from PAH patients. CONCLUSIONS: Endothelin-1 downregulates canonical BMPR2 signaling. This is related to decreased BMPR2 and increased anti-BMP gremlin expression associated with increased activation of p38MAPK and results in PA-SMC proliferation. J Heart Lung Transplant 2015;34:468–478 r 2015 International Society for Heart and Lung Transplantation. All rights reserved.
Reprint requests: Laurence Dewachter, PharmD, PhD, Laboratory of Physiology and Physiopathology, Faculty of Medicine, Université Libre de Bruxelles, CP604, 808 Lennik Rd, 1070 Brussels, Belgium. Telephone: þ32-2-555-4989. Fax: þ32-2-555-4124. E-mail address: [email protected]
The pathobiology of pulmonary arterial hypertension (PAH) is complex and remains incompletely understood.1,2 Even if the advent of disease-targeted therapy for severe PAH has reduced and delayed patient referral for lung
1053-2498/$ - see front matter r 2015 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2014.09.011
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transplant, transplantation remains an important option for most patients with advanced PAH.3 Endothelin (ET)-1 plays an important role in PAH because it is overexpressed in remodeled pulmonary arterioles of PAH patients, and ET receptor antagonists are efficacious therapy in the disease.3 Another essential piece in the pathobiologic puzzle of PAH is altered bone morphogenetic protein (BMP) type 2 receptor (BMPR2) signaling. Heterozygous germline mutations in the gene encoding BMPR2 have been reported in 75% of patients with heritable PAH and in 25% of patients with sporadic idiopathic PAH (iPAH)4 and are associated with an earlier and more aggressive disease compared with PAH patients without BMPR2 mutations.5 Reduced BMPR2 expression and activation have also been reported in patients with PAH in the absence of BMPR2 mutations.6,7 Increased expression of gremlin1, a secreted glycoprotein able to antagonize BMPR2 signaling through the binding of BMPs, was recently reported to contribute to the development of experimental murine hypoxic pulmonary hypertension.8 In that study, an argument in favor of an universal role of gremlin was provided by the observation of increased gremlin immunostaining in the pulmonary arterioles of lungs explanted from 2 patients with idiopathic PAH.8 ET-1 has been shown to present comitogenic action, potentiating the growth-promoting effects of other growth factors.9–11 There has been 1 report of an increased ET-1– induced mitosis and mitogen-activated protein kinase (MAPK) phosphorylation in the presence of BMP2 and BMP7 in pulmonary artery smooth muscle cells (PA-SMCs) from patients with iPAH.12 But how ET-1 and BMPR2 signaling pathways might interact and contribute together to PA-SMC proliferation in PAH remains uncertain. In the present study, we hypothesized that ET-1 impairs the activation of BMPR2/Smad signaling and, thereby, contributes to pulmonary arteriolar remodeling by a proliferation of PA-SMCs in patients with PAH.
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Table 1 Characteristics of Patients With Idiopathic Pulmonary Arterial Hypertension Variablesa Sex Female Male Age, years Mutation in BMPR2 Carrier Non-carrier NYHA Functional Class I II III IV 6-MWD test, meters Mean PAP, mm Hg PCWP, mm Hg Cardiac output, liters/min PVR, mm Hg/liters/min PAH therapy Endothelin receptor antagonists Phosphodiesterase 5 inhibitors Prostanoids Calcium channel blocker
iPAH patients (n ¼ 6) 5 1 48 ⫾ 5 0 6 0 0 4 2 340 ⫾ 56 56 ⫾ 7 10 ⫾ 2 3.66 ⫾ 0.35 12.7 ⫾ 1.4 6 5 6 0
6-MWD, 6-minute walking distance; BMPR2, bone morphogenetic protein type 2 receptor; IPAH, idiopathic pulmonary arterial hypertension; NYHA, New York Heart Association; PAH, pulmonary arterial hypertension; PAP, pulmonary artery pressure; PCWP, pulmonary capillary wedge pressure; PVR, pulmonary vascular resistance. a Continuous data are presented as mean ⫾ standard error of the mean and categoric data as number.
desmin, and vinculin. PA-SMCs were used for experiments from passages 3 to 6.
Lung BMPR2 and gremlin1 immunolocalization
Methods This study was approved by the local Institutional Ethics Committee at Erasmus University Hospital, Brussels, Belgium.
Study population Lung tissue and pulmonary arteries were sampled at lung transplantation in 6 patients with iPAH and during lobectomy or pneumectomy for a localized lung cancer in 14 control patients. Demographic and clinical characteristics of patients with iPAH are summarized in Table 1. The control patients (7 women, 7 men; aged 60 ⫾ 2 years) underwent transthoracic echocardiography preoperatively to rule out pulmonary hypertension, and lung specimens were sampled at a distance of tumor areas. None of the patients had BMPR2 or activin-like kinase type 1 mutations.
Culture of human PA-SMCs Human PA-SMCs were isolated and cultured, as previously described.7 To characterize the phenotype of cultured PA-SMCs, cells were assessed for expression of muscle-specific contractile and cytoskeletal proteins, including α-smooth muscle actin,
Lung specimens were fixed, embedded in paraffin, and immunohistochemically stained, as previously described.13 Primary antibodies for BMPR2 (goat polyclonal anti-human BMPR2; R&D Systems, Minneapolis, MN) and gremlin1 (goat polyclonal antimouse gremlin1; R&D Systems) were 1:100 diluted in phosphatebuffered saline and incubated overnight at 41C. Negative controls run without the primary antibody were tested.
Total messenger RNA extraction, complementary DNA preparation, and real-time quantitative polymerase chain reaction Expression of genes implicated in the regulation of BMP signaling were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR) total lung homogenates and in primary cultures of PA-SMCs isolated from iPAH patients and controls, as previously reported.7 Briefly, total RNA was extracted from snap-frozen human lung tissue by combining the method of Chomczynski and Sacchi14 using TRIzol reagent (Invitrogen, Merelbeke, Belgium) and further purified with the RNeasy Mini kit (Qiagen, Hilden, Germany). Total RNA was extracted from PA-SMCs using the RNeasy Mini kit. RNA concentration was determined by standard
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spectrophotometric techniques, and RNA integrity was assessed by visual inspection of GelRed (Biotium, Hayward, CA)-stained denaturing agarose gels. First-strand complementary DNA (cDNA) synthesis was done using SuperScript II Reverse Transcriptase System (Life Technologies, Carlsbad, CA), according to the manufacturer’s instructions. Sense and anti-sense primers for RT-qPCR were designed, using the Primer 3 program, for homo sapiens BMPR2, BMP4, gremlin1, gremlin2, cyclin D1, proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, inhibitor of DNA binding 1 (Id1) and hypoxanthine phosphoribosyltransferase (HPRT1) messenger (m)RNA sequences. To avoid inappropriate amplification of residual genomic DNA, intron-spanning primers were selected, analysis was run to check if primer pairs were only matching at the sequence of interest. For each sample, amplification reaction was performed in triplicate with SYBRGreen PCR Master mix (Quanta Biosciences, Gaithersburg, MA), specific primers, and diluted-template cDNA. Signal detection and analysis of results were performed using an iCycler system (BioRad Laboratories, Nazareth Eke, Belgium). Relative quantification was achieved with the comparative 2–ΔΔCt method by normalization with the housekeeping gene, HPRT1. Results were expressed as relative fold increase over the mean value of relative mRNA expression of the control lung group arbitrarily fixed to 1.
Effects of ET-1 on BMP signaling members PA-SMCs were seeded and allowed to adhere overnight in 10% fetal calf serum (FCS; Life Technologies, Grand Island, NY) supplemented Dulbecco’s modified Eagle medium (DMEM; Life Technologies). After 48-hour serum starvation, human recombinant ET-1 (10–8 to 10–6 mol/liter in log increments; Sigma-Aldrich, St. Louis, MO) or vehicle was added to PA-SMCs for 5 hours. Cells were washed with ice-cold PBS, harvested in specific lysis buffer, and used for RTq-PCR.
Effects of ET-1 and BMP2 on PA-SMC proliferation and apoptosis PA-SMC proliferation was assessed using 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (SigmaAldrich). Briefly, 3,000 PA-SMCs were seeded overnight in each well of a 96-well plate in DMEM containing 10% FCS (Life Technologies). After 48-hour serum starvation and 24-hour pretreatment with human recombinant ET-1 (10–7 and 10–6 mol/liter; Sigma-Aldrich), the culture medium was removed, and PA-SMCs were treated with human recombinant BMP2 (10 ng/ml; R&D Systems, Minneapolis, MN) and ET-1 (10–7 and 10–6 mol/liter) in 10% FCS supplemented DMEM during 72 hours. Finally, MTT was added to the medium at a final concentration of 5.10–7g/ml. After a 4-hour incubation, the medium was removed, and dimethyl sulfoxide was added to each well. The plate was gently rotated on an orbital shaker for 5 minutes to completely dissolve the precipitation, and absorbance was measured with a microplate reader (Bio-Rad, Hercules, CA) at 570 nm. Proliferation rates were expressed as relative fold increase over the mean value of the proliferative rate of the basal condition (corresponding to 0% FCS treated control PA-SMCs) arbitrarily fixed to 100%. To further characterize the effects of BMP2 and ET-1 on PASMC proliferation, the expression of genes implicated in cell-cycle progression, including cyclin D1 and PCNA, was also evaluated. Apoptosis activation was assessed by the determination of the proapoptotic Bax/Bcl-2 ratio. Therefore, PA-SMCs were seeded, serum-starved for 48 hours, and pre-treated with ET-1 (10–7 and
10–6 mol/liter) for 24 hours. The medium was removed, and PA-SMCs were treated for 5 hours with BMP2 (10 ng/ml) and ET1 (10–7 and 10–6 mol/L). mRNA extraction, cDNA synthesis, and RTq-PCR were performed as described above.
Effects of ET-1 and BMP2 on BMP signaling activation Activation of Smad1/5/8 and p38MAPK signaling was evaluated, as previously described.7 Briefly, PA-SMCs were seeded overnight in 10% FCS-DMEM medium. After serum starvation for 48 hours and ET-1 (10–6 mol/liter) pre-treatment for 24 hours, BMP2 (10 ng/ml) or vehicle was added to the cells for 20 minutes. Protein extracts were resolved on 4% to 12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA). After blocking with 5% non-fat milk in 0.1% Tween-Tris– buffered saline (Tris-HCl [pH 8.0]; NaCl 150 mmol/liter), membranes were incubated with monoclonal mouse anti-human phospho-p38MAPK (Thr180/Tyr182; 1:1000; Cell Signaling, Danvers, MA), polyclonal rabbit anti-human phosphoSmad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428; 1:1000; Cell Signaling), and polyclonal goat anti-human total Smad1/5/8 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with secondary horseradish-peroxidase–conjugated corresponding antibody (1:50,000; Thermo Fisher Scientific, Rockford, IL), immunoreactive bands were detected using SuperSignal WestPico Chemiluminescent substrate (Thermo Fisher Scientific) and quantified by laser densitometry. Relative quantification was performed by normalization with total Smad1/5/8 for phospho-Smad1/5/8 and β-actin (Sigma-Aldrich) for p38MAPK.
Statistical analysis Values are reported as mean ⫾ standard error of the mean. Statistical significance was tested using StatView 5.0 software (SAS Institute Inc, Cary, NC). Analysis of variance was used for comparisons between 2 groups. When the analysis of variance ratio reached a p-value of o0.05, the groups were compared using nonparametric Mann-Whitney U tests. The non-parametric Kruskal-Wallis test was performed to assess the effects of various treatments on PA-SMC proliferation, gene expression, or signaling activation. When the Kruskal-Wallis test showed a significant difference, the groups were further compared using a non-parametric Student-Newman-Keuls test. Values of p o 0.05 were accepted as significant.
Results BMP signaling in lungs In total lung homogenates from patients with iPAH, BMPR2 and BMP4 mRNA levels were similar as those of control lungs (Figure 1A and B). Gremlin1 expression was increased in total lung homogenates from iPAH patients compared with controls (Figure 1C), whereas gremlin2 expression was similar. Immunohistochemical staining showed BMPR2 and gremlin1 expression in the small intra-acinar pulmonary arterioles of iPAH patients in a distribution suggesting pulmonary arteriolar endothelial cell and SMC localization. BMPR2 staining was less marked in the remodeled pulmonary arterioles from iPAH patients than those from controls (Figure 1E), whereas gremlin1 staining was more marked in pulmonary arteries from iPAH patients
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Figure 1 Pulmonary expression and immunolocalization of bone morphogenetic protein (BMP) signaling members. Relative gene expression of (A) BMP receptor 2 (BMPR2), (B) BMP4, (C) gremlin1, and (D) gremlin2 in total lung homogenates from 6 patients with idiopathic pulmonary arterial hypertension (iPAH; black bars) and 10 controls (white bars). Values are expressed as mean ⫾ standard error of the mean. P o 0.05 compared with control lungs. Representative images of (E) BMPR2 and (F) gremlin1 immunostaining in pulmonary arterioles from iPAH patients and controls. Scale bars ¼ 20 µm.
(Figure 1F). Gremlin1 and BMPR2 were also expressed in the alveolar epithelial cells and lung fibroblasts.
BMP signaling in cultured PA-SMCs As illustrated in Figure 2, expression of BMPR2 (Figure 2A) and BMP4 (Figure 2B) was decreased in nonquiescent PA-SMCs isolated from iPAH patients, whereas levels of gremlin1 (Figure 2C) and gremlin2 (Figure 2D) were higher compared with controls. Expression of BMPR1A and BMPR1B did not differ between PA-SMCs isolated from iPAH patients and controls.
ET-1 alters the expression of BMP signaling members in control PA-SMCs As illustrated in Figure 3A and B, treatment of cultured serumstarved control PA-SMCs with increasing concentrations of human recombinant ET-1 (10–8, 10–7, and 10–6 mol/liter) caused a progressive decrease in BMPR2 and BMP4
expression, which was already significant with 10–8 mol/liter ET-1 for BMPR2 and with 10–7 mol/liter ET-1 for BMP4. In contrast, ET-1 treatment dose-dependently increased expression of gremlin1 and gremlin2 (Figure 3C and D). The expression levels reached after ET-1 treatment were similar to those observed in PA-SMCs from iPAH patients for BMPR2, BMP4, and gremlin1, whereas gremlin2 expression remained lower in control PA-SMCs treated with 10–6 mol/liter ET-1 compared with PA-SMCs from iPAH patients. In contrast to control PA-SMCs, ET-1 treatment did not affect the expression of BMPR2, BMP4, gremlin1, and gremlin2 in PA-SMCs from iPAH patients (Figure 3). ET-1 treatment did not significantly affect gene expression of BMPR1A and BMPR1B in PA-SMCs isolated from controls and iPAH patients. However, ET-1 dose-dependently reduced the ratio of BMPR1A/BMPR1B gene expression in control PA-SMCs (1.00 ⫾ 0.35, 0.78 ⫾ 0.24, and 0.51 ⫾ 0.14 in control PA-SMCs, respectively, treated with 0, 10–7, and 10–6 mol/liter ET-1 and 1.11 ⫾ 0.58, 0.91 ⫾ 0.63, and 0.77 ⫾ 0.17 in iPAH PA-SMCs treated with 0, 10–7, and 10–6 mol/liter ET-1).
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Figure 2 Expression of bone morphogenetic protein (BMP) signaling members in pulmonary artery smooth muscle cells (PA-SMCs). Relative gene expression of (A) BMP receptor 2 (BMPR2), (B) BMP4, (C) gremlin1, and (D) gremlin2 in 10% fetal calf serum-treated PA-SMCs isolated and cultured from 6 patients with idiopathic pulmonary arterial hypertension (iPAH; black bars) and 14 controls (white bars). Values are normalized relative to the corresponding expression of control lungs and are expressed as mean ⫾ standard error of the mean. *P o 0.05, ***p o 0.001 compared with control PA-SMCs.
ET-1 alone does not affect PA-SMC proliferation but alters BMP2-induced blunting effects on PA-SMC proliferation As illustrated in Figure 4, PA-SMCs isolated from iPAH patients exhibited increased proliferation induced by 10% FCS compared with controls. In PA-SMCs from iPAH patients and controls, treatment with ET-1 alone (10–7 and 10–6 mol/liter) did not affect PA-SMC proliferation, whereas ET-1 in presence of 10% FCS increased the proliferation rate compared with cells only treated with serum. The addition of BMP2 (10 ng/ml) reduced serum-induced proliferation in control PA-SMCs (by 41%), and to a lesser extent (by 25%) in cells from iPAH patients. In control PASMCs, pre-treatment with ET-1 (during 24 hours) restored (with 10–7 mol/liter ET-1) or even increased (with 10–6 mol/liter ET-1) the BMP2-induced decreased proliferation rate (Figure 4A). In PA-SMCs from iPAH, ET-1 pre-treatment dose-dependently restored proliferation reduced by BMP2 (Figure 4). In control cells, the proliferation rate obtained with 10–6 mol/liter ET-1 pre-treatment in presence with BMP2 and 10% serum remained lower than the rate observed in serumtreated PA-SMCs from iPAH patients (Figure 4). As shown in Figure 5A, BMP2 (10 ng/ml for 5 hours) decreased the expression of cyclin D1, a gene implicated in cell cycle progression, whereas ET-1 did not affect cyclin D1 expression. ET-1 pre-treatment (for 24 hours) restored cyclin D1 expression (with 10–7 mol/liter ET-1) or even increased cyclin D1 and PCNA expression (with 10–6 mol/liter ET-1) in control PA-SMCs treated with BMP2.
No treatment affected cyclin D1 and PCNA expression in PA-SMCs from iPAH patients (Figure 5A and B). BMP2 treatment increased the pro-apoptotic Bax-to-Bcl2 ratio in PA-SMCs from controls and iPAH patients. ET-1 pre-treatment dose-dependently decreased this increased pro-apoptotic Bax-to-Bcl-2 ratio in control PA-SMCs but did not in PA-SMCs from iPAH patients (Figure 5C).
ET-1 reduces activation of Smad1/5/8 signaling and activates p38MAPK signaling in control PA-SMCs To assess the downstream transmission of BMP signaling, we evaluated the expression of Id1, a BMP signaling target gene, and the activation (by phosphorylation) of Smad1/5/8 and p38MAPK signaling pathways. BMP2 treatment (10 ng/ml for 5 hours) increased the expression of Id1 in PA-SMCs from controls as well as from iPAH patients to lesser extent (Figure 6A), indicating that the transmission of BMP signaling was present but altered in PA-SMCs from iPAH patients. ET-1 pre-treatment (10–7 mol/liter and 10–6 mol/liter for 24 hours) dose-dependently reduced Id1 expression induced by BMP2 in control PA-SMCs but did not affect Id1 expression in PA-SMCs from iPAH patients. In control PA-SMCs pre-treated with ET-1 and treated with BMP2, levels of Id1 remained higher than in BMP2-treated PA-SMCs from iPAH patients (Figure 6A). ET-1 (10–7 mol/liter and 10–6 mol/liter for 24 hours) alone did not affect Id1 expression. BMP2 (10 ng/ml for 20 minutes) induced the activation (by phosphorylation) of Smad1/5/8 in PA-SMCs from iPAH patients and controls (Figure 6B). ET-1 pre-treatment
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Figure 3 Effects of endothelin (ET) -1 treatment on the expression of bone morphogenetic protein (BMP) signaling members in pulmonary artery smooth muscle cells (PA-SMCs). Relative gene expression of (A) BMP receptor 2 (BMPR2), (B) BMP4, (C) gremlin1, and (D) gremlin2 in 48-hour serum-starved PA-SMCs isolated and cultured from 6 patients with idiopathic pulmonary arterial hypertension (iPAH) and 14 controls, and treated with increasing concentrations of ET-1 (0, 10–8, 10–7 and 10–6 mol/liter) during 5 hours. Values are expressed as mean ⫾ standard error of the mean. *P o 0.05, ***p o 0.001 compared with corresponding condition in control PA-SMCs; †p o 0.05, ††p o 0.01 compared with basal condition in the same cell type.
(10–6 mol/liter during 24 hours) reduced BMP2-induced Smad1/5/8 signaling activation in control PA-SMCs, whereas no reduction was observed in PA-SMCs from iPAH patients. Pre-treatment with ET-1 alone did not affect Smad1/5/8 activation (Figure 6B). As illustrated in Figure 6C, BMP2 activated p38MAPK signaling in PA-SMCs from iPAH patients but not from controls. ET-1 pre-treatment increased the activation of p38MAPK signaling in control PA-SMCs at similar levels as those observed in BMP2-treated PA-SMCs from iPAH patients. ET-1 (10–6 mol/liter for 24 hours) alone increased the activation of p38MAPK signaling in control PA-SMCs but not from iPAH patients (Figure 6C).
Discussion The present results show that ET-1 downregulates BMP/ Smad signaling in control PA-SMCs and that this is mediated by a reduced BMPR2 expression and an induced overexpression of gremlins, associated to increased proliferation and decreased apoptosis associated to defective activation of Smad1/5/8 compensated by activated p38MAPK signaling. BMPR2 and gremlins were both expressed in the explanted lungs from patients with iPAH and controls with pulmonary arteriolar endothelial and smooth muscle localization. This is partly divergent with previous reports
of exclusive pulmonary endothelial cell localization of BMPR26 and gremlin1.8 However, more recent studies also reported a high expression of BMPR2 in PA-SMCs and pulmonary endothelial cells.7,15 Similarly, we showed increased gremlin1 expression in the pulmonary artery vessels in iPAH lungs, in a pattern compatible with endothelial and smooth muscle expression, suggesting that both cells types are able to produce gremlin1 in the small resistive vessel walls. In PA-SMCs cultured from patients with iPAH, expression of BMPR2 and BMP4 was decreased, whereas expression of the BMP antagonists, gremlin1 and gremlin2, was increased. This is in agreement with previous reports of decreased expression of BMPR2 in PA-SMCs of iPAH patients6,7 and adds essential elements of regulation by an agonist (BMP) and antagonist (gremlin) imbalance. In mice, selective deletion of BMPR2 specifically in PA-SMCs increases the susceptibility to pulmonary hypertension and pulmonary vascular remodeling.16 In contrast, BMPR2 overexpression protected mice against the development of pulmonary hypertension.17 Moreover, antagonizing gremlin1 improved experimental pulmonary hypertension induced by combined chronic hypoxia exposure and SU 5416 administration in rats, reducing pulmonary artery remodeling.18 In PA-SMCs, overexpression of gremlin1 has been shown to increase cell proliferation and migration, whereas
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Figure 4 Proliferation of pulmonary artery smooth muscle cells (PA-SMCs) pre-treated with endothelin (ET)-1 and treated with bone morphogenetic protein 2 (BMP2). PA-SMCs isolated and cultured from 10 controls and from 6 patients with idiopathic pulmonary arterial hypertension (iPAH) were serum-starved for 48 hours and pre-treated during 24 hours with ET-1 (10–7 and 10–6 mol/liter) before treatment with 10 ng/ml BMP2 in presence of 10% fetal calf serum. Proliferation was assessed using the MTT assay and normalized relative to the basal control PA-SMC proliferation rate. Values are expressed as mean ⫾ standard error of the mean. ***p o 0.001 compared with corresponding condition in control PA-SMCs; †p o 0.05, ††p o 0.01, †††p o 0.001 compared with 0% fetal calf serum-treated PA-SMCs in the same group; ‡p o 0.05, ‡‡p o 0.01 compared with corresponding ET-1 pre-treated PA-SMCs in the same group; §§p o 0.01, §§§p o 0.001 compared with BMP2-treated PA-SMCs in the same group; $$$p o 0.001 compared with PA-SMCs treated with lower ET-1 concentration.
gremlin1 gene silencing had the opposite effect.19 Increased gremlin expression and secretion in PA-SMCs from iPAH patients in the present study was optimally located to inhibit the BMPs-dependent maintenance of thin-walled relaxed pulmonary arterioles allowing for normally low pulmonary vascular resistance.20 This raises the possibility that the balanced action of BMPs and gremlins is essential for homeostatic maintenance of the pulmonary vascular structure and that alteration of this balance may contribute to decreased BMP signaling and pulmonary vascular remodeling in iPAH patients.20 BMP signaling in patients with iPAH was altered even in the absence of BMPR2 mutations, suggesting potential environmental, autocrine, and/or paracrine factors that may contribute to its decreased expression and function. The regulation of BMP signaling is poorly understood to date. Here, we show that ET-1 was able to decrease levels of BMPR2 and BMP4 and increase gremlin1 and gremlin2 expression in control PA-SMCs. The expression levels reached after ET-1 treatment were similar as those observed in cells from iPAH, suggesting the implication of ET-1 in the acquisition of altered PA-SMC phenotype in PAH. ET-1 treatment also affected the BMPR-1A/BMPR-1B ratio in favor of increased BMPR-1A expression. Paracrine factors coming from the endothelium, such as interleukin-621 and transforming growth factor-β22 have already been described as implicated in the decreased expression of BMPR2 in
PA-SMCs. Gremlin1 expression has also been shown to be regulated by growth factors such as transforming growth factor-β and platelet-derived growth factor.19 Moreover BMPs have been shown to stimulate endothelial production of ET-123 and to inhibit ET-1 release from PA-SMCs treated with cytokines.24 This strongly suggests a complex autocrine–paracrine feedback loop involving the balanced actions of ET-1 and BMP signaling operating together in the pulmonary vascular wall to maintain the normal vascular structure and function. This emphasizes the importance of the crosstalk existing between PA endothelial cells and SMCs in the pathogenesis of pulmonary vascular remodeling in PAH. We showed that ET-1 pre-treated control PA-SMCs were (depending on the dose) resistant to growth-suppressive effects of BMP2 and were even able to proliferate more than serum-stimulated cells. ET-1 has been shown to present mitogenic12 or comitogenic9–11 effects, which were more pronounced in PA-SMCs from patients with iPAH. In this study, mitogenic effects of ET-1 on PA-SMCs required the presence of serum. Taken together, our results suggest that the proliferative effects of ET-1 in PA-SMCs could be attributed (at least partly) to the downregulation of BMP/ Smad signaling, explaining why ET-1 alone fails to induce PA-SMC proliferation but not in presence of other mitogens (such as those present in serum). Moreover, we found that BMP2 activated the canonical BMP-signaling Smad1/5/8
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Figure 5 Effects of endothelin (ET)-1 pre-treatment and bone morphogenetic protein 2 (BMP2) treatment on the expression of genes implicated in the proliferation and the apoptosis of pulmonary artery smooth muscle cells (PA-SMCs). Relative gene expression of (A) cyclin D1, (B) proliferating cell nuclear antigen (PCNA), and the (C) Bax-to-Bcl-2 ratio in treated PA-SMCs isolated and cultured from 6 patients with idiopathic pulmonary arterial hypertension (iPAH) and 8 controls. PA-SMCs were serum-starved for 48 hours and pre-treated for 24 hours with ET-1 (10–7 and 10–6 mol/liter) before 5-hour treatment with 10 ng/ml BMP2. Values are expressed as mean ⫾ SEM. **P o 0.05 compared with corresponding condition in control PA-SMCs; †p o 0.05, †† p o 0.01, ††† p o 0.001 compared with basal condition in the same cell type; ‡p o 0.05 compared with corresponding ET-1 pre-treated PA-SMCs in the same group; §p o 0.01 compared with BMP2treated PA-SMCs in the same group.
and the Smad-independent p38MAPK in ET-1–treated control PA-SMCs, whereas only Smad1/5/8 signaling was activated in non-treated cells. BMP-mediated activation of p38MAPK has been shown to exert a pro-proliferative effect in PASMCs when Smad1/5/8 signaling is silenced.25 Although potentially anti-proliferative Smad1/5/8 and pro-proliferative p38MAPK signaling pathways were both activated in ET-1–treated control PA-SMCs, the functional evaluation in terms of proliferation and apoptosis was in favor of the pro-proliferative phenotype. The proliferation rate remained, however, lower in these ET-1–treated control
cells compared with the level observed in PA-SMCs from iPAH patients, suggesting the involvement of other genetic, autocrine, and/or paracrine factors in the acquisition of the altered PA-SMC phenotype in PAH. In PA-SMCs, ET-1 activates MAPKs, which in turn activate cyclins resulting in cell proliferation and subsequent PA-SMC hyperplasia in PAH.26,27 Antagonism of ET receptors is established as one of the most effective therapies in PAH patients.3 Our data showed that ET-1 upregulated PA-SMC proliferation only in the presence of other mitogens. The intracellular signaling pathway that
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Figure 6 Effects of endothelin (ET) -1 on the activation of bone morphogenic protein receptor 2 (BMPR2) signaling in pulmonary artery smooth muscle cells (PA-SMCs). (A) Relative gene expression of BMPR2 signaling target gene, the inhibitor of DNA binding (Id1) in 48-hour serum-starved, 24-hour ET-1 (10–6 mol/liter) pre-treated, and 5-hour BMP2 (10 ng/ml) treated PA-SMCs isolated and cultured from 6 patients with idiopathic pulmonary arterial hypertension (iPAH) and 8 controls. Representative immunoblots and corresponding densitometry analysis of (B) phosphorylated Smad1/5/8 (phosho-Smad1/5/8) and total Smad1/5/8 and (C) phosphorylated p38 mitogenactivated protein kinase (p38MAPK) and β-actin in 48-hour serum starved, 24-hour ET-1 (10–6 mol/liter) pre-treated, and 20-minute BMP2 (10 ng/ml)-treated PA-SMCs isolated and cultured from 3 patients with iPAH and 6 controls. Relative quantification was performed by normalization with (B) total Smad1/5/8 and (C) β-actin. Values are expressed as mean ⫾ standard error of the mean. *p o 0.05 compared with corresponding condition in control PA-SMCs; †p o 0.05, ††p o 0.01, †††p o 0.001 compared with basal condition in the same cell type; ‡p o 0.05, ‡‡p o 0.01 compared with corresponding ET-1 pre-treated PA-SMCs in the same group; §p o 0.05 compared with BMP2treated PA-SMCs in the same group.
mediates synergic pro-proliferative effects of ET-1 involves p38MAPK, which has been reported previously to contribute to the remodeling in PAH.25 This strongly supports the beneficial effect of the early use of ET receptor antagonists in the treatment of PAH patients in the context of multifactorial pathogenesis of PAH3 and to prevent the development of experimental PAH induced by systemic-topulmonary shunting in piglets.28,29 Indeed, ET receptor antagonists could also indirectly contribute to the restoration of BMPR2 signaling in PA-SMCs and therefore limit PA-SMC hyperplasia in normal PA-SMCs. In contrast, in PA-SMCs cultured from patients with end-stage iPAH, the functional effect of ET-1 pre-treatment in terms of
proliferation and apoptosis was totally negligible, suggesting that BMP signaling in these cells was constitutively altered independently of ET-1. In conclusion, the present study shows that ET-1 downregulates BMP/Smad signaling (Figure 7). ET-1 is, indeed, able to reduce the expression of BMPR2 and to over-express gremlins in control PA-SMCs, which is associated with increased proliferation of PA-SMCs. This mechanism is likely to contribute greatly to pulmonary vascular remodeling in PAH and may explain at least part of the therapeutic efficacy of ET receptor antagonists when they are used early in the pathologic processes associated with PAH.
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Figure 7 Schematic diagram of the proposed mechanism by which endothelin-1 and bone morphogenic protein (BMP) signaling interact in human control pulmonary artery smooth muscle cells (PA-SMCs). In control PA-SMCs, endothelin-1 reduces the expression of BMP receptor 2 (BMPR2) and BMP agonist (BMP4) but increases the expression of BMP antagonists (gremlin1 and gremin2). In these cells, endothelin-1 pretreatment reduces the activation of Smad1/5/8 signaling and the expression of inhibitor of DNA binding 1 (Id1) induced by BMP agonist (BMP2), but enhances p38 mitogen activated protein kinase (p38MAPK) signaling activation. This is associated with increased proliferation induced by growth factors and decreased expression of pro-apoptotic genes, probably contributing therefore to the acquisition of altered phenotype in PA-SMCs and pulmonary vascular remodeling observed in idiopathic pulmonary arterial hypertension (iPAH). ETR, endothelin receptor.
Disclosure statement The authors are grateful for technical assistance of Emilie Carette, Geoffrey De Medina, Marie-Thérèse Gauthier, and Inge Lanckriet. This work was supported by grants from the “Fonds de la Recherche Scientifique Medicale” (FNRS; Grant No. 3.4637.09) and the “Fonds pour la Chirurgie Cardiaque.” The present research work was also supported with unrestricted grants from Actelion and from Pfizer. C.D. was a “Fonds National de la Recherche Scientifique” (FNRS) doctoral fellow (“Aspirant FNRS”; Belgium). L.D. is a FNRS postdoctoral fellow (“Chargé de Recherches”; Belgium). None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.
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7. Dewachter L, Adnot S, Guignabert C, et al. Bone morphogenetic protein signalling in heritable versus idiopathic pulmonary hypertension. Eur Respir J 2009;34:1100-10. 8. Cahill E, Costello CM, Rowan SC, et al. Gremlin plays a key role in the pathogenesis of pulmonary hypertension. Circulation 2012;125:920-30. 9. Hafizi S, Allen SP, Goodwin AT, Chester AH, Yacoub MH. Endothelin-1 stimulates proliferation of human coronary smooth muscle cells via the ET(A) receptor and is co-mitogenic with growth factors. Atherosclerosis 1999;146:351-9. 10. Panettieri RA Jr, Goldie RG, Rigby PJ, Eszterhas AJ, Hay DW. Endothelin-1-induced potentiation of human airway smooth muscle proliferation: an ETA receptor-mediated phenomenon. Br J Pharmacol 1996;118:191-7. 11. Yang Z, Krasnici N, Luscher TF. Endothelin-1 potentiates human smooth muscle cell growth to PDGF: effects of ETA and ETB receptor blockade. Circulation 1999;100:5-8. 12. Yamanaka R, Otsuka F, Nakamura K, et al. Involvement of the bone morphogenetic protein system in endothelin- and aldosterone-induced cell proliferation of pulmonary arterial smooth muscle cells isolated from human patients with pulmonary arterial hypertension. Hypertens Res 2010;33:435-45. 13. Dewachter L, Adnot S, Fadel E, et al. Angiopoietin/tie2 pathway influences smooth muscle hyperplasia in idiopathic pulmonary hypertension. Am J Respir Crit Care Med 2006;174:1025-33. 14. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156-9. 15. Upton PD, Long L, Trembath RC, Morrell NW. Functional characterization of bone morphogenetic protein binding sites and Smad1/5 activation in human vascular cells. Molec Pharmacol 2008;73:539-52. 16. Song Y, Jones JE, Beppu H, Keaney JF Jr, Loscalzo J, Zhang YY. Increased susceptibility to pulmonary hypertension in heterozygous BMPR2-mutant mice. Circulation 2005;112:553-62. 17. Reynolds AM, Xia W, Holmes MD, et al. Bone morphogenetic protein type 2 receptor gene therapy attenuates hypoxic pulmonary
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hypertension. Am J Physiol Lung Cell Mol Physiol 2007;292: L1182-92. Ciuclan L, Sheppard K, Dong L, et al. Treatment with anti-gremlin 1 antibody ameliorates chronic hypoxia/SU5416-induced pulmonary arterial hypertension in mice. Am J Pathol 2013;183:1461-73. Maciel TT, Melo RS, Schor N, Campos AH. Gremlin promotes vascular smooth muscle cell proliferation and migration. J Mol Cell Cardiol 2008;44:370-9. Anderson L, Lowery JW, Frank DB, et al. Bmp2 and Bmp4 exert opposing effects in hypoxic pulmonary hypertension. Am J Physiol Regul Integr Comp Physiol 2010;298:R833-42. Brock M, Trenkmann M, Gay RE, et al. Interleukin-6 modulates the expression of the bone morphogenic protein receptor type ii through a novel Stat3-microrna cluster 17/92 pathway. Circ Res 2009;104: 1184-91. Upton PD, Davies RJ, Tajsic T, Morrell NW. Transforming growth factor-beta1 represses bone morphogenetic protein-mediated Smad signaling in pulmonary artery smooth muscle cells via Smad3. Am J Respir Cell Mol Biol 2013;49:1135-45. Star GP, Giovinazzo M, Langleben D. Bone morphogenic protein-9 stimulates endothelin-1 release from human pulmonary microvascular
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endothelial cells: a potential mechanism for elevated ET-1 levels in pulmonary arterial hypertension. Microvasc Res 2010;80:349-54. Din S, Sarathchandra P, Yacoub MH, Chester AH. Interaction between bone morphogenetic proteins and endothelin-1 in human pulmonary artery smooth muscle. Vasc Pharmacol 2009;51:344-9. Yang X, Long L, Southwood M, et al. Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension. Circ Res 2005;96:1053-63. Gallelli L, Pelaia G, D’Agostino B, et al. Endothelin-1 induces proliferation of human lung fibroblasts and il-11 secretion through an ET(A) receptordependent activation of MAP kinases. J Cell Biochem 2005;96:858-68. Yogi A, Callera GE, Montezano AC, et al. Endothelin-1, but not AngII, activates MAP kinases through c-Src independent Ras-Raf dependent pathways in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol 2007;27:1960-7. Rondelet B, Kerbaul F, Motte S, et al. Bosentan for the prevention of overcirculation-induced experimental pulmonary arterial hypertension. Circulation 2003;107:1329-35. Rondelet B, Kerbaul F, Vivian GF, et al. Sitaxsentan for the prevention of experimental shunt-induced pulmonary hypertension. Pediatr Res 2007;61:284-8.
Endothelin-Bone morphogenetic protein type 2 receptor interaction induces pulmonary artery smooth muscle cell hyperplasia in pulmonary arterial hypertension Hidekazu Maruyama, MD, PhD,a Céline Dewachter, MD, PhD,a Asmae Belhaj, MD,a,b,c Benoit Rondelet, MD, PhD,a,b,c Satoshi Sakai, MD, PhD,d Myriam Remmelink, MD, PhD,e Jean-Luc Vachiery, MD,f Robert Naeije, MD, PhD,a and Laurence Dewachter, PharmD, PhDa From the aLaboratory of Physiology and Physiopathology, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium; bDepartment of Thoracic Surgery, Erasmus University Hospital, Brussels, Belgium; cDepartment of CardioVascular and Thoracic Surgery and Lung Transplantation, Mont-Godinne University Hospital, Dinant, Belgium; d Cardiovascular Division, Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan; and the Departments of eAnatomopathology and fCardiology, Erasmus University Hospital, Brussels, Belgium.
KEYWORDS: Pulmonary arterial hypertension; endothelin; BMPR2; gremlin; pulmonary artery smooth muscle cell; proliferation
BACKGROUND: Endothelin receptor antagonists improve pulmonary arterial hypertension (PAH). Mutations in the bone morphogenetic protein (BMP) type 2 receptor (BMPR2) predispose to PAH. Here, we sought to determine whether there might exist interactions between these 2 signaling pathways and their effect on the acquisition of the altered phenotype of pulmonary artery smooth muscle cells (PA-SMCs) observed in PAH. METHODS: Expression of BMPR2, of the BMP agonist BMP4, and of the BMP antagonists gremlin1 and gremlin2 was evaluated in lungs and in PA-SMCs from 6 PAH patients and 14 controls treated with endothelin-1. Endothelin-1 pre-treated PA-SMCs were assessed for proliferation, apoptosis, and downstream signaling activation of Smad1/5/8 and p38 mitogen-activated protein kinase (p38MAPK) after BMP2 treatment. RESULTS: In PA-SMCs from PAH patients, expression of BMPR2 and BMP4 decreased, whereas expression of gremlin1 and gremlin2 increased compared with controls. Treatment of control PA-SMCs with endothelin-1 induced a dose-dependent increase in gremlin1 and gremlin2, whereas BMPR2 and BMP4 expression decreased, reaching similar levels as those observed in PAH cells. In control PA-SMCs, endothelin-1 pre-treatment reduced inhibitor of DNA binding 1 (Id1) expression and Smad1/5/8 activation induced by BMP2, whereas it enhanced p38MAPK activation. Moreover, BMP2 decreased serum-induced proliferation and increased the pro-apoptotic Bax/Bcl-2 ratio. These effects were attenuated by endothelin-1 pre-treatment. Endothelin-1 did not alter BMPR2 signaling in PA-SMCs from PAH patients. CONCLUSIONS: Endothelin-1 downregulates canonical BMPR2 signaling. This is related to decreased BMPR2 and increased anti-BMP gremlin expression associated with increased activation of p38MAPK and results in PA-SMC proliferation. J Heart Lung Transplant 2015;34:468–478 r 2015 International Society for Heart and Lung Transplantation. All rights reserved.
Reprint requests: Laurence Dewachter, PharmD, PhD, Laboratory of Physiology and Physiopathology, Faculty of Medicine, Université Libre de Bruxelles, CP604, 808 Lennik Rd, 1070 Brussels, Belgium. Telephone: þ32-2-555-4989. Fax: þ32-2-555-4124. E-mail address: [email protected]
The pathobiology of pulmonary arterial hypertension (PAH) is complex and remains incompletely understood.1,2 Even if the advent of disease-targeted therapy for severe PAH has reduced and delayed patient referral for lung
1053-2498/$ - see front matter r 2015 International Society for Heart and Lung Transplantation. All rights reserved. http://dx.doi.org/10.1016/j.healun.2014.09.011
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transplant, transplantation remains an important option for most patients with advanced PAH.3 Endothelin (ET)-1 plays an important role in PAH because it is overexpressed in remodeled pulmonary arterioles of PAH patients, and ET receptor antagonists are efficacious therapy in the disease.3 Another essential piece in the pathobiologic puzzle of PAH is altered bone morphogenetic protein (BMP) type 2 receptor (BMPR2) signaling. Heterozygous germline mutations in the gene encoding BMPR2 have been reported in 75% of patients with heritable PAH and in 25% of patients with sporadic idiopathic PAH (iPAH)4 and are associated with an earlier and more aggressive disease compared with PAH patients without BMPR2 mutations.5 Reduced BMPR2 expression and activation have also been reported in patients with PAH in the absence of BMPR2 mutations.6,7 Increased expression of gremlin1, a secreted glycoprotein able to antagonize BMPR2 signaling through the binding of BMPs, was recently reported to contribute to the development of experimental murine hypoxic pulmonary hypertension.8 In that study, an argument in favor of an universal role of gremlin was provided by the observation of increased gremlin immunostaining in the pulmonary arterioles of lungs explanted from 2 patients with idiopathic PAH.8 ET-1 has been shown to present comitogenic action, potentiating the growth-promoting effects of other growth factors.9–11 There has been 1 report of an increased ET-1– induced mitosis and mitogen-activated protein kinase (MAPK) phosphorylation in the presence of BMP2 and BMP7 in pulmonary artery smooth muscle cells (PA-SMCs) from patients with iPAH.12 But how ET-1 and BMPR2 signaling pathways might interact and contribute together to PA-SMC proliferation in PAH remains uncertain. In the present study, we hypothesized that ET-1 impairs the activation of BMPR2/Smad signaling and, thereby, contributes to pulmonary arteriolar remodeling by a proliferation of PA-SMCs in patients with PAH.
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Table 1 Characteristics of Patients With Idiopathic Pulmonary Arterial Hypertension Variablesa Sex Female Male Age, years Mutation in BMPR2 Carrier Non-carrier NYHA Functional Class I II III IV 6-MWD test, meters Mean PAP, mm Hg PCWP, mm Hg Cardiac output, liters/min PVR, mm Hg/liters/min PAH therapy Endothelin receptor antagonists Phosphodiesterase 5 inhibitors Prostanoids Calcium channel blocker
iPAH patients (n ¼ 6) 5 1 48 ⫾ 5 0 6 0 0 4 2 340 ⫾ 56 56 ⫾ 7 10 ⫾ 2 3.66 ⫾ 0.35 12.7 ⫾ 1.4 6 5 6 0
6-MWD, 6-minute walking distance; BMPR2, bone morphogenetic protein type 2 receptor; IPAH, idiopathic pulmonary arterial hypertension; NYHA, New York Heart Association; PAH, pulmonary arterial hypertension; PAP, pulmonary artery pressure; PCWP, pulmonary capillary wedge pressure; PVR, pulmonary vascular resistance. a Continuous data are presented as mean ⫾ standard error of the mean and categoric data as number.
desmin, and vinculin. PA-SMCs were used for experiments from passages 3 to 6.
Lung BMPR2 and gremlin1 immunolocalization
Methods This study was approved by the local Institutional Ethics Committee at Erasmus University Hospital, Brussels, Belgium.
Study population Lung tissue and pulmonary arteries were sampled at lung transplantation in 6 patients with iPAH and during lobectomy or pneumectomy for a localized lung cancer in 14 control patients. Demographic and clinical characteristics of patients with iPAH are summarized in Table 1. The control patients (7 women, 7 men; aged 60 ⫾ 2 years) underwent transthoracic echocardiography preoperatively to rule out pulmonary hypertension, and lung specimens were sampled at a distance of tumor areas. None of the patients had BMPR2 or activin-like kinase type 1 mutations.
Culture of human PA-SMCs Human PA-SMCs were isolated and cultured, as previously described.7 To characterize the phenotype of cultured PA-SMCs, cells were assessed for expression of muscle-specific contractile and cytoskeletal proteins, including α-smooth muscle actin,
Lung specimens were fixed, embedded in paraffin, and immunohistochemically stained, as previously described.13 Primary antibodies for BMPR2 (goat polyclonal anti-human BMPR2; R&D Systems, Minneapolis, MN) and gremlin1 (goat polyclonal antimouse gremlin1; R&D Systems) were 1:100 diluted in phosphatebuffered saline and incubated overnight at 41C. Negative controls run without the primary antibody were tested.
Total messenger RNA extraction, complementary DNA preparation, and real-time quantitative polymerase chain reaction Expression of genes implicated in the regulation of BMP signaling were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR) total lung homogenates and in primary cultures of PA-SMCs isolated from iPAH patients and controls, as previously reported.7 Briefly, total RNA was extracted from snap-frozen human lung tissue by combining the method of Chomczynski and Sacchi14 using TRIzol reagent (Invitrogen, Merelbeke, Belgium) and further purified with the RNeasy Mini kit (Qiagen, Hilden, Germany). Total RNA was extracted from PA-SMCs using the RNeasy Mini kit. RNA concentration was determined by standard
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spectrophotometric techniques, and RNA integrity was assessed by visual inspection of GelRed (Biotium, Hayward, CA)-stained denaturing agarose gels. First-strand complementary DNA (cDNA) synthesis was done using SuperScript II Reverse Transcriptase System (Life Technologies, Carlsbad, CA), according to the manufacturer’s instructions. Sense and anti-sense primers for RT-qPCR were designed, using the Primer 3 program, for homo sapiens BMPR2, BMP4, gremlin1, gremlin2, cyclin D1, proliferating cell nuclear antigen (PCNA), Bax, Bcl-2, inhibitor of DNA binding 1 (Id1) and hypoxanthine phosphoribosyltransferase (HPRT1) messenger (m)RNA sequences. To avoid inappropriate amplification of residual genomic DNA, intron-spanning primers were selected, analysis was run to check if primer pairs were only matching at the sequence of interest. For each sample, amplification reaction was performed in triplicate with SYBRGreen PCR Master mix (Quanta Biosciences, Gaithersburg, MA), specific primers, and diluted-template cDNA. Signal detection and analysis of results were performed using an iCycler system (BioRad Laboratories, Nazareth Eke, Belgium). Relative quantification was achieved with the comparative 2–ΔΔCt method by normalization with the housekeeping gene, HPRT1. Results were expressed as relative fold increase over the mean value of relative mRNA expression of the control lung group arbitrarily fixed to 1.
Effects of ET-1 on BMP signaling members PA-SMCs were seeded and allowed to adhere overnight in 10% fetal calf serum (FCS; Life Technologies, Grand Island, NY) supplemented Dulbecco’s modified Eagle medium (DMEM; Life Technologies). After 48-hour serum starvation, human recombinant ET-1 (10–8 to 10–6 mol/liter in log increments; Sigma-Aldrich, St. Louis, MO) or vehicle was added to PA-SMCs for 5 hours. Cells were washed with ice-cold PBS, harvested in specific lysis buffer, and used for RTq-PCR.
Effects of ET-1 and BMP2 on PA-SMC proliferation and apoptosis PA-SMC proliferation was assessed using 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (SigmaAldrich). Briefly, 3,000 PA-SMCs were seeded overnight in each well of a 96-well plate in DMEM containing 10% FCS (Life Technologies). After 48-hour serum starvation and 24-hour pretreatment with human recombinant ET-1 (10–7 and 10–6 mol/liter; Sigma-Aldrich), the culture medium was removed, and PA-SMCs were treated with human recombinant BMP2 (10 ng/ml; R&D Systems, Minneapolis, MN) and ET-1 (10–7 and 10–6 mol/liter) in 10% FCS supplemented DMEM during 72 hours. Finally, MTT was added to the medium at a final concentration of 5.10–7g/ml. After a 4-hour incubation, the medium was removed, and dimethyl sulfoxide was added to each well. The plate was gently rotated on an orbital shaker for 5 minutes to completely dissolve the precipitation, and absorbance was measured with a microplate reader (Bio-Rad, Hercules, CA) at 570 nm. Proliferation rates were expressed as relative fold increase over the mean value of the proliferative rate of the basal condition (corresponding to 0% FCS treated control PA-SMCs) arbitrarily fixed to 100%. To further characterize the effects of BMP2 and ET-1 on PASMC proliferation, the expression of genes implicated in cell-cycle progression, including cyclin D1 and PCNA, was also evaluated. Apoptosis activation was assessed by the determination of the proapoptotic Bax/Bcl-2 ratio. Therefore, PA-SMCs were seeded, serum-starved for 48 hours, and pre-treated with ET-1 (10–7 and
10–6 mol/liter) for 24 hours. The medium was removed, and PA-SMCs were treated for 5 hours with BMP2 (10 ng/ml) and ET1 (10–7 and 10–6 mol/L). mRNA extraction, cDNA synthesis, and RTq-PCR were performed as described above.
Effects of ET-1 and BMP2 on BMP signaling activation Activation of Smad1/5/8 and p38MAPK signaling was evaluated, as previously described.7 Briefly, PA-SMCs were seeded overnight in 10% FCS-DMEM medium. After serum starvation for 48 hours and ET-1 (10–6 mol/liter) pre-treatment for 24 hours, BMP2 (10 ng/ml) or vehicle was added to the cells for 20 minutes. Protein extracts were resolved on 4% to 12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA). After blocking with 5% non-fat milk in 0.1% Tween-Tris– buffered saline (Tris-HCl [pH 8.0]; NaCl 150 mmol/liter), membranes were incubated with monoclonal mouse anti-human phospho-p38MAPK (Thr180/Tyr182; 1:1000; Cell Signaling, Danvers, MA), polyclonal rabbit anti-human phosphoSmad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser426/428; 1:1000; Cell Signaling), and polyclonal goat anti-human total Smad1/5/8 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). After incubation with secondary horseradish-peroxidase–conjugated corresponding antibody (1:50,000; Thermo Fisher Scientific, Rockford, IL), immunoreactive bands were detected using SuperSignal WestPico Chemiluminescent substrate (Thermo Fisher Scientific) and quantified by laser densitometry. Relative quantification was performed by normalization with total Smad1/5/8 for phospho-Smad1/5/8 and β-actin (Sigma-Aldrich) for p38MAPK.
Statistical analysis Values are reported as mean ⫾ standard error of the mean. Statistical significance was tested using StatView 5.0 software (SAS Institute Inc, Cary, NC). Analysis of variance was used for comparisons between 2 groups. When the analysis of variance ratio reached a p-value of o0.05, the groups were compared using nonparametric Mann-Whitney U tests. The non-parametric Kruskal-Wallis test was performed to assess the effects of various treatments on PA-SMC proliferation, gene expression, or signaling activation. When the Kruskal-Wallis test showed a significant difference, the groups were further compared using a non-parametric Student-Newman-Keuls test. Values of p o 0.05 were accepted as significant.
Results BMP signaling in lungs In total lung homogenates from patients with iPAH, BMPR2 and BMP4 mRNA levels were similar as those of control lungs (Figure 1A and B). Gremlin1 expression was increased in total lung homogenates from iPAH patients compared with controls (Figure 1C), whereas gremlin2 expression was similar. Immunohistochemical staining showed BMPR2 and gremlin1 expression in the small intra-acinar pulmonary arterioles of iPAH patients in a distribution suggesting pulmonary arteriolar endothelial cell and SMC localization. BMPR2 staining was less marked in the remodeled pulmonary arterioles from iPAH patients than those from controls (Figure 1E), whereas gremlin1 staining was more marked in pulmonary arteries from iPAH patients
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Figure 1 Pulmonary expression and immunolocalization of bone morphogenetic protein (BMP) signaling members. Relative gene expression of (A) BMP receptor 2 (BMPR2), (B) BMP4, (C) gremlin1, and (D) gremlin2 in total lung homogenates from 6 patients with idiopathic pulmonary arterial hypertension (iPAH; black bars) and 10 controls (white bars). Values are expressed as mean ⫾ standard error of the mean. P o 0.05 compared with control lungs. Representative images of (E) BMPR2 and (F) gremlin1 immunostaining in pulmonary arterioles from iPAH patients and controls. Scale bars ¼ 20 µm.
(Figure 1F). Gremlin1 and BMPR2 were also expressed in the alveolar epithelial cells and lung fibroblasts.
BMP signaling in cultured PA-SMCs As illustrated in Figure 2, expression of BMPR2 (Figure 2A) and BMP4 (Figure 2B) was decreased in nonquiescent PA-SMCs isolated from iPAH patients, whereas levels of gremlin1 (Figure 2C) and gremlin2 (Figure 2D) were higher compared with controls. Expression of BMPR1A and BMPR1B did not differ between PA-SMCs isolated from iPAH patients and controls.
ET-1 alters the expression of BMP signaling members in control PA-SMCs As illustrated in Figure 3A and B, treatment of cultured serumstarved control PA-SMCs with increasing concentrations of human recombinant ET-1 (10–8, 10–7, and 10–6 mol/liter) caused a progressive decrease in BMPR2 and BMP4
expression, which was already significant with 10–8 mol/liter ET-1 for BMPR2 and with 10–7 mol/liter ET-1 for BMP4. In contrast, ET-1 treatment dose-dependently increased expression of gremlin1 and gremlin2 (Figure 3C and D). The expression levels reached after ET-1 treatment were similar to those observed in PA-SMCs from iPAH patients for BMPR2, BMP4, and gremlin1, whereas gremlin2 expression remained lower in control PA-SMCs treated with 10–6 mol/liter ET-1 compared with PA-SMCs from iPAH patients. In contrast to control PA-SMCs, ET-1 treatment did not affect the expression of BMPR2, BMP4, gremlin1, and gremlin2 in PA-SMCs from iPAH patients (Figure 3). ET-1 treatment did not significantly affect gene expression of BMPR1A and BMPR1B in PA-SMCs isolated from controls and iPAH patients. However, ET-1 dose-dependently reduced the ratio of BMPR1A/BMPR1B gene expression in control PA-SMCs (1.00 ⫾ 0.35, 0.78 ⫾ 0.24, and 0.51 ⫾ 0.14 in control PA-SMCs, respectively, treated with 0, 10–7, and 10–6 mol/liter ET-1 and 1.11 ⫾ 0.58, 0.91 ⫾ 0.63, and 0.77 ⫾ 0.17 in iPAH PA-SMCs treated with 0, 10–7, and 10–6 mol/liter ET-1).
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Figure 2 Expression of bone morphogenetic protein (BMP) signaling members in pulmonary artery smooth muscle cells (PA-SMCs). Relative gene expression of (A) BMP receptor 2 (BMPR2), (B) BMP4, (C) gremlin1, and (D) gremlin2 in 10% fetal calf serum-treated PA-SMCs isolated and cultured from 6 patients with idiopathic pulmonary arterial hypertension (iPAH; black bars) and 14 controls (white bars). Values are normalized relative to the corresponding expression of control lungs and are expressed as mean ⫾ standard error of the mean. *P o 0.05, ***p o 0.001 compared with control PA-SMCs.
ET-1 alone does not affect PA-SMC proliferation but alters BMP2-induced blunting effects on PA-SMC proliferation As illustrated in Figure 4, PA-SMCs isolated from iPAH patients exhibited increased proliferation induced by 10% FCS compared with controls. In PA-SMCs from iPAH patients and controls, treatment with ET-1 alone (10–7 and 10–6 mol/liter) did not affect PA-SMC proliferation, whereas ET-1 in presence of 10% FCS increased the proliferation rate compared with cells only treated with serum. The addition of BMP2 (10 ng/ml) reduced serum-induced proliferation in control PA-SMCs (by 41%), and to a lesser extent (by 25%) in cells from iPAH patients. In control PASMCs, pre-treatment with ET-1 (during 24 hours) restored (with 10–7 mol/liter ET-1) or even increased (with 10–6 mol/liter ET-1) the BMP2-induced decreased proliferation rate (Figure 4A). In PA-SMCs from iPAH, ET-1 pre-treatment dose-dependently restored proliferation reduced by BMP2 (Figure 4). In control cells, the proliferation rate obtained with 10–6 mol/liter ET-1 pre-treatment in presence with BMP2 and 10% serum remained lower than the rate observed in serumtreated PA-SMCs from iPAH patients (Figure 4). As shown in Figure 5A, BMP2 (10 ng/ml for 5 hours) decreased the expression of cyclin D1, a gene implicated in cell cycle progression, whereas ET-1 did not affect cyclin D1 expression. ET-1 pre-treatment (for 24 hours) restored cyclin D1 expression (with 10–7 mol/liter ET-1) or even increased cyclin D1 and PCNA expression (with 10–6 mol/liter ET-1) in control PA-SMCs treated with BMP2.
No treatment affected cyclin D1 and PCNA expression in PA-SMCs from iPAH patients (Figure 5A and B). BMP2 treatment increased the pro-apoptotic Bax-to-Bcl2 ratio in PA-SMCs from controls and iPAH patients. ET-1 pre-treatment dose-dependently decreased this increased pro-apoptotic Bax-to-Bcl-2 ratio in control PA-SMCs but did not in PA-SMCs from iPAH patients (Figure 5C).
ET-1 reduces activation of Smad1/5/8 signaling and activates p38MAPK signaling in control PA-SMCs To assess the downstream transmission of BMP signaling, we evaluated the expression of Id1, a BMP signaling target gene, and the activation (by phosphorylation) of Smad1/5/8 and p38MAPK signaling pathways. BMP2 treatment (10 ng/ml for 5 hours) increased the expression of Id1 in PA-SMCs from controls as well as from iPAH patients to lesser extent (Figure 6A), indicating that the transmission of BMP signaling was present but altered in PA-SMCs from iPAH patients. ET-1 pre-treatment (10–7 mol/liter and 10–6 mol/liter for 24 hours) dose-dependently reduced Id1 expression induced by BMP2 in control PA-SMCs but did not affect Id1 expression in PA-SMCs from iPAH patients. In control PA-SMCs pre-treated with ET-1 and treated with BMP2, levels of Id1 remained higher than in BMP2-treated PA-SMCs from iPAH patients (Figure 6A). ET-1 (10–7 mol/liter and 10–6 mol/liter for 24 hours) alone did not affect Id1 expression. BMP2 (10 ng/ml for 20 minutes) induced the activation (by phosphorylation) of Smad1/5/8 in PA-SMCs from iPAH patients and controls (Figure 6B). ET-1 pre-treatment
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Figure 3 Effects of endothelin (ET) -1 treatment on the expression of bone morphogenetic protein (BMP) signaling members in pulmonary artery smooth muscle cells (PA-SMCs). Relative gene expression of (A) BMP receptor 2 (BMPR2), (B) BMP4, (C) gremlin1, and (D) gremlin2 in 48-hour serum-starved PA-SMCs isolated and cultured from 6 patients with idiopathic pulmonary arterial hypertension (iPAH) and 14 controls, and treated with increasing concentrations of ET-1 (0, 10–8, 10–7 and 10–6 mol/liter) during 5 hours. Values are expressed as mean ⫾ standard error of the mean. *P o 0.05, ***p o 0.001 compared with corresponding condition in control PA-SMCs; †p o 0.05, ††p o 0.01 compared with basal condition in the same cell type.
(10–6 mol/liter during 24 hours) reduced BMP2-induced Smad1/5/8 signaling activation in control PA-SMCs, whereas no reduction was observed in PA-SMCs from iPAH patients. Pre-treatment with ET-1 alone did not affect Smad1/5/8 activation (Figure 6B). As illustrated in Figure 6C, BMP2 activated p38MAPK signaling in PA-SMCs from iPAH patients but not from controls. ET-1 pre-treatment increased the activation of p38MAPK signaling in control PA-SMCs at similar levels as those observed in BMP2-treated PA-SMCs from iPAH patients. ET-1 (10–6 mol/liter for 24 hours) alone increased the activation of p38MAPK signaling in control PA-SMCs but not from iPAH patients (Figure 6C).
Discussion The present results show that ET-1 downregulates BMP/ Smad signaling in control PA-SMCs and that this is mediated by a reduced BMPR2 expression and an induced overexpression of gremlins, associated to increased proliferation and decreased apoptosis associated to defective activation of Smad1/5/8 compensated by activated p38MAPK signaling. BMPR2 and gremlins were both expressed in the explanted lungs from patients with iPAH and controls with pulmonary arteriolar endothelial and smooth muscle localization. This is partly divergent with previous reports
of exclusive pulmonary endothelial cell localization of BMPR26 and gremlin1.8 However, more recent studies also reported a high expression of BMPR2 in PA-SMCs and pulmonary endothelial cells.7,15 Similarly, we showed increased gremlin1 expression in the pulmonary artery vessels in iPAH lungs, in a pattern compatible with endothelial and smooth muscle expression, suggesting that both cells types are able to produce gremlin1 in the small resistive vessel walls. In PA-SMCs cultured from patients with iPAH, expression of BMPR2 and BMP4 was decreased, whereas expression of the BMP antagonists, gremlin1 and gremlin2, was increased. This is in agreement with previous reports of decreased expression of BMPR2 in PA-SMCs of iPAH patients6,7 and adds essential elements of regulation by an agonist (BMP) and antagonist (gremlin) imbalance. In mice, selective deletion of BMPR2 specifically in PA-SMCs increases the susceptibility to pulmonary hypertension and pulmonary vascular remodeling.16 In contrast, BMPR2 overexpression protected mice against the development of pulmonary hypertension.17 Moreover, antagonizing gremlin1 improved experimental pulmonary hypertension induced by combined chronic hypoxia exposure and SU 5416 administration in rats, reducing pulmonary artery remodeling.18 In PA-SMCs, overexpression of gremlin1 has been shown to increase cell proliferation and migration, whereas
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Figure 4 Proliferation of pulmonary artery smooth muscle cells (PA-SMCs) pre-treated with endothelin (ET)-1 and treated with bone morphogenetic protein 2 (BMP2). PA-SMCs isolated and cultured from 10 controls and from 6 patients with idiopathic pulmonary arterial hypertension (iPAH) were serum-starved for 48 hours and pre-treated during 24 hours with ET-1 (10–7 and 10–6 mol/liter) before treatment with 10 ng/ml BMP2 in presence of 10% fetal calf serum. Proliferation was assessed using the MTT assay and normalized relative to the basal control PA-SMC proliferation rate. Values are expressed as mean ⫾ standard error of the mean. ***p o 0.001 compared with corresponding condition in control PA-SMCs; †p o 0.05, ††p o 0.01, †††p o 0.001 compared with 0% fetal calf serum-treated PA-SMCs in the same group; ‡p o 0.05, ‡‡p o 0.01 compared with corresponding ET-1 pre-treated PA-SMCs in the same group; §§p o 0.01, §§§p o 0.001 compared with BMP2-treated PA-SMCs in the same group; $$$p o 0.001 compared with PA-SMCs treated with lower ET-1 concentration.
gremlin1 gene silencing had the opposite effect.19 Increased gremlin expression and secretion in PA-SMCs from iPAH patients in the present study was optimally located to inhibit the BMPs-dependent maintenance of thin-walled relaxed pulmonary arterioles allowing for normally low pulmonary vascular resistance.20 This raises the possibility that the balanced action of BMPs and gremlins is essential for homeostatic maintenance of the pulmonary vascular structure and that alteration of this balance may contribute to decreased BMP signaling and pulmonary vascular remodeling in iPAH patients.20 BMP signaling in patients with iPAH was altered even in the absence of BMPR2 mutations, suggesting potential environmental, autocrine, and/or paracrine factors that may contribute to its decreased expression and function. The regulation of BMP signaling is poorly understood to date. Here, we show that ET-1 was able to decrease levels of BMPR2 and BMP4 and increase gremlin1 and gremlin2 expression in control PA-SMCs. The expression levels reached after ET-1 treatment were similar as those observed in cells from iPAH, suggesting the implication of ET-1 in the acquisition of altered PA-SMC phenotype in PAH. ET-1 treatment also affected the BMPR-1A/BMPR-1B ratio in favor of increased BMPR-1A expression. Paracrine factors coming from the endothelium, such as interleukin-621 and transforming growth factor-β22 have already been described as implicated in the decreased expression of BMPR2 in
PA-SMCs. Gremlin1 expression has also been shown to be regulated by growth factors such as transforming growth factor-β and platelet-derived growth factor.19 Moreover BMPs have been shown to stimulate endothelial production of ET-123 and to inhibit ET-1 release from PA-SMCs treated with cytokines.24 This strongly suggests a complex autocrine–paracrine feedback loop involving the balanced actions of ET-1 and BMP signaling operating together in the pulmonary vascular wall to maintain the normal vascular structure and function. This emphasizes the importance of the crosstalk existing between PA endothelial cells and SMCs in the pathogenesis of pulmonary vascular remodeling in PAH. We showed that ET-1 pre-treated control PA-SMCs were (depending on the dose) resistant to growth-suppressive effects of BMP2 and were even able to proliferate more than serum-stimulated cells. ET-1 has been shown to present mitogenic12 or comitogenic9–11 effects, which were more pronounced in PA-SMCs from patients with iPAH. In this study, mitogenic effects of ET-1 on PA-SMCs required the presence of serum. Taken together, our results suggest that the proliferative effects of ET-1 in PA-SMCs could be attributed (at least partly) to the downregulation of BMP/ Smad signaling, explaining why ET-1 alone fails to induce PA-SMC proliferation but not in presence of other mitogens (such as those present in serum). Moreover, we found that BMP2 activated the canonical BMP-signaling Smad1/5/8
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Figure 5 Effects of endothelin (ET)-1 pre-treatment and bone morphogenetic protein 2 (BMP2) treatment on the expression of genes implicated in the proliferation and the apoptosis of pulmonary artery smooth muscle cells (PA-SMCs). Relative gene expression of (A) cyclin D1, (B) proliferating cell nuclear antigen (PCNA), and the (C) Bax-to-Bcl-2 ratio in treated PA-SMCs isolated and cultured from 6 patients with idiopathic pulmonary arterial hypertension (iPAH) and 8 controls. PA-SMCs were serum-starved for 48 hours and pre-treated for 24 hours with ET-1 (10–7 and 10–6 mol/liter) before 5-hour treatment with 10 ng/ml BMP2. Values are expressed as mean ⫾ SEM. **P o 0.05 compared with corresponding condition in control PA-SMCs; †p o 0.05, †† p o 0.01, ††† p o 0.001 compared with basal condition in the same cell type; ‡p o 0.05 compared with corresponding ET-1 pre-treated PA-SMCs in the same group; §p o 0.01 compared with BMP2treated PA-SMCs in the same group.
and the Smad-independent p38MAPK in ET-1–treated control PA-SMCs, whereas only Smad1/5/8 signaling was activated in non-treated cells. BMP-mediated activation of p38MAPK has been shown to exert a pro-proliferative effect in PASMCs when Smad1/5/8 signaling is silenced.25 Although potentially anti-proliferative Smad1/5/8 and pro-proliferative p38MAPK signaling pathways were both activated in ET-1–treated control PA-SMCs, the functional evaluation in terms of proliferation and apoptosis was in favor of the pro-proliferative phenotype. The proliferation rate remained, however, lower in these ET-1–treated control
cells compared with the level observed in PA-SMCs from iPAH patients, suggesting the involvement of other genetic, autocrine, and/or paracrine factors in the acquisition of the altered PA-SMC phenotype in PAH. In PA-SMCs, ET-1 activates MAPKs, which in turn activate cyclins resulting in cell proliferation and subsequent PA-SMC hyperplasia in PAH.26,27 Antagonism of ET receptors is established as one of the most effective therapies in PAH patients.3 Our data showed that ET-1 upregulated PA-SMC proliferation only in the presence of other mitogens. The intracellular signaling pathway that
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Figure 6 Effects of endothelin (ET) -1 on the activation of bone morphogenic protein receptor 2 (BMPR2) signaling in pulmonary artery smooth muscle cells (PA-SMCs). (A) Relative gene expression of BMPR2 signaling target gene, the inhibitor of DNA binding (Id1) in 48-hour serum-starved, 24-hour ET-1 (10–6 mol/liter) pre-treated, and 5-hour BMP2 (10 ng/ml) treated PA-SMCs isolated and cultured from 6 patients with idiopathic pulmonary arterial hypertension (iPAH) and 8 controls. Representative immunoblots and corresponding densitometry analysis of (B) phosphorylated Smad1/5/8 (phosho-Smad1/5/8) and total Smad1/5/8 and (C) phosphorylated p38 mitogenactivated protein kinase (p38MAPK) and β-actin in 48-hour serum starved, 24-hour ET-1 (10–6 mol/liter) pre-treated, and 20-minute BMP2 (10 ng/ml)-treated PA-SMCs isolated and cultured from 3 patients with iPAH and 6 controls. Relative quantification was performed by normalization with (B) total Smad1/5/8 and (C) β-actin. Values are expressed as mean ⫾ standard error of the mean. *p o 0.05 compared with corresponding condition in control PA-SMCs; †p o 0.05, ††p o 0.01, †††p o 0.001 compared with basal condition in the same cell type; ‡p o 0.05, ‡‡p o 0.01 compared with corresponding ET-1 pre-treated PA-SMCs in the same group; §p o 0.05 compared with BMP2treated PA-SMCs in the same group.
mediates synergic pro-proliferative effects of ET-1 involves p38MAPK, which has been reported previously to contribute to the remodeling in PAH.25 This strongly supports the beneficial effect of the early use of ET receptor antagonists in the treatment of PAH patients in the context of multifactorial pathogenesis of PAH3 and to prevent the development of experimental PAH induced by systemic-topulmonary shunting in piglets.28,29 Indeed, ET receptor antagonists could also indirectly contribute to the restoration of BMPR2 signaling in PA-SMCs and therefore limit PA-SMC hyperplasia in normal PA-SMCs. In contrast, in PA-SMCs cultured from patients with end-stage iPAH, the functional effect of ET-1 pre-treatment in terms of
proliferation and apoptosis was totally negligible, suggesting that BMP signaling in these cells was constitutively altered independently of ET-1. In conclusion, the present study shows that ET-1 downregulates BMP/Smad signaling (Figure 7). ET-1 is, indeed, able to reduce the expression of BMPR2 and to over-express gremlins in control PA-SMCs, which is associated with increased proliferation of PA-SMCs. This mechanism is likely to contribute greatly to pulmonary vascular remodeling in PAH and may explain at least part of the therapeutic efficacy of ET receptor antagonists when they are used early in the pathologic processes associated with PAH.
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Figure 7 Schematic diagram of the proposed mechanism by which endothelin-1 and bone morphogenic protein (BMP) signaling interact in human control pulmonary artery smooth muscle cells (PA-SMCs). In control PA-SMCs, endothelin-1 reduces the expression of BMP receptor 2 (BMPR2) and BMP agonist (BMP4) but increases the expression of BMP antagonists (gremlin1 and gremin2). In these cells, endothelin-1 pretreatment reduces the activation of Smad1/5/8 signaling and the expression of inhibitor of DNA binding 1 (Id1) induced by BMP agonist (BMP2), but enhances p38 mitogen activated protein kinase (p38MAPK) signaling activation. This is associated with increased proliferation induced by growth factors and decreased expression of pro-apoptotic genes, probably contributing therefore to the acquisition of altered phenotype in PA-SMCs and pulmonary vascular remodeling observed in idiopathic pulmonary arterial hypertension (iPAH). ETR, endothelin receptor.
Disclosure statement The authors are grateful for technical assistance of Emilie Carette, Geoffrey De Medina, Marie-Thérèse Gauthier, and Inge Lanckriet. This work was supported by grants from the “Fonds de la Recherche Scientifique Medicale” (FNRS; Grant No. 3.4637.09) and the “Fonds pour la Chirurgie Cardiaque.” The present research work was also supported with unrestricted grants from Actelion and from Pfizer. C.D. was a “Fonds National de la Recherche Scientifique” (FNRS) doctoral fellow (“Aspirant FNRS”; Belgium). L.D. is a FNRS postdoctoral fellow (“Chargé de Recherches”; Belgium). None of the authors has a financial relationship with a commercial entity that has an interest in the subject of the presented manuscript or other conflicts of interest to disclose.
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