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Calcilytics enhance sildenafil-induced antiproliferation in idiopathic pulmonary arterial hypertension
European Journal of Pharmacology 784 (2016) 15–21
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Calcilytics enhance sildenafil-induced antiproliferation in idiopathic pulmonary arterial hypertension Aya Yamamura n, Satomi Yagi, Naoki Ohara, Kikuo Tsukamoto Department of Pharmacy, College of Pharmacy, Kinjo Gakuin University, Nagoya, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 31 March 2016 Received in revised form 22 April 2016 Accepted 28 April 2016 Available online 7 May 2016
Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and fatal disease of the pulmonary artery resulting from currently unidentified etiology. IPAH is pathologically characterized as sustained vasoconstriction and vascular remodeling of the pulmonary artery. Phosphodiesterase type 5 (PDE5) inhibitors have been clinically used in the treatment of IPAH. Recently, we have shown that Ca2 þ -sensing receptor (CaSR) antagonists, or calcilytics, inhibit excessive cell proliferation of pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. In this study, the additive or synergistic effect of calcilytics on antiproliferation following PDE5 inhibition was examined in IPAH-PASMCs by MTT assay. Treatment with sildenafil blocked the excessive cell proliferation of IPAH-PASMCs in a concentrationdependent manner with an IC50 value of 16.9 μM. However, sildenafil (0.03–100 μM) did not affect the cell growth of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Co-treatment with 0.3 μM NPS2143, a calcilytic, additively enhanced the antiproliferative effect induced by sildenafil (3 or 30 μM) in IPAH-PASMCs. Additionally, the inhibitory effect of calcilytics, NPS2143 or Calhex 231 (1 or 10 μM), on excessive cell proliferation of IPAH-PASMCs was synergistic increased in the presence of 1 μM sildenafil. Similar results were obtained by BrdU incorporation assay. These findings reveal that calcilytics additively/synergistically enhance the antiproliferative activity mediated by PDE5 inhibition, suggesting that a combination therapy of a PDE5 inhibitor with a calcilytic may be useful as a novel therapeutic approach for IPAH. & 2016 Elsevier B.V. All rights reserved.
Keywords: Pulmonary hypertension Calcium-sensing receptor Calcilytic Phosphodiesterase 5 inhibitor NPS2143 Sildenafil
1. Introduction Pulmonary arterial hypertension (PAH) is a progressive and fatal disease of the pulmonary artery. Pulmonary vasoconstriction and pulmonary vascular remodeling increase pulmonary arterial pressure. The pulmonary vascular remodeling is mainly due to enhanced cell proliferation and inhibited apoptosis of pulmonary arterial smooth muscle cells (PASMCs) in the media of pulmonary artery. Consequently, an increase of pulmonary arterial resistance leads to right heart failure and eventually death (McLaughlin et al., 2015). Pulmonary artery abnormalities, including vasoconstriction and vascular remodeling, in PAH patients are mostly triggered by an increase in cytosolic Ca2 þ concentration ([Ca2 þ ]cyt) (Morrell et al., 2009). Increased resting [Ca2 þ ]cyt and enhanced Ca2 þ influx have been reported in PASMCs from PAH patients. PASMCs express several Ca2 þ -permeable channels including voltage-dependent Ca2 þ channels (VDCCs), receptor-operated Ca2 þ (ROC) channels, n
Corresponding author. E-mail address: [email protected] (A. Yamamura).
http://dx.doi.org/10.1016/j.ejphar.2016.04.059 0014-2999/& 2016 Elsevier B.V. All rights reserved.
and store-operated Ca2 þ (SOC) channels (Fernandez et al., 2015; Firth et al., 2007; Guibert et al., 2007; Yamamura et al., 2011, 2014; Yang et al., 2010). ROC and SOC channels are upregulated in lung tissues and PASMCs from idiopathic PAH (IPAH) patients, compared with PASMCs from normal subjects and normotensive patients. These upregulations contribute to enhanced Ca2 þ signaling and excessive cell proliferation of PASMCs (Yu et al., 2004; Zhang et al., 2007). These channels are also upregulated in PASMCs during hypoxia (Lin et al., 2004; Smith et al., 2015; Wan et al., 2013; Wang et al., 2006). Recently, we have found that the extracellular Ca2 þ -sensing receptor (CaSR), a member of the G-protein-coupled receptor subfamily C (Magno et al., 2011), is upregulated in PASMCs from IPAH patients, compared with those from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). The upregulation of CaSRs is involved in the enhanced Ca2 þ response and subsequent excessive cell proliferation in IPAHPASMCs. Indeed, the blockade of CaSRs by calcilytics, or CaSR antagonists, attenuates excessive cell proliferation of IPAH-PASMCs, and inhibits the development of pulmonary hypertension in monocrotaline- or hypoxic-induced pulmonary hypertensive animals (Yamamura et al., 2012, 2015).
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Vascular remodeling of PASMCs is thought to be closely related to the dysfunction of prostacyclin, endothelin, and nitric oxide (NO) synthesis pathways. Thus, these signal pathways are considered as screening targets for antiproliferative drugs. Drug therapy for PAH has progressed in recent years with the development of several specific drugs targeting the pathological mechanisms of PAH. In addition to conventional VDCC blockers, three therapeutic options are currently used for the treatment of PAH patients: prostacyclins, endothelin receptor antagonists, and phosphodiesterase 5 (PDE5) inhibitors (Frumkin, 2012; McLaughlin et al., 2015; Seferian and Simonneau, 2013; Yamamura, 2014). In this study, we examined whether calcilytics (NPS2143 and Calhex 231) enhance the inhibitory effect of a clinically-used PDE5 inhibitor (sildenafil) on excessive cell proliferation of IPAHPASMCs using MTT and BrdU incorporation assays. We found that the PDE5 inhibition reduced excessive cell proliferation of IPAHPASMCs. Interestingly, the combination of calcilytic and PDE5 inhibitor acted additively or synergistically to block the excessive cell proliferation of IPAH-PASMCs.
2. Materials and methods 2.1. Cell culture Cell lines of PASMCs (passages 5–10) from normal subjects (Lonza, Walkersville, USA), IPAH patients (Yu et al., 2004), and CTEPH patients (Ogawa et al., 2009) were cultured in Medium 199 supplemented with 10% fetal bovine serum, 100 U/ml penicillin plus 100 μg/ml streptomycin (Invitrogen/GIBCO, Grand Island, USA), 50 μg/ml D-valine (Sigma-Aldrich, St. Louis, USA), and 20 μg/ ml endothelial cell growth supplement (BD Biosciences, Franklin Lakes, USA) at 37 °C. 2.2. MTT assay PASMCs were subcultured in 96-well plates ( 1 104 cells per well) and incubated at 37 °C for 6 h before assay. Then PASMCs were exposure to the culture medium including vehicle or drug (s) for 48 or 72 h. In the combination therapy, two different classes of drugs were applied into the medium at the same time. Cellular viability of PASMCs was evaluated using Cell Counting Kit-8 (Dojin, Kumamoto, Japan) based on MTT (3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2H-tetrazolium bromide) assay. The result was quantified colorimetrically as the absorbance at 450 nm (A450) using Benchmark Plus Microplate Reader and Microplate Manager (ver. 5.2; Bio-Rad Laboratories, Hercules, USA). 2.3. BrdU incorporation assay Cell preparation and drug application were performed in the same way as MTT assay. Cell proliferation of PASMCs was evaluated using Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Diagnostics, Mannheim, Germany) based on BrdU (bromodeoxyuridine) incorporation assay. The colorimetrical quantification was measured using the absorbance at 370 nm (A370) by a method similar to the MTT assay.
Fig. 1. Chemical structure of PDE5 inhibitor and calcilytics (CaSR antagonists). The chemical structures of a PDE5 inhibitor, sildenafil (A), and calcilytics, or negative allosteric modulators of CaSR, NPS2143 and Calhex 231 (B).
ethyl]amino]cyclohexyl]benzamide) (Sigma-Aldrich) are shown in Fig. 1. All hydrophobic compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 or 100 mM as a stock solution. The assay medium contained 0.1% DMSO throughout the experiments, regardless of the presence/absence of drug(s). It was confirmed that up to 0.1% DMSO did not affect these responses. 2.5. Statistical analysis Pooled data are shown as the mean 7S.E.M. The statistical significance of differences between two groups was determined by Student's t-test. The statistical significance of differences among groups was determined by Scheffé’s test following one-way analysis of variance (ANOVA). Significant differences are expressed in the figure as *Po 0.05 or **, ##Po0.01. The data of the relationship between drug concentrations and cell viability (Fig. 3B) were fitted using the following equation: MTT (A450)¼A1 (A1 A2)/{1 þ(Kd/ [drug])n), where Kd is the apparent dissociation constant of drug (IC50), [drug] is the concentration of drug, n is the Hill coefficient, A1 is the control value before the application of drug, and A2 is the component resistance to the drug.
3. Results 3.1. Excessive cell proliferation in PASMCs from IPAH patients
2.4. Drugs The chemical structures of sildenafil (1-[[3-(6,7-dihydro-1methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4ethoxyphenyl]sulfonyl]-4-methylpiperazine), NPS2143 (2-chloro6-[(2R)-2-hydroxy-3-[(2-methyl-1-naphthalen-2-ylpropan-2-yl) amino]propoxy]benzonitrile) (Tocris Bioscience, Ellisville, USA), and Calhex 231 (4-chloro-N-[(1S,2S)-2-[[(1R)-1-(1-naphthalenyl)
At first, the proliferation rates of PASMCs from normal subjects and patients with IPAH and CTEPH were analyzed by quantitative colorimetric assay based on the MTT test for cellular viability (Fig. 2), as reported previously (Yamamura et al., 2015). In normalPASMCs, the cell number was gradually increased until 72 h after subculture (A450 ¼0.58 70.02 at 48 h and 0.85 70.04 at 72 h, Po0.01 vs. 0.47 70.01 at 0 h, n ¼14). The proliferation rate in
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vs. sildenafilþ NPS2143, 84 75%, P40.05, n¼8; 30 μM sildenafil, 68 74% vs. sildenafil þNPS2143, 637 4%, P40.05, n¼ 8; Fig. 4A). Next, co-treatment with 0.3 μM NPS2143 was significantly enhanced the antiproliferative effect of 3 μM sildenafil in IPAHPASMCs (71 72%, P o0.01 vs. sildenafil alone, 85 72%, n¼16; Fig. 4B). Similarly, 30 μM sildenafil-induced inhibition of cell proliferation in IPAH-PASMCs was significantly enhanced by coadministration of 0.3 μM NPS2143 (57 73%, Po0.01 vs. sildenafil alone, 717 2%, n¼ 16). 3.4. Synergistic effect of sildenafil on calcilytic-induced antiproliferation in IPAH-PASMCs
Fig. 2. Excessive cell proliferation in PASMCs from IPAH patients. The cell proliferation of PASMCs from normal subjects, IPAH patients, and CTEPH patients were analyzed using quantitative colorimetric assay based on MTT assay for cellular viability. When PASMCs were subcultured in 96-well plates, the cell number was made up to 1 104 cells per well. Summarized data show the proliferation rates of PASMCs from normal subjects, IPAH patients, and CTEPH patients after 48 or 72 h culture. Data were obtained from 11 to 16 experiments. **Po 0.01 vs. normal-PASMCs.
IPAH-PASMCs was much higher than that in normal-PASMCs (0.99 70.07 at 48 h and 1.33 70.04 at 72 h, P o0.01 vs. 0.467 0.02 at 0 h, n¼11, and Po 0.01 vs. normal-PASMCs). On the other hand, the proliferation rate in CTEPH-PASMCs was similar to that in normal-PASMCs (0.57 7 0.02 at 48 h and 0.95 70.04 at 72 h, Po 0.01 vs. 0.40 70.03 at 0 h, n¼ 16, and P 40.05 vs. normalPASMCs). In this study, CTEPH-PASMCs were used as a negative control group for cell proliferation (Yamamura et al., 2012, 2015, 2013). 3.2. Inhibitory effect of sildenafil on excessive cell proliferation in IPAH-PASMCs The effect of sildenafil, a selective PDE5 inhibitor, on cell viability of normal-, IPAH-, and CTEPH-PASMCs was examined. Treatment with sildenafil at a concentration range between 0.03 and 100 μM for 48 h did not affect the cell survival (P4 0.05 vs. control of 0.51 70.03, n¼ 13; Fig. 3A). Similarly, the cell viability was not changed by treatment with sildenafil for 72 h (P 40.05 vs. control of 0.787 0.06, n¼ 10). In contrast, treatment with sildenafil for 48 h slightly reduced cell proliferation of IPAH-PASMCs (0.68 70.04 at 100 μM, Po 0.05 vs. control of 0.987 0.06, n¼7; Fig. 3B). Excessive cell proliferation of IPAH-PASMCs was attenuated by treatment with sildenafil for 72 h in a concentrationdependent manner (0.94 70.04 at 30 μM and 0.837 0.06 at 100 μM, Po 0.01 vs. control of 1.37 70.04, n ¼10). The IC50 value of sildenafil on the cell proliferation of IPAH-PASMCs was 16.9 μM, and the Hill coefficient was 0.99 (n ¼10). Conversely, the cell viability of CTEPH-PASMCs was not influenced by exposure to sildenafil for 48 or 72 h (P 40.05 vs. control, n¼12 or 8, respectively; Fig. 3C). 3.3. Additive effect of NPS2143 on sildenafil-induced antiproliferation in IPAH-PASMCs The modulation of NPS2143 on sildenafil-induced antiproliferation of IPAH-PASMCs was examined. NPS2143 is a calcilytic, or negative allosteric modulator of CaSRs, and therefore blocks CaSR function (Urwyler, 2011). As the inhibitory effect of sildenafil at 72 h culture was most prominent, cell proliferation of IPAH-PASMCs at 72 h was evaluated in subsequent experiments. When IPAH-PASMCs were co-treated with 0.1 μM NPS2143, in addition to 3 or 30 μM sildenafil, the antiproliferative effect of sildenafil was not modulated by NPS2143 (3 μM sildenafil, 92 75%
In the next set of experiments, the effect of sildenafil on NPS2143-induced antiproliferation in IPAH-PASMCs was examined. The co-addition of 1 μM sildenafil for 72 h markedly enhanced the inhibitory effect on excessive cell proliferation of 1 μM NPS2143 (58 73%, P o0.01 vs. NPS2143 alone, 71 72%, n¼16; Fig. 5A). Also 10 μM NPS2143-induced antiproliferative action was significantly enhanced by co-treatment with 1 μM sildenafil (26 71%, Po 0.01 vs. NPS2143 alone, 37 72%, n¼16). To confirm the synergistic effect of sildenafil on NPS2143-induced antiproliferation in IPAH-PASMCs, Calhex 231, a calcilytic structurally unrelated to NPS2143 (Urwyler, 2011) was used instead of NPS2143. Co-treatment with 1 μM sildenafil for 72 h in the presence of 1 μM (65 7 2%, Po0.01 vs. Calhex 231 alone, 73 72%, n ¼18) or 10 μM (28 71%, n¼18, Po0.01 vs. Calhex 231 alone, 397 2%, n ¼18) Calhex 231 significantly decreased excessive cell proliferation of IPAH-PASMCs compared to Calhex 231 treatment alone (Fig. 5B). 3.5. Antiproliferative effect by combination of NPS2143 and sildenafil using BrdU incorporation assay To confirm the results obtained from the MTT assay, another quantitative colorimetric assay based on the BrdU incorporation method for cell proliferation was carried out in IPAH-PASMCs. Excessive cell proliferation of IPAH-PASMCs was markedly reduced by treatment with 30 μM sildenafil for 72 h (72 73%, n¼13, Po0.01 vs. control (100%); Fig. 6). The sildenafil-induced antiproliferative effect was not affected by 0.1 μM NPS2143 (78 73%, n¼10, P4 0.05 vs. sildenafil alone). Treatment with 0.3 μM NPS2143 significantly inhibited excessive cell proliferation of IPAH-PASMCs (84 7 5%, n ¼13, Po 0.05 vs. control), as reported previously (Yamamura et al., 2015). Importantly, the antiproliferative effect of the combination of NPS2143 and sildenafil in IPAH-PASMCs was greater than that of each drugs individual effects (59 73%, n¼13, P o0.01 vs. control, sildenafil, or NPS2143).
4. Discussion Several specific therapeutic drugs such as prostacyclins (epoprostenol, treprostinil, and iloprost), endothelin receptor antagonists (bosentan, ambrisentan, and macitentan), and PDE5 inhibitors (sildenafil and tadalafil) have been developed for the treatment of PAH (Frumkin, 2012; McLaughlin et al., 2015; Seferian and Simonneau, 2013; Yamamura, 2014). Our previous work showed that CaSRs were upregulated in lung tissues and PASMCs from IPAH patients and that blockade of CaSRs inhibited excessive cell proliferation in PASMCs from IPAH patients (Yamamura et al., 2012, 2015). Therefore, in this study, the effects of a combination therapy of a calcilytic with another class of approved drug for the treatment of PAH were examined in IPAH-PASMCs. The findings reveal that sildenafil inhibits excessive cell proliferation of PASMCs from IPAH patients, and that, importantly, a combined treatment
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Fig. 3. Inhibition of excessive cell proliferation of IPAH-PASMCs by sildenafil. The effects of sildenafil, which is clinically used as a selective PDE5 inhibitor, on cell proliferation were examined in normal-, IPAH-, and CTEPH-PASMCs. Summarized data show the effect of sildenafil treatment for 48 or 72 h on cell proliferation at a concentration range between 0.03 and 100 μM in normal-PASMCs (A), IPAH-PASMCs (B), and CTEPH-PASMCs (C). The IC50 value of sildenafil for the cell proliferation of IPAH-PASMCs was 16.9 μM and the Hill coefficient was 0.99. Data were obtained from 7 to 13 experiments. *P o 0.05 or **Po 0.01 vs. control.
A. Yamamura et al. / European Journal of Pharmacology 784 (2016) 15–21
Fig. 4. Additive effect of NPS2143 on sildenafil-induced antiproliferation in IPAHPASMCs. The effects of NPS2143, which is widely used as a calcilytic (CaSR antagonist), on sildenafil-induced antiproliferation were examined in IPAH-PASMCs. The absorbance was normalized by the drug-free value (as 100%). Summarized data show the effect of 0.1 (A) or 0.3 (B) μM NPS2143 treatment for 72 h on the antiproliferative effect in the co-presence of 3 or 30 μM sildenafil in IPAH-PASMCs. Note that 0.3 μM NPS2143 had an additive influence on the sildenafil-induced antiproliferative effect in IPAH-PASMCs. Data were obtained from 8 to 16 experiments. **P o0.01 vs. sildenafil alone.
of calcilytics (NPS2143 and Calhex 231) with sildenafil is more effective in reducing proliferation of IPAH-PASMCs. In smooth muscles, PDE5 inhibition causes an increase in cyclic guanosine monophosphate (cGMP) levels and thereby activates cGMP-dependent protein kinase (PKG), resulting in relaxation of smooth muscles (Seferian and Simonneau, 2013). Sildenafil is well known to be a potent and selective PDE5 inhibitor that has been originally used to treat erectile dysfunction (Frumkin, 2012). So far, two PDE5 inhibitors, sildenafil and tadalafil, are approved for the treatment of PAH. PDE5 inhibition causes the dilation of pulmonary arterial smooth muscles through the cGMP pathway and thereby the reduction of pulmonary vascular resistance. PDE5 inhibitors have been used successfully in the treatment of PAH patients (Buckley et al., 2010; Frumkin, 2012; Seferian and Simonneau, 2013). Pulmonary arterial remodeling is closely associated with increased PASMC proliferation (McLaughlin et al., 2015; Morrell et al., 2009). Cell proliferation is triggered by an increase in [Ca2 þ ]cyt mediated by Ca2 þ -permeable channels in the plasma membrane. The Ca2 þ influx pathways are stimulated by vasoconstrictors, including serotonin and endothelin-1, and by growth factors, including platelet-derived growth factor (PDGF) and
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Fig. 5. Synergistic effect of sildenafil on calcilytic-induced antiproliferation in IPAHPASMCs. The effects of sildenafil on antiproliferation in the presence of calcilytics were examined in IPAH-PASMCs. The absorbance was normalized by the drug-free value (as 100%). (A) Summarized data show the effect of 1 μM sildenafil treatment for 72 h on the antiproliferative effect in the co-presence of 1 or 10 μM NPS2143 in IPAH-PASMCs. (B) Summarized data show the effect of 1 μM sildenafil treatment for 72 h on antiproliferative effect induced by 1 or 10 μM Calhex 231, which is another calcilytic structurally unrelated to NPS2143, in IPAH-PASMCs. Note that 1 μM sildenafil synergistically increased NPS2143- or Calhex 231-induced antiproliferative effects in IPAH-PASMCs. Data were obtained from 16 to 18 experiments. **Po 0.01 vs. absence of sildenafil.
epidermal growth factor (EGF) (Morrell et al., 2009). It has been reported that sildenafil blocks PASMC proliferation induced by PDGF (Li et al., 2007; Tantini et al., 2005), endothelin-1 (Wang et al., 2008), serum-deprivation (Wharton et al., 2005), and hypoxia (Wang et al., 2009). In addition, in this study, we demonstrate that sildenafil dose-dependently inhibits excessive cell proliferation of IPAH-PASMCs (IC50 ¼ 16.9 μM), whereas sildenafil has little effect on the cell growth of normal- and CTEPH-PASMCs. These results suggest that sildenafil acts specifically on abnormal PASMC proliferation, such as that in IPAH patients, indicating that sildenafil does not produce toxicity and/or side effect(s) at the range concentrations examined. To our knowledge, this is the first evidence showing the antiproliferative effect of sildenafil in IPAHPASMCs, although sildenafil has been clinically used for the treatment of PAH. NO/cGMP signaling can inhibit cell proliferation in addition to vasodilation in PASMCs (Frumkin, 2012; Seferian and Simonneau, 2013; Tantini et al., 2005; Wharton et al., 2005) and other types of vascular myocytes (Koyama et al., 2001). Sildenafil suppresses stimulated PASMC proliferation via upregulation of mitogen-
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Fig. 6. Combined treatment of sildenafil and NPS2143 on excessive cell proliferation of IPAH-PASMCs. The proliferation rates of PASMCs from IPAH patients were analyzed using quantitative colorimetric assay based on the BrdU incorporation method for cell proliferation. The effects of 30 μM sildenafil and 0.1 or 0.3 μM NPS2143 on excessive cell proliferation in a 72 h culture were examined in IPAH-PASMCs. The absorbance was normalized by the control value (as 100%). Note that, in IPAHPASMCs, the antiproliferative effect induced by the combination of NPS2143 and sildenafil was stronger than that of each drugs individual effect. Data were obtained from 10 to 16 experiments. *Po 0.05 or **P o0.01 vs. control (100%), and ##P o 0.01 vs. sildenafil or NPS2143.
activated protein kinase phosphatase-1 (MKP-1), degradation of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (Li et al., 2007; Tantini et al., 2005), and downregulation of transient potential receptor canonical subfamily 1 (TRPC1) channels followed by decrease in SOC entry and nuclear factor of activated T-cells (NFAT) activity (Wang et al., 2009, 2008). Treatment with sildenafil may have an antiproliferative effect on PASMCs through such signal pathways. It leads to a reduction in elevated pulmonary artery pressure resulting from pulmonary vascular remodeling in monocrotaline- (Itoh et al., 2004; Schermuly et al., 2004) and hypoxia- (Sebkhi et al., 2003; Zhao et al., 2001) induced pulmonary hypertensive animals. The therapeutic effect of sildenafil has been reported in PAH patients (Buckley et al., 2010; Galiè et al., 2005; Michelakis et al., 2002, 2003). Recently, we have found that cell proliferation of PASMCs is modulated by the activity of CaSRs, which are upregulated in PASMCs from IPAH patients. The finding reveals a novel pathogenic mechanism underlying the enhanced PASMC proliferation in IPAH patients. Inhibition of CaSRs by siRNA and calcilytics (CaSR antagonists) in IPAH-PASMCs attenuated excessive cell proliferation. Thus, calcilytics improved pulmonary vascular remodeling and inhibited the development of monocrotaline-induced pulmonary hypertension (Yamamura et al., 2012, 2015). CaSR regulates multiple signal pathways, including mitogen-activated protein kinase (MAPK) cascades: MAPK kinase (MEK), ERK1/2, and c-Jun N-terminal kinase (JNK). Recent reports suggest that CaSRs in vascular myocytes contribute to cell proliferation via MAPK and phospholipase C (PLC) cascades (Li et al., 2011; Magno et al., 2011; Molostvov et al., 2007; Smajilovic et al., 2006). The [Ca2 þ ]cyt increase mediated by CaSRs triggers MAPK cascades to accelerate PASMC proliferation under physiological and pathological conditions. Therefore, calcilytics are likely to block the excessive cell proliferation of IPAH-PASMCs. Medical management typically involves monotherapy of approved drugs (prostacyclins, endothelin receptor antagonists, and PDE5 inhibitors) for PAH patients who are not vasoactive, or who are vasoactive but not responding appropriately to VDCC blockers. If the clinical response, which is usually reassessed at 3–6 months after treatment begins, is not adequate, sequential combination therapy is considered. Two approved drugs with different action mechanisms are combined, in order to optimize clinical benefit
while minimizing side effects. In the case of inadequate clinical response with a double combination therapy, triple combination therapy is attempted (McLaughlin et al., 2015). Therefore, the pharmacological profiles of calcilytics and their combined use with other classes of approved drugs provide important information for developing therapeutic interventions for PAH. Treatment with sildenafil for 72 h inhibited excessive cell proliferation of IPAHPASMCs in a concentration-dependent manner with an IC50 value of 16.9 μM, as shown in Fig. 3B. The sildenafil-induced antiproliferation in IPAH-PASMCs was enhanced by the co-addition of 0.3 μM NPS2143 by 14% (Fig. 4B). Our previous report showed that the IC50 value of NPS2143 on excessive cell proliferation of IPAH-PASMCs at 72 h is 2.64 μM, and that 0.3 μM NPS2143 reduces it by 15.6% (Yamamura et al., 2015). Similarly, BrdU incorporation assay (Fig. 6) clearly shows that the inhibition rates on cell proliferation of IPAH-PASMCs by 30 μM sildenafil, 0.3 μM NPS2143, and the combination are 28%, 16%, and 41%, respectively. Therefore, the effect of NPS2143 is considered to be additive to the sildenafil-induced antiproliferation in IPAH-PASMCs. In addition, the antiproliferative effect by NPS2143 or Calhex 231 in IPAHPASMCs was enhanced in the presence of sildenafil at 1 μM (Fig. 5). Although 1 μM sildenafil did not show a significant antiproliferative activity in IPAH-PASMCs (2.2% decrease) as shown in Fig. 3B, 1 μM sildenafil markedly enhanced the antiproliferative effect induced by calcilytics. Therefore, the effect of sildenafil is supposed to be synergetic to calcilytic-induced antiproliferation in IPAH-PASMCs. Taken together, the antiproliferative effect of the combination therapy in IPAH-PASMCs was greater than each monotherapy. In conclusion, we showed excessive cell proliferation in PASMCs from IPAH patients was attenuated by PDE5 inhibition. Importantly, the combined treatment of PDE5 inhibitors with calcilytics is more effective in reducing proliferation of IPAHPASMCs. These results suggest a more effective strategy for improvement of pulmonary vascular remodeling in PAH patients.
Acknowledgements We thank Dr. Jason X.-J. Yuan (University of Arizona, Tucson, USA) for providing PASMC lines from IPAH and CTEPH patients. We also thank Dr. Hisao Yamamura (Nagoya City University, Nagoya, Japan) for valuable comments during the study. This work was supported by Grants-in-Aid for Young Scientists (B) (25860068 and 15K18881; to A. Yamamura) from the Japan Society for the Promotion of Science.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Cardiovascular pharmacology
Calcilytics enhance sildenafil-induced antiproliferation in idiopathic pulmonary arterial hypertension Aya Yamamura n, Satomi Yagi, Naoki Ohara, Kikuo Tsukamoto Department of Pharmacy, College of Pharmacy, Kinjo Gakuin University, Nagoya, Japan
art ic l e i nf o
a b s t r a c t
Article history: Received 31 March 2016 Received in revised form 22 April 2016 Accepted 28 April 2016 Available online 7 May 2016
Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and fatal disease of the pulmonary artery resulting from currently unidentified etiology. IPAH is pathologically characterized as sustained vasoconstriction and vascular remodeling of the pulmonary artery. Phosphodiesterase type 5 (PDE5) inhibitors have been clinically used in the treatment of IPAH. Recently, we have shown that Ca2 þ -sensing receptor (CaSR) antagonists, or calcilytics, inhibit excessive cell proliferation of pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. In this study, the additive or synergistic effect of calcilytics on antiproliferation following PDE5 inhibition was examined in IPAH-PASMCs by MTT assay. Treatment with sildenafil blocked the excessive cell proliferation of IPAH-PASMCs in a concentrationdependent manner with an IC50 value of 16.9 μM. However, sildenafil (0.03–100 μM) did not affect the cell growth of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Co-treatment with 0.3 μM NPS2143, a calcilytic, additively enhanced the antiproliferative effect induced by sildenafil (3 or 30 μM) in IPAH-PASMCs. Additionally, the inhibitory effect of calcilytics, NPS2143 or Calhex 231 (1 or 10 μM), on excessive cell proliferation of IPAH-PASMCs was synergistic increased in the presence of 1 μM sildenafil. Similar results were obtained by BrdU incorporation assay. These findings reveal that calcilytics additively/synergistically enhance the antiproliferative activity mediated by PDE5 inhibition, suggesting that a combination therapy of a PDE5 inhibitor with a calcilytic may be useful as a novel therapeutic approach for IPAH. & 2016 Elsevier B.V. All rights reserved.
Keywords: Pulmonary hypertension Calcium-sensing receptor Calcilytic Phosphodiesterase 5 inhibitor NPS2143 Sildenafil
1. Introduction Pulmonary arterial hypertension (PAH) is a progressive and fatal disease of the pulmonary artery. Pulmonary vasoconstriction and pulmonary vascular remodeling increase pulmonary arterial pressure. The pulmonary vascular remodeling is mainly due to enhanced cell proliferation and inhibited apoptosis of pulmonary arterial smooth muscle cells (PASMCs) in the media of pulmonary artery. Consequently, an increase of pulmonary arterial resistance leads to right heart failure and eventually death (McLaughlin et al., 2015). Pulmonary artery abnormalities, including vasoconstriction and vascular remodeling, in PAH patients are mostly triggered by an increase in cytosolic Ca2 þ concentration ([Ca2 þ ]cyt) (Morrell et al., 2009). Increased resting [Ca2 þ ]cyt and enhanced Ca2 þ influx have been reported in PASMCs from PAH patients. PASMCs express several Ca2 þ -permeable channels including voltage-dependent Ca2 þ channels (VDCCs), receptor-operated Ca2 þ (ROC) channels, n
Corresponding author. E-mail address: [email protected] (A. Yamamura).
http://dx.doi.org/10.1016/j.ejphar.2016.04.059 0014-2999/& 2016 Elsevier B.V. All rights reserved.
and store-operated Ca2 þ (SOC) channels (Fernandez et al., 2015; Firth et al., 2007; Guibert et al., 2007; Yamamura et al., 2011, 2014; Yang et al., 2010). ROC and SOC channels are upregulated in lung tissues and PASMCs from idiopathic PAH (IPAH) patients, compared with PASMCs from normal subjects and normotensive patients. These upregulations contribute to enhanced Ca2 þ signaling and excessive cell proliferation of PASMCs (Yu et al., 2004; Zhang et al., 2007). These channels are also upregulated in PASMCs during hypoxia (Lin et al., 2004; Smith et al., 2015; Wan et al., 2013; Wang et al., 2006). Recently, we have found that the extracellular Ca2 þ -sensing receptor (CaSR), a member of the G-protein-coupled receptor subfamily C (Magno et al., 2011), is upregulated in PASMCs from IPAH patients, compared with those from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). The upregulation of CaSRs is involved in the enhanced Ca2 þ response and subsequent excessive cell proliferation in IPAHPASMCs. Indeed, the blockade of CaSRs by calcilytics, or CaSR antagonists, attenuates excessive cell proliferation of IPAH-PASMCs, and inhibits the development of pulmonary hypertension in monocrotaline- or hypoxic-induced pulmonary hypertensive animals (Yamamura et al., 2012, 2015).
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Vascular remodeling of PASMCs is thought to be closely related to the dysfunction of prostacyclin, endothelin, and nitric oxide (NO) synthesis pathways. Thus, these signal pathways are considered as screening targets for antiproliferative drugs. Drug therapy for PAH has progressed in recent years with the development of several specific drugs targeting the pathological mechanisms of PAH. In addition to conventional VDCC blockers, three therapeutic options are currently used for the treatment of PAH patients: prostacyclins, endothelin receptor antagonists, and phosphodiesterase 5 (PDE5) inhibitors (Frumkin, 2012; McLaughlin et al., 2015; Seferian and Simonneau, 2013; Yamamura, 2014). In this study, we examined whether calcilytics (NPS2143 and Calhex 231) enhance the inhibitory effect of a clinically-used PDE5 inhibitor (sildenafil) on excessive cell proliferation of IPAHPASMCs using MTT and BrdU incorporation assays. We found that the PDE5 inhibition reduced excessive cell proliferation of IPAHPASMCs. Interestingly, the combination of calcilytic and PDE5 inhibitor acted additively or synergistically to block the excessive cell proliferation of IPAH-PASMCs.
2. Materials and methods 2.1. Cell culture Cell lines of PASMCs (passages 5–10) from normal subjects (Lonza, Walkersville, USA), IPAH patients (Yu et al., 2004), and CTEPH patients (Ogawa et al., 2009) were cultured in Medium 199 supplemented with 10% fetal bovine serum, 100 U/ml penicillin plus 100 μg/ml streptomycin (Invitrogen/GIBCO, Grand Island, USA), 50 μg/ml D-valine (Sigma-Aldrich, St. Louis, USA), and 20 μg/ ml endothelial cell growth supplement (BD Biosciences, Franklin Lakes, USA) at 37 °C. 2.2. MTT assay PASMCs were subcultured in 96-well plates ( 1 104 cells per well) and incubated at 37 °C for 6 h before assay. Then PASMCs were exposure to the culture medium including vehicle or drug (s) for 48 or 72 h. In the combination therapy, two different classes of drugs were applied into the medium at the same time. Cellular viability of PASMCs was evaluated using Cell Counting Kit-8 (Dojin, Kumamoto, Japan) based on MTT (3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2H-tetrazolium bromide) assay. The result was quantified colorimetrically as the absorbance at 450 nm (A450) using Benchmark Plus Microplate Reader and Microplate Manager (ver. 5.2; Bio-Rad Laboratories, Hercules, USA). 2.3. BrdU incorporation assay Cell preparation and drug application were performed in the same way as MTT assay. Cell proliferation of PASMCs was evaluated using Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Diagnostics, Mannheim, Germany) based on BrdU (bromodeoxyuridine) incorporation assay. The colorimetrical quantification was measured using the absorbance at 370 nm (A370) by a method similar to the MTT assay.
Fig. 1. Chemical structure of PDE5 inhibitor and calcilytics (CaSR antagonists). The chemical structures of a PDE5 inhibitor, sildenafil (A), and calcilytics, or negative allosteric modulators of CaSR, NPS2143 and Calhex 231 (B).
ethyl]amino]cyclohexyl]benzamide) (Sigma-Aldrich) are shown in Fig. 1. All hydrophobic compounds were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 or 100 mM as a stock solution. The assay medium contained 0.1% DMSO throughout the experiments, regardless of the presence/absence of drug(s). It was confirmed that up to 0.1% DMSO did not affect these responses. 2.5. Statistical analysis Pooled data are shown as the mean 7S.E.M. The statistical significance of differences between two groups was determined by Student's t-test. The statistical significance of differences among groups was determined by Scheffé’s test following one-way analysis of variance (ANOVA). Significant differences are expressed in the figure as *Po 0.05 or **, ##Po0.01. The data of the relationship between drug concentrations and cell viability (Fig. 3B) were fitted using the following equation: MTT (A450)¼A1 (A1 A2)/{1 þ(Kd/ [drug])n), where Kd is the apparent dissociation constant of drug (IC50), [drug] is the concentration of drug, n is the Hill coefficient, A1 is the control value before the application of drug, and A2 is the component resistance to the drug.
3. Results 3.1. Excessive cell proliferation in PASMCs from IPAH patients
2.4. Drugs The chemical structures of sildenafil (1-[[3-(6,7-dihydro-1methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4ethoxyphenyl]sulfonyl]-4-methylpiperazine), NPS2143 (2-chloro6-[(2R)-2-hydroxy-3-[(2-methyl-1-naphthalen-2-ylpropan-2-yl) amino]propoxy]benzonitrile) (Tocris Bioscience, Ellisville, USA), and Calhex 231 (4-chloro-N-[(1S,2S)-2-[[(1R)-1-(1-naphthalenyl)
At first, the proliferation rates of PASMCs from normal subjects and patients with IPAH and CTEPH were analyzed by quantitative colorimetric assay based on the MTT test for cellular viability (Fig. 2), as reported previously (Yamamura et al., 2015). In normalPASMCs, the cell number was gradually increased until 72 h after subculture (A450 ¼0.58 70.02 at 48 h and 0.85 70.04 at 72 h, Po0.01 vs. 0.47 70.01 at 0 h, n ¼14). The proliferation rate in
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vs. sildenafilþ NPS2143, 84 75%, P40.05, n¼8; 30 μM sildenafil, 68 74% vs. sildenafil þNPS2143, 637 4%, P40.05, n¼ 8; Fig. 4A). Next, co-treatment with 0.3 μM NPS2143 was significantly enhanced the antiproliferative effect of 3 μM sildenafil in IPAHPASMCs (71 72%, P o0.01 vs. sildenafil alone, 85 72%, n¼16; Fig. 4B). Similarly, 30 μM sildenafil-induced inhibition of cell proliferation in IPAH-PASMCs was significantly enhanced by coadministration of 0.3 μM NPS2143 (57 73%, Po0.01 vs. sildenafil alone, 717 2%, n¼ 16). 3.4. Synergistic effect of sildenafil on calcilytic-induced antiproliferation in IPAH-PASMCs
Fig. 2. Excessive cell proliferation in PASMCs from IPAH patients. The cell proliferation of PASMCs from normal subjects, IPAH patients, and CTEPH patients were analyzed using quantitative colorimetric assay based on MTT assay for cellular viability. When PASMCs were subcultured in 96-well plates, the cell number was made up to 1 104 cells per well. Summarized data show the proliferation rates of PASMCs from normal subjects, IPAH patients, and CTEPH patients after 48 or 72 h culture. Data were obtained from 11 to 16 experiments. **Po 0.01 vs. normal-PASMCs.
IPAH-PASMCs was much higher than that in normal-PASMCs (0.99 70.07 at 48 h and 1.33 70.04 at 72 h, P o0.01 vs. 0.467 0.02 at 0 h, n¼11, and Po 0.01 vs. normal-PASMCs). On the other hand, the proliferation rate in CTEPH-PASMCs was similar to that in normal-PASMCs (0.57 7 0.02 at 48 h and 0.95 70.04 at 72 h, Po 0.01 vs. 0.40 70.03 at 0 h, n¼ 16, and P 40.05 vs. normalPASMCs). In this study, CTEPH-PASMCs were used as a negative control group for cell proliferation (Yamamura et al., 2012, 2015, 2013). 3.2. Inhibitory effect of sildenafil on excessive cell proliferation in IPAH-PASMCs The effect of sildenafil, a selective PDE5 inhibitor, on cell viability of normal-, IPAH-, and CTEPH-PASMCs was examined. Treatment with sildenafil at a concentration range between 0.03 and 100 μM for 48 h did not affect the cell survival (P4 0.05 vs. control of 0.51 70.03, n¼ 13; Fig. 3A). Similarly, the cell viability was not changed by treatment with sildenafil for 72 h (P 40.05 vs. control of 0.787 0.06, n¼ 10). In contrast, treatment with sildenafil for 48 h slightly reduced cell proliferation of IPAH-PASMCs (0.68 70.04 at 100 μM, Po 0.05 vs. control of 0.987 0.06, n¼7; Fig. 3B). Excessive cell proliferation of IPAH-PASMCs was attenuated by treatment with sildenafil for 72 h in a concentrationdependent manner (0.94 70.04 at 30 μM and 0.837 0.06 at 100 μM, Po 0.01 vs. control of 1.37 70.04, n ¼10). The IC50 value of sildenafil on the cell proliferation of IPAH-PASMCs was 16.9 μM, and the Hill coefficient was 0.99 (n ¼10). Conversely, the cell viability of CTEPH-PASMCs was not influenced by exposure to sildenafil for 48 or 72 h (P 40.05 vs. control, n¼12 or 8, respectively; Fig. 3C). 3.3. Additive effect of NPS2143 on sildenafil-induced antiproliferation in IPAH-PASMCs The modulation of NPS2143 on sildenafil-induced antiproliferation of IPAH-PASMCs was examined. NPS2143 is a calcilytic, or negative allosteric modulator of CaSRs, and therefore blocks CaSR function (Urwyler, 2011). As the inhibitory effect of sildenafil at 72 h culture was most prominent, cell proliferation of IPAH-PASMCs at 72 h was evaluated in subsequent experiments. When IPAH-PASMCs were co-treated with 0.1 μM NPS2143, in addition to 3 or 30 μM sildenafil, the antiproliferative effect of sildenafil was not modulated by NPS2143 (3 μM sildenafil, 92 75%
In the next set of experiments, the effect of sildenafil on NPS2143-induced antiproliferation in IPAH-PASMCs was examined. The co-addition of 1 μM sildenafil for 72 h markedly enhanced the inhibitory effect on excessive cell proliferation of 1 μM NPS2143 (58 73%, P o0.01 vs. NPS2143 alone, 71 72%, n¼16; Fig. 5A). Also 10 μM NPS2143-induced antiproliferative action was significantly enhanced by co-treatment with 1 μM sildenafil (26 71%, Po 0.01 vs. NPS2143 alone, 37 72%, n¼16). To confirm the synergistic effect of sildenafil on NPS2143-induced antiproliferation in IPAH-PASMCs, Calhex 231, a calcilytic structurally unrelated to NPS2143 (Urwyler, 2011) was used instead of NPS2143. Co-treatment with 1 μM sildenafil for 72 h in the presence of 1 μM (65 7 2%, Po0.01 vs. Calhex 231 alone, 73 72%, n ¼18) or 10 μM (28 71%, n¼18, Po0.01 vs. Calhex 231 alone, 397 2%, n ¼18) Calhex 231 significantly decreased excessive cell proliferation of IPAH-PASMCs compared to Calhex 231 treatment alone (Fig. 5B). 3.5. Antiproliferative effect by combination of NPS2143 and sildenafil using BrdU incorporation assay To confirm the results obtained from the MTT assay, another quantitative colorimetric assay based on the BrdU incorporation method for cell proliferation was carried out in IPAH-PASMCs. Excessive cell proliferation of IPAH-PASMCs was markedly reduced by treatment with 30 μM sildenafil for 72 h (72 73%, n¼13, Po0.01 vs. control (100%); Fig. 6). The sildenafil-induced antiproliferative effect was not affected by 0.1 μM NPS2143 (78 73%, n¼10, P4 0.05 vs. sildenafil alone). Treatment with 0.3 μM NPS2143 significantly inhibited excessive cell proliferation of IPAH-PASMCs (84 7 5%, n ¼13, Po 0.05 vs. control), as reported previously (Yamamura et al., 2015). Importantly, the antiproliferative effect of the combination of NPS2143 and sildenafil in IPAH-PASMCs was greater than that of each drugs individual effects (59 73%, n¼13, P o0.01 vs. control, sildenafil, or NPS2143).
4. Discussion Several specific therapeutic drugs such as prostacyclins (epoprostenol, treprostinil, and iloprost), endothelin receptor antagonists (bosentan, ambrisentan, and macitentan), and PDE5 inhibitors (sildenafil and tadalafil) have been developed for the treatment of PAH (Frumkin, 2012; McLaughlin et al., 2015; Seferian and Simonneau, 2013; Yamamura, 2014). Our previous work showed that CaSRs were upregulated in lung tissues and PASMCs from IPAH patients and that blockade of CaSRs inhibited excessive cell proliferation in PASMCs from IPAH patients (Yamamura et al., 2012, 2015). Therefore, in this study, the effects of a combination therapy of a calcilytic with another class of approved drug for the treatment of PAH were examined in IPAH-PASMCs. The findings reveal that sildenafil inhibits excessive cell proliferation of PASMCs from IPAH patients, and that, importantly, a combined treatment
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Fig. 3. Inhibition of excessive cell proliferation of IPAH-PASMCs by sildenafil. The effects of sildenafil, which is clinically used as a selective PDE5 inhibitor, on cell proliferation were examined in normal-, IPAH-, and CTEPH-PASMCs. Summarized data show the effect of sildenafil treatment for 48 or 72 h on cell proliferation at a concentration range between 0.03 and 100 μM in normal-PASMCs (A), IPAH-PASMCs (B), and CTEPH-PASMCs (C). The IC50 value of sildenafil for the cell proliferation of IPAH-PASMCs was 16.9 μM and the Hill coefficient was 0.99. Data were obtained from 7 to 13 experiments. *P o 0.05 or **Po 0.01 vs. control.
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Fig. 4. Additive effect of NPS2143 on sildenafil-induced antiproliferation in IPAHPASMCs. The effects of NPS2143, which is widely used as a calcilytic (CaSR antagonist), on sildenafil-induced antiproliferation were examined in IPAH-PASMCs. The absorbance was normalized by the drug-free value (as 100%). Summarized data show the effect of 0.1 (A) or 0.3 (B) μM NPS2143 treatment for 72 h on the antiproliferative effect in the co-presence of 3 or 30 μM sildenafil in IPAH-PASMCs. Note that 0.3 μM NPS2143 had an additive influence on the sildenafil-induced antiproliferative effect in IPAH-PASMCs. Data were obtained from 8 to 16 experiments. **P o0.01 vs. sildenafil alone.
of calcilytics (NPS2143 and Calhex 231) with sildenafil is more effective in reducing proliferation of IPAH-PASMCs. In smooth muscles, PDE5 inhibition causes an increase in cyclic guanosine monophosphate (cGMP) levels and thereby activates cGMP-dependent protein kinase (PKG), resulting in relaxation of smooth muscles (Seferian and Simonneau, 2013). Sildenafil is well known to be a potent and selective PDE5 inhibitor that has been originally used to treat erectile dysfunction (Frumkin, 2012). So far, two PDE5 inhibitors, sildenafil and tadalafil, are approved for the treatment of PAH. PDE5 inhibition causes the dilation of pulmonary arterial smooth muscles through the cGMP pathway and thereby the reduction of pulmonary vascular resistance. PDE5 inhibitors have been used successfully in the treatment of PAH patients (Buckley et al., 2010; Frumkin, 2012; Seferian and Simonneau, 2013). Pulmonary arterial remodeling is closely associated with increased PASMC proliferation (McLaughlin et al., 2015; Morrell et al., 2009). Cell proliferation is triggered by an increase in [Ca2 þ ]cyt mediated by Ca2 þ -permeable channels in the plasma membrane. The Ca2 þ influx pathways are stimulated by vasoconstrictors, including serotonin and endothelin-1, and by growth factors, including platelet-derived growth factor (PDGF) and
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Fig. 5. Synergistic effect of sildenafil on calcilytic-induced antiproliferation in IPAHPASMCs. The effects of sildenafil on antiproliferation in the presence of calcilytics were examined in IPAH-PASMCs. The absorbance was normalized by the drug-free value (as 100%). (A) Summarized data show the effect of 1 μM sildenafil treatment for 72 h on the antiproliferative effect in the co-presence of 1 or 10 μM NPS2143 in IPAH-PASMCs. (B) Summarized data show the effect of 1 μM sildenafil treatment for 72 h on antiproliferative effect induced by 1 or 10 μM Calhex 231, which is another calcilytic structurally unrelated to NPS2143, in IPAH-PASMCs. Note that 1 μM sildenafil synergistically increased NPS2143- or Calhex 231-induced antiproliferative effects in IPAH-PASMCs. Data were obtained from 16 to 18 experiments. **Po 0.01 vs. absence of sildenafil.
epidermal growth factor (EGF) (Morrell et al., 2009). It has been reported that sildenafil blocks PASMC proliferation induced by PDGF (Li et al., 2007; Tantini et al., 2005), endothelin-1 (Wang et al., 2008), serum-deprivation (Wharton et al., 2005), and hypoxia (Wang et al., 2009). In addition, in this study, we demonstrate that sildenafil dose-dependently inhibits excessive cell proliferation of IPAH-PASMCs (IC50 ¼ 16.9 μM), whereas sildenafil has little effect on the cell growth of normal- and CTEPH-PASMCs. These results suggest that sildenafil acts specifically on abnormal PASMC proliferation, such as that in IPAH patients, indicating that sildenafil does not produce toxicity and/or side effect(s) at the range concentrations examined. To our knowledge, this is the first evidence showing the antiproliferative effect of sildenafil in IPAHPASMCs, although sildenafil has been clinically used for the treatment of PAH. NO/cGMP signaling can inhibit cell proliferation in addition to vasodilation in PASMCs (Frumkin, 2012; Seferian and Simonneau, 2013; Tantini et al., 2005; Wharton et al., 2005) and other types of vascular myocytes (Koyama et al., 2001). Sildenafil suppresses stimulated PASMC proliferation via upregulation of mitogen-
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Fig. 6. Combined treatment of sildenafil and NPS2143 on excessive cell proliferation of IPAH-PASMCs. The proliferation rates of PASMCs from IPAH patients were analyzed using quantitative colorimetric assay based on the BrdU incorporation method for cell proliferation. The effects of 30 μM sildenafil and 0.1 or 0.3 μM NPS2143 on excessive cell proliferation in a 72 h culture were examined in IPAH-PASMCs. The absorbance was normalized by the control value (as 100%). Note that, in IPAHPASMCs, the antiproliferative effect induced by the combination of NPS2143 and sildenafil was stronger than that of each drugs individual effect. Data were obtained from 10 to 16 experiments. *Po 0.05 or **P o0.01 vs. control (100%), and ##P o 0.01 vs. sildenafil or NPS2143.
activated protein kinase phosphatase-1 (MKP-1), degradation of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (Li et al., 2007; Tantini et al., 2005), and downregulation of transient potential receptor canonical subfamily 1 (TRPC1) channels followed by decrease in SOC entry and nuclear factor of activated T-cells (NFAT) activity (Wang et al., 2009, 2008). Treatment with sildenafil may have an antiproliferative effect on PASMCs through such signal pathways. It leads to a reduction in elevated pulmonary artery pressure resulting from pulmonary vascular remodeling in monocrotaline- (Itoh et al., 2004; Schermuly et al., 2004) and hypoxia- (Sebkhi et al., 2003; Zhao et al., 2001) induced pulmonary hypertensive animals. The therapeutic effect of sildenafil has been reported in PAH patients (Buckley et al., 2010; Galiè et al., 2005; Michelakis et al., 2002, 2003). Recently, we have found that cell proliferation of PASMCs is modulated by the activity of CaSRs, which are upregulated in PASMCs from IPAH patients. The finding reveals a novel pathogenic mechanism underlying the enhanced PASMC proliferation in IPAH patients. Inhibition of CaSRs by siRNA and calcilytics (CaSR antagonists) in IPAH-PASMCs attenuated excessive cell proliferation. Thus, calcilytics improved pulmonary vascular remodeling and inhibited the development of monocrotaline-induced pulmonary hypertension (Yamamura et al., 2012, 2015). CaSR regulates multiple signal pathways, including mitogen-activated protein kinase (MAPK) cascades: MAPK kinase (MEK), ERK1/2, and c-Jun N-terminal kinase (JNK). Recent reports suggest that CaSRs in vascular myocytes contribute to cell proliferation via MAPK and phospholipase C (PLC) cascades (Li et al., 2011; Magno et al., 2011; Molostvov et al., 2007; Smajilovic et al., 2006). The [Ca2 þ ]cyt increase mediated by CaSRs triggers MAPK cascades to accelerate PASMC proliferation under physiological and pathological conditions. Therefore, calcilytics are likely to block the excessive cell proliferation of IPAH-PASMCs. Medical management typically involves monotherapy of approved drugs (prostacyclins, endothelin receptor antagonists, and PDE5 inhibitors) for PAH patients who are not vasoactive, or who are vasoactive but not responding appropriately to VDCC blockers. If the clinical response, which is usually reassessed at 3–6 months after treatment begins, is not adequate, sequential combination therapy is considered. Two approved drugs with different action mechanisms are combined, in order to optimize clinical benefit
while minimizing side effects. In the case of inadequate clinical response with a double combination therapy, triple combination therapy is attempted (McLaughlin et al., 2015). Therefore, the pharmacological profiles of calcilytics and their combined use with other classes of approved drugs provide important information for developing therapeutic interventions for PAH. Treatment with sildenafil for 72 h inhibited excessive cell proliferation of IPAHPASMCs in a concentration-dependent manner with an IC50 value of 16.9 μM, as shown in Fig. 3B. The sildenafil-induced antiproliferation in IPAH-PASMCs was enhanced by the co-addition of 0.3 μM NPS2143 by 14% (Fig. 4B). Our previous report showed that the IC50 value of NPS2143 on excessive cell proliferation of IPAH-PASMCs at 72 h is 2.64 μM, and that 0.3 μM NPS2143 reduces it by 15.6% (Yamamura et al., 2015). Similarly, BrdU incorporation assay (Fig. 6) clearly shows that the inhibition rates on cell proliferation of IPAH-PASMCs by 30 μM sildenafil, 0.3 μM NPS2143, and the combination are 28%, 16%, and 41%, respectively. Therefore, the effect of NPS2143 is considered to be additive to the sildenafil-induced antiproliferation in IPAH-PASMCs. In addition, the antiproliferative effect by NPS2143 or Calhex 231 in IPAHPASMCs was enhanced in the presence of sildenafil at 1 μM (Fig. 5). Although 1 μM sildenafil did not show a significant antiproliferative activity in IPAH-PASMCs (2.2% decrease) as shown in Fig. 3B, 1 μM sildenafil markedly enhanced the antiproliferative effect induced by calcilytics. Therefore, the effect of sildenafil is supposed to be synergetic to calcilytic-induced antiproliferation in IPAH-PASMCs. Taken together, the antiproliferative effect of the combination therapy in IPAH-PASMCs was greater than each monotherapy. In conclusion, we showed excessive cell proliferation in PASMCs from IPAH patients was attenuated by PDE5 inhibition. Importantly, the combined treatment of PDE5 inhibitors with calcilytics is more effective in reducing proliferation of IPAHPASMCs. These results suggest a more effective strategy for improvement of pulmonary vascular remodeling in PAH patients.
Acknowledgements We thank Dr. Jason X.-J. Yuan (University of Arizona, Tucson, USA) for providing PASMC lines from IPAH and CTEPH patients. We also thank Dr. Hisao Yamamura (Nagoya City University, Nagoya, Japan) for valuable comments during the study. This work was supported by Grants-in-Aid for Young Scientists (B) (25860068 and 15K18881; to A. Yamamura) from the Japan Society for the Promotion of Science.
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