Inhibition of Excessive Cell Proliferation by Calcilytics in Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. Vascular remodeling due to excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a critical pathogenic event that leads to early morbidity and mortality. The excessive cell proliferation is closely linked to the augmented Ca2+ signaling in PASMCs. More recently, we have shown by an siRNA knockdown method that the Ca2+-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, involved in the enhanced Ca2+ response and subsequent excessive cell proliferation. In this study, we examined whether pharmacological blockade of CaSR attenuated the excessive proliferation of PASMCs from IPAH patients by MTT assay. The proliferation rate of PASMCs from IPAH patients was much higher (~1.5-fold) than that of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Treatment with NPS2143, an antagonist of CaSR or calcilytic, clearly suppressed the cell proliferation in a concentration-dependent manner (IC50 = 2.64 μM) in IPAH-PASMCs, but not in normal and CTEPH PASMCs. Another calcilytic, Calhex 231, which is structurally unrelated to NPS2143, also concentration-dependently inhibited the excessive proliferation of IPAH-PASMCs (IC50 = 1.89 μM). In contrast, R568, an activator of CaSR or calcimimetic, significantly facilitated the proliferation of IPAH-PASMCs (EC50 = 0.33 μM). Similar results were obtained by BrdU incorporation assay. These results reveal that the excessive PASMC proliferation was modulated by pharmacological tools of CaSR, showing us that calcilytics are useful for a novel therapeutic approach for pulmonary arterial hypertension.


Introduction
Pulmonary arterial hypertension (PAH) is caused by functional and structural changes in the pulmonary vasculature. Pulmonary vascular remodeling is triggered by a progressive elevation of pulmonary vascular resistance and pulmonary arterial pressure in patients with PAH. The elevated pulmonary arterial pressure induces extensive changes in heart structure followed by right heart failure, and eventually death. PAH is clinically defined by chronic increases of [25] and Cell Proliferation ELISA BrdU Colorimetric Kit (Roche Diagnostics, Mannheim, Germany) based on BrdU (bromodeoxyuridine) incorporation assay. PASMCs were subcultured in 96-well plates (approximately 1×10 4 cells per well) and incubated at 37°C for 24~72 h. Cellular viability was quantified colorimetrically as the absorbance at 450 nm (A 450 ) for MTT assay or 370 nm for BrdU incorporation assay using Benchmark Plus Microplate Reader and Microplate Manager (ver. 5.2; Bio-Rad Laboratories, Hercules, USA).

Drugs
The chemical structures of modulators for CaSR, NPS2143 (2-

Statistical analysis
Pooled data are shown as the mean±S.E. 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 after one-way analysis of variance (ANOVA). Significant differences are expressed in the figure as Ã p<0.05 or ÃÃ p<0.01. The data of the relationship between drug concentrations and cell proliferation were fitted using the following equations: relative value (%) = 100-(100-C)/{1+(K d /[drug]) n ) (for inhibition) and A max / {1+(K d /[drug]) n )+100 (for activation), where K d is the apparent dissociation constant of drug (IC 50 and EC 50 , respectively), [drug] is the concentration of drug, n is the Hill coefficient, C is the component resistant to drug, and A max is the maximum value of the response.

Excessive cell proliferation in PASMCs from IPAH patients
At first, we analyzed the proliferation rates of PASMCs from normal subjects and patients with IPAH and CTEPH by quantitative colorimetric assay based on the MTT test for cellular viability. In normal PASMCs, the cell number was gradually increased until 72 h after subculture (A 450 = 0.65±0.01 at 24 h, 0.78±0.01 at 48 h, and 1.08±0.03 at 72 h, n = 8 for each, p<0.01 vs. 0.50±0.02 at 0 h, n = 8; Fig 2). The proliferation rate in IPAH-PASMCs was much higher than that in normal PASMCs (0.81±0.02 at 24 h, 1.09±0.02 at 48 h, and 1.49±0.04 at 72 h, n = 8 for each, p<0.01 vs. 0.47±0.02 at 0 h, n = 8, and p<0.01 vs. normal PASMCs). On the other hand, the proliferation rate in CTEPH-PASMCs was similar to that in normal PASMCs (0.66±0.04 at 24 h, 0.79±0.04 at 48 h, and 1.12±0.05 at 72 h, n = 8 for each, p<0.01 vs. 0.44±0.03 at 0 h, n = 8, and p>0.05 vs. normal PASMCs). This result using a colorimetric cell counting kit was mostly consistent with our previous data using an automated cell counter [19].
Inhibitory effect of NPS2143 on excessive proliferation of PASMCs from IPAH patients NPS2143 is used as a calcilytic, or negative allosteric modulator of CaSR, and therefore blocks CaSR function. The cell survival of normal PASMCs was not affected by the treatment with NPS2143 regardless of culture time (98±7% at 10 μM and 72 h, n = 5, p>0.05 vs. control (100%), n = 5; Fig 3A). In contrast, the proliferation rate of IPAH-PASMCs was significantly reduced in the presence of 10 μM NPS2143 after 48 h (49% decrease, n = 3, p<0.01 vs. control) and 72 h (80% decrease, n = 10, p<0.01 vs. control) culture (Fig 3Ba). In the next set of experiments, the concentration dependence of the growth inhibition by NPS2143 was analyzed in IPAH--PASMCs. As the inhibitory effect by NPS2143 at 72 h culture was most prominent (Fig 3Ba), we Effect of Calcilytics in IPAH-PASMCs evaluated the dose-response relationship of PASMC proliferation upon 72 h culture. The NPS2143-induced growth inhibition occurred in a concentration-dependent manner (20±2% at 10 μM, n = 10, p<0.01; Fig 3Bb). The IC 50 value of NPS2143 was 2.64 μM, and the Hill coefficient was 1.00 (n = 7~13). On the other hand, the proliferation of CTEPH-PASMCs was not influenced by the presence of NPS2143 (89±9% at 10 μM and 72 h, n = 5, p>0.05 vs. control; Fig 3C).

Attenuation of excessive cell proliferation by Calhex 231 in PASMCs from IPAH patients
To confirm that the inhibitory effect of NPS2143 on excessive cell proliferation was mediated by CaSR antagonism in IPAH-PASMCs, the effect of Calhex 231, which is another calcilytic structurally unrelated to NPS2143, on cell proliferation was examined in normal, IPAH, and CTEPH PASMCs. The cell survival of normal PASMCs was not affected by the presence of Calhex 231 (89±4% at 10 μM and 72 h, n = 8, p>0.05 vs. control; Fig 4A). When IPAH-PASMCs were incubated with medium containing various concentrations of Calhex 231 for 72 h, Calhex 231 dramatically inhibited cell growth in a concentration-dependent manner (32±6% at 10 μM, n = 6, p<0.01 vs. control; Fig 4B) with an IC 50 value of 1.89 μM and a Hill coefficient of 1.39 (n = 6). In CTEPH-PASMCs, there was no significant difference in cell proliferation in the presence of Calhex 213 (95±6% at 10 μM, n = 5, p>0.05 vs. control; Fig 4C).

Enhancement of cell proliferation by calcimimetic in PASMCs from IPAH patients
Alternatively, the effect of the calcimimetic R568, a synthetic activator and positive allosteric modulator of CaSR, on cell proliferation was examined in normal, IPAH, and CTEPH

Effect of Calcilytics in IPAH-PASMCs
PASMCs. The proliferation rate of normal PASMCs was slightly increased by the treatment with R568 at the highest concentration of 10 μM after 48 h culture (118±6% at 48 h, n = 6, p = 0.022 vs. control; Fig 5A). The proliferation rate of IPAH-PASMCs was dramatically augmented in the presence of 10 μM R568 after 48 h culture. As the enhancing effect of R568 at 48 h was most prominent (Fig 5Ba), we evaluated the dose-response relationship of PASMC proliferation at 48 h. The R568-induced increase in cell number occurred in a concentrationdependent manner (185±15% at 10 μM, n = 11, p<0.01 vs. control; Fig 5Bb), with an EC 50 value of 0.33 μM and a Hill coefficient of 2.10 (n = 7~11). Similarly to normal PASMCs, the proliferation of CTEPH-PASMCs was slightly facilitated during the exposure to R568 for 48 h (117±4% at 10 μM, n = 5, p = 0.049 vs. control; Fig 5C).

Discussion
Our previous report showed that the excessive proliferation rate of IPAH-PASMCs was attenuated by siRNA knockdown of CaSR [19]; however, pharmacological modulators are required for the development of drug therapy for PAH. In this investigation, we have shown that calcilytics (NPS2143 and Calhex 231) inhibit the excessive proliferation of PASMCs from IPAH patients, whereas a calcimimetic (R568) enhances the proliferation of PASMCs from IPAH patients as well as from normal subjects and CTEPH patients. Similar results were obtained from two different experiments for cell proliferation based on MTT test and BrdU incorporation assay. These results indicate that the activity of CaSR contributes to the PASMC proliferation.
[Ca 2+ ] cyt plays an important role in regulation of the proliferation and migration of PASMCs. An increase in [Ca 2+ ] cyt in PASMCs is an important stimulus for PASMC proliferation and pulmonary vascular remodeling under physiological and pathological conditions [6,7,10]. We recently found that CaSR is upregulated in lung tissues and PASMCs isolated from IPAH patients, as well as in animals with experimental pulmonary hypertension [19,26,27]. This upregulated CaSR plays a pivotal role in the increased resting [Ca 2+ ] cyt and augmented Ca 2+ influx, as well as the enhanced cell proliferation in IPAH-PASMCs. Importantly, the intraperitoneal injection of the calcilytic NPS2143 prevented the development of pulmonary hypertension and right ventricular hypertrophy in monocrotaline-induced pulmonary hypertensive rats and hypoxia-induced pulmonary hypertensive mice. Functionally upregulated CaSR in PASMCs may be involved in a novel pathogenic mechanism underlying excessive pulmonary vascular remodeling in IPAH patients. In this study, we have direct evidence in vitro that calcilytics specifically inhibit the excessive proliferation of IPAH-PASMCs. Interestingly, calcilytics did not affect the cell growth of normal PASMCs. Pharmacological blockade of the upregulated CaSR with calcilytics may be a novel therapeutic approach for PAH patients who do not respond to conventional drug therapy. On the other hand, a calcimimetic enhanced the proliferation of IPAH-PASMCs. Even for normal and CTEPH PASMCs, cell growth was slightly but significantly increased by a calcimimetic. This result is in agreement with our previous observation that CaSR is expressed at low levels in the plasma membrane of normal PASMCs [19].
On the basis of our understanding of the pathological mechanisms of PAH, drug therapy for PAH has progressed in recent years via the development of several specific drugs that offer an effective alternative to voltage-dependent Ca 2+ channel blockers (calcium blockers), such as nifedipine and diltiazem [2,28]. Currently, three therapeutic classes are widely used for the treatment of PAH: endothelin receptor antagonists (bosentan and ambrisentan), phosphodiesterase type 5 inhibitors (sildenafil and tadalafil), and prostacyclins (also known as prostaglandin I 2 ) (epoprostenol, treprostinil, and iloprost). Moreover, numerous potential candidates have either been submitted for approval or are in development. Despite recent major therapeutic advances, current treatments of PAH do not achieve a cure of life-threatening disease. In this study, we have demonstrated that the PASMC proliferation is modulated by the activity of CaSR, which is upregulated in IPAH patients, and that this is a novel pathogenic mechanism underlying the augmented PASMC proliferation in IPAH patients. These results also provide an important pathway for developing therapeutic interventions for PAH. Calcilytics are potential drug candidates for the treatment of osteoporosis and other bone metabolism diseases [39,40]. Therefore, the accumulation of clinical data about calcilytics may be useful for clinical trials and the application of calcilytics as therapeutic drugs for PAH.
Our previous reports showed that a calcilytic inhibited the development of pulmonary hypertension in monocrotaline-induced pulmonary hypersensitive rats and hypoxia-induced pulmonary hypertensive mice [19,26]. In this study, we clearly demonstrate that calcilytics attenuate the excessive proliferation of PASMCs from IPAH patients, whereas calcilytics do not affect cell survival in PASMCs from normal subjects. Taken these findings together, calcilytics are a potential candidate for therapeutic drugs for PAH patients. Effect of Calcilytics in IPAH- PASMCs  Fig 6. Effect of CaSR modulators on cell proliferation using BrdU incorporation assay. The proliferation rates of PASMCs from normal subjects, IPAH patients, and CTEPH patients were analyzed using quantitative colorimetric assay based on the BrdU incorporation method for cell proliferation. The effects of NPS2143 (A), Calhex 231 (B), and R568 (C) on cell proliferation were examined in normal, IPAH, and CTEPH PASMCs. Summarized data (a) show the effect of CaSR modulators at a concentration of 3 μM on cell proliferation at 48 (for R568) or 72 (for NPS2143 and Calhex 231) h in normal, IPAH, and CTEPH PASMCs. Dose-response curve (b) of cell proliferation for NPS2143, Calhex 231, and R568 in IPAH-PASMCs. The absorbance was normalized by the value in the absence of drug (as 100%). The IC 50 values of NPS2143 and Calhex 231 for the proliferation of IPAH-PASMCs were 1.48 and 0.62 μM, respectively. The EC 50 value of R568 was 0.34 μM. Data were obtained from 7~14 experiments. *p<0.05 or **p<0.01 vs. control (100%). doi:10.1371/journal.pone.0138384.g006 Effect of Calcilytics in IPAH-PASMCs