Background/Aims: Pulmonary arterial hypertension (PAH) is a severe and debilitating disease characterized by remodeling of the pulmonary vessels, which is driven by excessive proliferation and migration and apoptosis resistance in pulmonary artery smooth muscle cells (PASMCs). The calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) signaling pathway is the most important downstream signaling pathway of store-operated Ca 2+ entry (SOCE), which is increased in PAH. CaN/NFAT has been

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... ted to contribute to abnormal proliferation in chronic hypoxia (CH)-induced PAH. However, the effect of CaN/NFAT signaling on PASMC proliferation, migration and apoptosis in monocrotaline (MCT)-induced PAH remains unclear. Methods: PAH rats were established by a single intraperitoneal injection of MCT for 21 days. PASMCs were isolated and cultured in normal and MCT-induced PAH Sprague-Dawley rat. PASMCs were treated with CsA targeting CaN and siRNA targeting NFATc2-4 gene respectively by liposome. We investigated the expression of calcineurin/NFAT signaling by immunofluorescence, qRT-PCR and Western blotting methods. Cell proliferation was monitored using MTS reagent or by assessing proliferating cell nuclear antigen (PCNA) expression. Cell apoptosis was evaluated with an Annexin V -FITC/propidium iodide (PI) apoptosis kit by flow cytometry. PASMC migration was assessed with a Transwell chamber. Results: MCT successfully induced PAH and pulmonary vascular remodeling in rats. CaN He et al.: Calcineurin/NFAT Signaling Modulates PASMC Proliferation, Migration and Apoptosis phosphatase activity and nuclear translocation of NFATc2-4 were increased in PASMCs derived from MCT-treated rats. In addition, CaNBβ/NFATc2-4 expression was amplified at the mRNA and protein levels. PASMC proliferation and migration were markedly inhibited in a dosedependent manner by cyclosporin A (CsA). Furthermore, siRNA targeting NFATc2 and NFATc4 attenuated the excessive proliferation and migration and apoptosis resistance in PASMCs derived from both CON and MCT-treated rats, while NFATc3 knockdown specifically affected MCT-PASMCs. Conclusion: Our results demonstrate that CaN/NFAT signaling is activated and involved in the modulation of PASMC proliferation, migration and apoptosis in MCT-induced PAH. He et al.: Calcineurin/NFAT Signaling Modulates PASMC Proliferation, Migration and Apoptosis then removed, and the weight of the left ventricle (LV), right ventricle (RV) and septum (S) and the right ventricular mass index (RVMI), which is the ratio of RV to LV plus S [RV/(LV+S)], were determined. Data were obtained from thirty-five rats in each group. Isolation and culture of PASMCs Rats were injected with heparin, anesthetized with urethane (1 g/kg), and exsanguinated. The lungs were then removed and transferred to a petri dish filled with cold HEPES-buffered salt solution (HBSS) containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl 2 , 1.5 CaCl 2 , 10 HEPES and 10 glucose, pH 7.2 (adjusted with NaOH). Under a dissection microscope, the right and left branches of the main pulmonary artery were first isolated from the whole lung, and then, the third-and fourth-generation pulmonary arteries (PAs) (~300 to 800 μm) were isolated and cleared of connective tissue. The endothelium was removed by gently rubbing the luminal surface with a cotton swab. PASMCs were then enzymatically isolated and transiently cultured as previously described [9, 42] . Briefly, arteries were allowed to recover for 30 min in cold HBSS, followed by 20 min in reduced-Ca 2+ (20 μM) HBSS at room temperature. The tissue was digested at 37°C for 20 min in 20 He et al.: Calcineurin/NFAT Signaling Modulates PASMC Proliferation, Migration and Apoptosis The above data, especially the increased CaN phosphatase activity and NFAT nuclear translocation, clearly verified the activation of CaN/NFAT signaling in PASMCs from MCTinduced PAH rats. MCT treatment enhanced PASMC proliferation and migration but inhibited apoptosis Compared with CON-PASMCs, MCT-PASMCs had significantly higher proliferation and migration rates and were more resistant to apoptosis. Enhanced MCT-PASMC proliferation was observed with an MTS assay. These results were confirmed by immunoblot analysis of PCNA, which showed a nearly two-fold increase in the MCT-treated group compared with the CON group (Fig. 3A) . In addition to proliferation, there was a 4-fold increase in MCT-PASMC migration, as determined by Transwell chamber experiments (Fig. 3B) . To further examine