Calcineurin/NFAT Signaling Molecules Regulate Proliferation,Migration And Apoptosis In PASMCs Of Pulmonary Hypertensive Rats

Pulmonary arterial hypertension(PAH) is a severe and debilitating disease characterized by increased resting intracellular free Ca2+ concentration([Ca2+]i) of pulmonary arterial smooth muscle cells(PASMCs), and progressive pulmonary remodeling. Recent studies have shown that increased store-operated calcium entry(SOCE) plays an important role to cytoplasm Ca2+ increasing in monocrotaline(MCT) induced pulmonary arterial hypertension rats. Increased intracellular Ca2+further activates calcineurin(Ca N, PP2B), and thus results in the dephosphorylation and nuclear translocation of nuclear factor of activated T cells(NFAT). Ca N-NFAT signaling has been found to participate in the progress of PAH. The present study aimed to examine the effects of Ca N-NFAT signaling on proliferation, migration and apoptosis of PASMCs in MCT-induced PAH rats.Firstly, rats developed severe PAH and right ventricular hypertrophy within three weeks after single intraperitoneal injection of 50mg/kg MCT. Upregulated m RNA and protein expression of Ca N-NFAT on PAs, as well as developed proliferation and migration and decreased apoptosis was observed in PASMCs of MCT-PAH rats.Secondly, using in vitro cultured PASMCs, we assessed the alteration of proliferation, apoptosis and migration when Ca N inhibitor cyclosporin A(Cs A) was used or NFATc2/3/4 expression is silenced by si RNA transfection. Finally, Cs A or Ginsenoside Rb1(Rb1) was administered to MCT-induced PAH rats for three weeks in order to clarify whether Cs A or Rb1 can interfere with cell function and the possible mechanism. Our resutls indentified the vital role of Ca N/NFAT signaling in PAH pathogenesis process, and provide a new target for PAH treatment.1. Alteration of Ca2+/Ca N/NFAT Signaling Pathway and Affect of Proliferation,Migration and Apoptosis in PASMCs of MCT-reduced PAH RatsObjective: To observe the pathogenic role of Ca2+/Ca N/NFAT signaling pathway and the change in the function of PASMCs in MCT-induced PAH rats.Methods: Sprague-Dawley rats developed severe PAH within three weeks after single intraperitoneal injection of MCT 50 mg/kg doses.(1) RVSP, RVP, RVMI and PAs morphology were measured with hemodynamics and hematoxylineosin staining.(2) Ca2+ influx in PASMCs following CPA stimulation was measured with Fluo-3fluorescence to determine SOCE activity.(3) Ca N and NFAT expression of PASMCs were detected by quantitative real-time PCR?western blot and immunofluorescence.(4) Cell viability and proliferation of PASMCs were determinated with MTS assay and PCNA expression.(5) PASMCs migration was tested by using transwell chambers.(6)Apoptotic rate in PASMCs was detected with Annexin V-FITC/PI apoptosis kit.Results: The following results were from MCT-induced PAH rats opposed to normal rats.(1)PAH rat model is created successfully. That was evidenced by significantly increased RVSP, RVP and RVMI, pulmonary arteriolar wall thicken and luminal stenosis obseved in MCT treated rats.(2) MRNA content and protein expressions of Ca NB?-NFATc2/3/4 were significantly increased.(3)After CPA induced Ca2+depletion in the sarcoplasmic reticulum, [Ca2+]i were greatly increased,,revealed enhanced SOCE in PASMCs.(4)MCT-induced PAH developed proliferation,migration and apoptosis resistance in PASMCs, which may result from NFAT up-regulation.Conclusion: MCT successfully induced rat PAH model. The mechanism of PAH may be related to SOCE enhancements and Ca NB?-NFATc2/3/4 over-expression in PASMCs, which caused cell proliferation, migration and anti-apoptosis in PASMCs.Then, pulmonary vascular remodeling occurred. Moreover, SOCE enhancement may be the start factor in the development of PAH.2. Effect of CaN/NFAT Signaling Pathway on the Proliferation, Migration and Apoptosis of PASMCsObjective: To further understand the function of NFAT signaling molecule in MCT-induced PAH rats, we observed the change of cellular proliferation, migration and apoptosis in PASMCs by depressing activity of Ca N and NFAT respectively.Methods: PASMCs isolated from normal or MCT-induced PAH rats were treated with Ca N inhibitor Cs A or NFATc2/3/4 si RNA.(1)The efficiency of RNA interference was determined by quantitative real-time PCR and immunofluorescence.(2)Cell viability and proliferation of PASMCs were measured by MTS assay and PCNA expression.(3)PASMCs migration was assessed by using transwell chambers.(4)The apoptosis of PASMCs was investigated by Annexin V-FITC/PI staining.Results:(1) The IC50 values of the growth inhibition of PASMCs treated with Cs A were 34.73 ± 2.23?M in CON group and 23.29 ± 2.23?M in MCT group,respectively. Accordingly, a dose of 15?M, 30?M or 60?M Cs A was used in the subsequent research.(2) Proliferation and migration of PASMCs were inhibited by Cs A in a concentration dependent manner, whereas PASMCs isolated from MCT-induced PAH rats seemed more sensitive to Cs A as compared with that in control. Those data suggested that Ca N as the effective point of Cs A might be a possible target of PASMCs proliferation in PAH.(3) The dose of 60?M Cs A decreased apoptosis rate of PASMCs particularly in normal cells and the other two doses had no effect on it. Those findings suggested that Ca N might involved in the proliferation in MCT-induced PAH.(4) NFATc2/3/4 expression was silenced with respective si RNA, compared with the corresponding MOCK group.(5) NFATc2/3/4m RNA and protein expression of PASMCs were significantly restrained in both CON and MCT rats, which meant successive RNA interference.(6) Proliferation,migration and apoptosis resistance of PASMCs were obviously inhibited by si RNA of NFATc2 and NFATc4 in both CON and MCT rats.(7) Pretreatment with si RNA of NFATc3 can obviously suppressed proliferation and migration, promoted apoptosis of PASMCs in MCT rats, however had no effect on PASMCs in CON rats.Conclusion: Ca N/NFAT signaling molecules may play a pivotal role in the pathogenesis process of PAH. Ca N is likely to participate in enhancement of cell viability and migration in PAH PASMCs. NFATc2 and NFATc4 are essential factors for PASMCs growth. Moreover, NFATc2/3/4 may contribute to pulmonary vascular remodeling in MCT- induced PAH.3. Cyclosporin A or Ginsenoside Rb1 Inhibited Proliferation, Migration and Promoted Apoptosis in PASMCs of MCT-induced PAH RatsObjective: To definite the effect of Cs A or Rb1 on the proliferation, apoptosis and migration of PASMCs from MCT-reduced PAH rats, and to further determine the role of Ca2+/Ca N/NFAT signaling pathway in pathogenesis process of PAH.Methods: Rats were single intraperitoneal injected with MCT, simultaneously they received a continuous intraperitoneal injection of 25mg/kg/day Cs A or Rb1 for 21 d.Pulmonary arteries were isolated from lungs and PASMCs were primarily cultured with 10% FBS.(1) RVSP, RVP, RVMI and PAs morphology were detected by hemodynamics and hematoxylineosin staining.(2) Ca2+ entry flow in PASMCs in response to CPA sitimulation was monitored by Fluo-3 fluorescence to determine SOCE activity.(3) Ca N and NFAT expression of PASMCs were detected by quantitative real-time PCR?western blot and immunofluorescence.(4) Cell viability and proliferation of PASMCs were determinated with MTS assay and PCNA expression.(5) PASMCs migration was tested with transwell chambers.(6) Rates of apoptosis in PASMCs were detected with Annexin V-FITC/PI apoptosis kit.Results: We obtained the following results from Cs A treated PAH rats.(1) RVSP,RVP and RVMI were significantly reduced. Decrease in the thick of pulmonary arteriolar walls as well as increase in the lumen, implied restraint of pulmonary artery remolding by Cs A.(2) Protein expression of NFATc3 and NFATc4 was decreased in Cs A treated groups.(3) Cs A intervention caused proliferative inhibition,migration restraining and apoptosis resistance.Rb1 was treated with MCT-induced PAH rats and compared with MCT group.(1)Rb1-treated group significantly reduced RVSP, RVP and RVMI, and resulted in pulmonary arteriolar wall thinning and lumen increasing. Those showed that Rb1 could inhibit MCT-induced PAH.(2) Rb1 reduced Ca NB?-NFATc2/3/4 expression in the m RNA and protein levels, NFATc1/5 expression is decreased in the m RNA level in PASMCs.(3) [Ca2+]i decreased, which stated Rb1 inhibited SOCE enhancement of PASMCs in PAH rats.(4) Rb1 suppressed proliferation and migration, promoted apoptosis in PASMCs.Conclusion: Cs A inhibited MCT-induced PAH and PASMCs proliferation,migration and anti-apoptosis. We supposed that the mechanism may be related with down-regulation of NFATc3/4 expression with Cs A intervention. Rb1 inhibited SOCE in PASMCs from MCT-induced PAH rats, resulted in the inhibition of Ca2+-Ca NB?-NFAT signaling pathway, and then caused the inhibition of proliferation, migration and apoptosis resistance. Thus, Cs A and Rb1 can be two candidates for PAH treatment and worth further investigation.In summary, Ca2+-Ca N-NFAT signaling pathway plays a key role in the pathogenesis process of MCT-induced PAH. NFATc2 and NFATc4 are principal factors for PASMCs growth. And, NFATc2/3/4 may contribute to pulmonary vascular remodeling in MCT- induced PAH. Rb1 attenuated PASMCs SOCE enhancement in MCT-induced PAH rats, also down-regulated the expression of Ca NB?-NFAT signal molecules. In addition, Rb1 impeded hyperproliferative and apoptosis-resistant phenotype of PASMCs, also reversed vascular remodeling of PAH. We further clarify vital role focusing on Ca N/NFAT signaling molecules for PAH pathogenesis process, also provide a new basis for PH treatment and new targets. However, Rb1 may become a new target drug for the treatment of PAH.