Previous studies have demonstrated that besides the classic canonical transient receptor potential channel family, Orai family and stromal interaction molecule 1 (STIM1) might also be involved in the regulation of store-operated calcium channels (SOCCs). An increase in cytosolic free Ca2+ concentration promoted by store-operated Ca2+ entry (SOCE) in pulmonary arterial smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation and migration of PASMCs. In this study, our data revealed the following: (1) in both rat distal pulmonary arteries and PASMCs, chronic hypoxia exposure upregulated the expression of Orai1 and Orai2, without affecting Orai3 and STIM1; (2) either heterozygous knockout of HIF-1α in mice or knockdown of HIF-1α in PASMCs abolished the hypoxic upregulation of Orai2, but not Orai1, suggesting the hypoxic upregulation of Orai2 depends on HIF-1α; and (3) using small interference RNA knockdown strategies, Orai1, 2, 3 and STIM1 were all shown to mediate SOCE in hypoxic PASMCs. Together, these results suggested that the components of SOCCs, including Orai1, 2, 3 and STIM1, may lead to novel therapeutic targets for the treatment of chronic hypoxia-induced pulmonary hypertension.

Danshen, the dried root of Salvia miltiorrhiza, is widely used in clinics in China for treating various diseases, including cardiovascular diseases. Sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of tanshinone IIA isolated as the major active component from Danshen, was recently reported to be effective in attenuating the characteristic pulmonary vascular changes associated with chronically hypoxic pulmonary hypertension (CHPH); however, the underlying detailed mechanisms are poorly understood. In this study, we investigated the effects of STS on basal intracellular Ca 21 concentration ([Ca 21 ] i ) and store-operated Ca 21 entry (SOCE) in distal pulmonary arterial smooth muscle cells (PASMCs) exposed to prolonged hypoxia or isolated from CHPH rats. SOCE measured by Mn 21 quenching of Fura-2 fluorescence in PASMCs from rats exposed to chronic hypoxia (10% O 2 , 21 d) was increased by 59%, and basal [Ca 21 ] i was increased by 119%; this effect was inhibited by intraperitoneal injection of STS. These inhibitory effects of STS on hypoxic increases of SOCE and basal [Ca 21 ] i were associated with reduced expression of canonical transient receptor potential (TRPC)1 and TRPC6 in distal pulmonary arterial smooth muscle and decreases on right ventricular pressure, right ventricular hypertrophy, and peripheral pulmonary vessel thickening. In ex vivo cultured distal PASMCs from normoxic rats, STS (0-25 mM) dose-dependently inhibited hypoxiainduced cell proliferation and migration, paralleled with attenuation in increases of basal [Ca 21 ] i , SOCE, mRNA, and protein expression of TRPC1 and TRPC6. STS also relieved right ventricular systolic pressure, right ventricular hypertrophy, and TRPC1 and TRPC6 protein expression in distal pulmonary arteries in a monocrotaline-induced rat model of pulmonary arterial hypertension. These results indicate that STS prevents pulmonary arterial hypertension development likely by inhibiting TRPC1 and TRPC6 expression, resulting in normalized basal [Ca 21 ] i and attenuated proliferation and migration of PASMCs.Keywords: STS; TRPC; SOCE; pulmonary hypertension Pulmonary arterial hypertension (PAH) is a rare yet life-threatening disease that affects 15 per 1 million adults, according to the most recent estimation in 2008 (1). It is physiologically defined by mean pulmonary arterial pressure of 25 mm Hg or greater at rest and is pathologically characterized by pulmonary vascular remodeling, including smooth muscle hypertrophy and intima thickening. Although significant progress has been made in the past decades in our understanding of PAH and in disease management, the prognosis is still poor, with a 1-year survival rate of 91.0% (2) and 3-year survival rate of 77% or less according to recent investigations (3-5). The principal treatments for PAH rely on approaches targeting the prostacyclin, endothelin, or NO pathways (phosphodiesterase inhibition) or, increasingly, on a combination of them (6-11). Few patients show indication for treatment with calcium channel...

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Transient receptor potential canonical (TRPC) proteins play important roles in chronically hypoxic pulmonary hypertension (CHPH). Previous results indicated that sildenafil inhibited TRPC1 and TRPC6 expression in rat distal pulmonary arteries (PAs). However, the underlying mechanisms remain unknown. We undertook this study to investigate the downstream signaling of sildenafil's regulation on TRPC1 and TRPC6 expression in pulmonary arterial smooth muscle cells (PASMCs). Hypoxia-exposed rats (10% O2 for 21 d) and rat distal PASMCs (4% O2 for 60 h) were taken as models to mimic CHPH. Real-time PCR, Western blotting, and Fura-2-based fluorescent microscopy were performed for mRNA, protein, and Ca(2+) measurements, respectively. The cellular cyclic guanosine monophosphate (cGMP) analogue 8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate sodium salt (CPT-cGMP) (100 μM) inhibited TRPC1 and TRPC6 expression, store-operated Ca(2+) entry (SOCE), and the proliferation and migration of PASMCs exposed to prolonged hypoxia. The inhibition of CPT-cGMP on TRPC1 and TRPC6 expression in PASMCs was relieved by either the inhibition or knockdown of cGMP-dependent protein kinase (PKG) and peroxisome proliferator-activated receptor γ (PPARγ) expression. Under hypoxic conditions, CPT-cGMP increased PPARγ expression. This increase was abolished by the PKG antagonists Rp8 or KT5823. PPARγ agonist GW1929 significantly decreased TRPC1 and TRPC6 expression in PASMCs. Moreover, hypoxia exposure decreased, whereas sildenafil treatment increased, PKG and PPARγ expression in PASMCs ex vivo, and in rat distal PAs in vivo. The suppressive effects of sildenafil on TRPC1 and TRPC6 in rat distal PAs and on the hemodynamic parameters of CHPH were inhibited by treatment with the PPARγ antagonist T0070907. We conclude that sildenafil inhibits TRPC1 and TRPC6 expression in PASMCs via cGMP-PKG-PPARγ-dependent signaling during CHPH.

BMP4 acts downstream of HIF-1 and mediates hypoxia-induced up-regulation of TRPC, leading to increased basal [Ca(2+)]i in PASMCs, promoting CHPH pathogenesis.

In this study, we investigated the role of peroxisome proliferator-activated receptor γ (PPARγ) on store-operated calcium entry (SOCE) and expression of the main store-operated calcium channels (SOCCs) components, canonical transient receptor potential (TRPC) in chronic hypoxia (CH) and monocrotaline (MCT)-induced PH rat models. siRNA knockdown and adenoviral overexpression strategies were constructed for both loss-of-function and gain-of-function experiments. PPARγ agonist rosiglitazone attenuates the pathogenesis of both CHPH and MCT-PH, suppresses Hif-1α, TRPC1, TRPC6 expression in the distal pulmonary artery (PA) and SOCE in freshly isolated rat distal pulmonary arterial smooth muscle cells (PASMCs). By comprehensive use of knockdown and overexpression studies, bioinformatically analysis of the TRPC gene promoter and luciferase reporter assay, we demonstrated that PPARγ exerts roles of anti-proliferation, anti-migration, and pro-apoptosis in PASMCs, likely by inhibiting the elevated SOCE and TRPC expression. These effects were inhibited under the conditions of hypoxia or Hif-1α accumulation. We also found that under hypoxia, accumulated Hif-1α protein acts as upstream of suppressed PPARγ level, however, targeted PPARγ rescue acts negative feedback on suppressing Hif-1α level and Hif-1α mediated signaling pathway. PPARγ inhibits PH by targeting SOCE and TRPC via inhibiting Hif-1α expression and signaling transduction.

The tumor-suppressive role of p53, a transcription factor that regulates the expression of many genes, has been linked to cell cycle arrest, apoptosis, and senescence. The noncanonical function or the pathogenic role of p53 has more recently been implicated in pulmonary vascular disease. We previously reported that rapid nuclear accumulation of hypoxia-inducible factor (HIF)-1α in pulmonary arterial smooth muscle cells (PASMCs) upregulates transient receptor potential channels and enhances Ca2+ entry to increase cytosolic Ca2+ concentration ([Ca2+]cyt). Also, we observed differences in HIF-1α/2α expression in PASMCs and pulmonary arterial endothelial cells (PAECs). Here we report that p53 is increased in PAECs, but decreased in PASMCs, isolated from mice with hypoxia-induced pulmonary hypertension (PH) and rats with monocrotaline (MCT)-induced PH (MCT-PH). The increased p53 in PAECs from rats with MCT-PH is associated with an increased ratio of Bax/Bcl-2, while the decreased p53 in PASMCs is associated with an increased HIF-1α. Furthermore, p53 is downregulated in PASMCs isolated from patients with idiopathic pulmonary arterial hypertension compared with PASMCs from normal subjects. Overexpression of p53 in normal PASMCs inhibits store-operated Ca2+ entry (SOCE) induced by passive depletion of intracellularly stored Ca2+ in the sarcoplasmic reticulum, while downregulation of p53 enhances SOCE. These data indicate that differentially regulated expression of p53 and HIF-1α/2α in PASMCs and PAECs and the cross talk between p53 and HIF-1α/2α in PASMCs and PAECs may play an important role in the development of PH via, at least in part, induction of PAEC apoptosis and PASMC proliferation.

Emerging studies have reported the mechanosensitive Piezo1 (piezo type mechanosensitive ion channel component 1) plays essential roles in regulating the vascular tone through mechanistic actions on intracellular calcium homeostasis. However, the specific roles of Piezo1 in pulmonary vessels remain incompletely understood. We aim to investigate whether and how Piezo1 regulates the intracellular calcium homeostasis in human pulmonary arterial smooth muscle cells (PASMCs) under normal and pulmonary arterial hypertension (PAH) conditions. Cultured human PASMCs isolated from both control donors and idiopathic PAH patients were used as cell models. Fura-2 based intracellular calcium imaging was performed to measure the intracellular free calcium concentration ([Ca 2+ ] i ). Results showed that activation of Piezo1 by Yoda1 increases [Ca 2+ ] i by inducing both intracellular calcium release from internal calcium stores through the intracellular (intra-) Piezo1 localized at the subcellular organelles, including endoplasmic reticulum/sarcoplasmic reticulum, mitochondria, and nucleus; as well as extracellular calcium influx through the plasma membrane-localized Piezo1 in a mechanism independent of the store-operated calcium entry. Moreover, the Piezo1-mediated increase of [Ca 2+ ] i is linked to increased contraction and proliferation of PASMCs. Yoda1 induces dose-dependent vasocontraction in endothelium-denuded rat intrapulmonary arteries. Significant upregulation and increased activity of Piezo1 were observed in idiopathic PAH-PASMCs versus donor-PASMCs, contributing to the increased [Ca 2+ ] i and excessive proliferation of idiopathic PAH-PASMCs. In summary, Piezo1 mediates the increase of [Ca 2+ ] i by triggering both intracellular calcium release and extracellular influx. The enhanced Piezo1 expression and activity accounts, at least partially, for the abnormally elevated [Ca 2+ ] i and proliferation in idiopathic PAH-PASMCs.