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.

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.

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