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Piezo is a mechanosensitive cation channel responsible for stretch-mediated Ca2+ and Na+ influx in multiple types of cells. Little is known about the functional role of Piezo1 in the lung vasculature and its potential pathogenic role in pulmonary arterial hypertension (PAH). Pulmonary arterial endothelial cells (PAECs) are constantly under mechanic stretch and shear stress that are sufficient to activate Piezo channels. Here, we report that Piezo1 is significantly upregulated in PAECs from patients with idiopathic PAH and animals with experimental pulmonary hypertension (PH) compared with normal controls. Membrane stretch by decreasing extracellular osmotic pressure or by cyclic stretch (18% CS) increases Ca2+-dependent phosphorylation (p) of AKT and ERK, and subsequently upregulates expression of Notch ligands, Jagged1/2 (Jag-1 and Jag-2), and Delta like-4 (DLL4) in PAECs. siRNA-mediated downregulation of Piezo1 significantly inhibited the stretch-mediated pAKT increase and Jag-1 upregulation, whereas downregulation of AKT by siRNA markedly attenuated the stretch-mediated Jag-1 upregulation in human PAECs. Furthermore, the mRNA and protein expression level of Piezo1 in the isolated pulmonary artery, which mainly contains pulmonary arterial smooth muscle cells (PASMCs), from animals with severe PH was also significantly higher than that from control animals. Intraperitoneal injection of a Piezo1 channel blocker, GsMTx4, ameliorated experimental PH in mice. Taken together, our study suggests that membrane stretch-mediated Ca2+ influx through Piezo1 is an important trigger for pAKT-mediated upregulation of Jag-1 in PAECs. Upregulation of the mechanosensitive channel Piezo1 and the resultant increase in the Notch ligands (Jag-1/2 and DLL4) in PAECs may play a critical pathogenic role in the development of pulmonary vascular remodeling in PAH and PH.

BACKGROUND AND PURPOSE: Halofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti-hypertrophic, anti-fibrotic and anti-proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia-induced pulmonary hypertension (HPH). EXPERIMENTAL APPROACH: Patch-clamp experiments were conducted to examine the activity of voltage-dependent Ca(2+) channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca(2+) concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O(2)) for 4 weeks were used as model of HPH for in vivo experiments. KEY RESULTS: Halofuginone increased voltage-gated K(+) (K(v)) currents in PASMCs and K(+) currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03–1 μM) inhibited receptor-operated Ca(2+) entry in HEK cells transfected with calcium-sensing receptor gene and attenuated store-operated Ca(2+) entry in PASMCs. Acute (3–5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia-induced pulmonary vasoconstriction dose-dependently (0.1–10 μM). Intraperitoneal administration of halofuginone (0.3 mg·kg(−1), for 2 weeks) partly reversed established PH in mice. CONCLUSION AND IMPLICATIONS: Halofuginone is a potent pulmonary vasodilator by activating K(v) channels and blocking VDCC and receptor-operated and store-operated Ca(2+) channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation–contraction coupling and anti-proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway.

The increase in cytosolic Ca2+ concentration ([Ca2+]cyt) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K+ (KV) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca2+ release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca2+ influx through receptor-operated and store-operated Ca2+ channels (SOC). Upon Ca2+ depletion from the SR, STIM forms clusters to mediate store-operated Ca2+ entry. Activity of KV channels, like KCNA5/KV1.5 and KCNA2/KV1.2, contributes to regulating membrane potential, and inhibition of KV channels results in membrane depolarization that increases [Ca2+]cyt by opening voltage-dependent Ca2+ channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca2+ influx through SOC channels. Extracellular Ca2+-mediated activation of CaSR increases [Ca2+]cyt in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca2+]cyt. Moreover, CaSR-mediated increase in [Ca2+]cyt was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K+ currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca2+]cyt by enhancing store-operated Ca2+ entry and inhibits KCNA5/KV1.5 and KCNA2/KV1.2, ultimately leading to voltage-activated Ca2+ entry.

Downregulated expression of K+ channels and decreased K+ currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K+ channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3′-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K+ channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K+ channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K+ currents and downregulated voltage-gated K+ channel 1.5 (KV1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K+ channel function and KV1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that KV1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca2+-activated K+ (BKCa) channel currents and downregulated BKCa channel β1 subunit (BKCaβ1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BKCa channel activity and BKCaβ1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of KV and BKCa channels in PASMC from patients with IPAH.

Calcifying fibrous tumors are rare lesions that occur throughout the body. A 35-year-old man presented with exertional dyspnea. Computed tomography showed a polypoid mass abutting pericardium, diaphragm, and left chest wall, and a pleural-based lesion adjacent to T12. Thoracoscopy revealed a large mass with diaphragmatic stalk, two pleural-based nodules, and four diaphragmatic nodules. Pathologic analysis of resected lesions showed calcifying fibrous tumors. Three lesions contained heterogenous mutations associated with this tumor type. The patient is disease free 1 year later. Calcifying fibrous tumors should be recognized as rare multifocal pleural and diaphragmatic-based tumors. Complete resection leads to excellent survival for patients with this uncommon disease.

The increase in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) and upregulation of calcium-sensing receptor (CaSR) and stromal interaction molecule 2 (STIM2) along with inhibition of voltage-gated K(+) (K(V)) channels in pulmonary arterial smooth muscle cells (PASMC) have been implicated in the development of pulmonary arterial hypertension; however, the precise upstream mechanisms remain elusive. Activation of CaSR, a G protein-coupled receptor (GPCR), results in Ca(2+) release from the endoplasmic/sarcoplasmic reticulum (ER/SR) and Ca(2+) influx through receptor-operated and store-operated Ca(2+) channels (SOC). Upon Ca(2+) depletion from the SR, STIM forms clusters to mediate store-operated Ca(2+) entry. Activity of K(V) channels, like KCNA5/K(V)1.5 and KCNA2/K(V)1.2, contributes to regulating membrane potential, and inhibition of K(V) channels results in membrane depolarization that increases [Ca(2+)](cyt) by opening voltage-dependent Ca(2+) channels. In this study, we show that activation of Notch by its ligand Jag-1 promotes the clustering of STIM2, and clustered STIM2 subsequently enhances the CaSR-induced Ca(2+) influx through SOC channels. Extracellular Ca(2+)-mediated activation of CaSR increases [Ca(2+)](cyt) in CASR-transfected HEK293 cells. Treatment of CASR-transfected cells with Jag-1 further enhances CaSR-mediated increase in [Ca(2+)](cyt). Moreover, CaSR-mediated increase in [Ca(2+)](cyt) was significantly augmented in cells co-transfected with CASR and STIM2. CaSR activation results in STIM2 clustering in CASR/STIM2-cotransfected cells. Notch activation also induces significant clustering of STIM2. Furthermore, activation of Notch attenuates whole cell K(+) currents in KCNA5- and KCNA2-transfected cells. Together, these results suggest that Notch activation enhances CaSR-mediated increases in [Ca(2+)](cyt) by enhancing store-operated Ca(2+) entry and inhibits KCNA5/K(V)1.5 and KCNA2/K(V)1.2, ultimately leading to voltage-activated Ca(2+) entry.

Downregulated expression of K(+) channels and decreased K(+) currents in pulmonary artery smooth muscle cells (PASMC) have been implicated in the development of sustained pulmonary vasoconstriction and vascular remodeling in patients with idiopathic pulmonary arterial hypertension (IPAH). However, it is unclear exactly how K(+) channels are downregulated in IPAH-PASMC. MicroRNAs (miRNAs) are small non-coding RNAs that are capable of posttranscriptionally regulating gene expression by binding to the 3′-untranslated regions of their targeted mRNAs. Here, we report that specific miRNAs are responsible for the decreased K(+) channel expression and function in IPAH-PASMC. We identified 3 miRNAs (miR-29b, miR-138, and miR-222) that were highly expressed in IPAH-PASMC in comparison to normal PASMC (>2.5-fold difference). Selectively upregulated miRNAs are correlated with the decreased expression and attenuated activity of K(+) channels. Overexpression of miR-29b, miR-138, or miR-222 in normal PASMC significantly decreased whole cell K(+) currents and downregulated voltage-gated K(+) channel 1.5 (K(V)1.5/KCNA5) in normal PASMC. Inhibition of miR-29b in IPAH-PASMC completely recovered K(+) channel function and K(V)1.5 expression, while miR-138 and miR-222 had a partial or no effect. Luciferase assays further revealed that K(V)1.5 is a direct target of miR-29b. Additionally, overexpression of miR-29b in normal PASMC decreased large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel currents and downregulated BK(Ca) channel β1 subunit (BK(Ca)β1 or KCNMB1) expression, while inhibition of miR-29b in IPAH-PASMC increased BK(Ca) channel activity and BK(Ca)β1 levels. These data indicate upregulated miR-29b contributes at least partially to the attenuated function and expression of K(V) and BK(Ca) channels in PASMC from patients with IPAH.