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Mechanosensitive cation channels and Ca2+ influx through these channels play an important role in the regulation of endothelial cell functions. Transient receptor potential canonical channel 6 (TRPC6) is a diacylglycerol-sensitive nonselective cation channel that forms receptor-operated Ca2+ channels in a variety of cell types. Piezo1 is a mechanosensitive cation channel activated by membrane stretch and shear stress in lung endothelial cells. In this study, we report that TRPC6 and Piezo1 channels both contribute to membrane stretch-mediated cation currents and Ca2+ influx or increase in cytosolic-free Ca2+ concentration ([Ca2+]cyt) in human pulmonary arterial endothelial cells (PAECs). The membrane stretch-mediated cation currents and increase in [Ca2+]cyt in human PAECs were significantly decreased by GsMTX4, a blocker of Piezo1 channels, and by BI-749327, a selective blocker of TRPC6 channels. Extracellular application of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of diacylglycerol, rapidly induced whole cell cation currents and increased [Ca2+]cyt in human PAECs and human embryonic kidney (HEK)-cells transiently transfected with the human TRPC6 gene. Furthermore, membrane stretch with hypo-osmotic or hypotonic solution enhances the cation currents in TRPC6-transfected HEK cells. In HEK cells transfected with the Piezo1 gene, however, OAG had little effect on the cation currents, but membrane stretch significantly enhanced the cation currents. These data indicate that, while both TRPC6 and Piezo1 are involved in generating mechanosensitive cation currents and increases in [Ca2+]cyt in human PAECs undergoing mechanical stimulation, only TRPC6 (but not Piezo1) is sensitive to the second messenger diacylglycerol. Selective blockers of these channels may help develop novel therapies for mechanotransduction-associated pulmonary vascular remodeling in patients with pulmonary arterial hypertension. [ABSTRACT FROM AUTHOR]

Concentric pulmonary vascular wall thickening due partially to increased pulmonary artery (PA) smooth muscle cell (PASMC) proliferation contributes to elevating pulmonary vascular resistance (PVR) in patients with pulmonary hypertension (PH). Although pulmonary vasoconstriction may be an early contributor to increasing PVR, the transition of contractile PASMCs to proliferative PASMCs may play an important role in the development and progression of pulmonary vascular remodeling in PH. A rise in cytosolic Ca2+ concentration ([Ca2+ ]cyt) is a trigger for PASMC contraction and proliferation. Here, we report that upregulation of Piezo1, a mechanosensitive cation channel, is involved in the contractile-to-proliferative phenotypic transition of PASMCs and potential development of pulmonary vascular remodeling. By comparing freshly isolated PA (contractile PASMCs) and primary cultured PASMCs (from the same rat) in a growth medium (proliferative PASMCs), we found that Piezo1, Notch2/3, and CaSR protein levels were significantly higher in proliferative PASMCs than in contractile PASMCs. Upregulated Piezo1 was associated with an increase in expression of PCNA, a marker for cell proliferation, whereas downregulation (with siRNA) or inhibition (with GsMTx4) of Piezo1 attenuated PASMC proliferation. Furthermore, Piezo1 in the remodeled PA from rats with experimental PH was upregulated compared with PA from control rats. These data indicate that PASMC contractile-to-proliferative phenotypic transition is associated with the transition or adaptation of membrane channels and receptors. Upregulated Piezo1 may play a critical role in PASMC phenotypic transition and PASMC proliferation. Upregulation of Piezo1 in proliferative PASMCs may likely be required to provide sufficient Ca2+ to assure nuclear/cell division and PASMC proliferation, contributing to the development and progression of pulmonary vascular remodeling in PH. [ABSTRACT FROM AUTHOR]

Endothelial-to-mesenchymal transition (EndMT) is a biological process that converts endothelial cells to mesenchymal cells with increased proliferative and migrative abilities. EndMT has been implicated in the development of pulmonary vascular remodeling in pulmonary arterial hypertension (PAH), a fatal and progressive lung vascular disease. Transforming growth factor β 1 (TGF-β 1 ), an inflammatory cytokine, is known to induce EndMT in many types of endothelial cells including lung vascular endothelial cells (LVEC). An increase in cytosolic free Ca 2+ concentration ([Ca 2+ ] cyt ) is a major stimulus for cellular proliferation and phenotypic transition, but it is unknown whether Ca 2+ signaling is involved in EndMT. In this study we tested the hypothesis that TGF-β 1 -induced EndMT in human LVEC is Ca 2+ -dependent. Treatment of LVEC with TGF-β 1 for 5-7 days resulted in increase in SNAI1/2 expression, induction of EndMT, upregulation of STIM/Orai1 and enhancement of store-operated Ca 2+ entry (SOCE). Removal (or chelation) of extracellular or intracellular Ca 2+ with EGTA or BAPTA-AM respectively abolished EndMT in response to TGF-β 1 . Moreover, EGTA diminished TGF-β 1 -induced increase in SNAI in a dose-dependent manner. Knockdown of either STIM1 or Orai1 was sufficient to prevent TGF-β-mediated increase in SNAI1/2 and EndMT, but did not rescue the continuous adherent junctions. Blockade of Orai1 channels by AnCoA4 inhibited TGF-β-mediated EndMT and restored PECAM1-positive continuous adherent junctions. In conclusion, intracellular Ca 2+ signaling plays a critical role in TGF-β-associated EndMT through enhanced SOCE and STIM1-Orai1 interaction. Thus, targeting Ca 2+ signaling pathways regulating EndMT may be a novel therapeutic approach to treat PAH and other forms of pre-capillary pulmonary hypertension., New & Noteworthy: EndMT has been reported to contribute to the pathogenesis of PH. In this study we aimed to determine the role of Ca 2+ signaling in the development of EndMT in human lung vascular endothelial cells. Our data suggest that TGF-β 1 requires store-operated Ca 2+ entry through STIM1/Orai channels to induce SNAI-mediated EndMT. For the first time we demonstrated that TGF-β 1 -induced EndMT is Ca 2+ -dependent event while inhibition of STIM1/Orai interaction attenuated EndMT in response to TGF-β 1 .