Store-operated Ca^{2 +} entry is involved in endothelium-to-mesenchymal transition in lung vascular endothelial cells

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 β₁ (TGF-β₁), an inflammatory cytokine, is known to induce EndMT in many types of endothelial cells including lung vascular endothelial cells (LVECs). 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-β₁-induced EndMT in human LVEC is Ca^{2 +} -dependent. Treatment of LVEC with TGF-β₁ 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-β₁. Moreover, EGTA diminished TGF-β₁-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 precapillary pulmonary hypertension.