Supplemental Figure-2

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 (LVEC). An increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) is a major stimulus for cellular proliferation and phenotypic transition, but it is unknown whether Ca²⁺ signaling is involved in EndMT. In this study we tested the hypothesis that TGF-β₁-induced EndMT in human LVEC is Ca²⁺-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 Ca²⁺ entry (SOCE). Removal (or chelation) of extracellular or intracellular Ca²⁺ 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 Ca²⁺ signaling plays a critical role in TGF-β-associated EndMT through enhanced SOCE and STIM1-Orai1 interaction. Thus, targeting Ca²⁺ signaling pathways regulating EndMT may be a novel therapeutic approach to treat PAH and other forms of pre-capillary pulmonary hypertension.内皮细胞向间充质细胞转化（Endothelial-to-mesenchymal transition, EndMT）是一类可将内皮细胞转化为间充质细胞的生物学过程，转化后的细胞可获得更强的增殖与迁移能力。EndMT已被证实与肺动脉高压（pulmonary arterial hypertension, PAH）中的肺血管重构密切相关，而PAH是一种致命且进行性的肺血管疾病。转化生长因子β₁（Transforming growth factor β₁, TGF-β₁）作为一种炎性细胞因子，可在包括肺血管内皮细胞（lung vascular endothelial cells, LVEC）在内的多种内皮细胞中诱导EndMT。胞质游离钙离子浓度（[Ca²+]cyt）升高是细胞增殖与表型转化的主要刺激因素之一，但目前尚不明确钙信号是否参与EndMT过程。本研究验证了如下假说：TGF-β₁诱导的人LVEC EndMT过程依赖于钙信号通路。将LVEC用TGF-β₁处理5~7天后，可观察到SNAI1/2表达上调、EndMT被诱导、STIM/Orai1表达水平升高以及钙池操纵性钙内流（store-operated Ca²+ entry, SOCE）增强。分别用乙二胺四乙酸（EGTA）螯合胞外钙离子、或用乙酰氧基甲酯化BAPTA（BAPTA-AM）螯合胞内钙离子，均可完全阻断TGF-β₁诱导的EndMT。此外，EGTA可呈剂量依赖性地削弱TGF-β₁诱导的SNAI表达升高。单独敲低STIM1或Orai1即可阻止TGF-β介导的SNAI1/2表达上调与EndMT，但无法恢复连续的黏着连接结构。使用AnCoA4阻断Orai1通道，可抑制TGF-β介导的EndMT，并恢复血小板内皮细胞黏附分子1（PECAM1）阳性的连续黏着连接。综上，胞内钙信号通过增强SOCE以及STIM1-Orai1相互作用，在TGF-β相关的EndMT过程中发挥关键调控作用。因此，靶向调控EndMT的钙信号通路，或可成为治疗PAH及其他类型毛细血管前肺动脉高压的新型治疗策略。