1 Vascular Research Laboratory, Providence Veterans Affairs Medical Center, Providence, Rhode Island.
2 Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island.
Am J Respir Cell Mol Biol. 2018 May;58(5):658-667. doi: 10.1165/rcmb.2016-0344OC.
Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH). Anoctamin (Ano)-1, a calcium-activated chloride channel, can regulate cell proliferation and cell cycle in multiple cell types. However, the expression and function of Ano1 in the pulmonary endothelium is unknown. We examined whether Ano1 was expressed in pulmonary ECs and if altering Ano1 activity would affect EC survival. Expression and localization of Ano1 in rat lung microvascular ECs (RLMVECs) was assessed using immunoblot, immunofluorescence, and subcellular fractionation. Cell counts, flow cytometry, and caspase-3 activity were used to assess changes in cell number and apoptosis in response to the small molecule Ano1 activator, Eact. Changes in mitochondrial membrane potential and mitochondrial reactive oxygen species (mtROS) were assessed using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine, iodide (mitochondrial membrane potential dye) and mitochondrial ROS dye, respectively. Ano1 is expressed in RLMVECs and is enriched in the mitochondria. Activation of Ano1 with Eact reduced RLMVEC counts through increased apoptosis. Ano1 knockdown blocked the effects of Eact. Ano1 activation increased mtROS, reduced mitochondrial membrane potential, increased p38 phosphorylation, and induced release of apoptosis-inducing factor. mtROS inhibition attenuated Eact-mediated p38 phosphorylation. Pulmonary artery ECs isolated from patients with idiopathic PAH (IPAH) had higher expression of Ano1 and increased cell counts compared with control subjects. Eact treatment reduced cell counts in IPAH cells, which was associated with increased apoptosis. In summary, Ano1 is expressed in lung EC mitochondria. Activation of Ano1 promotes apoptosis of pulmonary ECs and human IPAH-pulmonary artery ECs, likely via increased mtROS and p38 phosphorylation, leading to apoptosis.
高增殖性内皮细胞（ECs）在肺动脉高压（PAH）的发病机制中起着重要作用。Anoctamin（Ano）-1 是一种钙激活氯离子通道，可调节多种细胞类型的细胞增殖和细胞周期。然而，Ano1 在肺内皮细胞中的表达和功能尚不清楚。我们研究了 Ano1 是否在肺内皮细胞中表达，以及改变 Ano1 的活性是否会影响 EC 的存活。使用免疫印迹、免疫荧光和亚细胞分级分离来评估 Ano1 在大鼠肺微血管内皮细胞（RLMVECs）中的表达和定位。使用细胞计数、流式细胞术和 caspase-3 活性来评估小分子 Ano1 激活剂 Eact 对细胞数量和凋亡的影响。使用 5,5',6,6'-四氯-1,1',3,3'-四乙基苯并咪唑基碳菁碘化物（线粒体膜电位染料）和线粒体 ROS 染料分别评估线粒体膜电位和线粒体活性氧（mtROS）的变化。Ano1 在 RLMVECs 中表达，并富集在线粒体中。用 Eact 激活 Ano1 可通过增加凋亡来减少 RLMVEC 计数。Ano1 敲低可阻断 Eact 的作用。Ano1 的激活增加了 mtROS，降低了线粒体膜电位，增加了 p38 磷酸化，并诱导了凋亡诱导因子的释放。mtROS 抑制减弱了 Eact 介导的 p38 磷酸化。与对照相比，特发性肺动脉高压（IPAH）患者分离的肺动脉内皮细胞（PAECs）中 Ano1 的表达更高，细胞计数增加。Eact 处理减少了 IPAH 细胞的细胞计数，这与凋亡增加有关。总之，Ano1 在内皮细胞的线粒体中表达。激活 Ano1 可促进肺 EC 和人 IPAH-肺动脉 EC 的凋亡，可能通过增加 mtROS 和 p38 磷酸化来实现，从而导致凋亡。
