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 5,59,6,69-tetrachloro-1,19,3,39-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.
|Original language||English (US)|
|Number of pages||10|
|Journal||American journal of respiratory cell and molecular biology|
|State||Published - May 1 2018|
Bibliographical noteFunding Information:
This work was supported by Veterans Affairs Merit Awards 5IO1BX000711 and National Heart, Lung, and Blood Institute (NHLBI) 1R01HL128661 (G.C.), National Institutes of Health (NIH) grants 5R25GM083270 and 5T32GM077995 (A.M.A.), awards from American Heart Association (AHA) GRNT20460376 and National Institute of General Medical Sciences (NIGMS) U54GM115677 (R.T.C.), awards from NIH 1R01HL136757, 5P30GM1114750, AHA 16SDG27260248, Rhode Island Foundation grant 20164376, and American Physiological Society 2017 Shih-Chun Wang Young Investigator Award (J.O.-U.), NIH grant 5R01HL121796HL (D.T.), and by NIH grants U54GM115677 and 5P30GM1114750 (B.S.J.); the collection of human pulmonary arterial cells is supported by NIH grants HL060917 and HL081064.
Copyright © 2018 by the American Thoracic Society.
- Pulmonary arterial hypertension