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Arterioscler Thromb Vasc Biol. 2019 Jan 10:ATVBAHA118312280. doi: 10.1161/ATVBAHA.118.312280. [Epub ahead of print]

Spermine on Endothelial Extracellular Vesicles Mediates Smoking-Induced Pulmonary Hypertension Partially Through Calcium-Sensing Receptor.

Zhu L1,2, Xiao R1,2, Zhang X1,2, Lang Y1,2, Liu F1,2, Yu Z1,2, Zhang J2,3, Su Y2,3, Lu Y2,4, Wang T2,4, Luo S1,2, Wang J5, Liu ML6,7, Dupuis J8,9, Jing ZC10, Li T11, Xiong W2,4, Hu Q1,2.

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From the Department of Pathophysiology, School of Basic Medicine (L.Z., R.X., X.Z., Y.Lang, F.L., Z.Y., S.L., Q.H.).
Key Laboratory of Pulmonary Diseases of Ministry of Health (L.Z., R.X., X.Z., Y.Lang, F.L., Z.Y., J.Z., Y.S., Y.Lu, T.W., S.L., W.X., Q.H.).
Department of Pathology and Department of Respiratory and Critical Care Medicine, Union Hospital (J.Z., Y.S.).
Department of Pathology and Department of Respiratory and Critical Care Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China (Y.L., T.W., W.X.).
State Key Laboratory of Respiratory Diseases, Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, China (J.W.).
Department of Dermatology, Perelman School of Medicine, University of Pennsylvania, Philadelphia (M.-L.L.).
Philadelphia Veterans Administration Medical Center (M.-L.L.).
Montreal Heart Institute, Québec, Canada (J.D.).
Department of medicine, Université de Montréal, Québec, Canada (J.D.).
State Key Laboratory of Cardiovascular Disease, Fu Wai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing (Z.-C.J.).
Department of Heart Centre and Artificial Cell Engineering Technology Research Center of Public Health Ministry, Third Central Clinical College, Tianjin Medical University, China (T.L.).


Objective- This study aims to determine whether and how the enriched metabolites of endothelial extracellular vesicles (eEVs) are critical for cigarette smoke-induced direct injury of endothelial cells and the development of pulmonary hypertension, rarely explored in contrast to long-investigated mechanisms secondary to chronic hypoxemia. Approach and Results- Metabonomic screen of eEVs from cigarette-smoking human subjects reveals prominent elevation of spermine-a polyamine metabolite with potent agonist activity for the extracellular CaSR (calcium-sensing receptor). CaSR inhibition with the negative allosteric modulator Calhex231 or CaSR knockdown attenuates cigarette smoke-induced pulmonary hypertension in rats without emphysematous changes in lungs or chronic hypoxemia. Cigarette smoke exposure increases the generation of spermine-positive eEVs and their spermine content. Immunocytochemical staining and immunogold electron microscopy recognize the spermine enrichment not only within the cytosol but also on the outer surface of eEV membrane. The repression of spermine synthesis, the inhibitory analog of spermine, N1-dansyl-spermine, Calhex231, or CaSR knockdown profoundly suppresses eEV exposure-mobilized cytosolic calcium signaling, pulmonary artery constriction, and smooth muscle cell proliferation. Confocal imaging of immunohistochemical staining demonstrates the migration of spermine-positive eEVs from endothelium into smooth muscle cells in pulmonary arteries of cigarette smoke-exposed rats. The repression of spermine synthesis or CaSR knockout results in attenuated development of pulmonary hypertension induced by an intravascular administration of eEVs. Conclusions- Cigarette smoke enhances eEV generation with spermine enrichment at their outer surface and cytosol, which activates CaSR and subsequently causes smooth muscle cell constriction and proliferation, therefore, directly leading to the development of pulmonary hypertension.


cytosol; endothelium; hypoxia; smoking; spermine


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