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Reduced intracellular chloride concentration impairs angiogenesis by inhibiting oxidative stress-mediated VEGFR2 activation

Kai Li1, Ying-ying Liu1, Xiao-fei Lv1, Zhuo-miao Lin1, Ting-ting Zhang1, Fei-ran Zhang1, Jia-wei Guo1, Yu Hong1, Xiu Liu1, Xiao-chun Lin1, Jia-guo Zhou1,2,3,4,5,6, Qian-qian Wu7,8,9, Si-jia Liang1,2, Jin-yan Shang1,2
1 Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China
2 Program of Kidney and Cardiovascular Disease, The Fifth Affiliated Hospital, Sun Yat-Sen University, Zhuhai 519000, China
3 Guangdong Province Key Laboratory of Brain Function and Disease, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou 510080, China
4 Department of Cardiology, Sun Yat-sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510080, China
5 Department of Physiology, Key Laboratory of Cardiovascular disease, School of Basic Medical Sciences, Guangzhou Medical University, Guangzhou 511436, China
6 Guangzhou Institute of Cardiovascular Disease, The Second Affiliated Hospital, Guangzhou Medical University, Guangzhou 511436, China
7 Key Laboratory of Metabolic Cardiovascular Diseases Research of National Health Commission, Ningxia Medical University, Yinchuan 750004, China
8 Ningxia Key Laboratory of Vascular Injury and Repair Research, Ningxia Medical University, Yinchuan 750004, China
9 School of Basic Medical Sciences, Ningxia Medical University, Yinchuan 750004, China
Correspondence to: Si-jia Liang: liangsj5@mail.sysu.edu.cn, Jin-yan Shang: shangjy5@mail.sysu.edu.cn,
DOI: 10.1038/s41401-020-0458-7
Received: 10 November 2019
Accepted: 7 June 2020
Advance online: 21 July 2020

Abstract

Chloride (Cl−) homeostasis is of great significance in cardiovascular system. Serum Cl− level is inversely associated with the mortality of patients with heart failure. Considering the importance of angiogenesis in the progress of heart failure, this study aims to investigate whether and how reduced intracellular Cl− concentration ([Cl−]i) affects angiogenesis. Human umbilical endothelial cells (HUVECs) were treated with normal Cl− medium or low Cl− medium. We showed that reduction of [Cl−]i (from 33.2 to 16.18 mM) inhibited HUVEC proliferation, migration, cytoskeleton reorganization, tube formation, and subsequently suppressed angiogenesis under basal condition, and VEGF stimulation or hypoxia treatment. Moreover, VEGF-induced NADPH-mediated reactive oxygen species (ROS) generation and VEGFR2 axis activation were markedly attenuated in low Cl− medium. We revealed that lowering [Cl−]i inhibited the expression of the membrane-bound catalytic subunits of NADPH, i.e., p22phox and Nox2, and blunted the translocation of cytosolic regulatory subunits p47phox and p67phox, thereby restricting NADPH oxidase complex formation and activation. Furthermore, reduced [Cl−]i enhanced ROS-associated protein tyrosine phosphatase 1B (PTP1B) activity and increased the interaction of VEGFR2 and PTP1B. Pharmacological inhibition of PTP1B reversed the effect of lowering [Cl−]i on VEGFR2 phosphorylation and angiogenesis. In mouse hind limb ischemia model, blockade of Cl− efflux using Cl− channel inhibitors DIDS or DCPIB (10 mg/kg, i.m., every other day for 2 weeks) significantly enhanced blood flow recovery and new capillaries formation. In conclusion, decrease of [Cl−]i suppresses angiogenesis via inhibiting oxidase stress-mediated VEGFR2 signaling activation by preventing NADPH oxidase complex formation and promoting VEGFR2/PTP1B association, suggesting that modulation of [Cl−]i may be a novel therapeutic avenue for the treatment of angiogenic dysfunction-associated diseases.
Keywords: chloride; angiogenesis; oxidative stress; VEGFR2; PTP1B; PTP1B inhibitor II

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