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.
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Acknowledgements
This work was supported by National Key R&D Program of China (2017YFC0909302), National Natural Science Foundation of China (81525025, 91739104, 81473206, 81930106, 81803519, 81603103, 81603098, 81773723, 81903598), China Postdoctoral Science Foundation (2017M612829, 2018T110917), Natural Science Foundation of Guangdong Province (2019A1515011428, 2018A030310233, 2015A030312009), the Fundamental Research Funds for the Central Universities (19ykpy157, No. 18zxxt74), Science and Technology Program of Guangdong (2015TX01R159, 2015B090903063), and the Science and Technology Program of Guangzhou (201604010087).
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JYS, SJL, and JGZ conceived and designed the experiments; KL, YYL, XFL, TTZ, FRZ, JWG, and YH performed the experiments, especially KL, YYL, and XFL carried out most of the experiments; JYS, SJL, KL, YYL, XFL, and QQW analyzed the data; ZML, XL, and XCL contributed reagents/materials/analysis tools. JYS and SJL wrote the paper. All authors participated in the modification of paper.
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Li, K., Liu, Yy., Lv, Xf. et al. Reduced intracellular chloride concentration impairs angiogenesis by inhibiting oxidative stress-mediated VEGFR2 activation. Acta Pharmacol Sin 42, 560–572 (2021). https://doi.org/10.1038/s41401-020-0458-7
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DOI: https://doi.org/10.1038/s41401-020-0458-7
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