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Reduced intracellular chloride concentration impairs angiogenesis by inhibiting oxidative stress-mediated VEGFR2
Kai Li1, Ying-Ying Liu1, Xiao-Fei Lv1
1Department of Pharmacology, Cardiac and Cerebral Vascular Research Center, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, 510080, China.
Insights
Reduced intracellular chloride levels inhibit angiogenesis by impairing NADPH oxidase and VEGFR2 signaling. Modulating chloride may offer new treatments for diseases linked to poor blood vessel formation.
Area of Science:
- Cardiovascular physiology
- Cell biology
- Molecular medicine
Background:
- Chloride (Cl-) homeostasis is crucial for cardiovascular health, with low serum Cl- linked to heart failure mortality.
- Angiogenesis, the formation of new blood vessels, is vital in heart failure progression.
Purpose of the Study:
- To investigate the impact of reduced intracellular chloride concentration ([Cl-]i) on angiogenesis.
- To elucidate the underlying molecular mechanisms by which [Cl-]i affects endothelial cell function and blood vessel formation.
Main Methods:
- Human umbilical endothelial cells (HUVECs) were cultured in normal or low chloride media.
- Assays included proliferation, migration, cytoskeleton reorganization, and tube formation.
- Western blotting and pharmacological inhibitors were used to assess NADPH oxidase activity, ROS generation, and VEGFR2 signaling pathways.
- A mouse hind limb ischemia model was employed to evaluate in vivo angiogenesis.
Main Results:
- Reduced [Cl-]i significantly inhibited HUVEC proliferation, migration, cytoskeleton organization, and tube formation, suppressing angiogenesis.
- Low [Cl-]i attenuated VEGF-induced reactive oxygen species (ROS) generation and VEGFR2 activation by inhibiting NADPH oxidase complex formation.
- Reduced [Cl-]i increased protein tyrosine phosphatase 1B (PTP1B) activity, enhancing VEGFR2/PTP1B interaction, which was reversed by PTP1B inhibition.
- In vivo, chloride channel inhibition improved blood flow recovery and capillary formation in a mouse ischemia model.
Conclusions:
- Decreased intracellular chloride suppresses angiogenesis by inhibiting NADPH oxidase-mediated ROS generation and VEGFR2 signaling.
- The findings suggest that reduced [Cl-]i promotes VEGFR2/PTP1B association, hindering angiogenesis.
- Modulating intracellular chloride levels presents a potential therapeutic strategy for angiogenic dysfunction-associated diseases.
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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