Monitoring VEGF-Stimulated Calcium Ion Flux in Endothelial Cells

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2475; p. 113
• Main Authors: Critchley, William R, Fearnley, Gareth W F, Abdul-Zani, Izma, Molina-Paris, Carmen, Bendtsen, Claus, Zachary, Ian C, Harrison, Michael A, Ponnambalam, Sreenivasan
• Format: Journal Article
• Language: English
• Published: United States 2022
• Subjects: Animals; Calcium - metabolism; Cell Movement; Cells, Cultured; Endothelial Cells - metabolism; Neovascularization, Physiologic - physiology; Phosphorylation; Signal Transduction - physiology; Vascular Endothelial Growth Factor A - metabolism; Vascular Endothelial Growth Factor A - pharmacology; Vascular Endothelial Growth Factor Receptor-2 - metabolism; VEGFR2; NFATc2; Calcium; VEGF-A; Human umbilical vein endothelial cells (HUVECs); Endothelial cells
• DOI: 10.1007/978-1-0716-2217-9_7

The endothelial response to vascular endothelial growth factor A (VEGF-A) regulates many aspects of animal physiology in health and disease. Such VEGF-A-regulated phenomena include vasculogenesis, angiogenesis, tumor growth and progression. VEGF-A binding to receptor tyrosine kinases such as vascular endothelial growth factor receptor 2 (VEGFR2 ) activates multiple signal transduction pathways and changes in homeostasis, metabolism, gene expression, cell proliferation, migration, and survival. One such VEGF-A-regulated response is a rapid rise in cytosolic calcium ion levels which modulates different biochemical events and impacts on endothelial-specific responses. Here, we present a series of detailed and robust protocols for evaluating ligand-stimulated cytosolic calcium ion flux in endothelial cells. By monitoring an endogenous endothelial transcription factor (NFATc2 ) which displays calcium-sensitive redistribution, we can assess the relevance of cytosolic calcium to protein function. This protocol can be easily applied to both adherent and non-adherent cultured cells to evaluate calcium ion flux in response to exogenous stimuli such as VEGF-A.