Journal
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
Volume 35, Issue 9, Pages 1987-1994Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/ATVBAHA.115.305969
Keywords
calcium; cytosol; endothelial cells; Orai3 protein; vascular endothelial growth factor A
Categories
Funding
- Medical Research Council
- Wellcome Trust
- British Heart Foundation
- University of Leeds
- China Scholarship Council
- LTHT (Leeds Teaching Hospitals Trust) Charitable Foundation
- Cancer Research UK
- MRC [G0901203, G1002076] Funding Source: UKRI
- British Heart Foundation [FS/12/54/29671, FS/13/23/30122, FS/11/33/28831] Funding Source: researchfish
- Medical Research Council [G0901203, G1002076] Funding Source: researchfish
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Objective Vascular endothelial growth factor (VEGF) acts, in part, by triggering calcium ion (Ca2+) entry. Here, we sought understanding of a Synta66-resistant Ca2+ entry pathway activated by VEGF. Approach and Results Measurement of intracellular Ca2+ in human umbilical vein endothelial cells detected a Synta66-resistant component of VEGF-activated Ca2+ entry that occurred within 2 minutes after VEGF exposure. Knockdown of the channel-forming protein Orai3 suppressed this Ca2+ entry. Similar effects occurred in 3 further types of human endothelial cell. Orai3 knockdown was inhibitory for VEGF-dependent endothelial tube formation in Matrigel in vitro and in vivo in the mouse. Unexpectedly, immunofluorescence and biotinylation experiments showed that Orai3 was not at the surface membrane unless VEGF was applied, after which it accumulated in the membrane within 2 minutes. The signaling pathway coupling VEGF to the effect on Orai3 involved activation of phospholipase C1, Ca2+ release, cytosolic group IV phospholipase A2, arachidonic acid production, and, in part, microsomal glutathione S-transferase 2, an enzyme which catalyses the formation of leukotriene C-4 from arachidonic acid. Shear stress reduced microsomal glutathione S-transferase 2 expression while inducing expression of leukotriene C-4 synthase, suggesting reciprocal regulation of leukotriene C-4-synthesizing enzymes and greater role of microsomal glutathione S-transferase 2 in low shear stress. Conclusions VEGF signaling via arachidonic acid and arachidonic acid metabolism causes Orai3 to accumulate at the cell surface to mediate Ca2+ entry and downstream endothelial cell remodeling.
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