Abstract
Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks. Vascular endothelial growth factor (VEGF) is essential for angiogenesis and induces downstream signalling pathways including increased cytosolic calcium levels. Here we show that transmembrane protein 33 (tmem33), which has no known function in multicellular organisms, is essential to mediate effects of VEGF in both zebrafish and human ECs. We find that tmem33 localises to the endoplasmic reticulum in zebrafish ECs and is required for cytosolic calcium oscillations in response to Vegfa. tmem33-mediated endothelial calcium oscillations are critical for formation of endothelial tip cell filopodia and EC migration. Global or endothelial-cell-specific knockdown of tmem33 impairs multiple downstream effects of VEGF including ERK phosphorylation, Notch signalling and embryonic vascular development. These studies reveal a hitherto unsuspected role for tmem33 and calcium oscillations in the regulation of vascular development.
Highlights
Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks
Given the similar angiogenic defects induced by tmem[33] knockdown and SKF-96365 treatment, we examined whether inhibition of store-operated calcium entry (SOCE) could account for reduced EC number and migration previously observed in tmem[33] morphants (Fig. 4f–h, and Supplementary Fig. 2)
Increases in cytosolic Ca2+ are a well-established response to Vascular endothelial growth factor (VEGF) in ECs37, it remained unclear whether Ca2+ signalling was required for its effects in vivo and, if so, how Ca2+ co-ordinated EC behaviours during angiogenesis
Summary
Angiogenesis requires co-ordination of multiple signalling inputs to regulate the behaviour of endothelial cells (ECs) as they form vascular networks. Vascular endothelial growth factor (VEGF) is essential for angiogenesis and induces downstream signalling pathways including increased cytosolic calcium levels. Global or endothelial-cell-specific knockdown of tmem[33] impairs multiple downstream effects of VEGF including ERK phosphorylation, Notch signalling and embryonic vascular development. These studies reveal a hitherto unsuspected role for tmem[33] and calcium oscillations in the regulation of vascular development. Endothelial cells (ECs) line the inner lumen of blood vessels and their organisation into complex branching networks requires co-ordination of molecular outputs coupled to specific cellular behaviours via a process primarily orchestrated by signalling from vascular endothelial growth factor (VEGF)[1]. IP3 subsequently activates inositol triphosphate receptor (IP3R) Ca2+ channels within the endoplasmic reticulum (ER) to increase cytosolic Ca2+
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