Non-invading cells that coordinate with tip cells during angiogenic sprout initiation exhibit a molecular signature...

Angiogenesis requires endothelial cell (EC) plasticity and differentiation into either invasive, migratory tip cells or proliferative stalk cells. Using a transcriptional analysis that distinguished tip cells from non-invading cells in three-dimensions (3D), we observed that approximately half of the significantly upregulated transcripts in the non-invading population were direct targets of YAP and TAZ. Although YAP and TAZ were reported to control the decision to divide versus differentiate in other cell types, a careful investigation of the actions of YAP and TAZ in ECs has not been performed in 3D. Immunofluorescence confirmed that nuclear YAP and TAZ levels were elevated in non-invading versus tip cells. Non-invading cells also entered the cell cycle more readily than tip cells, and activation of YAP and TAZ increased S-phase entry and reduced cell invasion. Blockade of S-phase entry had no effect on invasion responses, indicating that driving cell division largely excluded sprouting, while blocking proliferation did not deter sprouting. Consistent with prior reports, silencing both YAP and TAZ reduced sprouting responses; however, individual silencing of YAP and TAZ revealed divergent functions. YAP silencing resulted in formation of significantly longer filopodia, while silencing TAZ decreased invasion and shortened filopodia. Conversely, YAP overexpression reduced filopodia formation. Altogether, this study reveals that YAP and TAZ localize more frequently to the nucleus in non-invading cells but remain more cytoplasmic in tip cells, consistent with a proliferative phenotype in non-tip cells. These results provide insights into how endothelial plasticity is regulated during angiogenic sprout initiation in 3D.