Noninvading 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 noninvading (NON) cells in three-dimensions (3-D), we observed that approximately half of the significantly upregulated transcripts in the noninvading population were direct targets of Yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (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 3-D. Immunofluorescence confirmed that nuclear YAP and TAZ levels were elevated in noninvading versus tip cells. Noninvading 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, whereas 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 the formation of significantly longer filopodia, whereas 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 noninvading 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 3-D.NEW & NOTEWORTHY Transcriptional analysis of early three-dimensional (3-D) angiogenic sprout initiation revealed that the cells that do not initiate sprouting exhibited a unique gene signature, robustly expressing Yes-associated protein 1 (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ) target genes. These cells exhibited higher nuclear YAP/TAZ and entered the cell cycle more readily than tip cells; YAP/TAZ activation also induced the noninvading (NON) phenotype. Finally, we uncovered divergent functions for YAP and TAZ in filopodia formation, gaining new insights into angiogenic sprout initiation and endothelial plasticity in 3-D.