Abstract 17103: Cdc42 Controls Vascular Network Assembly Through Protein Kinase Ciota
Vasculogenesis is the process in which endothelial precursor cells differentiate into endothelial cells and subsequently form the primitive vascular network. The signaling pathways that mediate this fundamental biological process are largely unknown. Here we demonstrate that Cdc42 ablation in embryonic stem cells blocks vascular network assembly but not endothelial lineage differentiation during embryoid body (EB) vasculogenesis. Stable expression of Cdc42 in mutant EBs largely rescues the vascular network, indicating that Cdc42 is essential for vascular network assembly. To explore the underlying mechanisms, we isolate endothelial cells from wild type and Cdc42-null EBs. The Cdc42-null endothelial cells are defective in directional migration and in network formation on Matrigel. In addition, PKCiota activation is abolished in the absence of Cdc42, and GSK3-β phosphorylation at Ser9 is significantly reduced by both Cdc42 ablation and inhibition of atypical PKCs. Targeted deletion of PKCiota blocks vascular network assembly, while expression of kinase-dead GSK3-β in Cdc42-null EBs promotes the formation of cord-like endothelial segments. These results suggest that PKCiota and GSK3-β are downstream of Cdc42 in mediating directional migration and/or endothelial network formation. Furthermore, we found that endothelial cell aggregates formed at the early step of vasculogenesis are associated with a nascent basement membrane. This basement membrane and VEGF synergistically activate Cdc42, and integrin β1 is required for the synergistic effect to occur. Altogether, our results suggest a model in which VEGF and basement membrane synergistically activate Cdc42, and the latter controls vascular network assembly through activating protein kinase Ciota.
- © 2010 by American Heart Association, Inc.