Laminin-Binding Integrins Induce Dll4 Expression and Notch Signaling in Endothelial Cells

Rationale: Integrins play a crucial role in controlling endothelial cell proliferation and migration during angiogenesis. The Delta-like 4 (Dll4)/Notch pathway establishes an adequate ratio between stalk and tip cell populations by restricting tip cell formation through "lateral inhibition" in response to a vascular endothelial growth factor gradient. Because angiogenesis requires a tight coordination of these cellular processes, we hypothesized that adhesion, vascular endothelial growth factor, and Notch signaling pathways are interconnected. Objective: This study was aimed at characterizing the cross-talk between integrin and Notch signaling in endothelial cells. Methods and Results: Adhesion of primary human endothelial cells to laminin-111 triggers Dll4 expression, leading to subsequent Notch pathway activation. SiRNA-mediated knockdown of alpha 2 beta 1 and alpha 6 beta 1 integrins abolishes Dll4 induction, which discloses a selective integrin signaling acting upstream of Notch pathway. The increase in Foxc2 transcription, triggered by alpha 2 beta 1 binding to laminin, is required but not sufficient per se for Dll4 expression. Furthermore, vascular endothelial growth factor stimulates laminin gamma 1 deposition, which leads to integrin signaling and Dll4 induction. Interestingly, loss of integrins alpha 2 or alpha 6 mimics the effects of Dll4 silencing and induces excessive network branching in an in vitro sprouting angiogenesis assay on three-dimensional matrigel. Conclusions: We show that, in endothelial cells, ligation of alpha 2 beta 1 and alpha 6 beta 1 integrins induces the Notch pathway, and we disclose a novel role of basement membrane proteins in the processes controlling tip vs stalk cell selection. (Circ Res. 2011;109:172-182.)