Abstract 3941: VEGF-A Induces Arterial Endothelial Differentiation By Increasing Both NOTCH-expression And NOTCH-related Signaling
It has previously been shown that VEGFA via a Notch-dependent signaling cascade critically controls endothelial differentiation with clear implications for vascular development and regeneration. In this study we investigated the potential instructive role of VEGF-receptor-R1 and VEGFR2-signaling on the expression of Notch-pathway members, their signaling and associated endothelial differentiation in human cardiac endothelial cells (ECs). Notch-expression, Notch-signaling and EC differentiation (i.e. Ephrin-B2 and COUP-TFII-expression) were studied in these cells by means of RT-qPCR, protein and luciferase promoter analysis for Notch-signaling (i.e. RBP-κJ related). In order to stimulate either VEGFR1 or VEGFR2-signaling, or both, cells were exposed to PlGF, VEGFE or VEGFA165, respectively. Only VEGFA165 and VEGFE, but not PlGF increased the expression of Notch-4 and the Notch ligand Delta-like-4 (DLL-4) along with the arterial marker Ephrin-B2, whereas the venous marker COUP-TFII was down regulated. The effect of VEGFA165 on endothelial differentiation could be blocked by specific inhibition of Notch-intracellular domain (NICD)-signaling and by the γ-secretase inhibitor (L685,458). Immobilized DLL-4 mimicked the effect of VEGFA165 in an NICD, HES-1 and γ-secretase dependent fashion. Furthermore, VEGFA165 was shown to increase the expression of ADAM10, a metalloprotease critically involved in activation of NICD-signaling. Interestingly, Notch-activation through DLL-4 was found to have a positive feedback on predominantly its own expression, potentially amplifying NICD-signaling. From our study it can be concluded that VEGFR2-signaling plays a principal role in cardiac arterial EC differentiation. VEGFR2-induced differentiation of the endothelium occurs through increase of both the expression and activation of Notch-family members, possibly involving upregulation of ADAM10.