Regulation of enfothelial cell cycle dynamics by Notch during angiogenesis

Abstract

The formation of new blood vessels by angiogenesis requires the precise coordination of endothelial cell differentiation, proliferation, migration and maturation to ensure that a proper vasculature is formed to satisfy the metabolic needs of the surrounding tissue. The Notch signaling pathway is a critical regulator of endothelial cell biology, participating in different processes of blood vessel formation from tipTcell specification to arterioTvenous differentiation and blood vessel stabilization. Inhibition of this pathway during angiogenesis has been associated with increased proliferation of endothelial cells. However, this seemingly antiproliferative effect of Notch activation does not adjust well to the current tipT stalk cell model of molecular control of angiogenesis, in which proliferative stalk cells are defined as having higher Notch activity. In this work we have tried to resolve this apparent paradox by characterizing in detail the effects of Notch on endothelial cell proliferation In vivo. We have used different pharmacological and classical genetic approaches in combination with newly developed mosaic genetic tools to evaluate, with high spatioTtemporal resolution, endothelial cell cycle dynamics. We have found that physiological Notch activation is required for maintaining endothelial cell proliferation by repressing excessive activation of proTproliferative molecular mechanism. Inhibition of Notch activity induces and hyperproliferative response that is partially dependent on ERK activation. This response is, however, not sustained in time and leads to proliferative arrest, partially mediated by ERK overactivation and p21 upregulation. This cellTcycle arrest is associated with an increase in the expression of genes characteristic of sprouting tip cells. Thus, Notch can control a mechanism to differentiate between a proliferative and migratory cellular response. Comparative trancriptomics revealed new putative NotchTrepressed genes that promote cell cycle progression like Myc and Odc1. Functional analysis of these genes showed their absolute requirement for normal endothelial cell proliferation.