Notch activation induces an endothelial-to-mesenchymal transformation

The Notch signalling pathway is an evolutionarily conserved intercellular signalling mechanism, and mutations in its components disrupt cell fate specification and embryonic development in organisms from nematodes to mammals. Various studies have demonstrated a critical role for Notch signalling in cardiovascular development. Patients with mutations of the Notch ligand, Jagged (JAG)l, demonstrate cardiac anomalies that are consistent with defects in endothelial-to-mesenchymal transformation that is essential for endocardial cushion formation. We demonstrate herein that constitutive activation of Notch in endothelial cells results in morphological and phenotypic changes consistent with this mesenchymal transformation. Specifically, we observe the downregulation of endothelial markers (such as vascular endothelial (VE)-cadherin) and the upregulation of mesenchymal proteins (such as alpha-smooth muscle actin (SMA)) in Notch-transformed endothelial cells. Moreover, we show that endothelial cells expressing activated Notch undergo a functional change characteristic of mesenchymal cells, in that they are capable of migrating towards platelet-derived growth factor (PDGF), but are no longer chemotactic towards vascular endothelial growth factor (VEGF). Our studies also reveal that the Notch-induced endothelial-to-mesenchymal transformation is cell autonomous, and independent of transforming growth factor (TGF)P signalling, which is an important regulator of this transformation in vivo. Furthermore, JAG1 stimulation of endothelial cells induces a similar mesenchymal transformation. Finally, we show that JAG1, Notch 1 and Notch4 are expressed in the ventricular outflow tract at the commencement of endocardial cushion formation. This is the first direct evidence that JAG 1-Notch interactions induce endothelial-to-mesenchymal transformation, and our findings suggest that the cardiovascular defects seen in patients with JAG1 mutations may be due to disruption of this process.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat