Mechanisms of DLL4/Notch and BMP9 Mediated Endothelial Cell Quiescence

Endothelial cell quiescence and homeostasis is required for maintaining vascular function, but various pathological states and therapeutic interventions disrupt this homeostasis and exacerbate vascular dysfunction and disease. Notch and BMP signaling are critical regulators of endothelial cells, and the ligands DLL4 and BMP9 are key ligands that cooperate in activating these pathways and inducing endothelial quiescence. The mechanisms of DLL4 and BMP9 cooperation are yet to be fully elucidated. The goal of this project was to uncover some of the mechanisms by which DLL4 and BMP9 signaling overlap for cooperativity, and the downstream effectors that mediate their homeostatic phenotype. Biochemical and molecular mechanisms were utilized for in vitro characterization of the effects of DLL4 and BMP9 stimulation of human endothelial cells, including changes in proliferation, cell cycle regulators, morphology, migration, feedback mechanisms, and cross-regulatory controls of the two pathways. P27KIP1 was identified as a major mediator of endothelial cell quiescence, and a suppression of the expression of multiple growth factor receptors was observed. A proteomic approach was taken to identify systematic changes in endothelial cells following DLL4 and BMP9 stimulation. We found changes in extracellular matrix proteins, among which thrombospondin-1 was identified as another major contributor to quiescence. To study these pathways in vivo, we characterized the blood vessels of a Dll4 heterozygous knockout mouse model (Dll4+/-). In the viable Dll4+/- mice we found splenomegaly, irregular patterning of the lung vasculature, and an upregulation of phospho-SMAD1/5/8 in endothelial cells, which was postulated to compensate for the decreased signaling from Dll4. Dll4+/- mouse aortae had increased endothelial cell density, enlarged lumens and thickened vessel walls. Together, our in vivo and in vitro results indicate that DLL4 and BMP9 mediate cooperative and interdependent signaling mechanisms which regulate multiple downstream effectors of quiescence. Future studies or therapies targeting DLL4 or BMP9 in endothelial cells or in the vasculature should take both pathways into consideration for increased efficacy and to prevent systemic loss of vascular homeostasis