PROJECT SUMMARY/ABSTRACT Vascular smooth muscle (SM) and endothelial cells (EC) coordinate the molecular signals governing arterial vasoreactivity. The balance of constrictor and relaxant signals establishes vessel tone and directly influences systemic blood pressure in health and disease. Our laboratory has uncovered novel and paradoxical roles for Notch signaling in the vessel wall that influence arterial function. Specifically, SM Notch receptors triggered by SM Jagged1 ligand promote myosin light chain kinase (MLCK) expression and Ca2+ sensitization inducing myosin light chain (MLC) phosphorylation, the molecular signature of force production. Mice lacking SM Jagged1 feature dysregulated blood pressure and pressor responses and arteries exhibit impaired force generation. In contrast, EC Dll4 ligand stimulates expression of SM MYPT1, the regulatory subunit of myosin phosphatase (MP), favoring dephosphorylation of MLC and a relaxant phenotype. Moreover, EC Dll4-deficient arteries yield vasorelaxation deficits in a novel NO-independent manner. Together, these findings underscore instructional Notch signaling through heterotypic (EC-SM) and homotypic (SM-SM) cell interactions in the vessel wall and provide new knowledge in the molecular determinants of vasoregulation. The overall goal in this new project proposal is to define the physiologically relevant Notch ligand/receptor that modulates distinct arterial functions. In Aim 1, we determine the contribution of both SM and EC Jagged1 ligand and Notch1 receptor in regulation of constrictor and hemodynamic responses in vascular health and disease animal models. Aim 2 delineates the physiological role and mechanism of EC Dll4 ligand in mediating arterial relaxation. Finally, Aim 3 examines the molecular basis for Notch signaling-dependent control of myosin phosphatase activity via Ca2+ sensitization and/or desensitization. Experimental approaches include a full spectrum of in vivo, ex vivo and in vitro vascular analyses necessary for understanding relevant physiology and novel molecular mechanisms through which Notch pathway components regulate arterial function.