PROJECT SUMMARY/ABSTRACT Precise and dynamic regulation of vascular barrier function, the ability of endothelial cells that line blood vessels to provide a selectively permeable barrier between the bloodstream and surrounding tissue, is universally important for maintaining tissue homeostasis. The pathological consequences of dysregulated barrier are evident in various cardiovascular diseases like atherosclerosis and chronic ischemia as well as inflammatory and neurodegenerative diseases. In homeostatic conditions, hemodynamic shear stress, the frictional drag force exerted by the flow of blood on endothelial cells, promotes vascular homeostasis and barrier function through remodeling and enhancement of cell-cell adherens junctions (AJs) and intrinsic actin cytoskeletal dynamics. The critical cell-cell adhesion molecule vascular endothelial (VE-) cadherin, the principle component of AJs, regulates junctional stability through its turnover and internalization and experiences significant changes in tension under shear stress. Additionally, the actin cytoskeleton regulates vascular barrier by maintaining a balance between dynamic pushing forces to maintain VE-cadherin and tensile forces which stabilize intracellular AJ complexes. However, the specific molecular sensors and transducers that link hemodynamic shear stress to the mechanical regulation of AJs and vascular barrier function remain poorly understood. Activation of the ubiquitously important Notch1 receptor was recently been found to modulate vascular barrier function in response to shear stress by complexing with VE-cadherin and stabilizing AJs. While previous work has determined how this Notch1 cortical pathway modulates vascular barrier function, it remains unclear how shear stress activates the Notch1 receptor. Building on preliminary data linking Notch1 to intrinsic cellular adhesive and cytoskeletal machinery, this proposal tests the hypothesis that intrinsic coupling of Notch1 to VE-cadherin and the cortical actin cytoskeleton regulates shear stress-mediated Notch1 activation. Interrogation of Notch1 activation in response to shear stress will be approached by completing two specific aims: (1) determine how VE-cadherin spatiotemporally regulates Notch1 and its ligand Dll4 to coordinate activation by shear stress and (2) identify the mechanical interplay between Notch1 and intrinsic actin cytoskeletal dynamics under shear stress. Throughout the course of the proposed research, I will gain training in 3D biomimetic models of the human microvasculature, super-resolution live cell microscopy, and mechanistic molecular approaches, while simultaneously enhancing career development through training in scientific communication, mentoring, and teaching. I have assembled an exceptional, complementary mentoring team to help me achieve my research and career goals: Dr. Matthew Kutys, an expert in organotypic tissue modeling and cell mechanics will be my primary sponsor and Dr. Diane Barber, a leader in cellular cytoskeletal dynamics, will be my co-sponsor. Ultimately, these findings will identify new mechanisms by which Notch1 is activated in response to shear stress and potentially identify new therapeutic targets for modulating barrier function and other vasculopathies where Notch1 is implicated.

PROJECT NARRATIVE Dysregulation of vascular barrier function, the selectively permeable barrier established by endothelial cells that line blood vessels to regulate the exchange of fluids, cells, and nutrients, contributes to a variety of cardiovascular, inflammatory, and neurodegenerative diseases. Despite a critical clinical demand, we lack interventional strategies to repair or modulate vascular barrier, largely due to a poor understanding of the master regulators within the vascular endothelium. This proposal aims to reveal new mechanisms of how the Notch1 receptor activates under shear stress, increasing barrier function, and potentially identify new therapeutic targets for modulating barrier function and other vasculopathies where Notch1 is implicated.