Wednesday, February 5, 2025
2/5/2025
PROJECT SUMMARY Vascular function and development are largely mediated by vascular endothelial cells (VECs) that line the inner wall of blood vessels. Fluid shear stress (FSS) generated by blood flow is a major determinant of their function and phenotype with major roles in development, physiology, and disease. VECs in healthy regions of arteries are under unidirectional laminar flow, where they align in the direction of flow and activate anti-inflammatory pathways which confers resistance to atherosclerosis. By contrast, VECs in curved or branched regions of arteries develop disturbances in flow patterns. These disturbed flow patterns fail to align VECs and activate inflammatory pathways, which correlates with susceptibility to form atherosclerotic plaque. FSS direction with respect to cell alignment also regulates inflammatory signaling outputs, which suggests cell polarity and flow direction sensing is important for the differential atheroprotective and atheroprone responses. Thus, how VECs sense and respond to flow direction is an important basic science question pertinent to human health, but the mechanism is unclear. A junctional flow-dependent complex comprising of VE-Cahderin, PECAM1, and VEGF is critical for integrating endothelial cell flow responses. Our lab recently discovered that the polarity adaptor protein, LGN, which binds directly to VE-Cahderin is important for proper endothelial cell alignment. Since LGN directly interacts with a flow dependent mechanosensitive complex and has an established role regulating cytoskeletal dynamics, I hypothesize LGN is important for flow direction sensing. I plan to address this hypothesis using the following specific aims: Aim 1: Characterize the mechanism by generating mutations in LGN’s functional domains to determine which sites are important for mediating endothelial cell flow dependent signaling. I will similarly examine the effects of known LGN interactors if they are shown to be crucial for flow mediated signaling. Aim 2: Examine the role of cell polarity in flow signaling. I will do this first by tracking the intracellular localization of LGN in response to flow and use patterned substrates to separately constrain cell and cytoskeletal polarity to determine which of these variables is important for LGN polarity and inflammatory vs. anti-inflammatory signaling. Aim 3: Determine the role of LGN in vivo by analyzing mice with endothelial deletion of LGN which will address the role of LGN in VEC alignment, inflammation, and resulting atherosclerosis in vivo. Together, these aims will reveal new mechanisms for endothelial flow sensing, vascular inflammation, and atherosclerotic disease.
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