Project Summary This project is based on the critical role of fluid shear stress from blood flow acting on the vascular endothelium in determining susceptibility to atherosclerosis. Physiological levels of laminar shear stress are actively anti-inflammatory and promote disease resistance whereas disturbed fluid shear stress amplifies inflammatory and metabolic risk factors to promote disease. Preliminary and published data implicate endothelial cell metabolism as critical in the differential effects of laminar vs disturbed flow, including a mitochondrial pathway activated through Piezo1 mechanosensitive channels, yet these effects, indeed, the entire area, is poorly understood. Our preliminary data also implicate cell cycle as a critical part of these regulatory effects, with a late G1 arrested state in which the cyclin-dependent kinase Cdk2 is active as a key inhibitor of atherogenic pathways. We therefore propose: 1) A thorough unbiased analysis of effects of laminar vs disturbed shear stress on endothelial metabolism, with in vivo analysis of Piezo1 loss and gain of function mutants, and extensive analysis to link metabolic effects to disease pathways; 2) In vitro mechanistic and in vivo pre-clinical analysis of the role of Cdk2 in endothelial inflammatory activation and disease, including links to metabolic pathways.