Abstract 3138: Atheroprone Flow, Aging, And Oxysterols Impair Endothelial Cell Nuclear Health Via Disruption Of Autoregulatory Perinuclear Cytoskeletal Dynamics

The vascular hemodynamic environment plays a well-known role with progressive pathological conditions such as atherosclerosis, hypercholesterolemia, and aging. Our lab has previously shown that the exposure of oxysterols to endothelial cells (ECs) causes increased cell stiffening. This stiffening effect is compounded when the hemodynamic environment changes from atheroprotective laminar flow to atheroprone oscillatory flow. The increasing EC stiffening demonstrates that these disruptive effects significantly alter cellular biomechanics. The complexity of events associated with these altered biomechanical states leaves many questions unanswered about spatiotemporal changes in the biomechanical structures of endothelial cells exposed to a varying hemodynamic environment and oxysterols. To address this void, we observed the effects of 7-Ketocholesterol exposure (a major oxysterol) on the primary cytoskeletal/biomechanical components (F-actin, Microtubules, Intermediate Filaments, etc.) of human aortic endothelial cells during various time points of laminar/oscillatory flow conditions through a combination of sophisticated multiplexed 6-color labeling, ultra-high resolution 3D microscopy, 3D computer vision, and machine learning. Additionally, we applied these techniques to extracted aortic tissues from young and aged mice (on low fat diet vs high fat diet) and focused on obtaining information from both the laminar flow areas (descending aorta) and the oscillatory flow areas (aortic arch). Our findings demonstrate that oxysterol exposure and aging cause significant remodeling & alteration of crucial endothelial cytoskeletal/biomechanical components and that these alterations are unique to each combinatorial condition. Notably, we find that cytoskeletal remodeling associated with endothelial stiffening is accompanied by major nuclear distortion, a hallmark of aging and senescence which subsequently causes DNA damage and genomic instability. We also find that this nuclear distortion is driven by dynamic biomechanical changes and mediated by the hemodynamic environment, where atheroprotective flow promotes cytoskeletal configurations protecting the nuclei, and atheroprone flow promotes cytoskeletal configurations that leave the nuclei unprotected.