Endothelial biomechanics plays a key role in multiple endothelial functions. Our earlier studies discovered that oxidized lipids in vitro and dyslipidemia in vivo induce significant endothelial stiffening via CD36 scavenger receptor and incorporation of oxysterols. Most recently, we found that endothelial stiffening in aging aortas critically depends on CD36 and the caveolar protein, Caveolin-1 (Cav1). Our long term goal is to elucidate the mechanisms responsible for age-induced changes in endothelial biomechanics and to determine the contribution of these mechanisms to endothelial dysfunction. In the current proposal, we address three goals: In Aim 1, we focus on elucidating the mechanism of age-induced EC stiffening. First (1A), we will determine whether endothelial-specific deletions of CD36 and/or Cav1 prevent endothelial stiffening in moderately aged (10-12 months old) and advanced aged (20-24 months old) mice and whether expression/membrane localization of these proteins is altered by age. Then (1B), we will provide a comprehensive lipidomics analysis of changes in lipid composition in arterial tissues with age and identify specific lipid species that accumulate with age and induce endothelial stiffening. In Aim 2, we focus on age-related actin remodeling and disruption of endothelial barrier integrity. First (aim 2A), we will determine the roles of endothelial CD36 and Cav1 and CD36/Cav1-mediated uptake of oxidized lipids in age-related actin remodeling, junctional morphology, endothelial permeability to macromolecules, and infiltration of monocytes. In the second part of the aim (2B), we will investigate the molecular mechanisms by which oxidized lipids, particularly oxysterols, induce actin remodeling, focusing on a novel hypothesis that oxysterols compete with RhoA for binding to a RhoA inhibitory protein, GDI-1. In Aim 3, these studies are extended to investigate the impact of CD36/Cav1/oxysterol- dependent endothelial stiffening in inducing distortion of nuclear morphology, DNA damage and nuclear translocation of mechanosensitive transcription factors (3A) and explore the hypothesis that oxysterol-induced endothelial stiffening results in nuclei distortion by disruption of a peri-nuclear structure called the actin cap (3B). These studies are then extended to an exploratory sub-aim (3C) of comparative analysis of transcriptomic changes in aged endothelium in vivo and in endothelial cells exposed to oxidized lipids that induce endothelial stiffening. These goals are achieved using endothelial-specific loss of function (CD36 and Cav1) and gain of function (Cav1) genetic mouse models. A combination of Atomic Force Microscopy, lipid mass-spectrometry, high resolution confocal imaging analyzed by machine-learning algorithms and other state- of-the-art experimental approaches. Taken together, these studies are expected to provide significant new insights into our understanding of the mechanisms responsible for endothelial stiffening in aging...