Endothelial cells respond to blood flow induced forces on a genetic, molecular, and structural level. How external mechanical stimuli such as fluid shear stress, are translated into various cellular responses, is not yet completely understood. Our ultimate goal is to fully delineate pathways by which endothelial cells sensed and respond to shear stress: from the apical surface to the cytoskeleton and the nucleus. We have already demonstrated that shear stress activates the glucocorticoid receptor (GR) to nuclear localize and regulate transcription. Using GR as a model, the proposed project will further examine the dynamics interactions of shear activated GR at the cell nucleus. Underlying the nuclear envelope is the lamina, a network of type V intermediate filaments, lamins, which provides structural support to the nucleus. Mechanical stress on lamin- deficient cells is hypothesized to disrupt normal cell mechanics and activities in disease mechanisms of laminopathies (e.g. muscular dystrophy, cardiomyopathy). This proposed project will further investigate the role of lamins in mechanotransduction at the nucleus of endothelial cells. To achieve this objective we set two specific aims: (1) to examine the role of endothelial lamina in nuclear import and transcription regulation by steroid or shear stress activated GR, (2) to characterize flow-induced structural changes of the nucleus following extended shearing. Using bovine aortic endothelial cells, we will first disrupt nuclear lamina using siRNA and examine subsequent GR nuclear import under flow, and second, utilize time-lapse fluorescent microscopy to analyze dynamics of nuclear movement under flow. Working from the central hypothesis the nuclear lamina plays an important role in maintaining nuclear structure under shearing forces - we want to investigate how GR activation under flow depends on the nuclear lamina, and if the nuclear lamina participates in nuclear shape and structural changes under extended shearing. Results from this investigation would not only yield a better understanding of the role of GR in cardiovascular inflammation and pathophysiology, but also provide a more comprehensive model of mechanotransduction pathways under shearing forces. Elucidating how endothelial cells respond to mechanical stresses due to blood flow will help identify markers of vascular diseases such as atherosclerosis and potential drug targets for their treatment and prevention. A major goal of this project is to determine how lamins in endothelial cells helps the nucleus sense and respond to shear stress. Mutations of the lamin gene in humans are implicated in a variety of diseases, including the Hutchinson-Gilford progeria syndrome - a premature aging syndrome often ending in fatal cardiovascular disease. Disease mechanisms of HGPS are not fully characterized. This study will expand mechanotransduction studies to the nucleus level in endothelial cells, and results from this investigation may yield new insights into not only cardiovascular pathophysiology, but also the molecular mechanisms of aging and the development of cardiovascular diseases. PUBLIC HEALTH RELEVANCE: Atherosclerotic lesions lead to heart attack and stroke, and together account for the leading cause of death in the United States and other developed countries. This study will further investigate how the endothelium lining of the blood vessel wall responds to hemodynamic forces, which is an integral part of the pathophysiology of cardiovascular diseases. Fully understanding the molecular pathways involved in endothelial responses to flow will impact identification of new drug target, methods of drug delivery, and developing new approaches to prevent and treat cardiovascular diseases