THE ROLE OF DAP-KINASE IN MODULATING VASCULAR ENDOTHELIAL CELL FUNCTION UNDER FLUID SHEAR STRESS

Abstract

Atherosclerosis preferentially develops in vascular regions of low or disturbed flow and high spatial gradients. Endothelial cells that line the vessel walls actively participate in translating mechanical stimuli, shear stress due to fluid flow, into intracellular signals to regulate cellular activities. Atherosclerosis is a chronic disease. During its development, a cascade of inflammatory signals alters the arterial endothelial homeostatic functions. Death-associated protein (DAP) kinase and its correlated pathway have been associated with cell apoptosis, turnover, and cytoskeleton remodeling in cellular networks, ultimately leading to changes in cell motility and vascular wall permeability. DAP-kinase is also highly regulated by inflammatory triggers such as TNF-α. This thesis investigates DAP-kinase modulation due to shear stress, and the role of DAP-kinase activity in endothelial responses toward applied shear stress. Using bovine aortic endothelial cells (BAEC), DAP-kinase expression is demonstrated in both sheared (10 dynes/cm2) and static conditions. Overall DAPK expression increased with extended shearing, while the presence of phosphorylated DAPK decreased with applied shear stress, as demonstrated in Western blot analysis. In correlation, DAPK RNA expression profiles were explored to understand pre-translational behavior and to understand just how shear stress influences DAPK expression over time. There is a temporal increase in DAPK mRNA, occurring at earlier time points when compared to DAPK protein expression, displaying the precedence of mRNA expression leading to increased translation into protein. From our apoptosis assay results, shear stress reduces apoptotic and late stage/necrotic cell fractions. The exposure of shear stress potentially plays a role in inhibiting apoptosis activation and TNF-α induced death cascade. Overall, the apoptosis activity influenced by shear further exhibits a possible connection between shear stress and apoptosis inhibition. The shear stress ultimately decreases overall apoptosis, while DAPK expression is increased. Therefore, DAPK may have a function in other possible mechano-transduction cascades, when endothelial cells are exposed to constant shear. Our data suggests shear stress modulation of DAP-kinase expression and activity, and the potential crosstalk of mechano-transduction and DAPK/apoptosis pathway, may lead to further understanding the responsibility of DAPK in endothelial cell function.