Cardiovascular diseases represent a major risk to global health, contributing to approximately one-third of all deaths worldwide. Inflammation and arterial stiffening are key in the pathogenesis of many cardiovascular diseases, including hypertension and atherosclerosis. However, little is known about their combined impact on vascular endothelial dysfunction. The central objective of this thesis is to investigate the contribution of arterial stiffness to endothelial cell stiffening and barrier loss in response to inflammation through actinomyosin contractility and reactive oxygen species. A novel dielectrophoretic (DEP) device for assessing deformability of micropatterned single attached cells was simulated, fabricated, and tested. The device was validated by showing that endothelial cells with cytoskeletal disruption and epithelial cells with induced tumorigenicity were softer by atomic force microscopy (AFM) and more deformable in response to directed DEP pushing force. Next, the effect of sub-endothelial stiffness on endothelial monolayer permeability in response to inflammation was explored ex vivo and in vitro. Elastin haploinsufficient (ELN+/-) mice exhibited stiffer sub-endothelium by AFM and increased vinculin at aortic endothelial cell-cell junctions. In vitro, porcine aortic endothelial cells (PAEC) on collagen-coated polyacrylamide gels of varying stiffness (6-50 kPa) showed increased vinculin localization to cell-cell junctions and junction loss in response to tumor necrosis factor alpha (TNF-α) and thrombin. These effects were abolished when cell contraction was inhibited. Substrate stiffness also enhanced functional barrier loss in response to thrombin, but not TNF-α; however, cells exhibited substrate-dependent stiffening following TNF-α exposure. Lastly, substrate stiffness enhanced endothelial reactive oxygen species (ROS) production in response to phorbol 12-myristate 13-acetate (PMA). On stiff substrates PMA-induced ROS elicited greater actin fiber formation and cell-cell junction loss, which was independent of cell contractility but prevented by ROS scavenging. Ex vivo, peripheral actin fiber formation was greater in the ELN+/- mouse abdominal aorta following exposure to PMA. These results demonstrate that sub-endothelial stiffness affects endothelial cell contractile and non-contractile (ROS-mediated) mechanisms of endothelial barrier dysfunction. This research supports studying the integrated effects of arterials stiffness and inflammation to develop new therapies to prevent endothelial dysfunction.Ph.D., Mechanical Engineering and Mechanics -- Drexel University, 201
