Arterial stiffness and inflammation are associated with atherosclerosis, and each have individually been shown to increase endothelial monolayer tension and permeability. The objective of this study was to determine if substrate stiffness enhanced endothelial monolayer tension and permeability in response to inflammatory cytokines. Porcine aortic endothelial cells were cultured at confluence on polyacrylamide gels of varying stiffness and treated with either tumor necrosis factor-α (TNFα) or thrombin. Monolayer tension was measured through vinculin localization at the cell membrane, traction force microscopy, and phosphorylated myosin light chain quantity and actin fiber colocalization. Cell permeability was measured by cell-cell junction confocal microscopy and a dextran permeability assay. When treated with TNFα or thrombin, endothelial monolayers on stiffer substrates showed increased traction forces, vinculin at the cell membrane, and vinculin phosphorylation, suggesting elevated monolayer tension. Interestingly, VE-cadherin shifted toward a smaller molecular weight in endothelial monolayers on softer substrates, which may relate to increased VE-cadherin endocytosis and degradation. Phosphorylated myosin light chain colocalization with actin stress fibers increased in endothelial monolayers treated with TNFα or thrombin on stiffer substrates, indicating elevated cell monolayer contractility. Endothelial monolayers also developed focal adherens intercellular junctions and became more permeable when cultured on stiffer substrates in the presence of the inflammatory cytokines. Whereas each of these effects was likely mitigated by Rho/ROCK, Rho/ROCK pathway inhibition via Y27632 disrupted cell-cell junction morphology, showing that cell contractility is required to maintain adherens junction integrity. These data suggest that stiff substrates change intercellular junction protein localization and degradation, which may counteract the inflammation-induced increase in endothelial monolayer tension and thereby moderate inflammation-induced junction loss and associated endothelial monolayer permeability on stiffer substrates.

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Arterial stiffness, which increases with aging and hypertension, is an independent cardiovascular risk factor. While stiffer substrates are known to affect single endothelial cell morphology and migration, the effect of substrate stiffness on endothelial monolayer function is less understood. The objective of this study was to determine if substrate stiffness increased endothelial monolayer reactive oxygen species (ROS) in response to protein kinase C (PKC) activation and if this oxidative stress then impacted adherens junction integrity. Porcine aortic endothelial cells were cultured on varied stiffness polyacrylamide gels and treated with phorbol 12-myristate 13-acetate (PMA), which stimulates PKC and ROS without increasing actinomyosin contractility. PMA-treated endothelial cells on stiffer substrates increased ROS and adherens junction loss without increased contractility. ROS scavengers abrogated PMA effects on cell-cell junctions, with a more profound effect in cells on stiffer substrates. Finally, endothelial cells in aortae from elastin haploinsufficient mice (Eln+/-), which were stiffer than aortae from wild-type mice, showed decreased VE-cadherin colocalization with peripheral actin following PMA treatment. These data suggest that oxidative stress may be enhanced in endothelial cells in stiffer vessels, which could contribute to the association between arterial stiffness and cardiovascular disease.

Dielectrophoresis (DEP), the force induced on a polarizable body by a non-uniform electric field, has been widely used to manipulate single cells in suspension and analyze their stiffness. However, most cell types do not naturally exist in suspension but instead require attachment to the tissue extracellular matrix in vivo. Cells alter their cytoskeletal structure when they attach to a substrate, which impacts cell stiffness. It is therefore critical to be able to measure mechanical properties of cells attached to a substrate. We present a novel inverted quadrupole dielectrophoretic device capable of measuring changes in the mechanics of single cells attached to a micropatterned polyacrylamide gel. The device is positioned over a cell of defined size, a directed DEP pushing force is applied, and cell centroid displacement is dynamically measured by optical microscopy. Using this device, single endothelial cells showed greater centroid displacement in response to applied DEP pushing force following actin cytoskeleton disruption by cytochalasin D. In addition, transformed mammary epithelial cell (MCF10A-NeuT) showed greater centroid displacement in response to applied DEP pushing force compared to untransformed cells (MCF10A). DEP device measurements were confirmed by showing that the cells with greater centroid displacement also had a lower elastic modulus by atomic force microscopy. The current study demonstrates that an inverted DEP device can determine changes in single attached cell mechanics on varied substrates.

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