performed the leukocyte adhesion and transmigration assays. results suggest that extracellular matrix stiffening alone, which occurs during aging, can lead to endothelial monolayer disruption and atherosclerosis pathogenesis. Because previous therapeutics designed to decrease vascular stiffness have been met with limited success, our findings could be the basis for the design of therapeutics that target the Rho-dependent cellular contractile response to matrix stiffening, rather than stiffness itself, to more effectively prevent atherosclerosis progression. == INTRODUCTION == Vascular stiffening accompanies a variety of cardiovascular pathologies including hypertension (1) and atherosclerosis (2). The blood vessel wall also Cbz-B3A stiffens with age (3). During aging, vessel stiffness increases owing to changes in the micro-scale architecture of the extracellular matrix (ECM) within the vessel wall, namely the increases in elastin fragmentation, collagen deposition, and matrix protein cross-linking (4,5). This matrix stiffening decreases arterial distensibility and capacitance and can increase mechanical strain on the heart. Macroscopic measurements of arterial stiffness are often used for clinical diagnosis and can independently predict cardiovascular events, such as coronary heart disease and stroke (6) and mortality in elderly patients (7). Although vascular stiffening occurs ubiquitously with age and is a predictor of cardiovascular risk, little is known about how vessel stiffness affects endothelial cells within blood vessels where arteriosclerosis initiates. Recent evidence suggests Cbz-B3A that matrix stiffness affects cell behaviors (8), including cell spreading and adhesion (9), migration (10), and differentiation (11). It has been shown to alter vascular smooth muscle cell phenotype Cbz-B3A (12) and promote intimal hyperplasia (13). Our own data suggest that changes in matrix stiffness can alter cell-cell contact (14). Because cell-cell contact is directly linked to monolayer integrity and permeability, we hypothesized that increased matrix stiffness with age may disrupt barrier function of the endothelium. Increased endothelial permeability to lipoproteins and immune cells is considered the initiating step of atherosclerosis pathogenesis, and the accumulation of debris in the intima results in the formation of atherosclerotic plaques (1517). Notably, decreasing permeability decreases plaque formation (18,19). Endothelial permeability is controlled in part by the dynamic opening and closing of endothelial cell-cell junctions (20), which are directly affected by interactions between endothelial cells and the ECM (21). Although the intima stiffens during aging and atherosclerosis progression, and endothelial permeability occurs with age and is regarded as one of the first steps in atherogenesis, the relationship between age-related blood-vessel stiffening, endothelial cell function, and monolayer integrity has not been investigated in depth. Because endothelial permeability is known as an early event in atherogenesis, we explored the effects of age-related matrix stiffness on endothelial barrier function using both in vitro and ex vivo models of intimal stiffening and aging. We found that stiffening of the matrix increased monolayer permeability. This increase in permeability was the result of upregulated cell contractility, which Cbz-B3A mechanically disrupted cell-cell junctions and promoted leukocyte transmigrationa critical step in atherosclerotic plaque formation. Pharmacological inhibition of cell contractility in vivo in mice reversed the effects of matrix stiffness on endothelial permeability by restoring tight cell-cell cIAP2 junctions, and also decreased leukocyte transmigration. These findings suggest that matrix stiffening alone, which occurs during Cbz-B3A the natural aging process, can directly cause endothelial cell permeability and atherosclerosis progression. == RESULTS == == Increased matrix stiffness promotes endothelial monolayer permeability by destabilizing cell-cell junctions == We first investigated the effect of substrate stiffness on the barrier function of endothelial monolayers. To mimic the stiffness of young and aged intima, synthetic hydrogel substrates (22) were fabricated that approximated and exceeded the Youngs Modulus of 2.7 1.1 kPa reported previously for the subendothelial matrix in bovine carotid arteries (23). Bovine.