Age-Related Intimal Stiffening Enhances Endothelial Permeability and Leukocyte Transmigration

Inhibiting endothelial cell contractility reverses the deleterious effects of age-related matrix stiffening on normal cell function, which could help prevent the development of atherosclerosis. Rock Your Heart Out According to novelist Thomas Bailey Aldrich, “To keep the heart unwrinkled, to be hopeful, kindly, cheerful, reverent, is to triumph over old age” (from Ponkapoag Papers). Unfortunately, despite a positive attitude, aging is accompanied by several changes of heart, at least at the cellular level. One age-related “wrinkle” is stiffening of the extracellular matrix that lines the blood vessels, a change that has been linked to atherosclerosis; yet, the cellular and mechanical features that couple the two conditions have remained elusive. Now, using a clever combination of biomaterials, cells, aortas, and mice, Huynh and colleagues have demystified the correlation between aging and atherosclerosis, showing that cell contractility is at the heart of it all. The authors first developed an in vitro system that mimicked the basic structures of both young and old blood vessels. Synthetic hydrogel matrices of varying stiffnesses were seeded with bovine aortic endothelial cells. By administering a solution of fluorescently labeled molecules to the cell-gel system and watching how the dye moved across the cell layer, Huynh et al. determined that permeability increased as a function of matrix stiffness, suggesting that age alone was a disruptive factor. These results were confirmed ex vivo by performing atomic force microscopy with decellularized thoracic aortas from both young (~10 weeks) and old (~92 weeks) mice. In both of these systems, the enhanced vessel permeability resulted from an increase in the distance—or junction—between neighboring cells. This increase in the so-called gap junction width also permitted the passage of leukocytes through the endothelial cell monolayer; along with leaky vasculature, cellular transmigration is a hallmark of atherosclerosis progression. Because the Rho signaling pathway is linked to the cellular cytoskeleton and, in turn, contractility, Huynh et al. hypothesized that they could reverse the effects of age-related intimal stiffening by inhibiting Rho-associated kinase (ROCK). By administering a pharmacological ROCK inhibitor (Y-27632) to their in vitro setup and to old mice, the authors showed that gap junction widths and endothelial cellular forces decreased. In vitro, the inhibitor also prevented leukocyte transmigration. These observations suggest that directly interfering with Rho signaling is a viable treatment option for age-related atherosclerosis. And because inhibitors of Rho signaling, such as fasudil, are already available in the clinic, one might say that physicians and researchers are ready to rock. Age is the most significant risk factor for atherosclerosis; however, the link between age and atherosclerosis is poorly understood. During both aging and atherosclerosis progression, the blood vessel wall stiffens owing to alterations in the extracellular matrix. Using in vitro and ex vivo models of vessel wall stiffness and aging, we show that stiffening of extracellular matrix within the intima promotes endothelial cell permeability—a hallmark of atherogenesis. When cultured on hydrogels fabricated to match the elasticity of young and aging intima, endothelial monolayers exhibit increased permeability and disrupted cell-cell junctions on stiffer matrices. In parallel experiments, we showed a corresponding increase in cell-cell junction width with age in ex vivo aortas from young (10 weeks) and old (21 to 25 months) healthy mice. To investigate the mechanism by which matrix stiffening alters monolayer integrity, we found that cell contractility increases with increased matrix stiffness, mechanically destabilizing cell-cell junctions. This increase in endothelial permeability results in increased leukocyte extravasation, which is a critical step in atherosclerotic plaque formation. Mild inhibition of Rho-dependent cell contractility using Y-27632, an inhibitor of Rho-associated kinase, or small interfering RNA restored monolayer integrity in vitro and in vivo. Our 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.