Biophysical Aspects of Leukocyte Transmigration through the Vascular Endothelium

Abstract

Leukocyte transmigration through the vascular endothelium is a key step in the immune response, and also in progression of the cardiovascular disease atherosclerosis. Much work has previously focused on the biological aspects of leukocyte transmigration, such as cytokine exposure, junctional protein organization in the endothelium, and signaling pathways. However, in recent years, many studies have identified links between the mechanical properties of the cellular microenvironment and cell behavior. This is relevant to the cardiovascular system in two ways: (1) it is likely that the mechanical properties of vasculature depend on both vessel size (large vessels versus microvasculature) and tissue type (soft brain versus stiffer muscle or tumor), and (2) both large vessels and microvasculature stiffen in atherosclerosis. For the first time, this dissertation provides a quantitative evaluation of the biophysical effects of vasculature stiffening on endothelial cell (EC) biomechanical properties, as well as leukocyte migration and transmigration.

A novel in vitro model of the vascular endothelium was created. This model mimics physiological conditions more closely than previous models, by taking into account the flexibility of the subendothelial matrix; previous models have mostly utilized glass or plastic substrates that are much stiffer than physiological. EC monolayers were formed on extracellular matrix (ECM) protein-coated hydrogels and activated with tumor necrosis factor-α or oxidized low density lipoprotein to induce an inflammatory response. We determined that three important components of the in vitro model (cell-cell adhesion, cytokine exposure, and subendothelial matrix stiffness) have significant effects on EC biomechanical properties. Next, we showed that neutrophils are mechanosensitive, as their migration is biphasic with substrate stiffness and depends on an interplay between substrate stiffness and ECM protein amount; these results suggest that any biomechanical changes which occur in vasculature may also affect the immune response. Finally, we discovered that neutrophil transmigration increases with subendothelial matrix stiffness, and we demonstrated that this effect is due to substrate stiffness-dependent EC contractile forces. These results indicate, for the first time, that the biophysical states of the endothelium and subendothelial matrix, which likely vary depending on size, location, and health of vasculature, are important regulators of the immune response.