Leukocyte Transmigration is Mediated by Endothelial Cell Contractile Forces and Substrate Stiffness

Abstract

Leukocyte transmigration through the vascular endothelium is a crucial step in the normal immune response. However, in the cardiovascular disease (CVD) of atherosclerosis, an excess of leukocytes adhere to and transmigrate through the endothelium, and progression of this disease is associated with arterial stiffening and variance in mechanical force transduction. In this study we investigated the mechanics of leukocyte transmigration in an in vitro model of the vascular endothelium. We modeled healthy versus diseased blood vessels through manipulation of substrate stiffness using polyacrylamide gels, coated with extracellular matrix protein and plated with human umbilical vein endothelial cell (HUVEC) monolayers. The HUVEC monolayers were activated with tumor necrosis factor-alpha to mimic inflammatory conditions. We observed that leukocyte transmigration through HUVEC monolayers increases with stiffness below the endothelium, and we hypothesized that substrate stiffness changes the biophysical properties of the endothelium to produce this effect. Using an array of biophysical techniques, we first evaluated the adhesion protein expression, stiffness, morphology, cytoskeletal arrangement, and cell-substrate adhesion of HUVEC monolayers as a function of substrate stiffness; however, none of these properties could account for the transmigration behavior. We also explored the role of endothelial cell-cell adhesion and myosin light chain kinase (MLCK)-dependent endothelial cell contraction. We observed that (1) decreasing cell-cell adhesion increases transmigration on soft substrates and (2) inhibition of MLCK and endothelial cell contraction normalizes the effects of substrate stiffness by reducing leukocyte transmigration on stiff substrates without affecting transmigration on soft substrates. These results provide strong evidence that neutrophil transmigration is regulated by MLCK-mediated generation of gaps at cell borders through endothelial cell contractile forces which depend on substrate stiffness.