Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces

Abstract

GP. Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces. Am J Physiol Cell Physiol 300: C146 -C154, 2011. First published September 22, 2010; doi:10.1152/ajpcell.00195.2010.-A hallmark of many, sometimes life-threatening, inflammatory diseases and disorders is vascular leakage. The extent and severity of vascular leakage is broadly mediated by the integrity of the endothelial cell (EC) monolayer, which is in turn governed by three major interactions: cell-cell and cell-substrate contacts, soluble mediators, and biomechanical forces. A potentially critical but essentially uninvestigated component mediating these interactions is the stiffness of the substrate to which the endothelial monolayer is adherent. Accordingly, we investigated the extent to which substrate stiffening influences endothelial monolayer disruption and the role of cell-cell and cell-substrate contacts, soluble mediators, and physical forces in that process. Traction force microscopy showed that forces between cell and cell and between cell and substrate were greater on stiffer substrates. On stiffer substrates, these forces were substantially enhanced by a hyperpermeability stimulus (thrombin, 1 U/ml), and gaps formed between cells. On softer substrates, by contrast, these forces were increased far less by thrombin, and gaps did not form between cells. This stiffness-dependent force enhancement was associated with increased Rho kinase activity, whereas inhibition of Rho kinase attenuated baseline forces and lessened thrombin-induced inter-EC gap formation. Our findings demonstrate a central role of physical forces in EC gap formation and highlight a novel physiological mechanism. Integrity of the endothelial monolayer is governed by its physical microenvironment, which in normal circumstances is compliant but during pathology becomes stiffer. contraction; human umbilical vein endothelial cells; permeability; traction force; cell-cell contact; cell-substrate contact; substrate stiffness; Rho kinase; vascular endothelial cadherin; thrombin THE OVERALL INTEGRITY and barrier properties of the endothelial cell (EC) monolayer are governed by three main categories of inputs: cell-cell and cell-substrate contacts, soluble mediators (e.g., thrombin, histamine, spingosphine 1-phosphate, and nitric oxide), and biomechanics (e.g., innate monolayer forces, shear forces, and stretch) (33). In vivo, these inputs are integrated by the EC monolayer to regulate its overall integrity and responses to inflammatory stimuli. Characterizing these inputs and their interrelationships is thus of central importance for understanding vascular biology and inflammation as a whole.A potentially critical, but entirely ignored, component of the EC environment that may influence the aforementioned inputs and their interactions is stiffness of the substrate to which the EC monolayer is adherent. This substrate stiffness varies greatly among diverse physiological settings (12,13,32,59), is enhanced with aging (21, 40), and is exacerbate...

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