Substrate stiffness and VE-cadherin mechano-transduction coordinate to regulate endothelial monolayer integrity

Abstract

The vascular endothelium is subject to diverse mechanical cues that regulate vascular endothelial barrier function. In addition to rigidity sensing through integrin adhesions, mechanical perturbations such as changes in fluid shear stress can also activate force transduction signals at intercellular junctions. This study investigated how extracellular matrix rigidity and intercellular force transduction, activated by vascular endothelial cadherin, coordinate to regulate the integrity of endothelial monolayers. Studies used complementary mechanical measurements of endothelial monolayers grown on patterned substrates of variable stiffness. Specifically perturbing VE-cadherin receptors activated intercellular force transduction signals that increased integrin-dependent cell contractility and disrupted cell-cell and cell-matrix adhesions. Further investigations of the impact of substrate rigidity on force transduction signaling demonstrated how cells integrate extracellular mechanics cues and intercellular force transduction signals, to regulate endothelial integrity and global tissue mechanics. VE-cadherin specific signaling increased focal adhesion remodeling and cell contractility, while sustaining the overall mechanical equilibrium at the mesoscale. Conversely, increased substrate rigidity exacerbates the disruptive effects of intercellular force transduction signals, by increasing heterogeneity in monolayer stress distributions. The results provide new insights into how substrate stiffness and intercellular force transduction coordinate to regulate endothelial monolayer integrity.

Introduction

The vasculature is a mechanically active environment. The endothelial lining is subject to a range of mechanical perturbations such as fluid shear stress and cyclic stretch associated with respiration. The endothelium experiences a variety of endogenous and exogenous chemo-mechanical inputs that finely tune vascular homeostasis. These inputs include cell-cell and cell-substrate interactions (e.g. via cadherins and integrins, respectively), soluble factors (e.g. thrombin, nitric oxide), and mechanical factors (e.g. blood flow, cyclic stretch). Exogenous mechanical forces such as fluid shear stress and the stiffness of the lamina intima also regulate vascular function [1], [2], [3], [4], and promote extracellular matrix (ECM) deposition and cross linking [5]. In vitro, physiological cyclic strain further coordinates with matrix stiffness to protect endothelial junctions against disruption by vasoactive agents such as thrombin [3], [4], [6].

The balance between cell contractility and tethering (adhesive) forces is postulated to regulate endothelial barrier function [7], [8]. This predicts that increased intercellular tension, due to elevated endogenous contractile forces, for example, would increase vascular leakage. An alternative view based on traction force microscopy measurements, is that force instability, rather than the force magnitude, predicts sites of endothelial disruption and gap formation [9]. Several factors regulate intercellular tension, such as matrix rigidity, cell contractility, and cytokines [8], [10], [11]. Perturbations to any of these inputs correlates with pathological responses, such as hypertension [12], [13] and atherosclerosis [14]. Intimal stiffening due to age related atherosclerosis [15] or collagen over-secretion and crosslinking [16] also correlate with leaky vessels in vivo [17].

Force fluctuations at cell-cell contacts activate signals that increase cell contractility and regulate vascular functions [10]. Fluid shear alignment (flow sensing), for example, involves force transduction complexes at interendothelial junctions that require platelet endothelial cell adhesion molecule one (PECAM-1), vascular endothelial growth factor 2 (VEGFR2), and vascular endothelial cadherin (VE-cadherin) [1], [18], [19], which is the main adhesion molecule at endothelial junctions. Besides fluid shear stress, other perturbations such as cyclic stretch in the lung appear to activate a similar signaling cascade [20]. In biophysical studies, we showed that directly perturbing VE-cadherin receptors on cell surfaces with VE-cadherin-modified magnetic beads activated similar signals as in flow sensing, but without PECAM-1 [21]. We further demonstrated that VE-cadherin-activated signals increase cell contractility, disrupt peripheral junctions, and even propagate mechanical perturbations 2–3 cell diameters from the stimulated cell [21]. Thus, force transduction signals at cell-cell junctions not only induce cytoskeletal remodeling, as during shear alignment [22], but they can also disrupt endothelial monolayer integrity.

Studies demonstrated that interendothelial force transduction triggers a kinase cascade that activates integrins at the basal plane [1]. Integrins in turn increase cell contractility through the Rho/ROCK associated protein kinase pathway [23], and are well known to increase cell contractility with increasing matrix rigidity [24]. Given the coordination between cadherin force-transduction and integrins [11], [25], [26], [27], we reasoned that mechanically sensitive endothelial processes that involve intercellular adhesions might also depend on substrate rigidity. Such information could enhance our understanding of the interplay between tissue mechanics and endothelial responses to perturbations that alter force at cell-cell contacts.

This study investigated the cooperation between intercellular force transduction signaling and substrate rigidity in regulating endothelial mechanics and monolayer integrity. Magnetic twisting cytometry was used to specifically activate VE-cadherin-mediated (intercellular) force transduction signals. In order to regulate the matrix rigidity, studies used micro-patterned substrates of variable, physiologically relevant stiffness. Mechanical measurements quantified the mechanical state of endothelial monolayers, and evaluated force-dependent, spatial and temporal changes in endothelial gap formation (disruption), cell tractions, and intercellular stress distributions. Our findings provide a detailed picture of the endothelial monolayer as a mechanically integrated mesoscale network. They further demonstrate how substrate rigidity modulates the impact of intercellular force transduction signaling on endothelial integrity.