Stiff Substrates Increase Inflammation-Induced Endothelial Monolayer Tension and Permeability

Abstract

Arterial stiffness and inflammation are associated with atherosclerosis, and each have individually been shown to increase endothelial monolayer tension and permeability. The objective of this study was to determine if substrate stiffness enhanced endothelial monolayer tension and permeability in response to inflammatory cytokines. Porcine aortic endothelial cells were cultured at confluence on polyacrylamide gels of varying stiffness and treated with either tumor necrosis factor-α (TNFα) or thrombin. Monolayer tension was measured through vinculin localization at the cell membrane, traction force microscopy, and phosphorylated myosin light chain quantity and actin fiber colocalization. Cell permeability was measured by cell-cell junction confocal microscopy and a dextran permeability assay. When treated with TNFα or thrombin, endothelial monolayers on stiffer substrates showed increased traction forces, vinculin at the cell membrane, and vinculin phosphorylation, suggesting elevated monolayer tension. Interestingly, VE-cadherin shifted toward a smaller molecular weight in endothelial monolayers on softer substrates, which may relate to increased VE-cadherin endocytosis and degradation. Phosphorylated myosin light chain colocalization with actin stress fibers increased in endothelial monolayers treated with TNFα or thrombin on stiffer substrates, indicating elevated cell monolayer contractility. Endothelial monolayers also developed focal adherens intercellular junctions and became more permeable when cultured on stiffer substrates in the presence of the inflammatory cytokines. Whereas each of these effects was likely mitigated by Rho/ROCK, Rho/ROCK pathway inhibition via Y27632 disrupted cell-cell junction morphology, showing that cell contractility is required to maintain adherens junction integrity. These data suggest that stiff substrates change intercellular junction protein localization and degradation, which may counteract the inflammation-induced increase in endothelial monolayer tension and thereby moderate inflammation-induced junction loss and associated endothelial monolayer permeability on stiffer substrates.

Matrix stiffness enhances VEGFR-2 internalization, signaling, and proliferation in endothelial cells

“…Altered matrix mechanics is known to influence cell behaviors [12,13] mainly through crosstalk between integrins and the Rho/ROCK pathway [14]. More specifically, matrix stiffness has been shown to affect endothelial gene expression [15,16], morphology [17–20], outgrowth [21], traction forces [22–24], and permeability [24–27]. …”

Matrix stiffness regulates vascular integrity through focal adhesion kinase activity

“…FAK phosphorylation on Tyr 379 creates a high affinity binding site for Src and triggers the activation of Src (25). The FAK:Src complex phosphorylates many components of focal adhesions, resulting in the initiation of downstream signaling cascades (7,16,25,27). Given the involvement of FAK and Src in endothelial barrier integrity, we hypothesized that stiffness-mediated FAK activation plays a major role in regulating endothelial integrity.…”

Signals of the Neuropilin-1–MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells

“…To unravel the molecular mechanism of this signaling pathway, the C-type lectin-related protein RCab, as the specific trigger of this signaling axis in ECs, was studied in vitro. RCab increased 1) mechanical tension between and within ECs, resulting in increased permeability of the vasculature, and 2) TEM of leukocytes at vascular sites of inflammation (9,20,43). Both processes are highly relevant in inflammatory diseases such as atherosclerosis and the life-threating SIRS (3,4).…”