Effect of stretch on structural integrity and micromechanics of human alveolar epithelial cell monolayers exposed to thrombin

Abstract

Trepat, Xavier, Ferranda Puig, Nuria Gavara, Jeffrey J. Fredberg, Ramon Farre, and Daniel Navajas. Effect of stretch on structural integrity and micromechanics of human alveolar epithelial cell monolayers exposed to thrombin. Alveolar epithelial cells in patients with acute lung injury subjected to mechanical ventilation are exposed to increased procoagulant activity and mechanical strain. Thrombin induces epithelial cell stiffening, contraction, and cytoskeletal remodeling, potentially compromising the balance of forces at the alveolar epithelium during cell stretching. This balance can be further compromised by the loss of integrity of cell-cell junctions in the injured epithelium. The aim of this work was to study the effect of stretch on the structural integrity and micromechanics of human alveolar epithelial cell monolayers exposed to thrombin. Confluent and subconfluent cells (A549) were cultured on collagen-coated elastic substrates. After exposure to thrombin (0.5 U/ml), a stepwise cell stretch (20%) was applied with a vacuum-driven system mounted on an inverted microscope. The structural integrity of the cell monolayers was assessed by comparing intercellular and intracellular strains within the monolayer. Strain was measured by tracking beads tightly bound to the cell surface. Simultaneously, cell viscoelasticity was measured using optical magnetic twisting cytometry. In confluent cells, thrombin did not induce significant changes in transmission of strain from the substrate to overlying cells. By contrast, thrombin dramatically impaired the ability of subconfluent cells to follow imposed substrate deformation. Upon substrate unstretching, thrombin-treated subconfluent cells exhibited compressive strain (9%). Stretch increased stiffness (56 -62%) and decreased cell hysteresivity (13-22%) of vehicle cells. By contrast, stretch did not increase stiffness of thrombin-treated cells, suggesting disruption of cytoskeletal structures. Our findings suggest that thrombin could exacerbate epithelial barrier dysfunction in injured lungs subjected to mechanical ventilation.

Tubulin Acetylation and Histone Deacetylase 6 Activity in the Lung under Cyclic Load

“…Many investigators have looked at the effects of stretch in alveolar epithelial cells (AECs) and have seen a number of structural changes consistent with the injury seen in ventilated lungs (4)(5)(6). Consequently, researchers have become interested in understanding what role mechanotransduction plays in the development of lung injury (7)(8)(9)(10).…”

Cellular traction forces increase during consecutive mechanical stretching following traction force attenuation

“…Alterations in the actin cytoskeleton have been reported to be involved in the attenuation of traction forces. With studies discussing, changes in actin polymerization, overstretching of stress fibers, rupturing and/or unfolding of actin filament cross-links and actomyosin cross-bridges as potential contributors to traction force attenuation (Costa et al, 2002;Gavara et al, 2008;Lim et al, 2012;Rosenblatt et al, 2007;Trepat et al, 2007;Trepat et al, 2006). However, no definitive evidence has been put forward to confirm these theories.…”

“…Rupturing and unfolding of both actin filament cross-links and actomyosin cross-bridges have been reported, as well as changes in actin polymerization following large strain mechanical stretching (Costa et al, 2002;Gavara et al, 2008;Lim et al, 2012;Rosenblatt et al, 2007;Trepat et al, 2007;Trepat et al, 2006). Increases in traction force depend on an intact actin cytoskeleton (Gavara et al, 2008;Lam et al, 2012;Munevar et al, 2004;Nagayama et al, 2011).…”

Postlactational Involution: Molecular Mechanisms and Relevance for Breast Cancer Development

“…Mechanical stress is a relevant factor to induce adaptive responses in multiple cell types [12][13][14][15][16]. Importantly, the signaling pathways triggered by this stimulus in those diferent examples also play a relevant role during mammary gland involution.…”