Stiffening and Contraction Induced by Dexamethasone in Alveolar Epithelial Cells

Abstract

Background The structural integrity of the alveolar monolayer, which is compromised during lung inflammation, is determined by the balance between cell-cell and cell-matrix tethering forces and the centripetal forces owing to cell viscoelasticity and contraction. Dexamethasone is an anti-inflammatory glucocorticoid with protective effects in lung injury. Aim To determine the effects of Dexamethasone on the stiffness and contractility of alveolar epithelial cells. Methods Cell stiffness (G′) and average traction exerted by the cell (T) were measured by magnetic twisting cytometry and by traction microscopy, respectively. A549 cells were treated 24 h with Dexamethasone (1 μM) or vehicle (control). G′ and T were measured before and 5 min after challenge with the inflammatory mediator Thrombin (0.5 U/ml). Changes induced by Dexamethasone in actin cytoskeleton polymerization were assessed by the fluorescent ratio between F-actin and G-actin obtained by staining cells with phalloidin and DNase I. Results Dexamethasone significantly increased G′ and T by 56% (n =11; p <0.01) and by 80% (n = 17; p <0.05), respectively. Dexamethasone also increased F/G-actin ratio from 2.68±0.07 to 2.96±0.09 (n=10; p<0.05). The relative increase in stiffness and contraction induced by Thrombin in control cells was significantly (p<0.05) reduced by Dexamethasone treatment: from 190 to 98% in G′ and from 318 to 105% in T. Conclusion The cytoskeleton remodelling and the increase in cell stiffness and contraction induced by Dexamethasone could account for its protective effect in the alveolar epithelium when subjected to inflammatory challenge.

Barrier-Protective Effects of Activated Protein C in Human Alveolar Epithelial Cells

“…Based on these findings and the fact that A549 cells can also express different proteins involved in the coagulation cascade [5], [22], [23] and can generate new APC in the presence of thrombin and protein C antigen [24], we considered the A549 cell line an appropriate model to study the effects of APC on the structural integrity of the alveolar epithelial monolayer subjected to thrombin. Moreover, the acute thrombin response found in A549 cell permeability is in line with several data showing that thrombin induces a more marked effect after five minutes of treatment [8], [10], [14], [18]. In agreement with previous studies, we found that thrombin stiffens and contracts alveolar epithelial cells, suggesting an increase in inward forces compromising the barrier integrity [8], [9], [18].…”

“…In this simple model, paracellular gaps form and the barrier is disrupted when adhesive cell-cell and cell-matrix tethering forces that regulate cell shape are unable to withstand cell tension forces exerted by cytoskeleton viscoelastic recoil and contraction of the actomyosin machinery [3]. In particular, thrombin can modulate this force balance [8]–[10], [18], acting near the site of its generation [19] through protease-activated receptors (PAR-1, PAR-3, and PAR-4).…”

“…Cell stiffness after thrombin challenge was computed as the average of data obtained between 4.5 and 5 min. This technique has been described in detail elsewhere [8], [18]. For each treatment, 10 wells were tested.…”

“…Cell contraction force was determined using traction microscopy (TM), as described elsewhere [9], [18]. Briefly, A549 cells were grown on a thin collagen-coated polyacrylamide gel disk with embedded fluorescent beads.…”

The Evolution of the Field of Biomechanics Through the Lens of Experimental Mechanics

“…Salsas-Escat et al [45] employed molecular simulations to illustrate the structural conformations of collagen and the inherent flexibility of certain regions of the molecular structure. In a cellular pharmacology study, Puig et al [46] demonstrated that the application of dexamethasone, an anti-inflammatory glucocorticoid that affects actin cytoskeleton remodelling, led to an increase in alveolar epithelial cell stiffness and average traction force as measured by magnetic twisting cytometry. Yuan et al [47] reported on a novel method to quantify the torque generated by the E. coli flagellar motor, powered by various cytoskeletal elements, near zero load.…”