Trepat Xavier, Puig Ferranda, Gavara Nuria, Fredberg Jeffrey J, Farre Ramon, Navajas Daniel
Unitat de Biofísica i Bioenginyeria, Facultat de Medicina, Casanova 143, 08036 Barcelona, Spain.
Am J Physiol Lung Cell Mol Physiol. 2006 Jun;290(6):L1104-10. doi: 10.1152/ajplung.00436.2005. Epub 2006 Jan 6.
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.
接受机械通气的急性肺损伤患者的肺泡上皮细胞会暴露于增强的促凝血活性和机械应变中。凝血酶可诱导上皮细胞变硬、收缩以及细胞骨架重塑，这可能会在细胞伸展过程中破坏肺泡上皮处的力平衡。受损上皮中细胞间连接完整性的丧失会进一步破坏这种平衡。本研究的目的是探讨拉伸对暴露于凝血酶的人肺泡上皮细胞单层结构完整性和微力学的影响。将融合和亚融合细胞（A549）培养在胶原包被的弹性基质上。在暴露于凝血酶（0.5 U/ml）后，使用安装在倒置显微镜上的真空驱动系统施加逐步的细胞拉伸（20%）。通过比较单层内的细胞间和细胞内应变来评估细胞单层的结构完整性。通过追踪紧密结合在细胞表面的珠子来测量应变。同时，使用光磁扭转细胞术测量细胞粘弹性。在融合细胞中，凝血酶不会诱导从基质到上层细胞的应变传递发生显著变化。相比之下，凝血酶显著损害了亚融合细胞跟随施加的基质变形的能力。在基质松弛后，经凝血酶处理的亚融合细胞表现出压缩应变（9%）。拉伸增加了对照细胞的硬度（56 - 62%）并降低了细胞滞后性（13 - 22%）。相比之下，拉伸并未增加经凝血酶处理的细胞的硬度，这表明细胞骨架结构受到破坏。我们的研究结果表明，凝血酶可能会加剧机械通气的损伤肺中的上皮屏障功能障碍。
