Influence of airway wall compliance on epithelial cell injury and adhesion during interfacial flows

Abstract

Higuita-Castro N, Mihai C, Hansford DJ, Ghadiali SN. Influence of airway wall compliance on epithelial cell injury and adhesion during interfacial flows. J Appl Physiol 117: 1231-1242, 2014. First published September 11, 2014 doi:10.1152/japplphysiol.00752.2013.-Interfacial flows during cyclic airway reopening are an important source of ventilator-induced lung injury. However, it is not known how changes in airway wall compliance influence cell injury during airway reopening. We used an in vitro model of airway reopening in a compliant microchannel to investigate how airway wall stiffness influences epithelial cell injury. Epithelial cells were grown on gel substrates with different rigidities, and cellular responses to substrate stiffness were evaluated in terms of metabolic activity, mechanics, morphology, and adhesion. Repeated microbubble propagations were used to simulate cyclic airway reopening, and cell injury and detachment were quantified via live/dead staining. Although cells cultured on softer gels exhibited a reduced elastic modulus, these cells experienced less plasma membrane rupture/necrosis. Cells on rigid gels exhibited a minor, but statistically significant, increase in the power law exponent and also exhibited a significantly larger height-to-length aspect ratio. Previous studies indicate that this change in morphology amplifies interfacial stresses and, therefore, correlates with the increased necrosis observed during airway reopening. Although cells cultured on stiff substrates exhibited more plasma membrane rupture, these cells experienced significantly less detachment and monolayer disruption during airway reopening. Western blotting and immunofluorescence indicate that this protection from detachment and monolayer disruption correlates with increased focal adhesion kinase and phosphorylated paxillin expression. Therefore, changes in cell morphology and focal adhesion structure may govern injury responses during compliant airway reopening. In addition, these results indicate that changes in airway compliance, as occurs during fibrosis or emphysema, may significantly influence cell injury during mechanical ventilation.ventilation-induced lung injury; airway compliance; airway reopening; cell mechanics; and cell injury THE ACUTE RESPIRATORY DISTRESS syndrome (ARDS) is a lifethreatening condition that involves disruption of the alveolarcapillary barrier and impaired gas exchange (46).1 Although mechanical ventilation of ARDS patients can restore normal oxygenation, these ventilators may also cause significant cellular injury and trigger proinflammatory signaling cascades (26, 31) in a process known as ventilator-induced lung injury (VILI). One form of VILI, known as volutrauma, involves the overdistension and cyclic stretching of lung tissue, which causes epithelial cell necrosis (43), disruption of the alveolarcapillary barrier (7), and inflammatory signaling (44). Presently, protective ventilation strategies, such as low tidal volumes, are the standard of care and seek to minimize volutrau...

Simvastatin Treatment Modulates Mechanically-Induced Injury and Inflammation in Respiratory Epithelial Cells

“…As described in our previous studies [17] and shown schematically in Fig 1, a Bioptechs FCS2 chamber (Bioptechs, Butler, PA) and a programmable PHD 2000 syringe pump (Harvard Apparatus, Holliston, MA) were used to expose cells to cyclic airway reopening conditions. Briefly, confluent samples were placed inside the chamber and the flow channel (35mm length, 10mm width, and 0.5mm height) was filled with phosphate-buffered saline (PBS).…”

“…Silicon nitride cantilevers with a nominal spring constant k = 0.01 N/m, length of 200 μm, and a regular four-sided pyramidal tip with an angle θ= 35° were used for these measurements (Veeco, Santa Barbara CA). Following protocols previously reported by our group [17, 34], 5–12 regions per sample were scanned in contact mode and the deflection error and height image was recorded. Height images were analyzed with the NanoScope Analysis software package (Bruker Instruments, Camarillo CA) to measure cell height, width and length.…”

“…Four to five 50μm x 50μm regions were scanned per sample and an average of 130 z vs. d curves were recorded per region at a specific indentation (δ). As described previously [17], the Young’s modulus (E) and the z- position where the cantilever tip contacts the cell surface (zc) were then estimated by fitting the raw z vs. d data with a modified Hertz model assuming incompressibility. Data analysis was restricted to the lower region of the force curve (0.05–0.5nN) to avoid noise in the <0.05nN range and nonlinearities associated with deep indentations.…”

“…an increase the power law structural dampening exponent (α), leads to dissipation of the applied interfacial stress and less cellular deformation/injury. Recently, we demonstrated that epithelial cells grown on compliant substrates are less susceptible to cellular injury during cyclic airway reopening [17]. In that study, the decreased injury susceptibility was due to morphological changes, where cells on softer substrates exhibit a decreased height-to-length aspect ratio, which according to computational studies [18], reduces the hydrodynamic stresses generated during airway reopening.…”

Modeling the Progression of Epithelial Leak Caused by Overdistension

“…In order to relate the parameters F s , F t , and k to dimensional values, we computed a reference force scale f based on Young’s modulus, E , of an alveolar epithelial cell, which has been measured with atomic-force microscopy to be around 1–5 kPa for human A549 cells in a confluent monolayer. 2,15,27 For this model, we assumed E = 1 kPa for the entire spring network. We simulated horizontal stretch of the 45-cell network and used the stress-strain relation E = σ/ε, where σ = f F s / A I (the applied force adjusted by the scaling force divided by the initial cross-sectional area over which the force is applied) and ε = Δ L / L 0 (the linear displacement of the network due to the force relative to its initial length), to compute the scaling force f for F s that yielded E = 1 kPa.…”