Influence of airway diameter and cell confluence on epithelial cell injury in an in vitro model of airway reopening

Abstract

Recent advances in the ventilation of patients with acute respiratory distress syndrome (ARDS), including ventilation at low lung volumes, have resulted in a decreased mortality rate. However, even low-lung volume ventilation may exacerbate lung injury due to the cyclic opening and closing of fluid-occluded airways. Specifically, the hydrodynamic stresses generated during airway reopening may result in epithelial cell (EpC) injury. We utilized an in vitro cell culture model of airway reopening to investigate the effect of reopening velocity, airway diameter, cell confluence, and cyclic closure/reopening on cellular injury. Reopening dynamics were simulated by propagating a constant-velocity air bubble in an adjustable-height parallel-plate flow chamber. This chamber was occluded with different types of fluids and contained either a confluent or a subconfluent monolayer of EpC. Fluorescence microscopy was used to quantify morphological properties and percentage of dead cells under different experimental conditions. Decreasing channel height and reopening velocity resulted in a larger percentage of dead cells due to an increase in the spatial pressure gradient applied to the EpC. These results indicate that distal regions of the lung are more prone to injury and that rapid inflation may be cytoprotective. Repeated reopening events and subconfluent conditions resulted in significant cellular detachment. In addition, we observed a larger percentage of dead cells under subconfluent conditions. Analysis of this data suggests that in addition to the magnitude of the hydrodynamic stresses generated during reopening, EpC morphological, biomechanical, and microstructural properties may also be important determinants of cell injury.

μ-PIV for the Analysis of Flow Fields near a Propagating Air-Liquid Interface

“…Follow-up studies by numerous groups have investigated this system and were found to be consistent with the theoretical predictions (Jacob and Gaver III 2005;Huh et al 2007;Yalcin et al 2007). Furthermore, Jacob and Gaver III (2005) included the influence of cell topography into the computational model, and found that the pressure gradient acting on the epithelial cells was even more pronounced than the original computations had demonstrated.…”

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

“…Although previous studies have used rigid-walled in vitro systems to investigate how interfacial flows influence cell injury patterns (3,30,35,47,48), lung tissue is highly compliant, and many clinical disorders are characterized by changes in lung/airway wall compliance (32,41). In this study, we investigated how changes in airway wall compliance influenced cellular injury during airway reopening by culturing lung epithelial cells on different stiffness substrates and exposing these cells to injurious interfacial flows.…”

“…Briefly, substrates with cultured A549 cells or HSAEpC were placed inside the FCS2 chamber, and silicone gaskets were used to create a parallel plate flow channel (35-mm length, 10-mm width, and 0.5-mm height). Similar to previous studies (47,48), this channel was then filled with a surfactant-deficient PBS fluid using a programmable PHD 2000 syringe pump (Harvard Apparatus, Holliston, MA). The fluid was then retracted from the channel at a flow rate (0.09 ml/min) to create an air bubble that propagated over the cell monolayer at a velocity of 0.3 mm/s (Fig.…”

“…These authors demonstrated that the large pressure gradient near the bubble front causes plasma membrane disruption and cell necrosis (3,30). Using a similar rigid airway model, Yalcin et al (48) demonstrated that the degree of cell injury during cyclic airway closure/reopening is inversely proportional to airway diameter, and that changes in the cell's cytoskeletal structure can be used to mitigate cell injury during reopening (47). Similarly, Oeckler et al (35) demonstrated that hypertonic treatment reduces cell injury by increasing plasma membrane-cytoskeleton adhesive interactions.…”

“…Under these conditions, the closure and reopening of fluid-filled airways generates interfacial and microbubble flows that can significantly damage the lung epithelium. Specifically, several investigators have used computational (13,14,23,25,29) and in vitro experimental (3,30,47,48) techniques to demonstrate that this interfacial flow generates complex mechanical forces that cause cell necrosis/plasma membrane disruption and detachment of cells from their substrate. In addition, mechanical forces associate with atelectrauma can also activate inflammatory signaling pathways (27,28).…”