Measurement of stretch-induced loss of alveolar epithelial barrier integrity with a novel in vitro method

Abstract

Mechanical ventilation with high tidal volumes has been shown to contribute to the formation or worsening of interstitial and alveolar edema. Previously we showed that application of large biaxial deformations in vitro perturbs the concentration and distribution of functional tight junction proteins in alveolar epithelial cells. Using a novel method, we determined that applied epithelial strain increases paracellular permeability in a dose- and rate-dependent manner. Primary rat alveolar epithelial cells were subjected to 12%, 25%, or 37% change in surface area (ΔSA) cyclic equibiaxial stretch for 1 h. Cells were also stretched noncyclically at 25% ΔSA for 1 h. During the experimental period, a fluorescently tagged ouabain derivative was added to the apical fluid. Evidence of binding indicated functional failure of the paracellular transport barrier. The percentage of field area stained was quantified from microscopic images. There was no significant evidence of basolateral fluorescent staining at 12% ΔSA or at 25% ΔSA applied cyclically or statically. However, cyclic stretch at 37% ΔSA resulted in significantly more staining than in unstretched cells ( P < 0.0001) or those stretched at either 12% ( P < 0.0001) or 25% cyclic ( P < 0.0005) or static ( P < 0.05) ΔSA. These results suggest that large cyclic tidal volumes may increase paracellular permeability, potentially resulting in alveolar flooding.

Stretch increases alveolar epithelial permeability to uncharged micromolecules

“…Although data exist indicating that the transport of molecules >30 Å in radius across the alveolar epithelium may occur via endocytosis or other active processes (33), the similarities between the monolayers and whole lung results support the extrapolation of our in vitro data to the in vivo milieu. The applicability of our monolayer data to the in vivo setting is further supported by our previous finding that stretch of cultured alveolar epithelial cells at 37% ΔSA increased the paracellular permeability of the monolayer to a ouabain derivative ~20 Å in radius (6). Finally, we only employed tracers with a neutral net charge.…”

“…This threshold stretch magnitude is the same level that we reported previously to disrupt tight junction structure and barrier function in similarly cultured cells (6,7), although barrier properties were assessed using a single larger tracer (radius = 15-20 Å). Tight junction properties in these two studies also were not affected by lower magnitudes of stretch, similar to what was observed in the current preparation.…”

“…This protocol was approved by the University of Pennsylvania Institutional Animal Care and Use Committee and has been described previously (6). After isolation, cells were suspended in minimum essential medium (MEM) with Earle's salts and supplemented with 10% fetal bovine serum, 25 μg/ml gentamicin, and 0.25 μg/ml amphotericin B (Life Technologies).…”

“…Our group (6,7) has previously shown that high yet physiological magnitudes of applied cyclic stretch decrease intracellular alveolar epithelial tight junction protein content and increase the tight junction permeability to a fluorescent ouabain derivative 15-20 Å in radius. However, the paracellular permeability of the stretched alveolar epithelium has not yet been measured directly because it has proven difficult to culture the cells onto a substrate that is flexible, permeable, and biocompatible.…”

Induced Physical Distension of Rat Mammary Glands Accelerates the Onset of Apoptosis and Involution

“…Furthermore, TJ have previously been reported to respond to mechanical tension as alterations in TJ strand arrangement and orientation occur when MEC are stretched in vitro [42]. In lung alveolar epithelial cells increased TJ permeability occurs during cyclic stretch [21,43,44]. The latter was associated with a decline in abundance of occludin protein, but no differences were detected in ZO-1 protein [21].…”