Atelectrauma disrupts pulmonary epithelial barrier integrity and alters the distribution of tight junction proteins ZO-1 and claudin 4

Abstract

Mechanical ventilation inevitably exposes the delicate tissues of the airways and alveoli to abnormal mechanical stresses that can induce pulmonary edema and exacerbate conditions such as acute respiratory distress syndrome. The goal of our research is to characterize the cellular trauma caused by the transient abnormal fluid mechanical stresses that arise when air is forced into a liquid-occluded airway (i.e., atelectrauma). Using a fluid-filled, parallel-plate flow chamber to model the "airway reopening" process, our in vitro study examined consequent increases in pulmonary epithelial plasma membrane rupture, paracellular permeability, and disruption of the tight junction (TJ) proteins zonula occludens-1 and claudin-4. Computational analysis predicts the normal and tangential surface stresses that develop between the basolateral epithelial membrane and underlying substrate due to the interfacial stresses acting on the apical cell membrane. These simulations demonstrate that decreasing the velocity of reopening causes a significant increase in basolateral surface stresses, particularly in the region between neighboring cells where TJs concentrate. Likewise, pulmonary epithelial wounding, paracellular permeability, and TJ protein disruption were significantly greater following slower reopening. This study thus demonstrates that maintaining a higher velocity of reopening, which reduces the damaging fluid stresses acting on the airway wall, decreases the mechanical stresses on the basolateral cell surface while protecting cells from plasma membrane rupture and promoting barrier integrity.

Fig. 1.

A: the reopening of fluid-filled, atelectatic airways generates abnormal fluid mechanical stresses and stress gradients that act along the airway walls (illustrated here by the shaded arrows) to alter both the structure and function of the lining cellular epithelium (as depicted within the circular windows below). B: this “airway reopening” process can be modeled using a parallel-plate flow chamber, as illustrated here in cross section. By culturing the base with pulmonary epithelial cells, this channel serves as a two-dimensional representation of a respiratory bronchiole with walls held in rigid apposition by a viscous liquid obstruction. “Reopening” occurs as a semi-infinite bubble of air is driven into the system at a velocity U_bubble, clearing the “airway” of its liquid occlusion.

Fig. 2.

Tight junctions (TJs) seal together neighboring cells to prevent fluid and protein passage across the pulmonary epithelial cell layer. TJ protein strands (claudin-4) weave together adjacent cell membranes and are linked to the internal actin cytoskeleton via cytosolic TJ proteins [zonula occludens-1 (ZO-1)].

Fig. 3.

Fluorescent images of pulmonary epithelial layers stained for paracellular permeability using BODIPY-ouabain (green) and for plasma membrane rupture using ethidium homodimer-1 (EthD-1) (red). Images were captured 1 h after a fast (U_F) or slow (U_S) reopening experiment or under control conditions. In the U_S image, representative areas of increased paracellular permeability that are relatively far removed from regions of cell wounding are indicated with white arrows. Scale bars are equivalent to 50 μm.

Fig. 4.

Percentage of the total field area stained with the green paracellular permeability indicator BODIPY-ouabain (shaded bars) or plasma membrane-compromised pulmonary epithelial cells with nuclei that stained positive (red) for EthD-1 (solid bars) 1 h post-airway reopening under U_F, U_S, or control conditions. Results are means ± SE (n = 8 flow chambers for all groups). Significant differences (P < 0.05) between results: *permeability tests and #percent wounded assay.

Fig. 5.

Distribution of the paracellular permeability indicator from wounded epithelial cells. Mean pixel intensities of the green paracellular permeability indicator BODIPY-ouabain (with a total intensity range of 0 to 1) after a single reopening event at either U_S (A) or U_F (B) is shown. The horizontal axis represents the radial distance from each green pixel to the nearest red-fluorescing, EthD-1-stained nucleus in an image, scaled by the average minimum radial distance between red-fluorescing nuclei in that image, and binned into 0.036 unit columns. Each inset depicts the same data unscaled and binned into 1.305-μm columns.

Fig. 6.

Pulmonary epithelial TJ protein immunofluorescence under control conditions (row 1) or 1 h after a fast (U_F, row 2) or slow (U_S, row 3) reopening experiment. Isolated expression of claudin-4 is provided in column 1, while column 2 contains the corresponding ZO-1 expression. Claudin 4 (red) and ZO-1 (green) are merged in column 3, along with cell nuclei (blue). Scale bars represent 50 μm.

Fig. 7.

Total intensity of red claudin-4 (shaded bars) and green ZO-1 (solid bars) relative to the total control intensities for the respective fluorophores 1 h post-airway reopening under fast (U_F), slow (U_S), or control conditions using a 37°C PBS occlusion fluid. Results are normalized by the total number of cells contained in each image field and presented as means ± SE (n = 8 flow chambers for all groups). Significant differences between results (P < 0.05): *claudin-4 and #ZO-1.

Fig. 8.

A: this computational study investigates the response of the incompressible, hyperelastic pulmonary epithelium to our parallel plate flow chamber model of airway reopening (not drawn to scale) using a neo-Hookean finite-element model. The cell cytoplasm and nucleus are treated as separate cell constituents with unique Young's and shear moduli (E_cyt, E_nuc and G_cyt, G_nuc, respectively), and the saline (PBS) fluid occlusion is characterized by a viscosity and surface tension of μ_PBS and γ_PBS, respectively. B: the model was implemented by applying the reopening-induced shear stress, τ_s(x_apical), and pressure, P(x_apical), fields to the apical cell surface (i.e., the plasma membrane), x_apical, as the reopening bubble swept through at a fast or slow velocity (U_bubble = U_F or U_S, respectively) using Eqs. 1–3. C: the meshed epithelial cell layer was free to deform in response to these reopening stresses over all but the basolateral cell surface, x_basolateral, which remained fixed to the rigid underlying substrate (i.e., glass coverslip). (dP/dx)_max, maximal pressure gradient.

Fig. 9.

Results of the airway reopening simulation collected once the semi-infinite bubble had passed entirely through the channel. A: this image compares the initial epithelium (depicted in gray) and its final deformed shape (outlined in black, with displacement values amplified by a factor of 100) following U_S reopening. The basolateral vector field represents the relative magnitude and direction of surface stresses exerted by the pulmonary epithelium on the underlying rigid substrate during U_S reopening. The graphs below present the distribution of the normal, T_y (B) and tangential, T_x (C) components of the surface stresses acting on the basolateral cell membrane, x_basolateral, due to either slow, U_S (solid line), or fast, U_F (dashed line), airway reopening. The vertical dotted lines indicate the location of the junctions between neighboring cells, and the abscissa is scaled by the diameter of a pulmonary epithelial cell in this study (L_cell = 31.25 μm).

Fig. 10.

Distribution of the paracellular permeability indicator from wounded epithelial cells. A: differences in the mean pixel intensities of the green paracellular permeability indicator BODIPY-ouabain (with a total intensity range of 0 to 1) after a single reopening event following U_S and U_F reopening (i.e., slow-fast results). The horizontal axis represents the radial distance from each green pixel to the nearest red-fluorescing, EthD-1-stained nucleus in an image, scaled by the average minimum radial distance between red-fluorescing nuclei in that image and binned into 0.036 unit columns. B: the cumulative quantity of BODIPY-ouabain that has passed through the epithelium during U_S (solid line) or U_F (dashed line) experiments.