Mechanical compression attenuates normal human bronchial epithelial wound healing

Abstract

Background: Airway narrowing associated with chronic asthma results in the transmission of injurious compressive forces to the bronchial epithelium and promotes the release of pro-inflammatory mediators and the denudation of the bronchial epithelium. While the individual effects of compression or denudation are well characterized, there is no data to elucidate how these cells respond to the application of mechanical compression in the presence of a compromised epithelial layer.

Methods: Accordingly, differentiated normal human bronchial epithelial cells were exposed to one of four conditions: 1) unperturbed control cells, 2) single scrape wound only, 3) static compression (6 hours of 30 cmH2O), and 4) 6 hours of static compression after a scrape wound. Following treatment, wound closure rate was recorded, media was assayed for mediator content and the cytoskeletal network was fluorescently labeled.

Results: We found that mechanical compression and scrape injury increase TGF-beta2 and endothelin-1 secretion, while EGF content in the media is attenuated with both injury modes. The application of compression after a pre-existing scrape wound augmented these observations, and also decreased PGE2 media content. Compression stimulated depolymerization of the actin cytoskeleton and significantly attenuated wound healing. Closure rate was partially restored with the addition of exogenous PGE2, but not EGF.

Conclusion: Our results suggest that mechanical compression reduces the capacity of the bronchial epithelium to close wounds, and is, in part, mediated by PGE2 and a compromised cytoskeleton.

Figure 1

Schematic demonstrating treatment and time course of measurements. Cells slated to undergo scrape and compression were scraped with a 200 μl pipette tip at time zero and washed 2× with cell culture media to remove debris and were imaged. The wells then underwent 30 cmH₂O mechanical compression for 6 hours. Following compression the cells were imaged and a representative well was fixed for imaging. 24 and 48 hours following the initial insult cells were again imaged to determine wound width, a representative well was fixed for imaging and media samples were taken for mediator content.

Figure 2

Representative images of wells that underwent scrape only (left) and scrape with 6 hours of mechanical compression (right) at 6, 24, and 48 hour time points. The black bars in each image represent three wound width measurements that were subsequently averaged, normalized to the initial wound width and treated as a single measurement (text).

Figure 3

Representative images of filamentous actin staining of fixed cells at 6, 24 and 48 hour time points that underwent a scrape wound (left), scrape and compression wound (middle), or cytochalasin-D treatment (right). Also included are images of microtubule structure (bottom row) at 6 hours.

Figure 4

Wound width normalized with respect to initial wound width as a function of time for cells that were scraped (circle, solid line) or scraped and underwent mechanical compression (square, dashed line). (* p < 0.05 between groups).

Figure 5

(A) Active TGF-β2, (B), Et-1, (C) EGF, and (D) PGE₂ media content for unstimulated cells (control), cells that underwent a scrape wound (scrape), cells that underwent mechanical compression (compression), and cells that underwent both a scrape wound and mechanical compression (both) at 24 (Black) and 48 hours (Gray). (* p < 0.05 compared to control at respective time point).

Figure 6

Wound width normalized with respect to initial wound width as a function of time (A) for cells that were scraped (circle, solid line), scraped and underwent mechanical compression (circle, dotted line), scraped, underwent compression and treated with PGE₂ (circle, dashed line) or scraped and treated with PGE₂ (circle, dash-dot). Wound width normalized with respect to initial wound width as a function of time (B) for cells that were scraped (circle, solid line or treated with Indomethacin (circle, dashed line). (* p < 0.05 compared to scrape only, # p < 0.05 compared to scrape and compression).