Balance of life and death in alveolar epithelial type II cells: proliferation, apoptosis, and the effects of cyclic stretch on wound healing

Abstract

After acute lung injury, repair of the alveolar epithelium occurs on a substrate undergoing cyclic mechanical deformation. While previous studies showed that mechanical stretch increased alveolar epithelial cell necrosis and apoptosis, the impact of cell death during repair was not determined. We examined epithelial repair during cyclic stretch (CS) in a scratch-wound model of primary rat alveolar type II (ATII) cells and found that CS altered the balance between proliferation and cell death. We measured cell migration, size, and density; intercellular gap formation; cell number, proliferation, and apoptosis; cytoskeletal organization; and focal adhesions in response to scratch wounding followed by CS for up to 24 h. Under static conditions, wounds were closed by 24 h, but repair was inhibited by CS. Wounding stimulated cell motility and proliferation, actin and vinculin redistribution, and focal adhesion formation at the wound edge, while CS impeded cell spreading, initiated apoptosis, stimulated cytoskeletal reorganization, and attenuated focal adhesion formation. CS also caused significant intercellular gap formation compared with static cells. Our results suggest that CS alters several mechanisms of epithelial repair and that an imbalance occurs between cell death and proliferation that must be overcome to restore the epithelial barrier.

Fig. 1.

Cyclic stretch (CS) impeded wound closure and caused gap formation. A–N: morphology of primary alveolar type II (ATII) cells under static conditions without wounding (St/U) or with wounding (St/W) and under CS conditions without wounding (CS/U) or with wounding (CS/W). Because cells were fixed at each time point, images do not represent a time lapse sequence from the same wells. Images of wounds at the initial time (0 h) were therefore used for static and stretched cells. Scale bar, 150 μm; ×4 bright-field objective. O: CS inhibited wound closure of ATII cells. *Significantly different from static at the indicated time. Error bars, SE.

Fig. 2.

Wounding caused changes in distribution of F-actin (red) and vinculin (green). Focal adhesions are indicated by overlap (yellow). A: St/U at 0 h. B–E: St/W at 0 h (B), 2 h (C), 12 h (D), and 24 h (E).

Fig. 3.

CS caused formation of gaps and redistribution of actin and vinculin. A and B: CS/U at 2 h (A) and 12 h (B). C–E: CS/W at 2 h (C), 12 h (D), and 24 h (E).

Fig. 4.

CS inhibited cell spreading in wounded monolayers. Cell area was measured in unwounded static (St/U) and CS (CS/U) cells and in cells within 650 μm of the wound edge [ST/W near (N) and CS/W N] and between 650 and 1,300 μm from the wound edge [St/W far (F) and CS/W F]. *Significantly different from static at the same time. +Significantly different from unwounded at the same time. #Significantly different from 0 h. Error bars, SE.

Fig. 5.

CS caused formation of gaps between wounded cells that remained unrepaired. *Significant difference between static and CS at the indicated time. #Significantly different from 0 h. +Significant difference between wounded and unwounded at the indicated time (P < 0.05). Error bars, SE.

Fig. 6.

Wounding significantly stimulated cell proliferation in primary ATII cells at the wound edge. A and B: cells near wound edge (A) and >1 field from wound edge (B) were stained with Click-iT ethidium deoxyuridine (EdU; green) to show S phase of replication and specifically stained with RTII70 anti-ATII cell antibody (red). Colocalization (yellow) of S phase and ATII cell-specific stain indicates proliferating cells near the wound edge (indicated by white line). Original (confocal) magnification, ×25. C: proliferative index was significantly increased in cells near the wound edge (0–30 cells from the edge), and CS diminished this response. *Significant difference between static and CS at the indicated time. #Significantly different from 0 h. +Significant difference between wounded and unwounded at the indicated time (P < 0.05). Error bars, SE. D: total cell counts. Total cell number was significantly different immediately following wounding (as expected) but was equivalent under all conditions by 24 h. *Significantly different from unwounded.

Fig. 7.

CS stimulated changes in morphology indicative of apoptosis. A: St/U at 0 h. B: St/U at 12 h. C: St/W ≤10 cells from the wound edge (white line indicated by arrow shows wound edge) at 12 h. D: St/W >10 cells from wound edge at 12 h. E: CS/U at 12 h. F: CS/W ≤10 cells from the wound edge (wound edge not shown) at 12 h. Thin arrows, apoptotic cells; thick arrow, apoptotic body. Images were obtained using confocal microscopy, CR590 and FAM fluorescence staining, ethidium bromide nuclear counterstaining, and 2-channel excitation at 488 and 543 nm, with ×40 objective + ×2 zoom.

Fig. 8.

CS stimulated apoptosis in primary ATII cells, and stimulation of apoptosis inhibited wound closure. A and B: changes in apoptotic index (AI, %) in static cells and cells subjected to CS. *Significant difference between static and CS. #Significantly different from 0 h. +Significant difference between wounded and unwounded (P < 0.05). Error bars, SE. C: treatment with sulindac sulfone stimulated apoptosis in cells near the wound edge at 24 h. *Significantly different from control. D: treatment with sulindac sulfone significantly inhibited wound closure. *Significantly different from untreated.