Alveolar cell apoptosis is dependent on p38 MAP kinase-mediated activation of xanthine oxidoreductase in ventilator-induced lung injury

Abstract

Signaling via p38 MAP kinase has been implicated in the mechanotransduction associated with mechanical stress and ventilator-induced lung injury (VILI). However, the critical downstream mediators of alveolar injury remain incompletely defined. We provide evidence that high-tidal volume mechanical ventilation (HVt MV) rapidly activates caspases within the lung, resulting in increased alveolar cell apoptosis. Antagonism of MV-induced p38 MAP kinase activity with SB-203580 suppresses both MV-induced caspase activity and alveolar apoptosis, placing p38 MAP kinase upstream of MV-induced caspase activation and programmed cell death. The reactive oxygen species (ROS)-producing enzyme xanthine oxidoreductase (XOR) is activated in a p38 MAP kinase-dependent manner following HVt MV. Allopurinol, a XOR inhibitor, also suppresses HVt MV-induced apoptosis, implicating HVt MV-induced ROS in the induction of alveolar cell apoptosis. Finally, systemic administration of the pan-caspase inhibitor, z-VAD-fmk, but not its inactive peptidyl analog, z-FA-fmk, blocks ventilator-induced apoptosis of alveolar cells and alveolar-capillary leak, indicating that caspase-dependent cell death is necessary for VILI-associated barrier dysfunction in vivo.

Fig. 1.

High-tidal volume mechanical ventilation (HVt MV) increased caspase activity within the lung in vivo. C57BL/6J mice were randomized to spontaneous breathing (sham) or to low-tidal volume (LVt; 7 ml/kg) or HVt MV (20 ml/kg) for 2 or 4 h. FU, fluorometric unit; Ctrl; control. A: total lung caspase-3/7 activity was significantly increased in response to HVt compared with sham or LVt MV after 2 and 4 h of MV. There was no significant change in caspase-3/7 activity between control and MV at LVt. *P < 0.05 vs. sham and 2-h LVt MV. †P < 0.05 vs. sham and 4-h LVt MV (n = 7–10 mice/group). B: the pan-caspase inhibitor z-VAD-fmk, but not its inactive peptidyl analog z-FA-fmk, suppressed HVt MV-induced caspase-3/7 activity in total lung homogenates. *P < 0.05 (n = 7–10 mice/group).

Fig. 2.

HVt MV-induced poly(adenosine-ribose) polymerase-1 (PARP-1) cleavage is antagonized by caspase, p38 MAP kinase, and xanthine oxidoreductase (XOR) inhibition. A: full-length (116 kDa) and cleaved fragment (89 kDa) PARP-1 were detected using immunoblot analysis of whole lung homogenates from animals exposed to sham therapy or HVt MV alone or with SB 203580, allopurinol, or z-VAD-fmk treatment. B: there was a significant increase in cleaved PARP-1 relative to actin after 4-h HVt MV. This was completely prevented by inhibition of p38 MAP kinase (by SB-203580) and XOR (by allopurinol). Cleavage of PARP-1 was caspase dependent and inhibited by z-VAD-fmk treatment. *P < 0.05 vs. all other conditions (n = 3–6 mice/group). Z-VAD, z-VAD-fmk peptide.

Fig. 3.

HVt MV induced DNA nicking within alveolar cells. A: C57BL/6J mice were randomized to spontaneous breathing (sham; a and b) or to LVt (7 ml/kg; c and d) or HVt MV (20 ml/kg; e and f) for 4 h. Nicked DNA was detected using terminal deoxynucleotidyl transferase-mediated dUDP nick-end labeling (TUNEL; green) within the lung parenchyma. Endothelial cells were recognized by thrombomodulin staining (white; long arrows); type I and II epithelial cells were recognized by staining with T1 alpha and surfactant protein C (SPC; red; short arrows). Apoptotic cells were quantified after nuclear staining (4′,6-diamidino-2-phenylindole; blue) as described in materials and methods. B: a subset of animals exposed to HVt MV were treated with z-VAD-fmk (g and h), SB 203580 (i and j), or allopurinol (k and l), all of which significantly prevented DNA nicking (TUNEL positivity; green).

Fig. 4.

Treatment with z-VAD-fmk prevented HVt MV-induced alveolar cell apoptosis. After 4 h of HVt MV, the number of cells exhibiting DNA fragmentation was significantly increased in all 3 cell types. There was no significant difference in DNA nicking between sham-treated mice and mice exposed to LVt MV or treated with z-VAD-fmk in any of the 3 cell types. *P < 0.05 vs. all other conditions (n = 7–10 mice/group).

Fig. 5.

Inhibition of p38 MAP kinase blocked HVt MV-induced caspase-dependent alveolar cell apoptosis. C57BL/6J mice were randomly exposed to spontaneous breathing (sham) or to HVt ventilation for 4 h. A: pretreatment with 2 mg/kg SB-203580 1 h before MV, but not its carrier, DMSO (vehicle), suppressed MV-induced caspase-3/7 activity in total lung homogenates. B: nicked DNA was detected using TUNEL staining as described in materials and methods. Treatment of mice with SB 203580 prevented HVt ventilator-induced DNA nicking compared with vehicle treatment in all 3 alveolar cell types. *P < 0.05 vs. vehicle and untreated mice exposed to MV (n = 7–10 mice/group).

Fig. 6.

Allopurinol blocked HVt MV-induced caspase-dependent alveolar cell apoptosis. C57BL/6J mice were randomly exposed to spontaneous breathing (sham) or to HVt ventilation for 4 h. A: pretreatment with allopurinol at 200 mg/kg suppressed MV-induced caspase-3/7 activity in total lung homogenates. B: nicked DNA was detected using TUNEL staining as described in materials and methods. Inhibition of XOR with allopurinol activity significantly decreased evidence of HVt ventilator-induced apoptosis in all 3 alveolar cell types. *P < 0.05 vs. vehicle-treated mice (n = 7–10 mice/group).

Fig. 7.

Caspase inhibition prevented ventilation-induced pulmonary capillary leakage. C57BL/6J mice were randomly exposed to spontaneous breathing (sham) or to HV_T ventilation for 4 h. A: there was a significant increase in the amount of Evans blue dye (EBD) in lung homogenates after HVt MV (1.68-fold higher than sham). Treatment with z-VAD-fmk (0.5 mg/mouse) significantly reduced EBD leakage to sham levels. In contrast, z-FA-fmk had no effect on capillary leakage. *P < 0.05 vs. sham and z-VAD-fmk (n = 6 mice/group). B: wet-to-dry lung weight ratio was significantly increased in mice after 4 h of HVt MV. This increase was completely prevented by z-VAD-fmk treatment but not with administration of the control peptide, z-FA-fmk. †P < 0.05 vs. sham and z-VAD-fmk (n = 4–6 mice/group).