Lung Injury Is Prevented in ASK-1 Knockout Mice
We compared the severity of lung injury in wild-type and ASK-1 knockout mice by evaluating lung histology (hematoxylin and eosin stains, ), composite lung injury scores (), lung injury subtype scores (), heart rate and oxygen saturation (), Cst (), total BALF cell counts (), and protein concentrations (). Lung sections of Control-WT () and Control-ko mice () showed normal lung parenchyma, whereas LIC-WT mice () showed severe and diffuse alveolar and interstitial edema, neutrophil infiltration, and especially hemorrhage. In contrast, LIC-ko mice () exhibited decreased alveolar and interstitial edema, neutrophil infiltration, and especially hemorrhages (). Although some injury was observed in the LIC-ko group, composite lung injury scores () were significantly lower than in the LIC-WT group, and did not differ statistically from either control group. The most significant differences were seen in alveolar edema and hemorrhage scores ().
Figure 1. The deletion of apoptosis signal–regulating kinase-1 (ASK-1) protects against lung injury in mice subjected to lung injury conditions (LIC; n = 3 mice per group). (A) Hematoxylin and eosin–stained, formalin-fixed, paraffin-embedded lung (more ...)
Figure 2. The deletion of ASK-1 reduces lung injury and barrier dysfunction in mice subjected to LIC (n = 3 mice per group). (A) Composite lung injury scores (LIS in D) represent the sum of the mean injury subtype scores (0–4) for each condition on a scale (more ...)
MEAN HEART RATES AND OXYGEN SATURATIONS OF LIC-WT AND LIC-KO AT 0 HOURS AND 4 HOURS OF VENTILATION
LIC-WT mice had significantly increased heart rates and reduced SaO2 during the 4 hours of mechanical ventilation, whereas in LIC-ko mice, both these parameters remained stable. Lung mechanics were similar in all experimental groups at 0 hours, but Cst significantly decreased in LIC-WT mice after 4 hours of mechanical ventilation, compared with both control groups and LIC-ko mice ().
LIC-WT mice showed a significant increase in total BALF cells () and protein concentrations () compared with control mice. In LIC-ko mice, concentrations of BALF protein were greater than in control mice, but significantly lower than in LIC-WT mice, whereas BALF cell counts did not differ significantly from those in control mice.
Lung Injury Conditions Activated ASK-1
To confirm the expression of ASK-1 protein, we documented that total ASK-1 was only present in wild-type lungs but not in ASK-1 knockouts (). The densitometric analysis of Western blots () indicated no significant change in the total expression of ASK-1 protein in LIC-WT compared with Control-WT mice, and no detectable ASK-1 protein in ASK-1 knockouts.
Figure 3. Protein expression and augmented threonine (Thr) phosphorylation of ASK-1 in whole-lung homogenates of wild-type mice subjected to LIC (n = 3 mice per group). (A) Protein expression of total ASK-1 in Control-WT (lane 1), Control-ko (lane 2), LIC-WT ( (more ...)
To determine whether lung injury was attributable to the activation of ASK-1, we needed to confirm the phosphorylation of ASK-1 to its active form (phosphorylation at threonine 845). No commercially available anti–phospho–ASK-1 (Thr845) antibodies provided adequate specificity. Therefore, we alternately immunoprecipitated whole-lung homogenates (control-WT and LIC-WT, as well as 4 hours of HV alone, 16 hours of HO alone, and N-acetylcysteine [NAC], 150 μg/g body weight, intraperitoneal, before LIC) with an antibody directed against phosphorylated threonine, followed by immunoblotting with anti–total ASK-1 antibody (). A densitometric analysis () indicated that LIC-WT increased ASK-1 threonine phosphorylation compared with all other groups. Minimal ASK-1 phosphorylation was seen with 16 hours of HO, whereas HV alone for 4 hours produced more than 16 hours of HO alone, but significantly less ASK-1 phosphorylation than LIC. Moreover, mice treated with the antioxidant NAC before LIC showed reduced ASK-1 phosphorylation. These results confirm that lung injury in this model was associated with the activation of ASK-1, and this activation was oxidant-dependent.
Deletion of ASK-1 Inhibited Apoptosis
To determine whether the deletion of ASK-1 reduces apoptosis during exposure to LIC, we performed Western blot analyses of cleaved caspase-3 and PARP-1 (). LIC-WT mice exhibited significantly increased cleavage of both caspase-3 () and PARP-1 (). In LIC-ko mice, the cleavage of caspase-3 and PARP-1 was completely inhibited.
Figure 4. Activation of executioner caspase, caspase-3, and its downstream substrate poly [ADP-ribose] polymerase 1 (PARP)-1 in wild-type and ASK-1–ko mice subjected to LIC (n = 3 mice per group). (A) Activation (cleavage) of caspase-3 and PARP-1 (numbers (more ...)
In addition, we performed TUNEL staining on fixed lung tissue from each group, to identify any evidence of apoptosis (). Minimal TUNEL-positive cells were seen in control groups, but significantly fewer in Control-ko mice compared with Control-WT mice (). LIC-WT mice showed significant and diffuse TUNEL-positive cells. LIC-ko mice exhibited significantly fewer TUNEL-positive cells, and did not differ significantly from Control-WT mice. LIC-ko mice had significantly more TUNEL-positive cells than did Control-ko mice.
Figure 5. Apoptotic cell death in wild-type and ASK-1–ko mice subjected to LIC (n = 3 mice per group). (A) Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay shows positive apoptotic cells (green) in lung sections of mice subjected (more ...)
Apoptosis Primarily Involved the Alveolar Epithelium
To determine the cell types in which apoptosis occurred, we performed immunofluorescence of lung sections exposed to antibodies specific for epithelial cells (cytokeratin 18), alveolar Type II cells (pro–SP-C), and endothelial cells (PECAM-1), and determined colocalization with TUNEL staining. shows that the majority of TUNEL-positive cells are alveolar Type II (positive for both cytokeratin 18 and pro–SP-C). A few TUNEL-positive cells were positive only for cytokeratin 18, indicating non–Type II epithelial cells. These were located in alveolar structures, indicating that they were Type I epithelial cells. Very few TUNEL-positive cells were observed that colocalized with PECAM-1, indicating a small number of endothelial cells undergoing apoptosis.
Figure 6. Cellular distribution of apoptosis in mice subjected to LIC. Immunofluorescence staining for TUNEL (green), prosurfactant C (pro–SP-C; yellow), platelet/endothelial cell adhesion molecule-1 (PECAM-1) (orange), and cytokeratin-18 (red) in lung (more ...)
Deletion of ASK-1 Did Not Affect VILI-Associated Release of Cytokines
To determine whether the deletion of ASK-1 affects the release of inflammatory mediators, we measured concentrations of IL-6, KC, MCP-1, and MIP-1 in BALF. Despite significant differences in indicators of lung injury, significant elevations of all four inflammatory mediators were seen in both LIC-WT and LIC-ko mice, compared with control mice ().
Figure 7. Concentration of inflammatory mediators in BALF of wild-type and ASK-1–ko mice subjected to LIC (n = 3 mice per group). (A–D) Protein expression (pg/ml) of (A) IL-6, (B) keratinocyte-derived chemokine (KC), (C) monocyte chemoattractant (more ...)