Male C57BL/6 mice, either wild-type Akt^+/+ or Akt^+/- on a C57BL/6 background, aged between 6 and 8 weeks, weighing between 20 and 25 g, were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and from the National Laboratory Animal Center (Taipei, Taiwan) as previously described 19. The study was performed in accordance with animal experimental guidelines of the National Institutes of Health and with approval from the local research committee.

We used our established mouse model of VILI as previously described 1320. A 20-gauge angiocatheter was introduced into the tracheotomy orifice of the mouse under general anesthesia with intraperitoneal ketamine (90 mg/kg) and xylazine (10 mg/kg). The mice were placed in a supine position on a heating blanket and were then attached to a Harvard apparatus ventilator (model 55-7058; Harvard Apparatus, Holliston, MA, USA), set to deliver either 6 ml/kg at a rate of 135 breaths/minute or 30 ml/kg at a rate of 65 breaths/minute for 1 and 5 hours while breathing room air with zero end-expiratory pressure. The mice then received 0.9% saline containing maintenance ketamine (0.1 mg/g/hour) and xylazine (0.01 mg/g/hour) at a rate of 0.09 ml/10 g/hour by a continuous intraperitoneal fluid pump.

The tidal volume delivered by the ventilator was checked by fluid displacement from an inverted calibration cylinder. Continuous monitoring of end-tidal carbon dioxide by a microcapnograph (Columbus Instruments, Columbus, OH, USA) was performed. The respiratory frequencies of 135 breaths/minute for 6 ml/kg tidal volume and 65 breaths/minute for 30 ml/kg tidal volume were chosen in the experiment, with end-tidal carbon dioxide between 30 and 40 mmHg. The airway peak inspiratory pressure was measured with a pressure transducer amplifier (Gould Instrument Systems, Valley View, OH, USA) connected to the tubing at the proximal end of the tracheostomy. The mean arterial pressure was monitored every hour during mechanical ventilation using the same pressure transducer amplifier connected to a 0.61 mm outer diameter (0.28 mm inner diameter) polyethylene catheter ending in the common carotid artery.

At the end of the study period, heparinized blood was taken from the arterial line for analysis of arterial blood gas and the mice were sacrificed. Control, nonventilated mice were anesthetized and sacrificed immediately. The experimental design and the number of animals in the study are summarized in Table 1.

The mice received a single dosage of 0.075 U bleomycin in 100 μl sterile normal saline (Sigma, St Louis, MO, USA) intratracheally, and were ventilated for 5 or 10 days after bleomycin administration 21.

The P38 inhibitor (SB203580, 16 mg/kg; Calbiochem, La Jolla, CA, USA) and the ERK1/2 inhibitor (PD98059, 2 mg/kg; Calbiochem) were given subcutaneously 30 minutes before ventilation based on previous in vivo studies 2223.

The lung tissues from control, nonventilated mice exposed to high-tidal-volume ventilation or low-tidal-volume ventilation for 5 hours while breathing room air were paraffin embedded, sliced at 4 μm, deparaffinized, and stained sequentially with Weigert's iron hematoxylin solution, Biebrish scarlet-acid fuchsin solution, and aniline blue solution according to the manufacturer's instruction of a trichrome kit (Sigma). A blue signal indicated positive staining of collagen.

The fibrotic grade of each lung field was assessed using the criteria of Ashcroft, ranging from grade 0 to grade 5 as follows: grade 0 = normal lung; grade 1 = minimal fibrous thickening of alveolar or bronchial walls; grade 2 = moderate thickening of walls without obvious damage to the lung architecture; grade 3 = increased fibrosis with definite damage to the lung structure and formation of fibrous bands or small fibrous masses; grade 4 = severe distortion of the structure and large fibrous areas (honeycomb lung); and grade 5 = total fibrous obliteration in the field 14. An average number of 10 nonoverlapping fields in Masson's trichrome staining of paraffin lung sections, six mice per group, were analyzed for each section by a single investigator blinded to the mouse genotype.

Lungs were homogenized in 2 ml PBS, and a 1 ml aliquot was desiccated and then hydrolyzed in 6 N HCl at 110°C for 12 hours. Twenty-five-microliter aliquots were added to 1 ml of 1.4% chloramine T (Sigma), 10% n-propanol, and 0.5 M sodium acetate, pH 6.0. After 20 min of incubation at room temperature, 1 ml Erlich's solution (1 M p-dimethylaminobenzaldehyde (Sigma) in 70% n-propanol, 20% perchloric acid) was added and a 15-minute incubation at 65°C was performed. Absorbance was measured at 550 nm and the amount of hydroxyproline was determined against a standard curve 21.

The lungs were homogenized in 3 ml lysis buffer (20 mM HEPES, pH 7.4, 1% Triton X-100, 10% glycerol, 2 mM ethylene glycol-bis (β-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 50 μM β-glycerophosphate, 1 mM sodium orthovanadate, 1 mM dithiotreitol, 400 μM aprotinin, and 400 μM phenylmethylsulfonyl fluoride), were transferred to eppendorf tubes and were placed on ice for 15 minutes. The tubes were centrifuged at 14,000 rpm for 10 minutes at 4°C and the supernatant was flash frozen. Crude cell lysates were matched for protein concentration, resolved on a 10% bis-acrylamide gel, and electrotransferred to Immobilon-P membranes (Millipore Corp., Bedford, MA, USA).

For assay of phosphorylation of JNK, p38, ERK1/2, and Akt protein expression, western blot analysis were performed with antibodies to phospho-JNK, phospho-p38, phospho-ERK1/2, and phospho-Akt (New England BioLabs, Beverly, MA, USA). For determination of total JNK, p38, ERK1/2, and Akt protein expression, western blot analysis was performed with the respective antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Blots were developed by enhanced chemiluminescence (NEN Life Science Products, Boston, MA, USA).

At the end of the study period, the lungs were lavaged via tracheostomy with a 20-gauge angiocatheter (sham instillation) three times with 0.6 ml of 0.9% normal saline. The effluents were pooled and centrifuged at 2,000 rpm for 10 minutes. Supernatants were frozen at -80°C for further analysis of cytokines. MIP-2 (1 pg/ml) and IP-10 (2.2 pg/ml) were measured in bronchoalveolar lavage fluid using a commercially available immunoassay kit containing antibodies cross-reactive with rat and mouse MIP-2 and IP-10 (Biosource International, Camarillo, CA, USA). Each sample was run in duplicate according to the manufacturer's instructions.

The lung tissues from control, nonventilated mice exposed to high-tidal-volume ventilation or low-tidal-volume ventilation for 5 hours while breathing room air were removed en bloc, and were filled with 10% neutral buffered formalin (pH 6.8 to 7.2) at 30 cmH₂O pressure via polyethylene tubing inserted into the trachea. The lungs were paraffin embedded, sliced at 4 μm, deparaffinized, antigen unmasked in 10 mM sodium citrate (pH 6.0), and incubated with phospho-Akt, phospho-ERK1/2 (1:100; New England BioLabs), S100A4 primary antibody (1:100; Thermo Fisher Scientific Anatomical Pathology, Fremont, CA, USA), and biotinylated goat anti-rabbit secondary antibody (1:100) of a immunohistochemical kit (Santa Cruz Biotechnology) according to the manufacturer's instructions. The specimens were further conjugated with horseradish peroxidase–streptoavidin complex, detected by diaminobenzidine substrate mixture, and counterstained by hematoxylin. A dark-brown diaminobenzidine signal indicated positive staining of phospho-Akt, phospho-ERK1/2, and S100A4 of epithelial cells or fibroblasts, while shades of light blue signified nonreactive cells.

Extravasation of Evans blue dye (Sigma Chemical) into the interstitium was used as a quantitative measure of changes of microvascular permeability in acute lung injury 13. Thirty minutes before the end of mechanical ventilation, 30 mg/kg Evans blue dye was injected through the internal jugular vein. At the time of sacrifice after 5 hours of mechanical ventilation, the lungs were perfused free of blood with 1 ml of 0.9% normal saline via the right ventricle and removed en bloc. Evans blue dye was extracted from lung tissue after homogenization for 2 minutes in 5 ml formamide (Sigma Chemical) and was incubated at 37°C overnight. The supernatant was separated by centrifugation at 5,000 × g for 30 minutes, and the amount was recorded.

Evans blue dye in the plasma and lung tissue was quantitated by dual-wavelength spectrophotometric analysis at 620 nm and 740 nm. The method corrects the specimen's absorbance at 620 nm for the absorbance of contaminating heme pigments, using the following formula: corrected absorbance at 620 = actual absorbance at 620 nm - [1.426 (absorbance at 740) + 0.03]. We calculated the Evans blue dye amount extracted from lung tissue and divided the amount by the weight of lung tissue.

Total RNA (1 μg) was reverse transcribed using a GeneAmp PCR system 9600 (PerkinElmer, Life Sciences, Inc., Boston, MA, USA), as previously described 20. The following primers were used for PCR: type I procollagen, forward primer 5'-TGTGCCACTCTGACTGGAAGA-3' and reverse primer 5'-CAGACGGCTGAGTAGGGAACA-3'; type III procollagen, forward primer 5'-GGAAAGGATGGAGAGTCAGGAA-3' and reverse primer 5'-CATTGCGTCCATCAAAGCCT-3'; and GAPDH (internal control), forward primer 5'-AATGCATCCTGCACCACCAA-3' and reverse primer 5'-GTAGCCATATTCATTGTCATA-3' (Integrated DNA Technologies, Inc., Coralville, IA, USA) 24.

The western blots were quantitated using a National Institutes of Health image analyzer (Image J 1.27z; National Institute of Health, Bethesda, MD, USA) and are presented as the ratio of phospho-MAPK to MAPK or the ratio of phospho-Akt to Akt (relative phosphorylation) in arbitrary units. Values are expressed as the mean ± standard deviation of at least six experiments.

The data for Evans blue dye, hydroxyproline, MIP-2, and IP-10 were analyzed using Statview 5.0 (SAS Institute, Inc., Cary, NC, USA).

All results of the western blot analyses were normalized to control, nonventilated wild-type bleomycin-treated mice breathing room air. Analysis of variance was used to assess the statistical significance of the differences, followed by multiple comparisons with a Scheffe test. P < 0.05 was considered statistically significant.

There were no statistical differences in the pH, the arterial carbon dioxide pressure, and the mean arterial pressure at the beginning versus the end of mechanical ventilation (Table 2). High-tidal-volume ventilation was injurious, with more carbon dioxide production than that of the low-tidal-volume group, and this increased the arterial carbon dioxide pressure in the high-tidal-volume group.

The inhibition of Akt/MAPK activation with Akt-deficient mice and pharmacological inhibitors reduced the high-tidal-volume-induced microvascular permeability, lung fibrosis, and chemokine production.

In a previous study, we have shown that high-tidal-volume ventilation caused more pulmonary edema than in control, nonventilated rats or in rats ventilated at low tidal volume 25. To measure the changes of microvascular permeability in VILI, we used the Evans blue dye assay (Figure 1a). The Evans blue dye levels significantly increased in mice receiving 30 ml/kg V_T mechanical ventilation compared with those either of mice receiving 6 ml/kg V_T or of control, nonventilated mice. The Evans blue dye levels were also significantly increased in 6 ml/kg V_T mice compared with control, nonventilated mice.

In another previous study we showed that normalizing the EBD as nanograms per milligram of lung may have underestimated the amount of Evans blue dye for the high-tidal-volume group, but the data of Evans blue dye not normalized with lung weight showed similar results 26. The data of hydroxyproline not normalized with lung weight showed a similar trend as those normalized with lung weight (control = 15.2 ± 0.9 μg; 6 ml/kg V_T = 24.6 ± 2.8 μg, 30 ml/kg VT = 46.2 ± 1.6 μg, 30 ml/kg VT with PD98059 = 22.1 ± 3.1 μg, 30 ml/kg VT with SB203580 = 23.8 ± 1.7 μg, and 30 ml/kg VT in Akt^+/- mice = 18.4 ± 1.2 μg, all P < 0.05 versus control).

To determine the effects of high-tidal-volume ventilation on pulmonary fibrosis, we measured the lung hydroxyproline content (Figure 1b) and performed Masson's trichrome staining and fibrosis scoring (Figure 2a,b,c,g). Five days after bleomycin treatment, an increase of peribronchiolar fibrosis and of parenchymal fibrosis was found in control nonventilated mice. The extent of fibrosis in mice ventilated at 30 ml/kg V_T was significantly elevated compared with control, nonventilated mice, and compared with mice ventilated at 6 ml/kg V_T. To determine the effects of high-tidal-volume ventilation on pulmonary fibrosis – which were measured by hydroxyproline content and Masson's trichrome staining, and were associated with upregulation of procollagen peptide – we measured type I and type III procollagen mRNA (Figure 3). Increases of type I and type III procollagen mRNA expressions were found in the 30 ml/kg V_T mice compared with those of control, nonventilated mice, of 6 ml/kg V_T mice, or of Akt-deficient mice.

To further define the cells types involved in the lung stretch-induced fibrogenesis, we measured S100A4/FSP1 using immunohistochemistry (Figure 4). The increased positive staining of S100A4/FSP1 in the epithelium of mice ventilated at 30 ml/kg V_T compared with that of control, nonventilated mice and of mice ventilated at 6 ml/kg V_T indicated the presence of a transition from epithelial cells to fibroblasts.

To explore the chemoattractants in VILI we measured MIP-2 and IP-10, involved in angiogenetic activity and angiostatic activity, respectively (Figure 1c,d). Increased production of MIP-2 but reduced production of IP-10 was found in mice ventilated at 30 ml/kg V_T compared with control, nonventilated mice and compared with mice ventilated at 6 ml/kg V_T. The mechanism regulating the increased chemokine production involved in pulmonary fibrosis needs to be further explored.

We have previously shown that high-tidal-volume ventilation induced neutrophil infiltration via MAPK pathways 20. We measured the activity of Akt and three members of the MAPK families – JNKs, p38, and ERK1/2 – to determine the stretch-induced kinase phosphorylation using 6 ml/kg V_T and 30 ml/kg V_T mechanical ventilation (Figure 5). There were dose-dependent increases in phosphorylation of Akt and ERK1/2 but there were no significant changes in the expression of total nonphosphorylated proteins of Akt and ERK1/2. Increased phosphorylation of P38 after either 6 ml/kg V_T or 30 ml/kg V_T mechanical ventilation occurred, but no significant changes in the expression of total nonphosphorylated protein P38 were found. There was no significant difference between 6 ml/kg V_T mice or 30 ml/kg V_T mice.

The phosphorylation of Akt, ERK1/2, and P38 was further increased after 5 hours of mechanical ventilation (relative phosphorylation of Akt: control = 1.0 ± 0.1; 30 ml/kg V_T, 1 hour = 1.4 ± 0.3* and 30 ml/kg V_T, 5 hours = 1.6 ± 0.4*; relative phosphorylation of ERK1/2: control = 1.0 ± 0.2; 30 ml/kg V_T, 1 hour = 1.6 ± 0.2* and 30 ml/kg V_T, 5 hours = 1.7 ± 0.3*; relative phosphorylation of p38: control = 1.0 ± 0.1; 30 ml/kg V_T, 1 hour = 1.2 ± 0.3* and 30 ml/kg V_T, 5 hours = 1.5 ± 0.4*; *P < 0.05 versus control). No significant increases of phosphorylation of JNK were found between control, nonventilated mice and mice ventilated at either 6 ml/kg V_T or 30 ml/kg V_T (control = 1.0 ± 0.2; 6 ml/kg V_T = 1.1 ± 0.3 and 30 ml/kg V_T = 0.9 ± 0.2, P = 0.12 versus control). The roles of Akt, ERK1/2, and P38 in the regulation of high-tidal-volume ventilation during lung fibrosis need to be further explored.

To determine the roles of Akt, EKR1/2, and P38 activation in stretch-induced lung fibrosis, we used Akt-deficient mice and pharmacological inhibitors of ERK1/2 and P38 (Figures 1 to 5). The type III procollagen mRNA expression, the microvascular permeability, the quantitative and qualitative evaluation of pulmonary fibrosis by hydroxyproline assay and Masson's trichrome staining, the positive S100A4/FSP1 staining of epithelium and fibroblasts in the interstitium, and the phosphorylation of Akt and ERK1/2 were significantly reduced after using Akt-deficient mice and pharmacological inhibition with PD98059 and SB203580. Reduced production of MIP-2 but increased production of IP-10 was found after using Akt-deficient mice and pharmacological inhibition with PD98059. Furthermore, PD98059 did not significantly decrease phosphorylation of Akt, but Akt-deficient mice reduced the phosphorylation of ERK1/2, suggesting that the Akt–ERK1/2 pathway was involved in the regulation of stretch-induced pulmonary fibrosis. Pharmacological inhibition with SB203580 significantly reduced the permeability, the lung fibrosis staining of collagen and fibroblasts, the phosphorylation of P38, and MIP-2 production, but not IP-10 production – suggesting that P38 played a less significant role than the Akt–ERK1/2 pathway in the mechanism of high-tidal-volume-induced pulmonary fibrosis.

There were no statistically significant differences between phosphorylation of Akt and ERK1/2 in bleomycin-treated mice exposed to 1 hour of 30 ml/kg V_T mechanical ventilation with or without pretreatment with vehicle (dimethyl sulfoxide) subcutaneously for 30 minutes (relative phosphorylation: control = 1.0 ± 0.1, Akt with vehicle = 1.37 ± 0.13 and Akt without vehicle = 1.42 ± 0.24, both P < 0.05 versus control; and control = 1.0 ± 0.11, ERK1/2 with vehicle = 1.51 ± 0.15 and ERK1/2 without vehicle = 1.64 ± 0.19, both P < 0.05 versus control). Using immunohistochemistry, we confirmed that high-tidal-volume ventilation induced Akt and ERK1/2 activation in bronchial epithelial cells (Figures 6 and 7).

To determine whether the Akt–ERK1/2 pathway was involved in the late fibroproliferative phase of VILI, mice exposed to 10 days of bleomycin administration were ventilated. Similar trends involving the Akt–ERK1/2 signaling pathway were found with higher grades of lung fibrosis (Figure 8).