PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of ajrccmIssue Featuring ArticlePublisher's Version of ArticleSubmissionsAmerican Thoracic SocietyAmerican Thoracic SocietyAmerican Journal of Respiratory and Critical Care Medicine
 
Am J Respir Crit Care Med. 2007 December 15; 176(12): 1222–1235.
Published online 2007 September 27. doi:  10.1164/rccm.200701-060OC
PMCID: PMC2176106

Genetic and Pharmacologic Evidence Links Oxidative Stress to Ventilator-induced Lung Injury in Mice

Abstract

Rationale: Mechanical ventilation (MV) is an indispensable therapy for critically ill patients with acute lung injury and the adult respiratory distress syndrome. However, the mechanisms by which conventional MV induces lung injury remain unclear.

Objectives: We hypothesized that disruption of the gene encoding Nrf2, a transcription factor that regulates the induction of several antioxidant enzymes, enhances susceptibility to ventilator-induced lung injury (VILI) and that antioxidant supplementation attenuates this effect.

Methods: To test our hypothesis and to examine the relevance of oxidative stress in VILI, we assessed lung injury and inflammatory responses in Nrf2-deficient (Nrf2−/−) mice and wild-type (Nrf2+/+) mice after an acute (2-h) injurious model of MV with or without administration of antioxidant.

Measurements and Main Results: Nrf2−/− mice displayed greater levels of lung alveolar and vascular permeability and inflammatory responses to MV as compared with Nrf2+/+ mice. Nrf2 deficiency enhances the levels of several proinflammatory cytokines implicated in the pathogenesis of VILI. We found diminished levels of critical antioxidant enzymes and redox imbalance by MV in the lungs of Nrf2−/− mice; however, antioxidant supplementation to Nrf2−/− mice remarkably attenuated VILI. When subjected to a clinically relevant prolong period of MV, Nrf2−/− mice displayed greater levels of VILI than Nrf2+/+ mice. Expression profiling revealed lack of induction of several VILI genes, stress response and solute carrier proteins, and phosphatases in Nrf2−/− mice.

Conclusions: Our data demonstrate for the first time a critical role for Nrf2 in VILI, which confers protection against cellular responses induced by MV by modulating oxidative stress.

Keywords: acute lung injury, antioxidant enzymes, mechanical ventilation, Nrf2, inflammation

AT A GLANCE COMMENTARY

Scientific Knowledge on the Subject

The mechanisms by which mechanical ventilation contributes to lung injury are not well understood.

What This Study Adds to this Field

This study suggests that the pathogenesis of ventilator-associated lung injury involves redox imbalance in the lungs and that the transcription factor Nrf2 has a key protective role by reducing oxidative stress.

The adult respiratory distress syndrome (ARDS) and its less severe form, acute lung injury (ALI), have an average incidence of 150,000 cases per year and carry substantial mortality ranging from 30 to 40% (1). Although mortality has improved over the past few years, patients with ALI/ARDS are faced with an increased risk of death, and survivors are left with significant decrements in physical function. Mechanical ventilation (MV) is the only known effective and cornerstone therapy used to support patients with ALI or ARDS. A lung-protective strategy of MV at low Vt (6 ml/kg) showed a beneficial effect in reducing mortality in patients with ALI/ARDS compared with conventional high Vt (HVt) (12 ml/kg), suggesting that mechanical and shear forces generated by HVt can enhance or perpetuate ventilator-induced lung injury (VILI) (2). Conventional MV at HVt causes excessive alveolar distention, resulting in lung injury and increased pulmonary vascular permeability and in an increase in the production of proinflammatory mediators (3). In vitro studies, using cyclic stretch that mimics the physical forces of MV, demonstrated that endothelial and epithelial cell deformation and physical disruption of plasma membrane integrity lead to alteration in the structure and function of these essential components of the alveolar–capillary membrane, along with activation of proinflammatory and prooxidant pathways (47). These events may serve to initiate and/or potentiate an inflammatory response, leading to a vicious cycle of inflammation locally or systemically, ultimately resulting in multisystem organ dysfunction and death (8). Cytokines and chemokines are potential effector molecules that modulate and regulate VILI (8). The accumulation of neutrophils in the lung tissue can also lead to enhanced levels of inflammatory cytokines, which play fundamental roles in the development of lung pathogenesis, including VILI (9, 10). Recent expression profiling using injurious and noninjurious MV in the presence and absence of inflammatory stimuli revealed the involvement of a complex network of genes encoding for matrix remodeling proteins, proinflammatory cytokines, and various transcription factors in VILI (1115). The exact molecular mechanisms controlling the expression levels of these genes in response to MV and their contribution to the development or susceptibility to VILI are unclear. Thus, further deciphering the mechanisms by which conventional MV exacerbates lung injury and inflammation is of considerable clinical significance, not only in alleviating the side effects of mechanical forces but also for the development of new therapeutic strategies.

Production of high levels of reactive oxygen species (ROS) and reactive nitrogen species is a condition that generally leads to oxidative stress. The induction of cytoprotective antioxidant enzymes in response to injurious insults or stressful stimuli is critical for cellular detoxification of ROS and reactive nitrogen species and in the maintenance of homeostasis of cellular redox. Emerging experimental evidence from animal models suggests that oxidative stress contributes to and/or exacerbates various clinical syndromes, such as ARDS, sepsis, shock, major surgery, and ALI (1618). However, the exact relevance of redox imbalance to the onset of MV-induced lung injury in vivo remains unclear. Recent studies have shown that cyclic stretch associated with conventional MV at HVt generates ROS and redox imbalance in lung epithelial and endothelial cells in vitro (1921). Consistent with the in vitro studies are a decrease in antioxidant activity in the lungs and enhanced levels of oxidative stress plasma markers, such as thiobarbituric reactive substances and malondialdehyde, that are observed in response to MV in vivo (22, 23). Based on these observations, we hypothesized that redox imbalance caused by conventional MV might initiate and/or amplify lung cellular injury and inflammatory responses, which promote and/or perpetuate the pathogenesis of VILI.

Nrf2 is a transcription factor and regulates cellular redox status via the antioxidant response element (24). Genetic disruption of Nrf2 enhances susceptibility to various experimental murine models of lung diseases produced by prooxidants (2528), mainly due to decreased levels of the basal and inducible expression of several critical antioxidant enzymes. Some of the critical enzymes include glutamate–cysteine ligase catalytic subunit (Gclc), glutamate cysteine ligase modifier subunit (Gclm), glutathione peroxidase 2 (Gpx2), and hemeoxygenase 1 (24). To test our hypothesis and to determine the contribution of oxidative stress to the pathogenesis of VILI in vivo, we compared lung injury and inflammatory responses in mice with disruption of the Nrf2 gene (Nrf2−/−) versus wild-type mice in response to injurious MV. Here we demonstrate that disruption of Nrf2 enhances susceptibility to MV-induced lung injury and inflammation and elevates levels of proinflammatory cytokine expression. We found diminished levels of antioxidant enzyme expression and redox imbalance in the lungs of Nrf2−/− mice subjected to MV as compared with wild-type animals. Finally, antioxidant supplementation attenuated MV-induced lung injury and inflammation in both genotypes, supporting a role for oxidative stress in the pathogenesis of VILI. Results from this work have previously been published in abstract form (29).

METHODS

Reagents

The Nrf2 wild-type (Nrf2+/+) and Nrf2-deficient (Nrf2−/−) CD-1/ICR strains of mice were generated as described (40). Mice were fed with normal diet and water ad libitum and were housed under controlled conditions (25 ± 2°C; 12-h light:dark periods). All experimental animal protocols were performed in accordance with guidelines approved by the animal care use committee at the Johns Hopkins University Bloomberg School of Public Health.

MV

Exposure to MV was performed as previously described (3032). Briefly, 6- to 8-week-old male mice (25–30 g) were used for these studies unless otherwise indicated. Mice were anesthetized with pentobarbital (intraperitoneally, 80 mg/kg body weight), and additional anesthetic was supplemented regularly at approximately 20 mg/kg per hour for maintaining the plane of anesthesia during the experiment period. Rectal temperature was continuously monitored and maintained at 37 ± 1°C. A neck midline incision was performed for exposure of the trachea to facilitate endotracheal intubation with a 20-gauge, 1-inch-long catheter (Johnson and Johnson, New Brunswick, NJ). The animals were subjected to MV (Model Inspira asv 55-7058; Harvard Apparatus, Boston, MA) with low Vt (LVt) and HVt at 12 ml/kg and 30 ml/kg, respectively, for 2 hours. The animals subjected to spontaneous ventilation (SpV) or MV with LVt (12 ml/kg) for 4 hours were supplied with phosphate-buffered saline (PBS) (1 μl/mg body weight) at 30-minute intervals.

The respiratory rate was set at 80 to 140 breaths per minute depending on Vt. The adequacy of MV settings on gas exchange was confirmed in preliminary experiments in which arterial blood gases obtained via catheterization of a femoral artery were analyzed by an automated blood gas analyzer (model 348 Blood Gas Analyzer; Chiron Diagnostics; Norwood, MA) as previously described (31, 32). Arterial blood gas analysis revealed stable levels of arterial oxygen (Pao2 of 55–90 mm Hg) and carbon dioxide (Paco2 of 30–50 mm Hg) (Table 1). Sham-operated, anesthetized, and SpV mice were used as control groups. Animals were given a lethal dose of the anesthetic agent before the lungs were harvested.

TABLE 1.
DIFFERENTIAL EXPRESSION PROFILING OF GENES ALTERED BY MECHANICAL VENTILATION IN THE LUNGS OF Nrf2-DEFICIENT (Nrf2−/−) AND WILD-TYPE (Nrf2+/+) MICE

Assessment of Lung Injury and Inflammation in Bronchoalveolar Lavage Fluid

Lung injury was assessed by alveolar and microvascular permeability. Lung inflammation was evaluated by differential cell counts in bronchoalveolar lavage fluid (BALF) as previously described (25). Lungs were instilled with 1.5 ml of sterile PBS, and cells in the BALF were counted using a hemocytometer. Differential cell counts were assessed with Diff-Quik stain set (Dade Behring, Inc., Newark, DE). The remainder of the BALF was centrifuged, and the supernatant was stored at −80°C. BALF protein concentration was measured by BCA Protein Assay kit (Pierce Chemical Co., Rockford, IL). Lung vascular permeability was determined using Evans blue injections as previously detailed (3032). Extravasated Evans blue concentration in lung homogenates was calculated against a standard curve and expressed as micrograms of Evans blue per milligram of lung tissue as previously described (3032).

Mouse Albumin ELISA

The albumin concentration in the BALF was determined by ELISA (E90-134; Bethyl Laboratories, Montgomery, TX) using a protocol recommended by the manufacturer.

Measurement of Reduced and Oxidized Glutathione Levels

A Bioxytech GSH/GSSG-412 kit (Oxis Health Products, Portland, OR) was used to measure reduced and oxidized glutathione in the lung homogenates as per the manufacturer's instructions and as previously described (21).

Supplementation of Antioxidant

To assess the effects of antioxidants on MV-induced pulmonary and vascular permeability and inflammation, mice received in some experiments a single intraperitoneal dose of the antioxidant N-acetyl-l-cysteine (NAC) (100 mg/kg body weight) or a similar volume of vehicle (PBS) 60 minutes before exposure to MV.

Immunohistochemical Analysis

At the end of 2 hours of MV, lungs were inflated to 25 cm of water pressure with 0.5% of low-melting agarose with 10% buffered formalin. Lungs were harvested and stored in 10% formalin for 24 hours at 4°C before embedding in paraffin. Sections (5-μm) were cut and stained with hematoxylin and eosin. For identification of neutrophils, deparaffinized tissue sections were incubated with rat antimouse neutrophil monoclonal antibody (Serotec, Raleigh, NC) followed by biotinylated antirat secondary antibody and horseradish peroxidase–streptavidin complex. Lung sections were acquired with a Nikon E 800 microscope (Nikon, Melville, NY). The number of neutrophils in the lung sections (n = 5 per group and 15 fields per lung section per animal) were counted manually by an investigator blinded to the various groups using a grid system. The number of neutrophils was normalized per field for each animal and expressed as mean ± SE as previously described (30).

Western Blot Analysis

Right lung tissues were homogenized, and the protein concentration of cytosolic fraction of each sample was estimated using BCA reagent (Pierce) and stored at −70°C. Equal amounts of protein were separated and immunoblotted with specific antibodies as indicated. The blots were visualized with the ECL Western blot detection system. Autoradiogram signals were quantified by densitometry scanning (Molecular Dynamics, Sunnyvale, CA), and values were normalized to those of housekeeping gene β-actin and Hsp70.

Gene Expression Profiling

Nrf2+/+ and Nrf2−/− mice were subjected to MV (HVt) or SpV for 2 hours. Lungs were immediately removed and processed for total RNA isolation using TRIzol reagent (LifeTechnologies, Grand Island, NY). The isolated RNA was applied to Murine Genome UoA GeneChip arrays (Affymetrix, Santa Clara, CA), which contain probes for detecting approximately 14,500 well characterized genes and 4,371 expressed sequence tags according to standard microarray protocol. Scanned output files were analyzed by using Affymetrix GeneChip Operating Software and were independently normalized to an average intensity of 500. Further analyses were done by performing nine pairwise comparisons for each group (Nrf2+/+ SpV [n = 3] vs. Nrf2+/+ MV [n = 3]; Nrf2−/− SpV [n = 3], Nrf2−/− MV [n = 3], and Nrf2+/+ SpV [n = 3] vs. Nrf2−/− SpV [n = 3]). To minimize the number of false positives, only altered genes that showed a change of [gt-or-equal, slanted]1.5-fold and appeared in at least six of the nine comparisons were selected as described previously (27, 28). In addition, the Mann-Whitney pairwise comparison test was performed to rank the results by concordance as an indication of the significance (P < 0.05) of each identified change in gene expression.

Real-Time Polymerase Chain Reaction

The mRNA levels of genes encoding mouse Gpx2, Gclc, Gclm, IL-6, amphiregulin, early growth response gene (Egr)-1, glyceraldehyde-3-phosphate dehydrogenase (Gapdh), and actin-beta (β-actin) in the lungs of mice (n = 3–4 per group) were quantified in triplicate by TaqMan gene expression assays according to the supplier's recommendations (Applied Biosystems, Foster City, CA) as previously described (21). The absolute values for each gene were normalized to that of Gapdh/β-actin.

Measurement of Cytokines in the Lung Tissue

Lung tissues were homogenized at 4°C in cell lysis buffer (50 mM Tris HCl [pH 7.4], 150 mM NaCl, 1% Nonidet-P40, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO5, 1 mM PMSF) and 1X protease inhibitor cocktail (Cat. #P8340; Sigma Chemical Co., St. Louis, MO) using a Brinkman polytron. The homogenates were centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatants were collected, and protein concentration was determined by BCA method (Pierce Biochemicals, Rockford, IL). Up to 100 μg of total protein was used for the cytokine multiplex bead-based immunoassay kit as per the manufacturer's recommendations (Bio-Rad Laboratories, Inc., Hercules, CA). Each supernatant sample was incubated with a mixture of all microbead types for 90 minutes at room temperature. Samples were then washed, incubated with a mixture of secondary biotinylated antibodies directed against each target for 30 minutes at room temperature, washed again, and incubated with a streptavidin-coupled phycoerythrin reporter system for 10 minutes at room temperature. After a final wash, the samples were suspended in buffer and subjected to flow-cytometric analysis. The data were analyzed using Bio-Plex Manager 3.0 software (Bio-Rad Laboratories, Inc.). For measurement of macrophage inflammatory protein (MIP)-2, extracts of lung tissue prepared as described previously were analyzed with a commercially available ELISA kit for mouse MIP-2 according to the manufacturer's suggestions (R&D Systems, Minneapolis, MN).

Statistical Analysis

All data involving animal experimentation were collected in a double-blind fashion. Data were expressed as the mean ± SEM (n = 3–5 for each condition). Analysis of variance was used to compare means of multiple groups. For paired data, Student's t test was used. Significance in all cases was defined as P [less-than-or-eq, slant] 0.05.

RESULTS

Nrf2 Deficiency Enhances MV-induced Lung Permeability and Neutrophil Infiltration

One of the objectives of this study was to determine whether oxidative stress associated with conventional MV alone contributed to and/or exacerbated VILI and, if so, whether antioxidant supplementation could rescue these phenotypic effects. To examine the role of oxidative stress modifier Nrf2 in VILI, wild-type (Nrf2+/+) and Nrf2−/− mice were subjected to MV at LVt (12 ml/kg) or HVt (30 ml/kg) for 2 hours. Sham-operated, anesthetized, and SpV Nrf2+/+ and Nrf2−/− mice were used as respective control groups. We found no trend differences in peak airway pressures between the wild-type and Nrf2−/− mice subjected to LVt or HVt (data not shown). There was a small increase in airway pressure (average of 1 and 3 cm H2O for LVt and HVt, respectively) over time (at 60 and 120 min) compared with baseline (time 0) value in both genotypes. Arterial blood gas analysis revealed no significant differences in the levels of arterial oxygen (Pao2 of 55–90 mm Hg) and carbon dioxide (Paco2 of 30–50 mm Hg) (data not shown). We have previously reported no significant differences in alveolar diameter, alveolar cell proliferation, ultrastructural organization, and total lung capacity between Nrf2+/+ mice and Nrf2−/− mice (27).

The effects MV on lung alveolar permeability and neutrophil accumulation in BALF were compared in both genotypes subjected to LVt and HVt (Figure 1). There was no difference in BALF protein levels in Nrf2+/+ and Nrf2−/− mice in response to LVt as compared with sham-operated (SpV) control groups (Figure 1A). However, we found statistically significant effects of MV for total protein accumulation in the BALF of wild-type and Nrf2−/− mice as compared with the respective SpV control groups (P < 0.05). We also measured the concentration of albumin, as a measure of vascular leak, in the BALF. We did not find significant differences in serum albumin concentration between the two groups (data not shown).

Figure 1.
Effect of mechanical ventilation (MV) on lung injury and inflammatory responses in Nrf2−/− mice. (A) Mice (n = 3–5 per group) were subjected to MV at low Vt (LVt, 12 ml/kg) and high Vt (HVt, 30 ml/kg) for 2 hours and allowed ...

This assay revealed significant differences in the amount of albumin content in BALF between the two genotypes in response to HVt but not to LVt. Compared with genotype-matched SpV controls, HVt caused a statistically significant increase in mean albumin concentration in Nrf2+/+ mice (25.7 ± 3 vs. 20.4 ± 2.0 μg/mg in HVt compared with the SpV group; P < 0.04) and Nrf2−/− mice (32.4 ± 1.29 vs. 21.04 ± 2.0 μg/mg in HVt compared with the SpV group; P < 0.001). However, the amount of albumin found in the BALF of Nrf2−/− mice was significantly higher than that in Nrf2+/+ mice subjected to HVt (32.4 ± 1.29 vs. 25.7 ± 3, respectively; P < 0.001) (Figure 1B).

Differential cell count analysis revealed a significantly higher percentage of neutrophils in the BALF of Nrf2−/− mice (21%) than wild-type animals (11%) (Figure 1C) after HVt. We also found significantly higher levels of neutrophils in Nrf2−/− mice subjected to MV at LVt (8%) as compared with mice subjected to SpV (3%). No appreciable change in the levels of neutrophils was found in wild-type animals in response to LVt (2%) as compared with SpV (1%). These results demonstrate that disruption of Nrf2 enhances susceptibility to MV-induced lung injury and inflammation.

Disruption of Nrf2 Augments MV-induced Lung Vascular Leak and Neutrophil Infiltration

Lung vascular leak and leukocyte infiltration is one of the hallmarks of ARDS. To assess the effects of MV on lung leukocyte infiltration, mice were subjected to HVt and SpV, after which lungs were fixed with formalin and embedded in paraffin. Tissue sections were stained with antineutrophil antibody (Figure 2A), and the number of neutrophils present in the alveolar septa was quantified (Figure 2B) as detailed in Methods. Immunohistochemical analysis showed no statistically significant difference in the number of neutrophils in both genotypes subjected to SpV (Figures 2A and 2B). Although there was a statistically significant increased number of neutrophils present in the lungs of Nrf2+/+ mice subjected to HVt (10.4 ± 1.9 cells) compared with the SpV control group (6.57 ± 0.82 cells), Nrf2−/− mice subjected to HVt had greater number of neutrophils compared with the SpV control group (16.7 ± 2.6 vs. 8.2 ± 0.7 cells, respectively; P < 0.01). There was a greater number of neutrophils (P < 0.04) in the lungs of Nrf2−/− mice than in Nrf2+/+ mice in response to MV.

Figure 2.Figure 2.Figure 2.
Effect of mechanical ventilation (MV) on pulmonary neutrophil infiltration and vascular protein leakage in Nrf2−/− mice. (A) Assessment of neutrophil infiltration in lung parenchyma of Nrf2+/+ and Nrf2−/− ...

To assess whether Nrf2 deficiency enhances susceptibility to vascular leak in response to MV, a separate group of Nrf2+/+ and Nrf2−/− mice was subjected to HVt, and the results were compared with those of respective SpV control groups. We assessed lung vascular permeability using the Evans blue dye technique as previously described (3032). Exposure to MV at LVt caused no significant increase in extravasation of Evans blue in both genotypes compared with the respective SpV control groups (results not shown). However, exposure to HVt caused significant accumulation of Evans blue in the lungs of Nrf2+/+ (0.366 ± 0.074 vs. 0.172 ± 0.03 μg/mg in HVt compared with the SpV group, respectively; P < 0.02) and Nrf2−/− mice (0.428 ± 0.056 vs. 0.191 ± 0.06 μg/mg in HVt compared with the SpV group, respectively; P < 0.001) (Figure 2C). Although there was no significant difference between these genotypes exposed to HVt, there was a trend toward greater levels of Evans blue accumulation in the lungs of Nrf2−/− mice as compared with Nrf2+/+ mice. Taken together, these results demonstrate that deficiency of the oxidative stress modifier Nrf2 may greatly enhance susceptibility to VILI.

Nrf2 Deficiency Augments MV-induced Lung Proinflammatory Cytokines Expression

Cytokines and chemokines are potential effector molecules that modulate and regulate VILI (8). We therefore assessed the levels of various cytokines present in the lungs of Nrf2+/+ and Nrf2−/− mice subjected to HVt using multiplex cytokine assays as detailed in Methods. Sham-operated (SpV) respective control groups were used for basal level expression. Cytokine assays revealed high levels of IL-6, KC (keratinocyte-derived chemokine), MIP-2, granulocyte colony-stimulating factor (G-CSF), IL-1β, monocyte chemotactic protein–1, IL-12 (p70), and MIP-1α cytokines in the lung tissues of mice subjected to HVt as compared with SpV control in both genotypes (Figure 3). However, we found significantly greater levels of IL-6, KC, and G-CSF (Figure 3A) in the lungs of Nrf2−/− compared with Nrf2+/+ mice (Figure 3A). MV-induced levels of IL-12 (p40) were almost 3-fold higher in Nrf2−/− mice and were only modestly altered in Nrf2+/+ mice (Figure 3A). In contrast, IL-1β, monocyte chemotactic protein–1, MIP-1α, and MIP-2 levels did not differ significantly between the two genotypes (Figure 3B). There were no statistical differences in the levels of IL-3, IL-4, IFN-γ, and IL-12 (p70) cytokines (data not shown) in either genotype in response to MV as compared with their respective SpV control groups. These experiments suggest that elevated levels of IL-6, KC, IL-12 (p40), and G-CSF may play a role in perpetuating VILI in Nrf2−/− mice.

Figure 3.
Effect of mechanical ventilation (MV) on cytokine expression in the lungs of Nrf2+/+ and Nrf2−/− mice. Lung tissues of Nrf2+/+ and Nrf2−/− mice subjected to spontaneous ventilation (SpV) ...

MV Induces Redox Imbalance in the Lungs of Nrf2−/− Mice

Oxidant and antioxidant (redox) imbalance seems to regulate the pathogenesis of various lung diseases (17). Several studies, including ours, recently demonstrated that exposure to cyclic stretch causes reactive oxygen species (ROS) generation in a magnitude-dependent manner in lung epithelial cells, suggesting a role for redox imbalance in VILI (1921). Therefore, we examined whether Nrf2 deficiency leads to redox imbalance, thereby resulting in an enhanced susceptibility to VILI. To address this aspect, we first assessed the effects of MV on antioxidant enzyme (mRNA and protein) expression in the lungs of Nrf2+/+ and Nrf2−/− mice subjected to HVt or sham-operated conditions (Figure 4A). Total lung RNA was isolated, and the expression of Gclc, Gclm, and Gpx2 was determined by quantitative real-time polymerase chain reaction. We chose these antioxidant enzymes because they are well characterized prototropic targets of Nrf2 and play key roles in maintaining cellular redox balance (24). Constitutive expression of these enzymes in sham-treated Nrf2−/− mice was lower than in their counterpart Nrf2+/+ mice (Figure 4A). MV significantly increased the mRNA expression of Gclc, Gclm, and GPx2 in the Nrf2+/+ mice over basal levels (Figure 4A). In contrast, the inducible expression of these enzymes in Nrf2−/− mice subjected to MV was considerably lower than in their counterpart wild-type animals.

Figure 4.Figure 4.Figure 4.
Effects of mechanical ventilation (MV) on antioxidant gene expression and reduced glutathione/oxidized glutathione (GSH/GSSG) levels. (A) Mice were subjected to spontaneous breathing (sham-treated) or MV at HVt for 2 hours, and total RNA and protein were ...

In a separate group of animals, total lung protein was isolated from the lungs of mice subjected to MV or SpV. An equal amount of protein was used for immunoblot analysis with antibodies specific for glutathione-S-transferase (GST) isoenzymes GSTa1, GSTa2, and GSTm1. Constitutive expression of these enzymes was significantly lower in the lungs of Nrf2−/− mice than in Nrf2+/+ mice (Figure 4B). The levels of GSTa1, GSTa2, and GSTm1 were not significantly altered by MV in both genotypes (Figure 4C). The total GST (Figure 4D) and GPx activity (Figure 4E) in the lungs of Nrf2+/+ mice subjected to air or MV (72 h) was significantly higher (1.6- to ~2.2-fold) compared with Nrf2−/− mice. These results suggest that MV induces redox imbalance in the lungs of Nrf2−/− mice. To confirm this notion, we analyzed the levels of reduced glutathione (GSH) and oxidized glutathione (GSSG) as a measure of the antioxidant imbalance, in lung tissue homogenates of mice subjected to HVt and sham-operated conditions (Figure 4F). In response to MV stimulus, there was a significant increase in the levels of GSH oxidation (i.e., a decrease of GSH/GSSG ratio) in the lungs of Nrf2−/− mice subjected to MV (bar 4) compared with the SpV control group (bar 2) (Figure 4F). In contrast, there was no significant change in GSH/GSSG ratio in wild-type animals subjected to MV.

Antioxidant Supplementation Attenuates VILI in Mice

A decrease in GSH/GSSG ratio and lack of a significant induction of Nrf2-dependent cytoprotective response suggested that redox imbalance may contribute to the greater levels of lung injury and inflammatory response in Nrf2−/− mice. To validate this notion, we investigated whether exogenous antioxidant supplementation could provide protection against MV-induced lung injury and inflammation in Nrf2−/− mice. Nrf2−/− mice were supplemented with NAC 60 minutes before MV exposure. PBS was used as vehicle control. After NAC supplementation, mice were randomly assigned to SpV or MV with HVt for 2 hours. Lung inflammation and injury were analyzed as detailed previously. MV induced an increase in total number of cells (Figure 5A) and neutrophils (Figure 5B) in the BALF of vehicle-treated groups. However, NAC therapy before MV greatly attenuated MV-induced lung inflammatory responses in Nrf2−/− and wild-type animals. There was no significant effect of NAC, as compared with PBS (vehicle), on neutrophil influx in mice subjected to SpV. The dosage of NAC in these experiments, which is three times higher than that used clinically, may have some unwarranted nonspecific effects, such as autooxidation.

Figure 5.
Effects of exogenous antioxidant on mechanical ventilation (MV)-induced lung inflammation and injury. (A and B) Wild-type and Nrf2−/− mice were pretreated for 30 minutes with N-acetyl-l-cysteine (NAC) (100 mg/kg body weight) before MV. ...

We also examined the effect of NAC on MV-induced pulmonary vascular leak as detailed previously. Supplementation of NAC had a significant effect on MV-induced albumin leak in BALF (Figure 5C) and pulmonary extravasation of Evans blue (Figure 5D) in both genotypes as compared with vehicle-treated groups. Taken together, these results strongly support that oxidative stress regulates the pathogenesis of VILI.

Nrf2−/− Mice Subjected to Prolonged MV with LVt Have Greater Susceptibility than Wild-Type Mice

Our results indicate that Nrf2−/− mice subjected to HVt for 2 hours display greater levels of lung injury and inflammation as compared with wild-type mice when exposed to MV. To determine whether disruption of Nrf2 enhances susceptibility to clinically relevant tidal volumes for longer ventilatory periods, Nrf2+/+ and Nrf2−/− mice were subjected to LVt and SpV for 4 hours, after which lungs were fixed with formalin and embedded in paraffin or used for collection of BALF for differential cell counts and protein estimation. Compared with genotype-matched SpV controls, a longer period of MV at LVt caused a statistically significant increase in mean protein accumulation in Nrf2+/+ and Nrf2−/− mice. However, the amount of protein found in the BALF of Nrf2−/− mice subjected to LVt was higher than that in Nrf2+/+ mice subjected to identical Vt (P < 0.06) (Figure 6A). Differential cell count analysis revealed significantly higher levels of neutrophils in Nrf2−/− and Nrf2+/+ mice subjected to MV at LVt as compared with respective SpV control groups. We found significantly higher number of neutrophils in the BALF of Nrf2−/− mice than Nrf2+/+ mice (Figure 6B; P < 0.0001) after LVt. Longer periods of MV at LVt markedly increased total cells and macrophages in BALF of Nrf2+/+ and Nrf2−/− mice, but no significant differences were noted between Nrf2+/+ mice and Nrf2−/− mice. To assess the effects of MV on neutrophil infiltration, lung tissue sections were stained with antineutrophil antibody, and the number of neutrophils present in the alveolar septa was quantified as in Figure 2B. The analysis revealed no statistically significant difference in the number of neutrophils in both genotypes subjected to SpV (Figure 6E). There was a significant increase in the number of neutrophils present in the lungs of Nrf2+/+ mice (17 vs. 11 cells; P < 0.001) and Nrf2−/− mice (22 vs. 14 cells, respectively; P < 0.004) subjected to LVt as compared with the respective SpV control groups. However, Nrf2−/− mice had significantly more neutrophils compared with Nrf2+/+ mice subjected to LVt (22 vs.17 cells; P < 0.002).

Figure 6.Figure 6.
Lung injury and inflammatory response of Nrf2−/− and Nrf2+/+ mice subjected to mechanical ventilation (MV) at low Vt (LVt) for 4 hours. (A) Mice (n = 5–6 mice per group) were subjected to MV at LVt or spontaneous ...

We assessed vascular leakage in a separate group of Nrf2+/+ and Nrf2−/− mice subjected to LVt for 4 hours, and the results were compared with those of respective SpV control groups. MV at LVt for 4 hours caused a significant increase in extravasation of Evans blue in both genotypes compared with their respective SpV control groups (Figure 6F). However, exposure to LVt caused greater accumulation of Evans blue in lungs of Nrf2−/− mice compared with Nrf2+/+ mice (P < 0.05). There was no significant difference between these genotypes exposed to SpV. Taken together, these results demonstrate that a longer period (4 h) of MV at LVt, in contrast to a shorter period (2 h), induces lung injury and inflammation and that the lack of Nrf2 transcriptional response further enhances the severity of VILI.

Expression Profiling Genes Regulated by Nrf2 in Response to MV

To further define the mechanisms by which Nrf2 modulates MV-induced inflammatory responses, global gene expression profiles were examined in the lungs of Nrf2+/+ and Nrf2−/− mice subjected to MV at HVt or SpV for 2 hours. The full list of genes that were up- or down-regulated by MV in the lungs of Nrf2+/+ and Nrf2−/− mice (see Tables E1–E4 in the on-line supplement) and validation real time polymerase chain reaction data for some of these genes (Figure E1) is presented in the online supplement. Expression profiling revealed 197 up-regulated (Table E1) and 70 down-regulated genes (Table E2) in the lungs of Nrf2+/+ mice and 193 up-regulated (Table E3) and 85 down-regulated genes (Table E4) in the lungs of Nrf2−/− mice after MV. These genes were further analyzed to dissect out the Nrf2-regulated transcriptional programs and the differences in the expression profiles between Nrf2+/+ and Nrf2−/− mice that are known to be altered by MV and associated with ventilator-induced lung injury/ventilator-associated lung injury (VILI/VALI), as shown in Table 1. The expression profiles of genes encoding several redox regulators, stress response proteins, transcription factors, kinases, phosphatases, and solute transporter proteins are also shown in Table 1.

VALI genes.

Our results, consistent with previous reports (11, 15, 3335), demonstrated an inducible expression of a majority of VALI genes in wild-type (Nrf2+/+) mice (Table 1); however, we found a lack of induction of several of these genes in the lungs of Nrf2−/− mice. For example, several genes that modulate tissue matrix remodeling and blood coagulation were significantly induced in Nrf2+/+ mice but not in Nrf2−/− mice. These genes include Adamts1, Thbs1, Plaur, coagulation factor III (F3), Cyr61, and proteinase inhibitors Serpine 1 and Timp3. MV markedly induced the expression of Ptgs2 (Cox2), but no significant difference in the inducible levels of this enzyme between the two genotypes was observed. Although MV significantly induced the levels of amphiregulin, an EGFR ligand, in both genotypes, the induction was markedly lower in Nrf2−/− mice as compared with Nrf2+/+ mice. VALI genes that regulate lung inflammation, such as Tnsfr12a, IL-6, Cxcl1, and Socs3, were elevated by MV in Nrf2+/+ mice but not in Nrf2−/− mice. The mRNA levels of these genes were not altered by MV in Nrf2−/− mice. On the contrary, we found that Il-1β and Cxcl2 were down-regulated by MV in Nrf2−/− mice. We also found diminished levels of Tnfrsf19 and Traf1 in Nrf2+/+ mice in response to MV. Members of the IFN family were distinctly altered by MV in wild-type and Nrf2−/− mice. MV strongly induced expression of IFN-related developmental regulator 1 (Irf1), whereas that of IFN-induced protein with tetratricopeptide repeats 2 (Ifit2) and IFN-inducible GTPase 1 (Iigp1) was down-regulated in wild-type mice. In contrast, we found elevated levels of IFN-activated gene 203 (Ifit203) in Nrf2−/− mice exposed to MV but not in Nrf2+/+ mice.

Transcription factors.

Several genes that regulate transcription, such as Atf3, Cebpd, Egr1, Egr2, Fos, Gadd 45γ, Nr4a1, Nr4a2, Srf, and Maff, were significantly induced in Nrf2+/+ mice, but their induction is very low or not induced significantly above basal levels in the lungs of Nrf2−/− mice. The inducible levels of Ppargc1α expression did not differ significantly between Nrf2+/+ and Nrf2−/− mice. MV significantly induces the expression of several other transcription factors that have not been directly linked to VILI. The expression levels of Fosb, Klf6, Klf7, Trib1, and Smad1 were significantly induced in Nrf2+/+ but not in Nrf2−/− mice. In contrast, MV down-regulated the expression levels of Sox7, Sox18, and Stat5b levels in Nrf2+/+ mice, but these levels were unchanged in Nrf2−/− mice.

Stress response genes.

Nrf2 regulates the expression of several antioxidant genes. We found markedly lower expression of Gst family members in the lungs of Nrf2−/− mice than in Nrf2+/+ mice in SpV control groups. The expression of these genes was not altered significantly in Nrf2+/+ mice after MV challenge. However, the level of Gclc and Txrdn1 expression was significantly higher in Nrf2+/+ mice than in Nrf2−/− mice after MV exposure. The expression levels of members of the NADPH oxidase family, such as neutrophil cytosolic factor-2 (Ncf2 or p67-phox), neutrophil cytosolic factor-4 (Ncf4 or p47-phox), and Nox4 (NADPH oxidase), were significantly lower in the lungs of Nrf2−/− mice after MV. The expression levels of these genes were unchanged in the lungs of Nrf2+/+ mice in response to MV. Metallothionenin-1 and -2 gene expression was markedly up-regulated by MV in Nrf2+/+ but not in Nrf2−/− mice.

We found down-regulation of expression of several heat shock protein (Hsp) family members in the lungs of Nrf2−/− mice after MV. These genes include Hapa1a (Hsp70 kD protein 1a), Hspa1b (Hsp70 kD protein 1b), Hspa8 (Hsp70 kD protein 8), Dnaja1 (Hsp40 kD protein 4), and Dnajb1 (Hsp40 kD protein 4). Carhsp1 (calcium-regulated heat-stable protein of 24 kD) was markedly down-regulated in the lungs of Nrf2−/− mice. In contrast, MV down-regulated expression levels of Hsp105 and Hspa12b in Nrf2+/+ mice but not in Nrf2−/− mice.

Signal transducers.

Kinases and phosphatases play key roles in the modulation of signal transduction induced by external stimuli, such as MV. These genes were differentially expressed between wild-type and Nrf2−/− mice after MV (Table 1). Dual-specificity phosphate (Dusp)-1, Dusp8, and Dusp16 were up-regulated by MV only in wild-type mice. Likewise, the expression of protein tyrosine phosphatase 4a1 (Ptp4a1) and protein tyrosine phosphatase, nonreceptor type 2 (Ptpn2) was significantly induced in Nrf2+/+ mice. The mRNA expression of various kinases, such as hexokinase (Hk2), pyruvate dehydrogenase kinase isoenzyme 4, and receptor-interacting serine threonine kinase 4, and a nonreceptor tyrosine kinase BMX, was elevated in Nrf2+/+ but not in Nrf2−/− mice. Ptp4a1 and protein phosphatase 2 regulatory subunit B (PR 52), alpha isoform (Ppp2r2a), were only induced by MV in the lungs of Nrf2−/− mice.

Solute transporters.

Profiling experiments revealed differential expression of several members of the solute carrier (Slc) family of proteins between the lungs of wild-type and Nrf2−/− mice (Table 1). Genes encoding for Slc family 23 member 2 (Slc23a2) (nucleobase transporter), Slc family 2 member 1 (Slc2a1, facilitated glucose transporter), Slc family 20 member 1 (Slc20a1, sodium/hydrogen exchanger), Slc family 7 member 5 (Slc7a5, cationic amino acid transporter), and Slc family 38 members 2 (Slc38 a2) and 4 (Slc38a4) were up-regulated by MV in wild-type animals. The expression levels of these genes, except Slc38 a2, were not altered significantly in Nrf2−/− mice. However, slc25a4 expression was elevated in Nrf2−/− mice by MV as compared with the SpV control group. Slc4a1 expression was down-regulated by MV in the lungs of both genotypes.

DISCUSSION

The present study clearly establishes that disruption of the oxidative stress modifier Nrf2 exacerbates MV-induced lung permeability and inflammation (i.e., neutrophil accumulation) and is accompanied by greater levels of several proinflammatory cytokines in lung tissue. These changes were associated with a lack of induction of critical antioxidant enzymes essential for GSH biosynthesis and redox imbalance in Nrf2−/− mice after MV. Antioxidant intervention significantly reduced lung alveolar and vascular permeability as well as inflammatory responses in Nrf2−/− mice. Collectively, our data demonstrate for the first time that Nrf2-dependent antioxidant transcriptional responses act in concert to counteract MV-induced oxidative stress, which otherwise contributes to the pathogenesis of VILI.

The loss of airway epithelial and endothelial integrity resulting in enhanced levels of pulmonary edema is a hallmark of ALI and ARDS (36). Similarly, the influx of protein leak in the lungs of mice exposed to MV and various oxidants (hyperoxia or acid aspiration) and toxins (endotoxin) is well documented in various experimental models of ALI/ARDS (1). In our studies, lung vascular permeability was significantly enhanced in Nrf2−/− mice as compared with Nrf2+/+ mice after MV. Consistent with these results, MV-induced albumin leakage was markedly higher in Nrf2−/− mice than Nrf2+/+ mice. We did not find significant lung injury, as assessed by alveolar protein or albumin content in the BALF (see Figures 1A and 1B), in Nrf2−/− mice subjected to MV at LVt in our acute (2-h) experimental model of VILI, although we noticed greater levels of neutrophils in Nrf2−/− mice as compared with SpV control mice. This result suggests that neutrophil accumulation is not dependent on lung epithelial injury in our acute model of VILI. This phenomenon has been observed in various models of ALI by other laboratories (3739). Our experiments revealed that MV at LVt for longer periods (4 h) produced significant lung injury and vascular leak, which was associated with elevated levels of lung neutrophil infiltration in wild-type and Nrf2−/− mice (Figure 6).

Inflammatory response induced by injurious MV contributes to VILI (8). We found that MV causes a greater increase in the total number of neutrophils in BALF and in lung alveolar septa of Nrf2−/− mice than Nrf2+/+ mice, suggesting that Nrf2-dependent transcriptional response may limit inflammatory responses induced by MV. The accumulation of neutrophils in the lung tissue can lead to enhanced levels of inflammatory cytokines, which play fundamental roles in the development of lung pathogenesis, including VALI (9, 10). Multiplex cytokine assays revealed significantly greater levels of KC (human homolog of CXCL1), IL-6, and G-CSF concentrations in the lungs of Nrf2−/− mice compared with Nrf2+/+ mice (see Figure 3A). KC and MIP-2 chemokines are potent neutrophil chemoattractants and play a critical role in lung inflammatory responses induced by MV (8). Several studies have shown that exposure to MV or cyclic stretch increases the expression levels of KC (4042). Treatment of mice with antibodies specific for KC or disruption of CXCR2, the KC receptor, markedly attenuate neutrophil sequestration and lung injury in a murine model of VILI suggesting that KC-CXCR2 activated signaling regulates pathogenesis in this syndrome (42). Elevated levels of KC in the lungs of Nrf2−/− mice are consistent with these studies and suggest that Nrf2 deficiency enhances the expression levels of KC, thereby resulting in enhanced neutrophil accumulation and lung injury. We also found enhanced levels of GCSF, which is important for neutrophil accumulation and activation in the lungs (43). High levels of GCSF correlate with ARDS in humans (4446), and administration of GCSF induces ALI in animals (43, 47). We also found elevated levels of IL-6 in the lungs of Nrf2−/− mice compared with Nrf2+/+ mice in response to MV, suggesting that this cytokine may play a role in causing VILI. Consistent with this notion, elevated levels of IL-6 have been detected in various experimental models of VILI (48, 49) and in BALF of patients with ARDS on MV (2, 50). Further studies are warranted to dissect temporal induction of these proinflammatory cytokines and their contribution to the development or susceptibility to VILI in Nrf2−/− mice in our experimental conditions. The mechanisms by which Nrf2 deficiency enhances the expression levels of KC, G-CSF, and IL-6 cytokines in response to MV are unclear. However, we have recently shown that disruption of Nrf2 potentiates nuclear factor (NF)-κB activation in a murine model of sepsis (28). NF-κB is critical for the induction of various cytokines in several experimental models of lung injury. MV has been to shown to activate NF-κB and other transcription factors, such as AP-1 (41). Whether Nrf2 directly or indirectly, through the modulation of NF-κB, AP-1, and/or other factors, regulates the expression levels of proinflammatory cytokines in response to MV warrants further study.

GCLC and GCLM are essential for the biosynthesis of GSH, a major cellular antioxidant. GPx2 scavenges hydrogen peroxide and organic hydroperoxides. These enzymes play key roles in the maintenance of intracellular redox balance (24). Our experiments revealed (1) failure to increase levels of Gclc, Gclm, and Gpx2; (2) reduced levels of GST and Gpx2 enzyme activities; and (3) greater redox imbalance (i.e., decreased in GSH/GSSG ratio) in Nrf2−/− mice compared with wild-type mice in response to MV. In contrast, MV stimulates the expression of Gclc, Gclm, and Gpx2 without a significant effect on the lung redox status in wild-type animals, which are less susceptible to VILI than their Nrf2−/− counterparts. We have previously reported that C57BL/6 mice subjected to MV at a Vt of 20 ml/kg for 2 hours demonstrate significant lung injury and inflammation when compared with animals exposed to Vt at 7 ml/kg or sham-operated controls (31, 32). However, we found that the CD1/ICR strain of mice (wild-type and Nrf2−/−) were more resistant to HVt in terms of developing VILI (as assessed by protein leakage in BALF), as compared with C57BL/6, and thus required larger Vt to cause injury. Consistent with these results, we have previously reported that CD1/ICR mice have intrinsic resistance to oxidative stress–mediated emphysema compared with C57BL/6 mice (27). This resistance was mainly attributed to high-level induction of antioxidant genes in CD1/ICR mice compared with C57BL/6J mice in response to prooxidants, such as cigarette smoke (51). CD1/ICR mice, when subjected to a longer ventilatory period of MV at LVt, display significant lung injury and inflammation.

Neutrophil accumulation in lung tissue can lead to enhanced levels of ROS, and antioxidant enzymes are essential in the response to ROS generated by neutrophils (52). Recently, we have shown that Nrf2 deficiency diminishes the levels of several antioxidant enzymes and enhances levels of ROS in neutrophils in response to LPS (53). We and others have also shown that exposure to pathologically relevant cyclic stretch leads to high levels of ROS and redox imbalance in lung epithelial cells in vitro (20, 21). Thus, it is likely that ROS generated by pulmonary cell types in response to MV may contribute to redox imbalance in Nrf2−/− mice. Expression profiling from various laboratories (including ours) demonstrated that Nrf2 regulates the expression of several genes, in addition to antioxidant enzymes (24). However, the present study demonstrates that antioxidant intervention remarkably attenuates MV-induced lung vascular injury and neutrophil accumulation in Nrf2−/− mice, suggesting that oxidative stress mainly contributes to VILI, which is mitigated by an antioxidant response. In preliminary results, Syrika and colleagues showed protective effects of NAC against MV-induced neutrophil influx in a rat model of VILI (54). Recently, we have shown that specific inhibition of xanthine oxidoreductase activity, which is required for ROS generation, attenuates MV-induced lung injury and inflammation (32), further underscoring a role for oxidative stress in the development and/or perpetuation of lung injury.

Expression profiling demonstrated that disruption of Nrf2 diminishes the inducible expression of several VALI genes (Table 1) that are known to be induced by MV in different experimental models of VILI (11, 15, 3335). The induction of several genes that modulate tissue matrix remodeling and blood coagulation in Nrf2+/+ mice but not in Nrf2−/− mice strongly suggests that the Nrf2 transcription factor is critical for up-regulating gene expression in response to MV (Table 1). There was no significant difference in the expression of proinflammatory genes between the genotypes, with the exception of IL-6 and Cxcl1 (up-regulated in Nrf2+/+ mice) and Il-1β and Cxcl2 (down-regulated in Nrf2−/− mice). Ptgs2 (Cox2) regulates lung inflammation; however, we found no significant differences in the expression levels of this enzyme between the two genotypes subjected to MV. Our results suggest that dysregulation of cytokine and tissue matrix remodeling proteins may a play role in enhancing the susceptibility of Nrf2−/− mice to VILI. In contrast to mRNA expression profiling, we found greater levels of proinflammatory cytokines, such as KC, IL-6, and G-CSF (measured by multiplex cytokine assays), in the lungs of in Nrf2−/− mice (see Figure 3A). Cytokine assays and microarray experiments were performed in lung tissues harvested at the termination of MV at 2 hours. It is well known that various cytokines are induced, at the transcriptional level, within 30 to 60 minutes in response to various stimuli in lung tissues/cell types. For example, we recently reported a robust induction of proinflammatory genes, such as IL-6, MIP-1α (Ccl3), MIP1β (Ccl4), MIP2 (IL-8), and KC (Cxcl or Gro-α), in the lungs of Nrf2−/− mice after 30 minutes of LPS administration, as compared with wild-type mice (28). Thus, the observed discordance between mRNA and protein expression profiling may be at least in part attributable to temporal induction of patterns of cytokines. Elaborative lung mRNA and protein expression profiling studies of Nrf2+/+ and Nrf2−/− mice subjected to MV at LVt and HVt, performed at earlier time points (30–60 min), are warranted to further decipher Nrf2-regulated inflammatory cytokine and chemokine transcriptional programs.

Nrf2 deficiency affects the induction of several antioxidant genes. Although a lack of induction of antioxidant genes was anticipated, expression profiling revealed that deficiency of Nrf2 results in down-regulation of several HSPs after MV. HSPs, by acting as molecular chaperones, limit protein aggregation and facilitate protein folding and transport of other proteins, thereby providing cytoprotection and maintenance of cellular homeostasis. The down-regulation of the HSP family members Hspa1a, Hspa1b, and Hspa8 by MV in Nrf2−/− mice but not in wild-type mice suggests that the Nrf2 transcriptional response is critical for the maintenance of HSP levels in the lung after mechanical stimuli. In support of this notion, Weiss and colleagues demonstrated that overexpression of Hsp70 in pulmonary epithelium reduces mortality and lung injury in a model of cecal ligation and puncture (55). Hsp70 induction was also associated with decreased pulmonary fibrosis (56), whereas ablation of the heat shock factor-1, which regulates HSP family members expression, increases susceptibility to hyperoxia in experimental models of ALI in mice (57). DnaJ (Hsp40) family proteins, Dnaja1 and Dnajb1, which are down-regulated by MV in the absence of Nrf2, by acting as co-chaperones modulate Hsp70 activity (58). Carhsp1, a calcium-regulated, heat-stable protein, is involved in calcium-mediated signal transduction (59). The expression of this gene is also markedly lower in Nrf2−/− mice subjected to MV. Thus, it is likely that Nrf2 up-regulates the expression of several HSP family members to maintain cellular homeostasis in response to MV.

The microarray results suggest that Nrf2 regulates signal transduction pathways by controlling the expression of several transcription factors, kinases, and phosphatases. Some of these proteins have been linked to VILI. We found striking differences in the expression levels of VALI genes encoding ATF3, Gadd45γ, Fos, EGR1, and Nr4a1 transcription factors. Up-regulation of these transcripts was found by various laboratories in different experimental models of VILI (11, 14, 15, 3335). Gadd45γ and ATF3 transcription factors are known to regulate gene expression involved in cellular stress, whereas EGR1 and Fos are immediate early response genes important for the expression of various cytokines and growth factors. We found that Nrf2 is critical for the induction of Klf6, Klf7, and Trib1 by MV. Trib1 differentially modulates the duration and the specificity of MAP kinase activation (60, 61). We found a marked induction of Trib1 in wild-type mice but not in Nrf2−/− mice, suggesting that Nrf2 response modulates MV-induced signaling in the lung. Consistent with this result, various genes encoding protein phosphatases, such as Dusp1, Dusp8, Dusp16, Ptp4a1, and Ptpn2, are induced by MV in the presence of Nrf2 but not in its absence. Previous studies have demonstrated that inhibition of P13K-Akt signaling and JNK1/2 kinases attenuates VILI in mice (62, 63). Although the exact roles of these protein phosphatases in the development of lung pathogenesis induced by MV and other stressful stimuli remain unclear, it is likely that these proteins may play a role in modulating the duration and/or magnitude of the activity of various kinases induced by MV.

We found striking differences in the expression levels of various Slc proteins in the lungs of wild-type and Nrf2−/− mice subjected to MV. There are at least 108 Slc proteins reported that are selectively expressed in various tissues, including the lung (64). These proteins play key roles in the transport of various substrates, such as saccharides, amino acids, ions, metals, and peptides. The differential induction of Slc2a1 (facilitated glucose transporter), Slc7a5 (L-type amino acid transporter), Slc20a1 (phosphate transporter), Slc23a2 (vitamin C transporter), Slc25a4 (adenine nucleotide translocator), and Slc38a2 and Slc38a4 (amino acid transporters) suggests that Nrf2 deficiency may result in a dysregulated absorption and distribution of various substrates, thereby enhancing the severity of VILI. NCF2 and NCF4 are critical for activation of the NOX4 (NADPH oxidase), which transfers electrons from NADPH to molecular oxygen, thereby resulting in the production of reactive superoxide. Paradoxically, we found that NCF2 and NCF4 are down-regulated in the lungs of Nrf2−/− mice in response to MV, raising the possibility that a lack of Nrf2 may result in activation of compensatory mechanisms that suppress the expression of these enzymes, probably to dampen high levels of superoxide generation. Although the majority of VALI genes are induced by MV wild-type mice (CD1/ICR strain) in our experimental conditions, we found certain discrepancies in the expression levels of several genes. For example, in our experiments we found the induction of GADD45γ, instead of the GADD45α isoform as reported by others (34, 35). We attribute this and other variations to differences in the genetic background of mice or slight variations in experimental conditions related to MV.

In summary, our data suggest that maintenance of a proper redox balance regulated by an Nrf2-dependent transcriptional program might be essential for the integrity and function of different lung cellular components and the outcome of VILI. We found functional polymorphisms in the promoter region of human NRF2, which were associated with an increased risk of ALI in a population of patients with major trauma (65), further underscoring a critical role for Nrf2-dependent signaling in ALI/ARDS. We therefore speculate that dysregulation or variation in Nrf2 signaling pathway(s) may enhance susceptibility to VILI due to lack of a proper redox balance in the lungs. Consistent with this notion, expression profiling studies revealed that Nrf2 regulates, in addition to VALI genes, several novel networks, such as stress proteins, transcription factors, phosphatases, and solute carrier proteins. Analyzing the functional roles of candidate target genes and signal transduction pathways regulated by Nrf2 in response to MV may provide additional insight into the factors that promote or perpetuate VILI and help in developing novel therapies targeted at oxidative stress and inflammation in this syndrome.

Supplementary Material

[Online Supplement]

Acknowledgments

The authors thank Genomics, Biomarkers/Proteomics, and Pathology Cores of the ALI SCCOR for performing microarray cytokine ELISA analysis and assisting in immunohistochemical and histopathological analysis, respectively. The authors thank Hannah Lee for help in microarray analysis.

Notes

Supported by NIH/NHLBI SCCOR grant P50 HL073994 (S.P.R.).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200701-060OC on September 27, 2007

Conflict of Interest Statement: S.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.R.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. N.M.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.M.D.-O. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.T.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. D.N.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. K.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.M.T. received an unrestricted postdoctoral support grant from Quark Biotech for studies involving RTP801 in cigarette smoke–induced emphysema. He received $2,500 for speaker fees in an international conference sponsored by AstraZeneca. He received $1,500 from the Rush Medical Center's CME speakers' training workshop titled Simply Speaking PAH: An Expert Educators CME Lecture Series. M.Y. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. T.W.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. W.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. P.M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.P.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

References

1. Matthay MA, Zimmerman GA. Acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management. Am J Respir Cell Mol Biol 2005;33:319–327. [PMC free article] [PubMed]
2. The Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 2000;342:1301–1308. [PubMed]
3. Ware LB. Pathophysiology of acute lung injury and the acute respiratory distress syndrome. Semin Respir Crit Care Med 2006;27:337–349. [PubMed]
4. Tschumperlin DJ, Oswari J, Margulies AS. Deformation-induced injury of alveolar epithelial cells: effect of frequency, duration, and amplitude. Am J Respir Crit Care Med 2000;162:357–362. [PubMed]
5. Waters CM, Sporn PH, Liu M, Fredberg JJ. Cellular biomechanics in the lung. Am J Physiol Lung Cell Mol Physiol 2002;283:L503–L509. [PubMed]
6. Vlahakis NE, Hubmayr RD. Cellular stress failure in ventilator-injured lungs. Am J Respir Crit Care Med 2005;171:1328–1342. [PMC free article] [PubMed]
7. Han B, Lodyga M, Liu M. Ventilator-induced lung injury: role of protein–protein interaction in mechanosensation. Proc Am Thorac Soc 2005;2:181–187.
8. Belperio JA, Keane MP, Lynch JP III, Strieter RM. The role of cytokines during the pathogenesis of ventilator-associated and ventilator-induced lung injury. Semin Respir Crit Care Med 2006;27:350–364. [PubMed]
9. Halbertsma FJ, Vaneker M, Scheffer GJ, van der Hoeven JG. Cytokines and biotrauma in ventilator-induced lung injury: a critical review of the literature. Neth J Med 2005;63:382–392. [PubMed]
10. Goodman RB, Pugin J, Lee JS, Matthay MA. Cytokine-mediated inflammation in acute lung injury. Cytokine Growth Factor Rev 2003;14:523–535. [PubMed]
11. Ma S-F, Grigoryev DN, Taylor AD, Nonas S, Sammani S, Ye SQ, Garcia JGN. Bioinformatic identification of novel early stress response genes in rodent models of lung injury. Am J Physiol Lung Cell Mol Physiol 2005;289:L468–L477. [PubMed]
12. Grigoryev DN, Ma SF, Simon BA, Irizarry RA, Ye SQ, Garcia JG. In vitro identification and in silico utilization of interspecies sequence similarities using GeneChip technology. BMC Genomics 2005;6:62. [PMC free article] [PubMed]
13. Altemeier WA, Matute-Bello G, Gharib SA, Glenny RW, Martin TR, Liles WC. Modulation of lipopolysaccharide-induced gene transcription and promotion of lung injury by mechanical ventilation. J Immunol 2005;175:3369–3376. [PubMed]
14. Gharib SA, Liles WC, Matute-Bello G, Glenny RW, Martin TR, Altemeier WA. Computational identification of key biological modules and transcription factors in acute lung injury. Am J Respir Crit Care Med 2006;173:653–658. [PubMed]
15. Dolinay T, Kaminski N, Felgendreher M, Kim HP, Reynolds P, Watkins SC, Karp D, Uhlig S, Choi AM. Gene expression profiling of target genes in ventilator-induced lung injury. Physiol Genomics 2006;26:68–75. [PubMed]
16. Halliwell B, Gutteridge JM, Cross CE. Free radicals, antioxidants, and human disease: where are we now? J Lab Clin Med 1992;119:598–620. [PubMed]
17. MacNee W. Oxidative stress and lung inflammation in airways disease. Eur J Pharmacol 2001;429:195–207. [PubMed]
18. MacNee W. Oxidants/antioxidants and chronic obstructive pulmonary disease: pathogenesis to therapy. Novartis Found Symp 2001;234:169–185. [PubMed]
19. Chess PR, O'Reilly MA, Sachs F, Finkelstein JN. Reactive oxidant and p42/44 MAP kinase signaling is necessary for mechanical strain-induced proliferation in pulmonary epithelial cells. J Appl Physiol 2005;99:1226–1232. [PubMed]
20. Chapman KE, Sinclair SE, Zhuang D, Hassid A, Desai LP, Waters CM. Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells. Am J Physiol Lung Cell Mol Physiol 2005;289:L834–L841. [PubMed]
21. Papaiahgari S, Yerrapureddy A, Hassoun PM, Garcia JG, Birukov KG, Reddy SP. EGFR-activated signaling and actin remodeling regulate cyclic stretch-induced Nrf2-ARE activation. Am J Respir Cell Mol Biol 2006;36:304–312. [PMC free article] [PubMed]
22. Cheng YJ, Chan KC, Chien CT, Sun WZ, Lin CJ. Oxidative stress during 1-lung ventilation. J Thorac Cardiovasc Surg 2006;132:513–518. [PubMed]
23. Collard KJ, Godeck S, Holley JE, Quinn MW. Pulmonary antioxidant concentrations and oxidative damage in ventilated premature babies. Arch Dis Child Fetal Neonatal Ed 2004;89:F412–F416. [PMC free article] [PubMed]
24. Cho HY, Reddy SP, Kleeberger SR. Nrf2 defends the lung from oxidative stress. Antioxid Redox Signal 2006;8:76–87. [PubMed]
25. Cho HY, Jedlicka AE, Reddy SP, Zhang LY, Kensler TW, Kleeberger SR. Linkage analysis of susceptibility to hyperoxia. Nrf2 is a candidate gene. Am J Respir Cell Mol Biol 2002;26:42–51. [PubMed]
26. Cho HY, Reddy SP, Yamamoto M, Kleeberger SR. The transcription factor NRF2 protects against pulmonary fibrosis. FASEB J 2004;18:1258–1260. [PubMed]
27. Rangasamy T, Cho CY, Thimmulappa RK, Zhen L, Srisuma SS, Kensler TW, Yamamoto M, Petrache I, Tuder RM, Biswal S. Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest 2004;114:1248–1259. [PMC free article] [PubMed]
28. Thimmulappa RK, Lee H, Rangasamy T, Reddy SP, Yamamoto M, Kensler TW, Biswal S. Nrf2 is a critical regulator of the innate immune response and survival during experimental sepsis. J Clin Invest 2006;116:984–995. [PMC free article] [PubMed]
29. Reddy SP, Hassoun PM, Garcia JG, Kleeberger SR, Yamamoto M, Papaiahgari S. Nrf2 transcription factor confers protection against mechanical ventilation induced lung injury. Am J Respir Crit Care Med 2005;2:A318.
30. Peng X, Hassoun PM, Sammani S, McVerry BJ, Burne MJ, Rabb H, Pearse D, Tuder RM, Garcia JG. Protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury. Am J Respir Crit Care Med 2004;169:1245–1251. [PubMed]
31. Peng X, Abdulnour RE, Sammani S, Ma SF, Han EJ, Hasan EJ, Tuder R, Garcia JG, Hassoun PM. Inducible nitric oxide synthase contributes to ventilator-induced lung injury. Am J Respir Crit Care Med 2005;172:470–479. [PMC free article] [PubMed]
32. Abdulnour RE, Peng X, Finigan JH, Han EJ, Hasan EJ, Birukov KG, Reddy SP, Watkins JE III, Kayyali US, Garcia JG, et al. Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways. Am J Physiol Lung Cell Mol Physiol 2006;291:L345–L353. [PubMed]
33. Copland IB, Kavanagh BP, Engelberts D, McKerlie C, Belik J, Post M. Early changes in lung gene expression due to high tidal volume. Am J Respir Crit Care Med 2003;168:1051–1059. [PubMed]
34. Grigoryev DN, Ma SF, Irizarry RA, Ye SQ, Quackenbush J, Garcia JG. Orthologous gene-expression profiling in multi-species models: search for candidate genes. Genome Biol 2004;5:R34. [PMC free article] [PubMed]
35. Altemeier WA, Matute-Bello G, Frevert CW, Kawata Y, Kajikawa O, Martin TR, Glenny RW. Mechanical ventilation with moderate tidal volumes synergistically increases lung cytokine response to systemic endotoxin. Am J Physiol Lung Cell Mol Physiol 2004;287:L533–L542. [PubMed]
36. Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med 2000;342:1334–1349. [PubMed]
37. Martin TR, Pistorese BP, Chi EY, Goodman RB, Matthay MA. Effects of leukotriene B4 in the human lung: recruitment of neutrophils into the alveolar spaces without a change in protein permeability. J Clin Invest 1989;84:1609–1619. [PMC free article] [PubMed]
38. Wiener-Kronish JP, Albertine KH, Matthay MA. Differential responses of the endothelial and epithelial barriers of the lung in sheep to Escherichia coli endotoxin. J Clin Invest 1991;88:864–875. [PMC free article] [PubMed]
39. Walker DC, Behzad AR, Chu F. Neutrophil migration through preexisting holes in the basal laminae of alveolar capillaries and epithelium during streptococcal pneumonia. Microvasc Res 1995;50:397–416. [PubMed]
40. Tremblay L, Valenza F, Ribeiro SP, Li J, Slutsky AS. Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model. J Clin Invest 1997;99:944–952. [PMC free article] [PubMed]
41. Held HD, Boettcher S, Hamann L, Uhlig S. Ventilation-induced chemokine and cytokine release is associated with activation of nuclear factor-kappaB and is blocked by steroids. Am J Respir Crit Care Med 2001;163:711–716. [PubMed]
42. Belperio JA, Keane MP, Burdick MD, Londhe V, Xue YY, Li K, Phillips RJ, Strieter RM. Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J Clin Invest 2002;110:1703–1716. [PMC free article] [PubMed]
43. Hierholzer C, Kelly E, Lyons V, Roedling E, Davies P, Billiar TR, Tweardy DJ. G-CSF instillation into rat lungs mediates neutrophil recruitment, pulmonary edema, and hypoxia. J Leukoc Biol 1998;63:169–174. [PubMed]
44. Matute-Bello G, Liles WC, Radella F II, Steinberg KP, Ruzinski JT, Hudson LD, Martin TR. Modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome. Crit Care Med 2000;28:1–7. [PubMed]
45. Aggarwal A, Baker CS, Evans TW, Haslam PL. G-CSF and IL-8 but not GM-CSF correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome. Eur Respir J 2000;15:895–901. [PubMed]
46. Wiedermann FJ, Mayr AJ, Hobisch-Hagen P, Fuchs D, Schobersberger W. Association of endogenous G-CSF with anti-inflammatory mediators in patients with acute respiratory distress syndrome. J Interferon Cytokine Res 2003;23:729–736. [PubMed]
47. Arimura K, Inoue H, Kukita T, Matsushita K, Akimot M, Kawamata N, Yamaguchi A, Kawada H, Ozak A, Arima N, et al. Acute lung injury in a healthy donor during mobilization of peripheral blood stem cells using granulocyte-colony stimulating factor alone. Haematologica 2005;90:ECR10. [PubMed]
48. Rich PB, Douillet CD, Hurd H, Boucher RC. Effect of ventilatory rate on airway cytokine levels and lung injury. J Surg Res 2003;113:139–145. [PubMed]
49. Steinberg JM, Schiller HJ, Halter JM, Gatto LA, Lee HM, Pavone LA, Nieman GF. Alveolar instability causes early ventilator-induced lung injury independent of neutrophils. Am J Respir Crit Care Med 2004;169:57–63. [PubMed]
50. Ranieri VM, Suter PM, Tortorella C, De Tullio R, Dayer JM, Brienza A, Bruno F, Slutsky AS. Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized controlled trial. JAMA 1999;282:54–61. [PubMed]
51. Cavarra E, Bartalesi B, Lucattelli M, Fineschi S, Lunghi B, Gambelli F, Ortiz LA, Martorana PA, Lungarella G. Effects of cigarette smoke in mice with different levels of α1-proteinase inhibitor and sensitivity to oxidants. Am J Respir Crit Care Med 2001;164:886–890. [PubMed]
52. Chabot F, Mitchell JA, Gutteridge JM, Evans TW. Reactive oxygen species in acute lung injury. Eur Respir J 1998;11:745–757. [PubMed]
53. Thimmulappa RK, Scollick C, Traore K, Yates M, Trush MA, Liby KT, Sporn MB, Yamamoto M, Kensler TW, Biswal S. Nrf2-dependent protection from LPS induced inflammatory response and mortality by CDDO-imidazolide. Biochem Biophys Res Commun 2006;351:883–889. [PMC free article] [PubMed]
54. Syrkina OL, Quinn DA, Moufarrej R, Hales CA. N-acetylcysteine (NAC), an antioxidant, decreased lung neutrophil influx in a rat model of ventilator-induced lung injury (VILI) [abstract]. FASEB J 2002;16:A410.
55. Weiss YG, Maloyan A, Tazelaar J, Raj N, Deutschman CS. Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome. J Clin Invest 2002;110:801–806. [PMC free article] [PubMed]
56. Hagiwara S, Iwasaka H, Matsumoto S, Noguchi T, Yoshioka H. Association between heat stress protein 70 induction and decreased pulmonary fibrosis in an animal model of acute lung injury. Lung 2007;185:287–293. [PubMed]
57. Malhotra V, Kooy NW, Denenberg AG, Dunsmore KE, Wong HR. Ablation of the heat shock factor-1 increases susceptibility to hyperoxia-mediated cellular injury. Exp Lung Res 2002;28:609–622. [PubMed]
58. Shaner L, Morano KA. All in the family: atypical Hsp70 chaperones are conserved modulators of Hsp70 activity. Cell Stress Chaperones 2007;12:1–8. [PMC free article] [PubMed]
59. Groblewski GE, Yoshida M, Bragado MJ, Ernst SA, Leykam J, Williams JA. Purification and characterization of a novel physiological substrate for calcineurin in mammalian cells. J Biol Chem 1998;273:22738–22744. [PubMed]
60. Kiss-Toth E, Bagstaff SM, Sung HY, Jozsa V, Dempsey C, Caunt JC, Oxley KM, Wyllie DH, Polgar T, Harte M, et al. Human tribbles, a protein family controlling mitogen-activated protein kinase cascades. J Biol Chem 2004;279:42703–42708. [PubMed]
61. Hegedus Z, Czibula A, Kiss-Toth E. Tribbles: a family of kinase-like proteins with potent signalling regulatory function. Cell Signal 2007;19:238–250. [PubMed]
62. Uhlig U, Fehrenbach H, Lachmann RA, Goldmann T, Lachmann B, Vollmer E, Uhlig S. Phosphoinositide 3-OH kinase inhibition prevents ventilation-induced lung cell activation. Am J Respir Crit Care Med 2004;169:201–208. [PubMed]
63. Li LF, Yu L, Quinn DA. Ventilation-induced neutrophil infiltration depends on c-Jun N-terminal kinase. Am J Respir Crit Care Med 2004;169:518–524. [PubMed]
64. Nishimura M, Naito S. Tissue-specific mRNA expression profiles of human Toll-like receptors and related genes. Biol Pharm Bull 2005;28:886–892. [PubMed]
65. Marzec JM, Christie JD, Reddy SP, Jedlicka AE, Vuong H, Lanken PN, Aplenc R, Yamamoto T, Yamamoto M, Cho HY, et al. Functional polymorphisms in the transcription factor NRF2 in humans increase the risk of acute lung injury. FASEB J 2007;21:2237–2246. [PubMed]

Articles from American Journal of Respiratory and Critical Care Medicine are provided here courtesy of American Thoracic Society