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Published ahead of print on June 19, 2003, doi:10.1164/rccm.200208-964OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 1051-1059, (2003)
© 2003 American Thoracic Society

Early Changes in Lung Gene Expression due to High Tidal Volume

Ian B. Copland, Brian P. Kavanagh, Doreen Engelberts, Colin McKerlie, Jaques Belik and Martin Post

Program in Lung Biology and Department of Critical Care Medicine, The Hospital for Sick Children, and Departments of Laboratory Medicine and Pathobiology, and Pediatrics, University of Toronto, Toronto, Ontario, Canada

Correspondence and requests for reprints should be addressed to Martin Post, Ph.D., Lung Biology Program, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8, Canada. E-mail: martin.post{at}sickkids.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The purpose of this study was to use gene expression profiling to understand how adult rat lung responds to high tidal volume (HV) ventilation in vivo. HV ventilation for 30 minutes did not cause discernable lung injury (in terms of altered mechanics or histology) but caused obvious injury when continued for 90 minutes. However, at 30-minute ventilation, HV caused significant upregulation of 10 genes and suppression of 12 genes. Among the upregulated genes were transcription factors, stress proteins, and inflammatory mediators; the downregulated genes were exemplified by metabolic regulatory genes. On the basis of cluster analysis, we studied Egr-1, c-Jun, heat shock protein 70, and interleukin (IL)-1ß in further detail. Temporal studies demonstrated that Egr-1 and c-Jun were increased early and before heat shock protein 70 and IL-1ß. Spatial studies using in situ hybridization and laser capture microscopy revealed that all four genes were upregulated primarily in the bronchiolar airway epithelium. Furthermore, at 90 minutes of HV ventilation, a significant increase in intracellular IL-1ß protein was observed. Although there are limitations to gene array methodology, the current data suggest a global hypothesis that (1) the effects of HV are cumulative; (2) specific patterns of gene activation and suppression precede lung injury; and (3) alteration of gene expression after mechanical stretch is pathogenic.

Key Words: ventilation • gene expression profiling

Our knowledge about the role played by specific mediators in the pathogenesis of stretch-induced lung injury is rapidly expanding but is not comprehensive (1–4). The precise role of individual mediators is confounded by the use of ex vivo models that induce extreme degrees of injury or that involve pretreatment with either bacterial products or saline lavage. In addition, the exploration of specific mediators has been driven by the a priori knowledge of currently identified cytokines as well as by the availability of validated assays. Thus, several issues are unresolved. First, there is fundamental disagreement in terms of which mediators are released (1–4). Second, although stretch is clearly associated with release of several mediators, the temporal relationship (a requirement for inferring cause and effect) of these mediators has not been clearly defined. Third, current data are based on subjective expectations and availability of specific assays. Finally, although studies have focused on established or evolving injury, no studies to date have reported on the gene activation induced by mechanical stretch that occurs in the absence of physiologic or structural impairment. Therefore, in an effort to better understand mechanotransduction in the lung in vivo, we investigated the changes in gene expression in the lungs of adult rats exposed to high tidal volume (HV) ventilation, which did not initially cause overt lung injury.

Gene array technology affords the ability to screen thousands of genes for evidence of activation or suppression after a specific stimulus, without a priori bias about which genes (i.e., gene products) may be important. We therefore used gene arrays to develop a global picture of changes in gene expression after 30 minutes of HV ventilation in previously healthy in vivo adult rats. Additional experimentation confirmed time dependence, messenger RNA (mRNA) tissue localization, as well as increased production of protein corresponding to one of the exemplary genes. We conclude that these findings provide a biologically plausible hypothesis that the pattern of gene activation that precedes HV-induced injury plays a role in the pathogenesis of HV-induced lung injury.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Male Sprague-Dawley rats (350–400 g) were used. After anesthesia and tracheostomy, animals were allocated either to a control group (not ventilated) or to a HV protocol (VT 25 ml/kg, positive end-expiratory pressure 0 cm H2O, frequency 30/minute.) and ventilated for 5–180 minutes. Impaired compliance was determined by static inflation pressure curves after incremental 1.0 ml volume injections (5).

Histologic Lung Injury
Three groups were studied: control, HV for 30 minutes, and HV for more than 90 minutes. The lungs were fixed and histologic scoring (six sagittal sections, each left lung) performed by a pathologist blinded to group allocation.

Macroarray Experiments
Presentation of array data is as per minimum information about a microarray experiment criteria (6). Using fresh nylon arrays (Atlas Rat cDNA Arrays; Clontech, Palo Alto, CA), three independent hybridizations were performed for each group on each of the two macroarrays (Atlas Rat 1.2 Array and Atlas Rat 1.2 Array II; Clontech). Total RNA was extracted using Trizol (In Vitrogen, Burlington, ON, Canada) and DNase treated (Roche, PQ, Canada) before reverse transcription. Pooled RNA (2 µg; n = 4 for each group) was reverse transcribed into 32P-labeled first-strand complementary DNA probes using gene-specific (array specific) primer mixes. Hybridization (overnight, 68°C) was followed by stringency washes, phosphorimaging exposure, and scanning. Gene stimulation (or suppression) was then determined using Atlas Image and significance analysis of microarrays (SAM) (7). Only genes demonstrating the following expression differences versus control lungs were considered altered by HV: absolute difference greater than 5,000 AU; differential ratio greater than or equal to 2 (Atlas Image analysis); and {Delta} >= 0.8 (SAM). Ward's hierarchical and k-means clustering were performed using JMP (Version 5) statistical analysis program (8).

Northern Blotting
Total RNA (10 µg) was gel fractionated, transferred to a nylon blot, and hybridized with 32P-deoxycytidine triphosphate-labeled polymerase chain reaction (PCR)–generated probes. Equal loading of RNA was confirmed by 32P-labeled ribosomal 18S protein and blots quantified by densitometry.

Real-time PCR
Total RNA (2 µg) from whole lungs was reverse transcribed (37°C) using random hexamers (Applied Biosystems, Foster City, CA), and the resulting templates quantified by real-time PCR (ABI Prism 7700) using SybrGreen. The relative quantification method was used to compare the data (9).

In Situ Hybridization
Rat-specific complementary DNAs inserted in PCRII vector were used to generate nonradioactive sense and antisense dioxygenin–labeled complementary RNA probes. The RNA probes were hybridized (60°C) to paraformaldehyde-fixed, paraplast-embedded lung tissue sections (10 µm). After high-stringency washing, slides were incubated with an alkaline phosphatase–conjugated sheep anti-dioxygenin (DIG) antibody to catalyze a 5-bromo-4-chloro-indolyl-phosphatase (BCIP) substrate color reaction (Roche).

Laser Capture Microdissection
Optimal cutting temperature compound (OTC)-frozen lung sections (8 µm) from control (n = 4) and 30-minute HV (n = 4) groups were fixed in 75% ethanol, rehydrated, stained, and dehydrated (10). Bronchiolar and alveolar epithelial cells were dissected using a PixCell II System (Arcturus Engineering, Mountain View, CA) and the RNA extracted.

Western Blotting
Proteins were extracted from whole lung homogenate and immunoblotting performed as described previously (11).

Data Presentation and Statistical Analysis
Gene expression was normalized to 18S RNA levels in each sample. The mean value of these ratios for the HV-treated animals was expressed as mean fold difference ± SD compared with nonventilated control group. Significance was determined using analysis of variance.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Physiologic Assessment
Figure 1A demonstrates the static compliance of control animals (Ctrl), and animals ventilated for either 30 minutes or for more than 90 minutes with HV. The compliance, presented as static inflation pressure, was similar in the control and 30-minute HV groups but was significantly worse in the 90-minute HV group (p < 0.05: 90-minute HV group vs. control group, 30-minute HV). Table 1 outlines the histologic lung injury scores in each group. The injury score was similar in the control and 30-minute HV groups (i.e., uninjured) but was significantly worse in the 90-minute HV group (p < 0.05: 90-minute HV group vs. control group, 30-minute HV). Figure 1B illustrates representative histologic changes. In addition, no ultrastructural differences were observed between the control group versus 30-minute group (see Figure E1 in the online supplement).



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  		Figure 1. Compliance (A) and histologic (B) assessment in control (filled squares), 30-minute high tidal volume (HV) (open diamonds), and 90-minute HV (open triangles ) groups. There was no difference in static inflation pressure between the control and 30-minute HV groups, but the compliance was significantly worse in the 90-minute HV group (p < 0.05; vs. control and 30-minute HV groups). Representative lung images of histologic sections from control, 30-minute HV, and 90-minute HV animals are shown (B). Perivascular, interstitial, and alveolar edema (indicated by arrows) was present in 90-minute HV but absent in the control and 30-minute HV groups. (Magnification—i, ii, iii: x100; iv, v, vi: x400).

 

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  		TABLE 1. Histologic evaluation demonstrated that lung injury was absent in both the 30-MINUTE high tidal volume (hv) and control groups. injury score was significantly greater in the 90-MINUTE hv group

 
Gene Profiling Using Complementary DNA Macroarrays
Figure E2 (see online supplement) illustrates a typical result from one of our array experiments represented as a dark field image. Regions with obvious visual differences in intensity of genes between control (A) and 30-minute HV (B) groups are highlighted. After array analysis (n = 3) for each group with the Atlas Image Analysis and SAM programs, we identified 22 genes, wherein expression was significantly altered by 30-minute HV ventilation. Table 2 outlines the genes that were found to be significantly upregulated (or downregulated) together with their average signal intensities, standard deviations, and fold changes compared with control group. In contrast to previous data demonstrating that HV ventilation increased tumor necrosis factor–{alpha} and interleukin (IL)-6 protein content (3), our arrays showed that tumor necrosis factor–{alpha} and IL-6 mRNAs expression were not changed at 30-minute HV.


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  		TABLE 2. List of significantly altered genes on the basis of atlas image and sam analysis, including: average signal intensity of control and high tidal volume groups and difference in signal intensity and fold change

 
Figure 2 shows the hierarchical clustering of the altered genes. On the basis of these analyses, the upregulated genes were categorized into six main clusters. In the largest cluster, IL-1ß clustered with Egr-1 and the intracellular calcium-binding protein migration inhibition factor-related protein 14 (MRP14). The next branch of the tree is the transcription factor c-Jun. Two stress proteins (heat shock protein 70 [HSP70] and I{kappa}B) also clustered closely. Among the downregulated genes, the largest cluster contains six genes, including metabolic enzymes (myoadenylate deaminase, endoplasmic reticulum {alpha}-mannosidase, and phosphoethanolamine cytidylyltransferase), a stress protein (heme oxygenase-3), and two functionally unclassified proteins (brain–kidney protein and secreted phosphoprotein-24). Using k-means clustering (15 or 20 clusters) of the entire normalized data set, c-Jun, Egr-1, IL-1ß, and MRP14 clustered into one small group, whereas HSP70 clustered closely but separately from I{kappa}B (see Figure E3 in the online supplement). The remaining genes that were significantly altered on the basis of the macroarray analysis clustered into nodes containing multiple genes that were not significantly altered.



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  		Figure 2. Ward's hierarchical clustering of genes determined to be significantly altered, on the basis of a twofold change in intensity, a minimum alteration of 5,000 intensity units, and a {Delta} of 0.8. Within the upregulated genes (green to red) six main clusters (clusters are defined by similar color) are identified, whereas in the downregulated genes (red to green) four main clusters are seen.

 
Confirmation of Selected Upregulated Genes
On the basis of the clustering analysis and evidence in the literature for mechanical stress-induced alterations in gene expression (3, 12–15), we focused our attention on Egr-1, c-Jun, HSP70, and IL-1ß. Using reverse transcription (RT)-PCR we initially demonstrated that Egr-1, c-Jun, HSP70, and IL-1ß mRNA expression were all increased in 30-minute HV group versus control group (data not shown). Furthermore, RT-PCR for tumor necrosis factor–{alpha} and IL-6 showed no difference between 30-minute HV group versus control group, validating the specificity of our macroarray data (data not shown).

To rule out potential skewing effects of outliers associated with the use of pooled RNA samples from several animals, we analyzed RNA from individual samples. Northern blotting analysis for Egr-1, c-Jun, HSP70, and IL-1ß (Figure 3A) demonstrated significant increases in mRNA for each gene in all animals ventilated with 30-minute HV compared with control lungs (Figure 3B). Both Egr-1 and IL-1ß were increased approximately 10-fold, whereas c-Jun and HSP70 mRNA levels were elevated 1.7- and 2.4-fold, respectively. These increases correlate closely with the array analysis (see Table 2).



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  		Figure 3. (A) Northern blotting confirmed increased expression of Egr-1, c-Jun, heat shock protein 70 (HSP70), and interleukin (IL)-1ß in 30-minute high tidal volume (HV) versus control animals (Ctrl). (B) Quantitation was performed using 18S as a normalizer and revealed that Egr-1, c-Jun, HSP70, and IL-1ß mitochondrial RNA expression are all significantly increased (*p < 0.05; 30-minute HV group vs. control group).

 
Effect of HV Ventilation Gene Expression
To validate that the increased gene expression was the result of HV ventilation (as opposed to mechanical ventilation per se) we subjected rats (n = 3–4) to either low tidal volume (VT 5 ml/kg, positive end-expiratory pressure 1 cm H2O) or HV (VT 25 ml/kg, positive end-expiratory pressure 0 cm H2O) ventilation for 30 minutes. Total RNA was then extracted from the lungs, and the expression of selected upregulated and downregulated genes was analyzed by real-time PCR. Egr-1, Nur77, c-Jun, HSP70, and IL-1ß mRNA were all significantly increased in our HV group (Figure 4) . We also found that three genes (heme oxygenase–3, B/K, and Rabin3), which were downregulated on the basis of our array analyses, were also significantly downregulated in our HV group compared with low tidal volume group (Figure 4).



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  		Figure 4. Effects of low tidal volume (5 ml/kg; LV) versus high tidal volume (HV) (25 ml/kg were compared using real-time polymerase chain reaction. Genes that were found to be upregulated in the HV group compared with the low tidal volume group were Egr-1, c-Jun, heat shock protein 70, interleukin-1ß, and Nur77, whereas genes that were found to be downregulated in the HV group compared with low tidal volume were Rabin3, B/K protein, and heme oxygenase–3 (*p < 0.05).

 
Temporal Effect of HV Ventilation on Gene Expression
To determine the temporal pattern of Egr-1, c-Jun, HSP70, and IL-1ß mRNA expression, animals were ventilated with HV for 5, 15, 30, or 90 minutes. Real-time PCR results from individual samples (n = 3–4) defining the temporal mRNA expression are shown in Figure 5 . Consistent with the previously mentioned confirmation experiments, all four genes were significantly increased after 30 minutes of ventilation (Figure 5). However, the temporal pattern of gene induction fell into two separate categories. Egr-1 and c-Jun expression were significantly increased by 15 minutes after the onset of HV ventilation, whereas the induction of HSP70 and IL-1ß mRNA expression was not significantly elevated until 30 minutes. By 90 minutes, all four genes are significantly elevated. At 90 minutes of HV, all four genes continued to be increased to a significantly greater extent compared with 30 minutes of HV (Figure 5).



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  		Figure 5. Duration of high tidal volume (HV) ventilation on gene expression of Egr-1, c-Jun, heat shock protein 70 (HSP70), and interleukin (IL)-1ß. Real-time polymerase chain reaction demonstrated that both Egr-1 and c-Jun expression were significantly increased within 15 minutes after the onset of HV ventilation, whereas the induction of HSP70 and IL-1ß mitochondrial RNA expression was not significantly elevated until 30 minutes. By 90 minutes, all four genes are significantly elevated compared with their levels at 30 minutes of HV ventilation. ({dagger}p < 0.05; vs. 0 [Ctrl]; {ddagger}p < 0.05 vs. 30-minute HV.)

 
Cellular Localization of Gene Expression Induced by HV Ventilation
Because the expression of Egr-1, c-Jun, HSP70, and IL-1ß mRNA was consistently upregulated after 90 minutes of HV ventilation, we choose this time point to determine in which tissue compartment of the lung gene expression was upregulated. Using nonradioactive in situ hybridization, we found that message for all four genes localized to the bronchiolar epithelium of HV ventilated lung (Figures 6 and 7) . No message for Egr-1 (Figure 6i, 6iii), c-Jun (Figure 6ii, 6iv), or IL-1ß (Figure 7ii, 7iv) was detected in the alveolar epithelium. In contrast, modest expression of HSP70 mRNA was observed in the alveolar region after 90-minutes of HV ventilation (Figure 7i, 7iii). Using laser capture microdissection, we demonstrated upregulated gene expression of IL-1ß in the bronchiolar epithelium within 30 minutes after the onset of HV ventilation (Figure 8) .



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  		Figure 6. Cellular localization of Egr-1 and c-Jun mitochondrial RNA in lungs subjected to high tidal volume (HV) ventilation. Using nonradioactive in situ hybridization, the cellular expression of Egr-1 (i, iii, v) and c-Jun (ii, iv, vi) were compared between control (i, ii) and HV animals (iii, iv). At 90 minutes, strong positive staining (purple) for both Egr-1 (iii) and c-Jun (iv) was localized to the bronchiolar epithelium of HV animals when compared with control animals (i, ii). In both control and HV animals there was no appreciable staining in the alveolar and smooth muscle regions or vasculature. Hybridization with sense probes resulted in minimal nonspecific staining (v, vi).

 


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  		Figure 7. Cellular localization of heat shock protein 70 (HSP70) and interleukin (IL)-1ß mitochondrial RNA in lungs subjected to high tidal volume (HV) ventilation. Using nonradioactive in situ hybridization, the cellular expression of HSP70 (i, iii, v) and IL-1ß (ii, iv, vi) were compared between control (i, ii) and HV animals (iii, iv). At 90 minutes, strong positive staining (purple) for both HSP70 (arrows) (iii) and IL-1ß (iv) was localized to the bronchiolar epithelium of HV animals when compared with control animals (i, ii). In both control and HV animals, there was no appreciable staining noted for IL-1ß in the alveolar and smooth muscle regions or vasculature. Positive staining was apparent in the alveolar region for HSP70 (arrows) (iii) but not in the smooth muscle or vasculature. Hybridization with sense probes resulted in minimal nonspecific staining (v, vi).

 


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  		Figure 8. Confirmation of cellular localization and quantitation of mitochondrial RNA levels for interleukin (IL)-1ß in bronchiolar epithelium. Laser-capture microdissection followed by real-time polymerase chain reaction demonstrated that IL-1ß expression was significantly greater in the bronchiolar epithelium of animals exposed to high tidal volume ventilation compared with control animals (*p < 0.05).

 
IL-1ß Protein Expression
To determine whether IL-1ß protein content (as distinct from only mRNA) was increased after HV ventilation, we performed Western blot analysis. There was a progressive increase in pro–IL-1ß (37 kD) content with duration of HV ventilation (Figure 9A) . Forty minutes after the onset of HV ventilation there was a small increase in lung pro–IL-1ß content when compared with nonventilated control animals. At 90 minutes of HV ventilation, lung pro–IL-1ß protein levels were significantly increased versus control group (Figure 9B). No appreciable levels of active secreted IL-1ß, ranging from 13 and 19 kD, were detected after 90 minutes of HV ventilation.



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  		Figure 9. Expression of interleukin (IL)-1ß protein with duration of high tidal volume (HV) ventilation. Western blotting (A) and densitometric analysis (B) demonstrated a significant effect (*p < 0.05) of duration of HV ventilation on increased lung pro–IL-1ß protein content (0 minutes, 40 minutes, 90 minutes; n = 3 per group).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The current study highlights the potential of gene screening methodologies to explore the molecular mechanisms of ventilator-assisted lung injury (VALI). Using this approach, we demonstrated that altered gene (and protein) expression occurs before demonstrable lung injury, these alterations are time and stretch dependent and have characteristic spatial distributions.

Analysis of the array data identified three members of the nerve growth factor–induced protein family (Egr-1, Nur77, and BTG2) in the HV group, suggesting a common upstream mechanism linking mechanotransduction with growth factor signaling. Currently, several signaling pathways are linked to mechanotransduction as well as to growth factor signaling (16). In particular, mechanical stress (17) and certain growth factors activate phospholipase C with subsequent activation of protein kinase C (18). Previous work from our laboratory demonstrated that mechanical strain of mixed fetal rat lung cells activates phospholipases, followed by protein kinase C activation, within 5–15 minutes of stretch initiation (19). Growth factors also have the ability to activate the mitogen-activated protein kinase pathway (20), and recent studies have demonstrated that stretching human bronchial epithelial BEAS-2B cells results in rapid (5–10 minutes) activation of the three main mitogen-activated protein kinases (21). This mechanism appears to be conserved in primary cells and is at least partially regulated by G-proteins (22). When taken together, these data suggest a unifying hypothesis, whereby mechanical stretch activates phospholipase C and through protein kinase C activates the mitogen-activated protein kinase pathway via Raf, resulting in increased transcription of nerve growth factor–inducible genes (e.g., Egr1, Nur77, or BTG2).

A second potential mechanism of VALI may involve the secretion of preformed proteins. Multiple reports demonstrate that cyclic stretch of lung cells is associated with increased secretion of inflammatory mediators (21, 23), surfactant (24, 25), growth factors (26, 27), and several extracellular martix proteins (28). The current study demonstrated downregulation of two members of the Rab-signaling family (i.e., Rabin3, Rab7), which is consistent with the report of Basdra and coworkers (29), who demonstrated that mechanical stress can alter Rab expression. Because the Rab family of guanosine triphosphatases are important regulators of intracellular vesicle trafficking (30), these data suggest a possible link among mechanical stretch, altered Rab expression, intracellular vesicular trafficking, and secretion.

Although two potential mechanisms of stretch-induced injury are suggested by altered expression in gene families, use of cluster analysis, which discovers the natural grouping in such data, can suggest additional pathways (8). As others have reported in alternative models of lung disease (31–34), we clustered our gene expression data in an attempt to identify less obvious linkages. Both hierarchical and k-means clustering suggested that Egr-1, c-Jun, and IL-1ß are linked. Supporting this contention is the fact that both c-Jun and Egr-1 expression are increased before IL-1ß in the current model and have the ability to increase IL-1ß expression (35, 36). This linkage is further supported by the coexpression of all three genes in the bronchial epithelium and the time-dependent increase in IL-1ß protein content. These findings may be important as the presence of IL-1ß has been associated with stretch-induced injury in many in vitro (37), ex vivo (3), in vivo (13), and clinical (14) studies. Also, persistent elevation of plasma IL-1ß in mechanically ventilated patients with adult respiratory distress syndrome predicts adverse outcome (38), whereas pretreatment with an IL-1ß antagonist can attenuate experimental lung injury (39).

Globally, data from the current study also supports the idea that HV ventilation initiates all the essential elements of a classic stress response. Typically, the classical stress response involves cytoprotection, decreased protein synthesis, and altered apoptosis (40). Specifically, we identified upregulation of HSP70, a critical mediator for cytoprotection (41), in the bronchiolar and alveolar epithelium. We also identified several downregulated genes that are associated with reduced energy metabolism (e.g., glucose-6-phosphatase; adenosine kinase and myoadenylate deaminase) as well as attenuated protein modification (e.g., endoplasmic reticulum {alpha}-mannosidase). Finally, our analysis revealed that high stretch is associated with increased expression of Nur77, a gene that has a conclusive link to apoptosis (42). Thus, as has been suggested previously (15), targeting elements of the stress response may represent therapeutic goals in the prevention of ventilator-associated lung injury.

Limitations of Experiments
There are several limitations to the current data. As with all gene screening experiments there is considerable potential for false-positive and false-negative results. Thus, we analyzed our data by using several approaches. Our initial threshold, using Atlas Image, was set at twofold or greater change in array expression and a minimum difference of 5,000 AU to maximize sensitivity. To add further power to our analysis, we processed our normalized data through SAM, a statistical program that measures the strength of the relationship between gene expression and the treatment conditions (e.g., mechanical ventilation vs. control conditions) (7). The cutoff for significance in this approach is determined by the parameter {Delta} (difference in expression vs. control animals), as opposed to fold changes. We chose a {Delta} of 0.80, and this yielded a false discovery rate of around 1%. SAM identified many more genes as significantly altered compared with our analysis using Atlas Image; however, to maximize our specificity, only those genes that demonstrated a twofold change in expression, a difference of 5,000 AU, and had a {Delta} of 0.8 or greater were deemed true-positives. The use of pooled samples is associated with serious concerns. For example, we found that pooled samples of MRP14 suggested significant alteration of expression of that gene, but confirmation demonstrated that the overall gene array signal was attributable to a single extreme outlying case. Use of pooled samples has considerable merit, especially in initial pilot analyses, where the underlying mechanisms of a disorder are not well described. However, to make these data useful, confirmation studies need to be performed on individual animals or tissues.

CONCLUSIONS
The current study globally assessed the early changes in gene expression resulting from in vivo HV ventilation and confirms time dependency, tissue localization, and increased gene product production. These data suggest that the nerve growth factor–inducible family of proteins and the Rab family of proteins may be key mechanisms in the pathogenesis of stretch-induced lung injury. Furthermore, we suggest a linkage between two early transcription factors (Egr-1 and c-Jun) and the production of IL-1ß, and finally, we outline potential mediators for each of the three components of the stress response. Taken together, the patterns of gene expression we find help generate biologically plausible hypotheses regarding mechanistic pathways that precede stretch-induced injury and may play a causative role therein.


   FOOTNOTES
 
Supported by Canadian Institutes of Health Research.

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

Conflict of Interest Statemet: I.B.C. has no declared conflict of interest; B.P.K. has no declared conflict of interest; D.E. has no declared conflict of interest; C.McK. has no declared conflict of interest; J.B. has no declared conflict of interest; M.P. has no declared conflict of interest.

Received in original form August 29, 2002; accepted in final form June 12, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Verbrugge SJ, Uhlig S, Neggers SJ, Martin C, Held HD, Haitsma JJ, Lachmann B. Different ventilation strategies affect lung function but do not increase tumor necrosis factor-alpha and prostacyclin production in lavaged rat lungs in vivo. Anesthesiology 1999;91:1834–1843.[CrossRef][Medline]
  2. von Bethmann AN, Brasch F, Nusing R, Vogt K, Volk HD, Muller KM, Wendel A, Uhlig S. Hyperventilation induces release of cytokines from perfused mouse lung. Am J Respir Crit Care Med 1998;157:263–272.
  3. 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.[Medline]
  4. Ricard JD, Dreyfuss D, Saumon G. Production of inflammatory cytokines in ventilator-induced lung injury: a reappraisal. Am J Respir Crit Care Med 2001;163:1176–1180.[Abstract/Free Full Text]
  5. Duggan M, McCaul CL, McNamara PJ, Engelberts D, Ackerley C, Kavanagh BP. Atelectasis Causes Vascular Leak and Lethal Right Ventricular Failure in Uninjured Rat Lungs. Am J Respir Crit Care Med 2003;167:1633–1640.[Abstract/Free Full Text]
  6. Brazma A, Hingamp P, Quackenbush J, Sherlock G, Spellman P, Stoeckert C, Aach J, Ansorge W, Ball CA, Causton HC, et al. Minimum information about a microarray experiment (MIAME)—toward standards for microarray data. Nat Genet 2001;29:365–371.[CrossRef][Medline]
  7. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 2001;98:5116–5121.[Abstract/Free Full Text]
  8. Valafar F. Pattern recognition techniques in microarray data analysis: a survey. Ann N Y Acad Sci 2002;980:41–64.[Abstract/Free Full Text]
  9. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:402–408.[CrossRef][Medline]
  10. Fink L, Seeger W, Ermert L, Hanze J, Stahl U, Grimminger F, Kummer W, Bohle RM. Real-time quantitative RT-PCR after laser-assisted cell picking. Nat Med 1998;4:1329–1333.[CrossRef][Medline]
  11. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–685.[CrossRef][Medline]
  12. Morawietz H, Ma YH, Vives F, Wilson E, Sukhatme VP, Holtz J, Ives HE. Rapid induction and translocation of Egr-1 in response to mechanical strain in vascular smooth muscle cells. Circ Res 1999;84:678–687.[Abstract/Free Full Text]
  13. Chiumello D, Pristine G, Slutsky AS. Mechanical ventilation affects local and systemic cytokines in an animal model of acute respiratory distress syndrome. Am J Respir Crit Care Med 1999;160:109–116.[Abstract/Free Full Text]
  14. Pugin J, Verghese G, Widmer MC, Matthay MA. The alveolar space is the site of intense inflammatory and profibrotic reactions in the early phase of acute respiratory distress syndrome. Crit Care Med 1999;27:304–312.[CrossRef][Medline]
  15. Ribeiro SP, Rhee K, Tremblay L, Veldhuizen R, Lewis JF, Slutsky AS. Heat stress attenuates ventilator-induced lung dysfunction in an ex vivo rat lung model. Am J Respir Crit Care Med 2001;163:1451–1456.[Abstract/Free Full Text]
  16. Eliceiri BP. Integrin and growth factor receptor crosstalk. Circ Res 2001;89:1104–1110.[Abstract/Free Full Text]
  17. Li C, Xu Q. Mechanical stress-initiated signal transductions in vascular smooth muscle cells. Cell Signal 2000;12:435–445.[CrossRef][Medline]
  18. Azzi A, Boscoboinik D, Hensey C. The protein kinase C family. Eur J Biochem 1992;208:547–557.[Medline]
  19. Liu M, Xu J, Liu J, Kraw ME, Tanswell AK, Post M. Mechanical strain-enhanced fetal lung cell proliferation is mediated by phospholipase C and D and protein kinase C. Am J Physiol 1995;268:L729–L738.
  20. Edwards DR. Cell signalling and the control of gene transcription. Trends Pharmacol Sci 1994;15:239–244.[CrossRef][Medline]
  21. Oudin S, Pugin J. Role of MAP Kinase activation in interleukin-8 production by human BEAS- 2B bronchial epithelial cells submitted to cyclic stretch. Am J Respir Cell Mol Biol 2002;27:107–114.[Abstract/Free Full Text]
  22. Correa-Meyer E, Pesce L, Guerrero C, Sznajder JI. Cyclic stretch activates ERK1/2 via G proteins and EGFR in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 2002;282:L883–L891.[Abstract/Free Full Text]
  23. Vlahakis NE, Schroeder MA, Limper AH, Hubmayr RD. Stretch induces cytokine release by alveolar epithelial cells in vitro. Am J Physiol 1999;277:L167–L173.
  24. Wirtz HR, Dobbs LG. Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 1990;250:1266–1269.[Abstract/Free Full Text]
  25. Rose F, Zwick K, Ghofrani HA, Sibelius U, Seeger W, Walmrath D, Grimminger F. Prostacyclin enhances stretch-induced surfactant secretion in alveolar epithelial type II cells. Am J Respir Crit Care Med 1999;160:846–851.[Abstract/Free Full Text]
  26. Yamamoto H, Teramoto H, Uetani K, Igawa K, Shimizu E. Stretch induces a growth factor in alveolar cells via protein kinase. Respir Physiol 2001;127:105–111.[CrossRef][Medline]
  27. Yamamoto H, Teramoto H, Uetani K, Igawa K, Shimizu E. Cyclic stretch upregulates interleukin-8 and transforming growth factor-beta1 production through a protein kinase C-dependent pathway in alveolar epithelial cells. Respirology 2002;7:103–109.[CrossRef][Medline]
  28. Liu M, Post M. Invited review: mechanochemical signal transduction in the fetal lung. J Appl Physiol 2000;89:2078–2084.[Abstract/Free Full Text]
  29. Basdra EK, Papavassiliou AG, Huber LA. Rab and rho GTPases are involved in specific response of periodontal ligament fibroblasts to mechanical stretching. Biochim Biophys Acta 1995;1268:209–213.[Medline]
  30. Novick P, Zerial M. The diversity of Rab proteins in vesicle transport. Curr Opin Cell Biol 1997;9:496–504.[CrossRef][Medline]
  31. Geraci MW, Moore M, Gesell T, Yeager ME, Alger L, Golpon H, Gao B, Loyd JE, Tuder RM, Voelkel NF. Gene expression patterns in the lungs of patients with primary pulmonary hypertension: a gene microarray analysis. Circ Res 2001;88:555–562.[Abstract/Free Full Text]
  32. Sugita M, Geraci M, Gao B, Powell RL, Hirsch FR, Johnson G, Lapadat R, Gabrielson E, Bremnes R, Bunn PA, et al. Combined use of oligonucleotide and tissue microarrays identifies cancer/testis antigens as biomarkers in lung carcinoma. Cancer Res 2002;62:3971–3979.[Abstract/Free Full Text]
  33. Miura K, Bowman ED, Simon R, Peng AC, Robles AI, Jones RT, Katagiri T, He P, Mizukami H, Charboneau L, et al. Laser capture microdissection and microarray expression analysis of lung adenocarcinoma reveals tobacco smoking- and prognosis-related molecular profiles. Cancer Res 2002;62:3244–3250.[Abstract/Free Full Text]
  34. Zou J, Young S, Zhu F, Gheyas F, Skeans S, Wan Y, Wang L, Ding W, Billah M, McClanahan T, et al. Microarray profile of differentially expressed genes in a monkey model of allergic asthma. Genome Biol [serial online] 2002[April 11, 2002];3(5):research0020.1–0020.13. Available from: http://genomebiology.com/2002/3/5/research/0200.
  35. Serkkola E, Hurme M. Synergism between protein-kinase C and cAMP-dependent pathways in the expression of the interleukin-1 beta gene is mediated via the activator- protein-1 (AP-1) enhancer activity. Eur J Biochem 1993;213:243–249.[Medline]
  36. Husmann M, Jehnichen P, Jahn B, Schlosshan D, Romahn E, Marx J. A novel SP-1 site in the human interleukin-1 beta promoter confers preferential transcriptional activity in keratinocytes. Eur J Immunol 1996;26:3008–3014.[Medline]
  37. Schwachtgen JL, Houston P, Campbell C, Sukhatme V, Braddock M. Fluid shear stress activation of egr-1 transcription in cultured human endothelial and epithelial cells is mediated via the extracellular signal-related kinase 1/2 mitogen-activated protein kinase pathway. J Clin Invest 1998;101:2540–2549.[Medline]
  38. Meduri GU, Headley S, Kohler G, Stentz F, Tolley E, Umberger R, Leeper K. Persistent elevation of inflammatory cytokines predicts a poor outcome in ARDS. Plasma IL-1 beta and IL-6 levels are consistent and efficient predictors of outcome over time. Chest 1995;107:1062–1073.[Abstract/Free Full Text]
  39. Narimanbekov IO, Rozycki HJ. Effect of IL-1 blockade on inflammatory manifestations of acute ventilator-induced lung injury in a rabbit model. Exp Lung Res 1995;21:239–254.[Medline]
  40. Wong HR, Wispe JR. The stress response and the lung. Am J Physiol 1997;273:L1–L9.
  41. Beere HM, Green DR. Stress management—heat shock protein-70 and the regulation of apoptosis. Trends Cell Biol 2001;11:6–10.[CrossRef][Medline]
  42. Slagsvold HH, Ostvold AC, Fallgren AB, Paulsen RE. Nuclear receptor and apoptosis initiator NGFI-B is a substrate for kinase ERK2. Biochem Biophys Res Commun 2002;291:1146–1150.[CrossRef][Medline]



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