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Am J Physiol Lung Cell Mol Physiol 291: L851-L861, 2006. First published June 16, 2006; doi:10.1152/ajplung.00463.2005
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TRANSLATIONAL PHYSIOLOGY

Microarray analysis of regional cellular responses to local mechanical stress in acute lung injury

Brett A. Simon,1 R. Blaine Easley,1 Dmitry N. Grigoryev,2 Shwu-Fan Ma,2 Shui Q. Ye,2 Tera Lavoie,2 Rubin M. Tuder,3 and Joe G. N. Garcia2

Departments of 1Anesthesiology and Critical Care Medicine, 2Pulmonary and Critical Care Medicine, and 3Pathology, The Johns Hopkins University, Baltimore, Maryland

Submitted 2 November 2005 ; accepted in final form 9 June 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Human acute lung injury is characterized by heterogeneous tissue involvement, leading to the potential for extremes of mechanical stress and tissue injury when mechanical ventilation, required to support critically ill patients, is employed. Our goal was to establish whether regional cellular responses to these disparate local mechanical conditions could be determined as a novel approach toward understanding the mechanism of development of ventilator-associated lung injury. We utilized cross-species genomic microarrays in a unilateral model of ventilator-associated lung injury in anesthetized dogs to assess regional cellular responses to local mechanical conditions that potentially contribute pathogenic mechanisms of injury. Highly significant regional differences in gene expression were observed between lung apex/base regions as well as between gravitationally dependent/nondependent regions of the base, with 367 and 1,544 genes differentially regulated between these regions, respectively. Major functional groupings of differentially regulated genes included inflammation and immune responses, cell proliferation, adhesion, signaling, and apoptosis. Expression of genes encoding both acute lung injury-associated inflammatory cytokines and protective acute response genes were markedly different in the nondependent compared with the dependent regions of the lung base. We conclude that there are significant differences in the local responses to stress within the lung, and consequently, insights into the cellular responses that contribute to ventilator-associated lung injury development must be sought in the context of the mechanical heterogeneity that characterizes this syndrome.

adult respiratory distress syndrome; mechanical ventilation; canine; computed tomography; cross-species microarray

IN RECENT YEARS, THE FOCUS of research into acute lung injury (ALI) has shifted from primarily mechanical and supportive management towards investigation into the cellular and molecular basis of the disease process. However, despite a great deal of data suggesting interactions between mechanical stress, inflammation, and the development of lung injury, the pathogenesis of ventilator-associated lung injury (VALI) is not well understood. A hallmark of human ALI that is not captured in many small animal models is the marked heterogeneity of tissue involvement (19). Mechanical and biological phenomena potentially contributing to VALI vary widely throughout the lung and may be highly dependent on particular supportive interventions. Recognizing this, management strategies have implicitly sought to reduce this heterogeneity so as to minimize the presumed "injurious" mechanical events (such as overdistension or air space opening/closing) while maintaining adequate gas exchange for life support (21).

We believe that VALI does in fact have its origin in inflammatory and other cellular responses in lung tissue exposed to (and possibly, predisposed to injury from) mechanical stress and hypothesize that these responses will vary throughout the heterogeneous injured lung in relation to local mechanical events. To explore this hypothesis, we assessed regional cellular responses by genomic microarray analysis, correlating changes in gene expression with the specific regional mechanical stresses imposed on that local tissue, in a novel canine model of unilateral lung injury. With each animal serving as its own control, the microarray-based genomic approaches (cross-species hybridization to the Affymetrix Human U133A GeneChip) provide broad characterization of regional cellular responses which, combined with functional computed tomography (CT) imaging for the noninvasive measurement of regional mechanical stress, makes possible the investigation of in vivo local cellular responses in the lung associated with heterogeneous VALI. Our goal was to establish whether regional cellular responses to local conditions could be determined and provide a novel window into understanding the mechanism of development of VALI.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal Preparation and Experimental Protocol

All experimental procedures were approved by the Johns Hopkins University Animal Care and Use Committee. Three mongrel dogs (weight 21.3 ± 1.5 kg) were anesthetized with 25 mg/kg pentobarbital intravenously and instrumented with femoral arterial and venous catheters. Anesthesia was maintained with additional pentobarbital (5 mg/kg iv every hour and when indicated), and muscle relaxation was provided by pancuronium (3 mg bolus and 0.5 mg hourly iv). A 39 or 41 French double-lumen endobronchial tube (Mallinkrodt, St. Louis, MO) was placed via a tracheostomy, and position was confirmed by fiber-optic bronchoscopy. The animals were ventilated with a "dual piston" large animal ventilator (Harvard Apparatus, Holliston, MA) permitting independent control of tidal volume (Vt), inspired oxygen fraction (FIO2 1.0), positive end-expiratory pressure (PEEP), measurement of airway opening pressure (Paw), and end-tidal partial pressure of carbon dioxide (ETPCO2) for each lung. Oxygen saturation (SaO2) was continuously measured using a pulse oximeter applied to the tongue or ear, and ETPCO2, arterial blood pressure (Pa), Paw, and esophageal pressure (Pes) were continuously recorded. An infusion of lactated ringers (LR, 5–10 ml·kg–1·h–1) was given for maintenance fluid replacement. Rectal or pulmonary artery temperature was maintained at 36 ± 1°C with radiant heat lamps. At the conclusion of the study, the animals were killed by exsanguination after supplemental pentobarbital (10 mg/kg iv).

After instrumentation, the individual lung Vt were adjusted to provide an ETPCO2 of 30–35 mmHg for each lung at a respiratory rate of 20 breaths/min. This procedure resulted in individual lung Vt (means ± SD) of 8.7 ± 0.3 ml/kg for the right lung and 6.1 ± 0.4 ml/kg for the left, approximating the normal distribution of lung volume. The left lung was then mildly injured by repeated lavage with warmed saline, 20 ml/kg repeated four times while switching the animal's position between right and left lateral decubitus, with gravity drainage. For comparison, typical lung lavage injury protocols use 40–60 ml/kg (both lungs) for six to eight washes to achieve an arterial partial pressure of oxygen (PaO2) of ~100 mmHg. Ventilation continued at baseline settings with the addition of 5 cmH2O PEEP to the control right lung for 5 h. At that time, the animal was killed by exsanguination, the chest was opened, and eight tissue samples were taken from four corresponding regions in both lungs (apex dependent and nondependent, base dependent and nondependent). The lung tissue samples were immersed in RNA later (Ambion, Austin, TX) and frozen for subsequent analysis. In some animals, additional tissue samples were taken and stored in 10% formalin for histology.

CT Imaging and Analysis

One animal was transported to a Toshiba Aquilion 16 multidetector CT scanner for imaging at 4 h postinjury. During the 15-min transport, both lungs were ventilated with a single transport ventilator, but independent lung ventilation was reestablished before imaging. Whole lung image sets gated to end-expiration and end-inspiration were obtained during steady-state ventilation, four 2-mm contiguous slices per breath, using the ECG-gating capability of the scanner and timing signals generated by a computer running LabView 6.1 (National Instruments, Austin, TX). Image density distributions were analyzed using Pulmonary Analysis Software Suite (27) developed at the University of Iowa Division of Physiologic Imaging.

Sample Preparation and Microarray Analysis

Expression data analysis. Each regional tissue sample was run on a separate Affymetrix U133A GeneChip. The signal intensity fluorescent images produced during Affymetrix GeneChip hybridizations were read using the Agilent Gene Array Scanner and converted into GeneChip probe result files (CEL) using MAS 5.0 software (Affymetrix, Santa Clara, CA). The analysis of the probe level data (available in the CEL files) was performed using the Bioconductor affy package (31) as described previously (22). Briefly, the MAS5 (30) module of this package was used for background correction, across array global normalization, and extraction of the probe level data. Poorly performing probe pairs revealed by probe level analysis were masked before converting CEL files into gene expression values (22, 23). This approach increased present call of a transcript (P < 0.04) on average by 45% compared with unadjusted probe set processing. The remaining absent calls (P > 0.06) for transcript abundance were assumed to be a result of undetectable message concentration [<1 pM (9)] rather than technical or detecting errors. Therefore, absent calls for each GeneChip were averaged and all absent calls for a given chip were replaced with the average value. This modified data set was used for further analysis.

Selecting significant gene expression changes. The gene expression profiles of control and injured lungs were paired by animal and lung region from which samples were collected. Signal intensity values for each pair were log transformed (base 2) and raw-wise normalized, thus rendering each profile pair an independent data module composed of normalized hybridization signals of corresponding control and injured samples. For apex/base comparisons, there were six modules in each group (dependent and nondependent samples for each apex and base location in 3 animals), and there were three modules in each group for dependent base/nondependent base comparisons. These modules were grouped and tested for similarities in gene expression either between base and apex regions (12 apex vs. 12 base) or dependent and nondependent areas of the lung base (6 nondependent vs. 6 dependent) using Significance Analysis of Microarray [SAM (53)]. To evaluate the significance of differences in gene expression between apex/base or nondependent/dependent regions, the control/injury modules were converted into injured/uninjured fold-change values (log base 2) and analyzed using SAM, comparing six apex vs. six base or three nondependent vs. three dependent, respectively. The injured/uninjured fold-change ratio for individual genes was computed by SAM as the antilog of the mean log-transformed individual dog-injured/uninjured ratios. All data are thus reported as the absolute (non-log-transformed) fold-change ratio. Genes with 70% change (±1.70-fold) in expression and false discovery rate (FDR) <2% were considered affected by lung injury based on the sample size analysis described below.

To determine the appropriate fold-change cutoff, we utilized a new concept for sample size determination in microarray experiments for class comparisons (11). The median ({sigma}2 = 0.169) of signal intensity log ratios in dog lung tissues was calculated from 10 arrays generated for uninjured lungs and was utilized as a combined variance following the recommendations of Yang and Speed (59). This derived variance value for inbred dogs is fairly typical for models employing homogeneous subject populations [e.g., inbred mice strains demonstrated {sigma}2 = ~0.25 (11)]. We applied {sigma}2 = 0.169, 2% FDR ({alpha} = 0.02), and number of replicates n = 3 to the single-labeled microarray sample size-identifying formula and obtained a significance cut-off fold-change value {delta} = ± 1.70 for changes in gene expression between dependent and nondependent base regions:

Formula 1(1)
where n = number of predicted arrays; z{alpha}/2 and zbeta are the 100{alpha}/2th and 100betath percentiles of the normal distribution, respectively; {delta} is the distance between the class means that the study is being powered to detect; and {sigma}g2 is the combined gene expression variances (biological variance due to heterogeneity from individual to individual of gene expression within the phenotype and experimental error variance due to technical inaccuracies in the microarray measurement variances). Despite this theoretical approach to determine an appropriate fold-change cut-off, it should be recognized that the actual value used remains somewhat arbitrary and, because of the small number of animals in the study, data interpretation should be with caution.

Semiquantitative Duplex RT-PCR

Total RNAs (0.5 µg) from each tissue sample were used as a starting material for the reverse transcriptase (RT) reaction. Primers for PBEF, THBS1, GADD45a, DEADH, GGH, and ribosomal protein S18 (housekeeping gene control) were designed based on published GenBank mRNA sequences (NM_005746.1, NM_003246.1, NM_001924.2, R0068, NM_003878, and NM_022551.2, respectively). One tenth of RT product was used in a typical 25-cycle PCR run. The PCR products were separated on 2% agarose gel electrophoresis and visualized by ethidium bromide staining. The image was captured on thermal paper (Fotodyne System, Hartland, WI) and scanned into Adobe Photoshop 7.0.1 using an HP Scanjet 7400c scanner, and band intensities were analyzed with AlphaEase FC Software (Alpha Innotech, San Leandro, CA). Results were expressed as a fold-change of injured tissues vs. control tissue.


   RESULTS
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 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Unilateral Canine Lung Injury Model

Over the course of the 5-h observation period, the peak inspiratory pressure of the injured lungs approximately doubled, whereas that of the control lungs remained unchanged. SaO2 remained at 99–100% at all times despite unilateral lung injury because of the use of a high FIO2 and the presence of the control lung. All animals remained normotensive throughout the study without intervention. Lung wet/dry weight ratios (w/d, means ± SD) were lower in control lungs (5.76 ± 1.01) compared with injured lungs (8.08 ± 1.86, P < 0.01), with w/d in injured lungs greater in base regions (9.14 ± 1.37) than apex regions (7.01 ± 1.70, P = 0.02). There was no significant difference in w/d between base and apex regions in the control lungs (6.15 ± 1.20 vs. 5.38 ± 0.66, P = 0.13).

Representative tissue histological sections show severe acute inflammation in the left (injured) lung base, with reduced inflammation in the left nondependent base (Fig. 1, A and B). In comparison, the right (control) lung sections revealed minimal inflammatory changes (Fig. 1, C and D). Apical sections of both control and injured lungs showed minimal inflammation.


Figure 1
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  		Fig. 1. Histological sections from injured and control lung base. Lung sections shown are representative of the left lung (injured), base-dependent region (A); left lung, base-nondependent region (B); right lung (control), base-dependent region (C); and right lung, base-nondependent region (D). Note the severe acute inflammation in the left base-dependent region, with accumulation of neutrophils in bronchioles and centrilobular alveoli, whereas there is significantly lesser inflammation in the left base-nondependent region (B) when compared with A. The dependent areas of the control right lung (C) show some mild increase in septal inflammatory cells, whereas the nondependent areas are preserved (D) (hematoxylin and eosin, bar = 150 µm).

 
CT imaging in one animal revealed increased lung density throughout the lavaged left lung, with preservation of normal aeration in the control right lung with the exception of mild dependent atelectasis (Fig. 2A). Apex-to-base profiles of average lung density from the cross-sectional images for the control lung showed a normal distribution of density for a supine dog (36), ranging from 80% air content at the apex to 50–60% toward the lung base. In contrast, the injured lung exhibited severe loss of aeration throughout except for regions toward the apex (Fig. 2D). Examining vertical gradients of lung density in subvolumes from the midapex and midbase regions showed steep aeration gradients in both injured and control lungs. At the apex, both lungs were well aerated in nondependent regions, but with the injured lung losing aeration much more rapidly toward the dependent regions. At the base, the injured lung was poorly aerated throughout (Fig. 2B). Voxel density histograms from dependent and nondependent regions of both lungs from the apex and base further illustrate the mechanical heterogeneity of these lung regions (Fig. 2C). This distribution of lung density, with greatest loss of aeration in dependent base regions, is consistent with patterns seen in humans with ALI/acute respiratory distress syndrome (ARDS) (45) as well as with the pattern of long-term injury seen in the lungs of ARDS survivors (18).


Figure 2
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  		Fig. 2. Computed tomography (CT) data from unilateral lung injury model. A: end-expiratory axial images from the lung apex and base and a coronal reconstruction through a level just anterior to the spine. Circles mark approximate location of tissue sampling for microarray analysis. B: vertical gradient of lung density in control and injured lungs from the apex (top) and base (bottom) CT images. Abscissa, vertical position (cm). C: end-expiratory lung density histograms from dependent (Dep) and nondependent (Non-Dep) regions of the control and injured lungs from the apex (top) and base (bottom) CT images. D: base to apex profile of average density of control and injured lungs. Solid lines indicate location of images used to determine apex and base data presented in B and C.

 
Regional Microarray Analysis

A total of 24 samples from three animals were successfully hybridized to 24 Affymetrix HG-U133A microarray chips and are included in this analysis. Since samples were taken from corresponding regions of the injured and control lungs of each animal, results from each regional injury sample were normalized to the within-animal control, a unique feature of this unilateral injury model. Initial analysis of expression profiles generated using Affymetrix MicroArray Suite (MAS 5.0) showed that ~14% (~3,000 of >22,000 probe sets analyzing 18,000 transcripts and variants) of the canine genome hybridized to corresponding human probe sets with an efficiency similar to human mRNA. Sequence comparison showed that this fraction of canine mRNAs represented canine genes that were highly homologous to their human counterparts. Overall transcript detection by the cross-species hybridization of canine mRNA to the HG-U133A chip was much less sensitive (14%) than hybridization of human mRNA (typically 48%). However, application of canine-specific masking techniques (22, 23) in which nonhomologous probe sets between species are excluded from the usual multiprobe set matching requirements increased transcript detection to >65%.

Comparing regions, there were 308 and 595 genes significantly changed [defined as FDR <2% and average regional injured/control fold-change (FC) >1.7 or <–1.7] in the apex and base, respectively, of which 213 (69.2%) increased expression in the apex and 238 (40.0%) increased in the base; 367 genes were significantly differentially expressed between the apex and base (FDR <2% for apex/base difference and either apex and/or base injured/control FC >1.7 or <–1.7). Cluster analysis of significantly changed genes revealed groupings of differential regional gene expression changes, along with the most represented gene ontologies (12) (Fig. 3).


Figure 3
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  		Fig. 3. Hierarchical clustering of apex-base changes in gene expression; 255 genes whose expression was either significantly affected by injury in both apex and base regions or was significantly different between these regions were combined and clustered using MeV (46). Each column represents an experimental condition of corresponding sample location. Regional hierarchical clustering (top dendrogram) correctly grouped experimental conditions into 2 large apex and base clusters. Hierarchical clustering of genes identified 6 major clusters (blue triangles), of which 4 clusters demonstrated clear differences or similarities in gene expression between apex and base regions. Genes from these clusters were linked to gene ontology terms using MAPPFinder (12), and the most representative gene ontologies for each cluster are listed (right). Fractions of genes in the cluster linked to corresponding ontologies were calculated and expressed in percentages. Red color indicates upregulation, and green color indicates downregulation, of gene expression relative to corresponding region of control lung in same animal, with color intensity corresponding to the fold-change amplitude (fold-change scale shown at top). Sample coding: A, apex; B, base; 1, 2, 3, number of the animal; D, dependent; ND, nondependent.

 
Regional gene expression differences were even more marked between dependent and nondependent samples from the lung base. Of the 1,438 genes with changed expression (FDR <2% and injured/control FC >1.7 or <–1.7) in the dependent samples, 55.1% had increased expression relative to control compared with 45.5% of the 1,221 changed genes in the nondependent samples. Expression was significantly different between the dependent and nondependent base regions in 1,544 genes and changed in opposite directions (FC >1.7 and <–1.7 in the 2 regions) in 63 genes (Table 1). These differences were not due to regional variation in the control samples, as there were no significant dependent/nondependent differences in control lung gene expression and only 18 apex/base differences, none of which appeared in any of the results described here. Cluster analysis of genes significantly changed between apex and base, along with their gene ontologies, are presented in Fig. 4.


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  		Table 1. Summary of gene expression changes in genes with significant expression differences (FDR <2%) between dependent and nondependent base regions

 

Figure 4
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  		Fig. 4. Hierarchical clustering of dependent and nondependent base changes in gene expression; 221 genes whose expression was either significantly affected by injury in both dependent and nondependent regions or was significantly different between these regions were combined and clustered using MeV (46). Each column represents an experimental condition of corresponding sample location. Regional hierarchical clustering (top dendrogram) correctly grouped experimental conditions into 2 large dependent and nondependent clusters. Hierarchical clustering of genes identified 7 major clusters (blue triangles), of which 4 clusters demonstrated clear differences or similarities in gene expression between dependent and nondependent regions (vertical bars). Genes from these clusters were linked to gene ontology terms using MAPPFinder (12), and the most representative gene ontologies for each cluster are listed (right). Fractions of genes in the cluster linked to corresponding ontologies were calculated and expressed in percentages. Red color indicates upregulation, and green color indicates downregulation, of gene expression relative to corresponding region of control lung in same animal, with color intensity corresponding to the fold-change amplitude (fold-change scale shown at top). Sample coding: D, dependent; ND, nondependent; 1, 2, 3, number of the animal.

 
The gene ontologies (12) most significantly changed between dependent and nondependent base samples are presented in Table 2. We identified 226 genes in the literature related to ALI (3, 10, 13, 22, 24, 29, 35, 60) or modified by cyclic stretch in an alveolar epithelial cell model (13) and compared them to the regional expression results. From this list of ALI candidate genes (including closely related genes from the same families), 87 genes exhibited differential gene expression between dependent and nondependent regions within the same lung lobe in these animals. A selection of 50 of these ALI-associated genes, along with additional highly changed genes (FDR < 2% and FC <–2.5 or >2.5) in our model, presented in Fig. 5, represents an extended list of cellular processes altered by the regional environment (see Supplemental Table 1 for the complete referenced list of these 50 differentially changed ALI-related or highly changed genes and Supplemental Table 2 for the complete list of 294 ALI-related or highly changed genes online at the AJP-Lung Cellular and Molecular Physiology web site.). In addition, MIAME compliant microarray data have been deposited at http://www.ncbi.nlm.nih.gov/geo under accession no. GSE1935.


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  		Table 2. Ontology of altered gene expression between dependent and nondependent base regions

 

Figure 5
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  		Fig. 5. Regional differences in gene expression within the lung base. Differential gene expression (fold-change injured/uninjured) between dependent and nondependent regions of the lung base for 50 oligonucleotide probe sets targeting genes either previously identified as acute lung injury (ALI) associated (*) or highly changed in our model. Each color-coded column represents results from an individual animal with mean numerical fold-change values (n = 3) to the right. (See online data Supplemental Table 1 for additional details about these 50 genes and Supplemental Table 2 for the complete list of ALI-associated or highly changed genes at the AJP-Lung Cellular and Molecular Physiology web site.) HSPA4 or heat shock 70-kDa protein 4 is represented by 2 oligonucleotide probe sets on the HG-U133A GeneChip.

 
Validation Studies

GeneChip results for five different upregulated genes were validated using semiquantitative RT-PCR, showing good agreement in fold-change injured/control between samples taken from apex (n = 6) and base (n = 6) regions (Fig. 6). There were no statistically significant differences between microarray and RT-PCR measured fold-changes by paired t-test (P range 0.39–0.57).


Figure 6
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  		Fig. 6. RT-PCR validation of microarray results, expressed as fold-change of injured/control samples, for 5 significantly upregulated genes. Each symbol represents means ± SD for 6 injured and 6 control samples from the apex (solid symbols) or base (open symbols).

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Ventilator management has been associated with patient outcome in ALI since the original description of ARDS by Ashbaugh and colleagues in 1967 (5). Since that time, the advent of imaging studies using CT revealed the heterogeneous mechanical nature of ARDS (19, 20) and provided insight into the impact of ventilator management on regional mechanical events. Meanwhile, basic research in rodent models identified a relationship between presumed mechanical events, such as end-expiratory airways opening/closing and end-inspiratory overdistension, and the release of inflammatory cytokines (8, 44, 51) that could potentially account for the observation that most patients with ARDS die of multiorgan system failure (39), and further, that subjects with sepsis, trauma, and systemic syndromes appear susceptible to developing VALI (1, 33). The association between ALI, ventilator management, outcome, and inflammation has been strengthened in many subsequent basic and clinical studies (7, 25, 49, 52), including several that utilized genomic microarrays to explore broad mechanisms of cellular activation in ALI (3, 10, 13, 22, 35). Several physiological systems have been identified as important in the evolution of VALI, including coagulation, surfactant, stress response, cytoskeletal structure, oxidative injury, and endothelial/epithelial barrier dysfunction (2, 10, 16, 17, 22, 47). Despite these advances, understanding of the relationship between specific mechanical events in the lung and the cellular responses that result in the development or progression of VALI remains incomplete.

Our goal was to establish whether regional cellular responses to local mechanical conditions could be determined as a first step towards using these regional events as a novel window into understanding the mechanism of development of VALI. We developed a unique canine model of unilateral lung injury and mechanical ventilation to allow the comparison of control and injured tissue samples from the same animal. We chose the canine saline lavage model of ALI because the insult is primarily mechanical and serves to amplify the effects of mechanical ventilation by removing surfactant and reducing stability of peripheral air spaces, although a secondary inflammatory response does ensue (48). The progressive increase in peak inspiratory pressure over the 5 h seen only in the lavage lung indicates the interaction between mechanical ventilation and the initial lavage insult that is characteristic of ventilator-associated lung injury. Previous experience with this model had demonstrated significant mechanical heterogeneity (14, 34) not unlike that observed in patients with ARDS (18, 20), with dependent, basal flooding and air space collapse, intermediate zones of apparent air space opening and closing, nondependent overdistension, and relatively preserved apical regions, behavior confirmed by our CT data from one animal. Furthermore, successful cross-species hybridization to human microarrays had been described (38, 40, 57) and canine-specific microarrays were under development (4, 6, 26, 50). Finally, the canine model is of sufficient size to allow lung isolation and independent treatment, permitting the use of each subject as its own control to reduce the baseline variation inherent in unbred subjects. The benefit of decreased intersubject variation from this control scheme should offset possible changes to the control lung from systemic "spillover" of inflammatory or other mediators from the injured lung (49, 51), although the possibility remains of false negative results from this effect.

Although it is known that there is regional mechanical heterogeneity within the injured lung (19), and we documented regional differences in tissue aeration, w/d ratio, and inflammation, there are potentially other factors that could influence these results. In addition to differences in regional ventilation, there are likely differences in regional perfusion within the injured lung. In the lavaged lung, dependent, poorly aerated regions reduce their perfusion via the homeostatic hypoxic pulmonary vasoconstriction response (15). Similarly, although postural repositioning was used in an attempt to uniformly distribute the saline lavage, the removal of surfactant and distribution of residual fluid may be nonuniform. Finally, the number and type of migratory inflammatory cells differ between regions. Thus the stimulus for the differential regional responses found may not be purely the local mechanical stresses but could reflect contributions of other heterogeneous aspects of this large animal injury model.

The extension of microarray technology to large animal models of disease has been limited by the lack of availability of species-specific gene chips, and thus cross-species hybridization has been increasingly utilized for this purpose (38, 40, 57). This approach is useful for gene products with an adequate degree of homology to their human counterparts, whereas failure to detect an expressed gene cannot distinguish between lack of stimulus effect vs. failure to hybridize because of species-specific sequence differences. The present call rate can be increased using masking techniques that either eliminate poorly functioning probe sets or relax the stringent criteria for detecting all the probe set sequences in Affymetrix-type arrays, since differences between species may be limited to only portions of the sequence (22, 23, 26). As our purpose was to demonstrate regional differences in gene expression in response to different mechanical stresses, false negatives are irrelevant with the acknowledgment that these results are but a subset of the gene expression changes that have occurred. Recent announcement of the availability of a commercial canine microarray from Affymetrix as well as the development of custom canine cDNA arrays (4, 6, 50) will facilitate more specific and exhaustive analysis in the future. In any event, the use of microarray data in this manner should be considered exploratory and any specific mechanistic inferences should be validated with more precise techniques.

Analysis of the microarray data for the injured vs. control lung showed significantly changed expression in 472 genes, of which 46% were increased. The functional groupings (ontologies) of these genes were similar to those we have previously described (22, 24), including angiogenesis, cell cycle arrest, inflammation, coagulation, and immune response. Confirming our initial hypothesis, there were large numbers of genes differentially regulated between the apex and base (Fig. 3) and between dependent and nondependent regions within the base (Table 1 and Figs. 4 and 5). These lung regions experience strikingly different mechanical environments, as evidenced by their CT density distributions (Fig. 2). Furthermore, imaging studies of ARDS survivors have shown that chronic lung damage, mostly bullous lesions, occurs predominantly in dependent and basal lung regions, whereas apical regions are relatively spared (18). The mechanical events related to artificial ventilation most commonly invoked as injurious to the lung are overdistension, airways repeatedly opening and closing with each breath, and to a lesser extent tissue collapse and/or flooding, although the data supporting these relationships in patients remain controversial (28). In our model, which used no PEEP in the injured lung, there was minimal evidence of overdistension based on the end-inspiratory criterion of >90% aeration by CT (54), and any overdistension present occurred toward the apex. Of course, it is also possible that differences in other regional properties, such as perfusion or migratory inflammatory cells, influenced the observed changes in gene expression.

Examination of the highly differentially regulated genes between apex and base regions reveals several genes commonly associated with ALI (vascular endothelial growth factor C, THBS1, tranforming growth factor-beta) as well as novel genes not previously described in the ALI literature. For example, the gene encoding the proposed proinflammatory cytokine Pre-B cell colony enhancing factor (PBEF) was one of the most upregulated genes in the injured lung. PBEF was originally described for its role in the maturation of B cell precursors (42) and was subsequently found to be upregulated in amniotic membranes from patients undergoing premature labor (especially with amniotic infections) (37). Epithelial cells from amniotic membranes also upregulate PBEF expression when subjected to stretching in vitro (41), and human fetal membrane explants exposed to PBEF produce inflammatory cytokines and chemokines (43). Furthermore, PBEF was recently identified as a cell cycle regulator, delaying neutrophil apoptosis in experimental inflammation and clinical sepsis (32) and thus functions as a regulator of the inflammatory response. Thus PBEF represents a proinflammatory cytokine, with proposed roles in immune regulation and oxidative metabolism and known association to infected or stretched epithelial cells, which has now been identified as significantly upregulated in a primarily mechanical model of VALI. These observations led us to pursue in-depth investigation of its role in other ALI disease models and in patients with sepsis-related ALI, revealing a consistent upregulation of PBEF in murine and canine LPS-induced ALI, in cytokine and cyclic stretch lung endothelial cells, and increased PBEF protein in both bronchoalveolar lavage and serum of patients with ALI (60). Furthermore, analysis of two single nucleotide polymorphisms in patients with sepsis-associated ALI suggests that a susceptible haplotype (GC) in the PBEF promoter region is associated with a 7.7-fold higher risk of developing ALI (60), strongly supporting a role for PBEF both in the pathogenesis of ALI and as a biomarker for ALI susceptibility.

Among the genes differentially regulated within the lung base were several stress response proteins, notably the inducible 70-kDa heat shock protein (HSP) whose expression has been associated with protection from development of ALI (55). Three HSP family genes, along with related chaperonins CCT7 and CCT8 and early response genes EGR3 and IER5, were all upregulated in both dependent and nondependent base regions. The increased expression of all these genes was significantly greater in the nondependent regions (2.3- to 6.5-fold) compared with dependent regions (1.0- to 1.7-fold) (Fig. 5). In keeping with the reported protective and anti-inflammatory actions of these proteins (55, 58), expression of cytokines (TNF-{alpha}, IL-2, IL-6 receptor, and IL-7) and chemokines (IL-8, CCL, and CXCL families) was decreased in the nondependent regions (–1.1- to –2.0-fold), whereas it increased in dependent regions (1.0- to 2.0-fold). Thus, in the dependent regions of the lung base, which are flooded and collapsed with minimal penetration of air throughout the respiratory cycle (Fig. 2), there is an enhanced inflammatory response. In contrast, the well-aerated and potentially overdistended nondependent regions experience a robust stress response along with reduced cytokine and chemokine expression. These disparate responses are consistent with the results of a recent study in rats in which bacterial endotoxin plus injurious ventilation revealed a similar inverse relationship between HSP70 levels and IL-6 and IL-1beta mRNA expression (56). Studies examining the time course of these changes and further modulating mechanical conditions will help determine how these factors contribute to protection vs. injury in VALI.

To further illustrate the significance of these regional differences in gene expression, we compared our list of genes differentially regulated between dependent and nondependent regions of the lung base with a list of candidate genes identified from the literature (3, 10, 13, 22, 24, 29, 35, 60). Of the ~226 ALI candidate genes identified, 87 (including closely related genes in the same family) met our criteria for significantly differentially changed between dependent and nondependent regions. Remarkably, expression of 19 of these genes were changed in opposite directions, mostly upregulated in the dependent regions while downregulated in the nondependent region of the same lung in the same animal. Thus, the particular pattern of gene expression in the injured lung is critically dependent on the sampling location. Whether these regional differences are specifically due to the different mechanical stresses, the population of cell types in these regions or some other aspect of the local physiology such as perfusion remains unknown. It should be apparent, however, that sampling approaches that average expression patterns across both apex and base, dependent and nondependent regions are likely to underestimate the impact of genes that exhibit regional-specific expression. Averaging out of regional differences may explain the increasing numbers of differentially regulated genes detected as the regions examined become more localized. Furthermore, insight into the earliest cellular responses constituting VALI, and the contribution of different regional mechanical phenomena to these responses (that may be modified by patient and ventilator management), must be sought in the context of the mechanical heterogeneity that characterizes human ALI.

In summary, we have demonstrated significant differences in gene expression between different regions of the lung in a canine model of ALI, regions that undergo very different mechanical stresses during mechanical ventilation. These findings have important implications for the design and interpretation of studies investigating the mechanisms of VALI. A hallmark of human ALI is the heterogeneity of tissue involvement, and management strategies that reduce heterogeneity, in particular extremes of mechanical behavior, are the focus of current clinical investigations. Correlating regional cellular responses with the local mechanical and biological milieu may help us to not only understand how different mechanical stresses cause or propagate existing lung injury but also identify therapeutic targets, associate specific biomarkers with different aspects of evolving injury, and find surrogate endpoints to better predict outcomes and guide therapy.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-64368, National Institutes of Health Acute Lung Injury SCCOR HL-073994, and the Johns Hopkins HopGene Program in Genomic Applications UO1HL66583.


   ACKNOWLEDGMENTS
 
We thank Dr. Eric A. Hoffman and Dr. Junfeng Guo of the Department of Radiology, Division of Physiologic Imaging, University of Iowa, for providing the PASS software.


   FOOTNOTES
 

Address for reprint requests and other correspondence: B. A. Simon, Dept. of Anesthesiology and Critical Medicine, Tower 711, Johns Hopkins Hospital, Baltimore, MD 21287-8711 (e-mail: bsimon{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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