Comments
Abstract
The aim of this study was to assess the association between regional tidal volume (Vt), regional functional residual capacity (FRC), and the expression of genes linked with ventilator-induced lung injury. Two groups of BALB/c mice (n = 8 per group) were ventilated for 2 hours using a protective or injurious ventilation strategy, with free-breathing mice used as control animals. Regional Vt and FRC of the ventilated mice was determined by analysis of high-resolution four-dimensional computed tomographic images taken at baseline and after 2 hours of ventilation and corrected for the volume of the region (i.e., specific [s]Vt and specific [s]FRC). RNA concentrations of 21 genes in 10 different lung regions were quantified using a quantitative PCR array. sFRC at baseline varied regionally, independent of ventilation strategy, whereas sVt varied regionally depending on ventilation strategy. The expression of IL-6 (P = 0.04), Ccl2 (P < 0.01), and Ang-2 (P < 0.05) was associated with sVt but not sFRC. The expression of seven other genes varied regionally (IL-1β and RAGE [receptor for advanced glycation end products]) or depended on ventilation strategy (Nfe2l2 [nuclear factor erythroid-derived 2 factor 2], c-fos, and Wnt1) or both (TNF-α and Cxcl2), but it was not associated with regional sFRC or sVt. These observations suggest that regional inflammatory responses to mechanical ventilation are driven primarily by tidal stretch.
Acute respiratory distress syndrome (ARDS) has a high mortality rate (1–3). Although the severity of ARDS is characterized by the degree of hypoxia (4), only a low percentage of patients with ARDS died of hypoxemia, with the majority dying as a result of multisystem organ failure (5). Unfortunately, although mechanical ventilation is necessary for patients with ARDS, it may also directly contribute to multisystem organ failure by inducing inflammation (6, 7).
Mechanical ventilation in ARDS aims to provide adequate gas exchange while minimizing lung injury; however, it may damage the lung in a process known as “ventilator-induced lung injury” (VILI) (8–10). The impact of high tidal volume (high Vt; overdistention) has been clearly demonstrated by the ARDSnet (NHLBI ARDS Network) study, which showed 22% lower mortality in patients ventilated at 6 ml/kg than in those receiving a traditional Vt of 12 ml/kg (11). However, only one intervention has proved to be effective in reducing mortality in ARDS since this study: prone positioning for patients with severe ARDS (1, 2, 12, 13). This highlights the importance of the lung mechanical response to ventilation in determining patient outcomes.
Identifying the optimum ventilation strategy to minimize VILI is complicated by the heterogeneous nature of ARDS (14). In addition, both overdistention and atelectrauma can occur within the same lung (15, 16), and both can trigger an inflammatory response (biotrauma) (17–20). However, their relative contribution to overall biotrauma within an individual lung remains poorly understood (21). At the whole-lung level, we have previously shown an association between overdistention and the presence of pulmonary edema with protein leak and macrophage infiltration (21). However, we were unable to assess how these responses varied spatially, which may be an important determinant of outcome because studies have shown that ventilation causes a heterogeneous pattern of lung damage (15, 22) due to the inhomogeneous distribution of regional tidal strain (23).
The aim of this study was to assess the association between regional functional residual capacity (FRC) and regional Vt and the expression of markers of lung injury in response to mechanical ventilation. We investigated this in a mouse model by combining gene expression analysis with a recently developed lung-imaging technology that allows image capture at high speed and high resolution over the entire breathing cycle (24).
Six- to 9-week-old female BALB/c mice (Animal Research Platform, Monash University) were provided food and water ad libitum and housed in a 12-h/12-h light/dark cycle. All experiments were approved by the Monash University Animal Ethics Committees.
Mice were prepared as described previously (25). Briefly, they were anesthetized, tracheostomized, and mounted upright on a rotating stage (Zaber Technologies) in a customized holder. They were ventilated for 2 hours according to one of two protocols: 1) protective (n = 16; 225 breaths/min; peak inspiratory pressure, 12 cm H2O; PEEP, 2 cm H2O) or 2) injurious (n = 16; 144 breaths/min; peak inspiratory pressure, 20 cm H2O; positive end-expiratory pressure [PEEP], 0 cm H2O). Lung images of the ventilated groups were taken at baseline (H0) and after 2 hours (H2) of ventilation. Mice were killed by overdose with sodium pentobarbitone (200 mg/kg) before processing of the lung tissue for gene expression (n = 8 per group) or immunohistochemistry (n = 8 per group). A separate group of mice (n = 8) served as unventilated control animals for gene expression.
For X-ray imaging, we used the liquid MetalJet X-Ray Source Technology (Excillum AB), enabling high-brightness and high-resolution imaging (26, 27). A high-speed detector (PaxScan; Varian Medical Systems) was used to capture projection images at 30 frames per second for four-dimensional computed tomographic reconstructions (28).
We applied a three-dimensional velocimetry technique to measure the regional Vt (24, 28, 29) by analyzing 400 frames per computed tomographic scan to calculate regional tissue expansion (24, 28, 29). To determine the regional FRC, we used the grayscale values (intensity), which were converted to Hounsfield units to determine the fraction of air for each voxel.
To assess the regional Vt and FRC, the scans were segmented into 10 regions (Figure 1). The airway tree was segmented (30) using a centerline tree extracted from an image of the airways filtered to accentuate cylindrical structures (31). The airway tree geometry was then used to segment the lung into five regions (left lobe and four right lobes [R1–R4]) by assigning voxels to the nearest supplying airway (see References 24 and 29). The left lobe data were further segmented into six (L1–L6) volumes (Figure 1).
Figure 1. Regional lung segmentation. Schematic showing the segmentation approach for the assessment of regional lung volumes. This segmentation corresponded to the tissue sampling process for the assessment of gene expression. L = left lobe; R = right lobe.
[More] [Minimize]After segmentation of the lung imaging data, tissue expansion and volume of gas for each region were summed to provide the regional values of Vt and FRC, respectively. To correct for variation in regional lung volumes, we calculated regional specific functional residual capacity (sFRC = regional FRC/regional lung volume), specific tidal volume (sVt = regional Vt/regional lung volume), and regional lung distention (sFRC + sVt). Global values for these indices were also calculated. Data are presented as means(SD).
After mice were killed, their lungs were removed en bloc and divided into 10 regions corresponding to the image segmentation (Figure 1). Each lung region was stored in RNAlater (Sigma-Aldrich), and RNA extraction was performed with a miRNeasy Mini Kit (Qiagen).
The expression of 21 genes was assessed using reverse transcription–quantitative PCR (qPCR) arrays according to the manufacturer’s instructions (Qiagen). VILI-related genes with known roles in inflammation, surfactant production, epithelial and mesenchymal responses, transcription/cell signaling, and coagulation were selected (Table 1). qPCRs were performed on a LightCycler 480 II instrument (Roche) in 96-well qPCR array plates. Gene expression relative to a housekeeping gene (Rpl37) was calculated using the comparative cycle threshold method and expressed as fold change relative to the average gene expression of the L1 region in the free-breathing control group.
| Gene Function | Gene Name | Ventilation | Region | Ventilation × Region |
|---|---|---|---|---|
| Inflammation | Ccl2 | 0.07 | <0.001 | <0.001 |
| Cxcl2 | <0.001 | 0.04 | 0.48 | |
| Elane | 0.12 | 0.30 | 0.13 | |
| IL-1β | 0.48 | 0.02 | 0.72 | |
| IL-6 | <0.001 | 0.08 | 0.02 | |
| Mpo | 0.14 | 0.35 | 0.70 | |
| Tnf-α | <0.01 | <0.01 | 0.86 | |
| RAGE | 0.34 | 0.01 | 0.86 | |
| Ang-2 | 0.73 | <0.001 | 0.75 | |
| Surfactant | Sftpb | 0.67 | 0.95 | 0.81 |
| Epithelial–mesenchymal response | Cdh1 | 0.81 | 0.33 | 0.88 |
| Ctnnb1 | 0.65 | 0.87 | 0.78 | |
| Egfr | 0.22 | 0.24 | 0.88 | |
| Tgfb1 | 0.39 | 0.67 | 0.90 | |
| Vim | 0.88 | 0.08 | 0.78 | |
| Wnt1 | <0.001 | 0.16 | 0.29 | |
| Transcription, cell signaling | c-fos | <0.01 | 0.59 | 0.93 |
| Mapk1 | 0.99 | 0.37 | 0.54 | |
| Nfe2l2 | <0.001 | 0.81 | 0.87 | |
| Nfkb1 | 0.78 | 0.51 | 0.94 | |
| Anticoagulant | Plat | 0.14 | 0.14 | 0.97 |
In a separate group of mice, after they were killed, lungs were instillation fixed in situ with 10% formalin at a transrespiratory pressure of 10 cm H2O for 1 hour. The trachea was ligated, and the lung was removed en bloc and submerged in formalin before transfer to 70% ethanol. The fixed lungs were embedded in paraffin, and 5-μm coronal sections were taken at the midline. Immunohistochemistry was performed using a horseradish peroxidase/diaminobenzidine (DAB) (ABC) detection immunohistochemistry kit (Abcam). Antigen retrieval was performed in a pressure cooker using a solution of 1 mM EDTA in citrate buffer (pH 6.0) for 10 minutes. Sections were stained using the following antibodies: anti–IL-6 (10 ng/μl, Ab208113; Abcam), anti–MCP-1 (anti–monocyte chemoattractant protein 1, 5 ng/μl; Abcam), anti-p53 (0.5 ng/μl; Abcam), antineutrophil (NIMP-R14, 5 ng/μl; Abcam), or antirabbit IgG polyclonal (isotype control, 5 ng/μl; Abcam). The antigen–antibody reaction was visualized after the application of DAB substrate in DAB chromogen solution for 5 minutes and counterstaining with hematoxylin. Slides were dehydrated and clear mounted for light microscopy. Images of the entire section were captured and analyzed using ImageJ software. The regional concentrations of IL-6, Ccl2 (MCP-1), and p53 in the left lobe were estimated by calculating the proportion of the positive stain per unit area of tissue within each region after subtracting nonspecific staining based on the intensity of isotype control stain in the adjacent section. The regional number of neutrophils was counted in randomly selected images within each lung region and quantified as the number of cells per unit area.
Differences in sFRC, sVt, distention, and relative gene expression, both between regions and between ventilation protocols, were assessed using two-way repeated measures ANOVA with Holm-Šidák post hoc tests (SigmaPlot version 12.5; Systat Software). Between-regions protein expression data (immunohistochemistry) were assessed by one-way ANOVA with Holm-Šidák post hoc tests. Data were transformed when necessary to satisfy the assumptions of normality and homoscedasticity of the variances. Associations between regional sFRC, sVt, distention, and regional RNA concentrations were assessed using linear regression analysis.
Qualitatively, lung stretch in the mice ventilated with the injurious protocol was heterogeneous and changed between baseline (H0) and after 2 hours (H2) of ventilation (Figures 2A and 2B), whereas lung stretch with the protective protocol was more homogeneous (Figures 2C and 2D).
Figure 2. Imaging of lung motion during ventilation. Representative transverse images from the apex (top) to the base (bottom) of a mouse ventilated with an injurious strategy (A) at baseline and (B) after 2 hours of ventilation, as well as a mouse ventilated with a protective strategy (C) at baseline and (D) after 2 hours of ventilation. Tidal volume contours (arbitrary units) were normalized to the size of the whole lung. Scale bar: 5 mm.
[More] [Minimize]At H0, global and regional sFRC values were higher in the protective group than in the injurious group (P < 0.01) (Figure 3A). For both ventilation strategies, there were regional differences in sFRC (P < 0.001), where sFRC was lower in the more distal regions (L2, R2, and R4; P < 0.05). In general, regional sVt was higher in the injurious group in most regions, with the exception of R4 (Figure 3B). In animals receiving injurious ventilation, regional sVt was higher in proximal lung regions (L1 and R1; P < 0.001), whereas protective ventilation showed homogeneous regional sVt at baseline (Figure 3B). Distention (sFRC + sVt) followed a pattern similar to that of sVt with higher distention in the injurious ventilation group, with the exception of the three lower regions (L5, P = 0.51; L6, P = 0.54; R4, P = 0.76) (Figure 3C).
Figure 3. Effect of different ventilation strategies on lung volumes. Box plots (median, interquartile range, and 10th to 90th percentiles) for (A, B, and C, respectively) sFRC, sVt, and sFRC + sVt at baseline and the proportional change in (D, E, and F, respectively) sFRC, sVt, and sFRC + sVt after 2 hours of ventilation relative to baseline for each of 10 lung regions in mice ventilated with a protective or injurious strategy. *P < 0.05 and **P < 0.001 between ventilation strategies. †P < 0.05 and ††P < 0.001 compared with the same region in the protective group. n = 8 mice per group. FRC = functional residual capacity; sFRC = specific FRC; sVt = specific tidal volume.
[More] [Minimize]After 2 hours of ventilation, there was no change in sFRC (average change, 0.8%), sVt (Δ = 3.2%), or regional distention (Δ = 1.1%) in the protective group (Figures 3D–3F) compared with baseline. In contrast, sFRC was reduced in all lung regions in the injurious group (Δ = 8.6%) (Figure 3D), whereas the change in global sVt was minimal (Δ = 1.2%) (Figure 3E). On average, there was no significant change in global distention in the injurious group (Δ = 5.5%; P = 0.76) (Figure 3F). There were, however, significant regional differences in sVt and distention in the injurious group (P < 0.001) (Figures 3E and 3F) between H0 and H2.
The expression of IL-6 (P = 0.02) and Ccl2 (P < 0.001) varied regionally, depending on the ventilation strategy (Table 1). In contrast, the expression levels of Tnf-α and Cxcl2 varied independently with both region and ventilation strategy (Table 1). The expression of IL-1β, Ang-2 (angiopoietin 2), and RAGE (receptor for advanced glycation end products) varied regionally, but there was no influence of ventilation strategy on the expression of these genes. Expression levels of three genes (Wnt1, P < 0.001; c-fos, P < 0.01; Nfe2l2 [nuclear factor erythroid-derived 2 factor 2], P < 0.001) varied depending on ventilation strategy, but there were no significant differences in levels between lung regions (Table 1). There were no regional differences or associations with mechanical ventilation for the other genes measured (Elane [elastase neutrophil expressed], Mpo [myeloperoxidase], SftpB [surfactant-associated protein B], Cdh1 [cadherin 1], Ctnnb1 [catenin-β1], Egfr [epidermal growth factor receptor], TGFb1 [transforming growth factor-β], Vim [vimentin], Mapk1 [mitogen-activated protein kinase], Nfkb1 [NF-κB], and Plat [plasminogen activator, tissue]) (Table 1).
The expression of IL-6 in the protective group was higher than in the free-breathing group, whereas the expression in the injurious group was higher again. However, regional differences were limited to the injurious group, with higher expression in the proximal (L1, L3, and R2) lung regions (Figure 4A). For Ccl2, the expression was elevated in some regions (R2, P = 0.03; L3, P < 0.01; L1, P = 0.03), but only in the injurious group.
Figure 4. Regional gene expression measured by quantitative PCR array. Relative fold change of RNA concentrations was calculated using the comparative cycle threshold method relative to the housekeeping gene (Rlp37) and average cycle threshold of the L1 region in the free-breathing group for (A) IL-6, (B) Ccl2, (C) TNF-α, and (D) IL-1β. *P < 0.05. Data are shown as mean (SD). n = 8 per group.
[More] [Minimize]Ccl2 was expressed in the airway epithelial cells and inflammatory cells (primarily macrophages) and diffusely throughout the lung parenchyma (Figures 5A and 5B). The concentration of Ccl2 was highest in L1 and L3 (P < 0.05) (Figure 6A) in the injurious group, which was consistent with the gene expression data (i.e., L1 and L3 had the highest Ccl2 gene expression) (Figure 4B). Interestingly, we saw the same qualitative pattern in p53 protein expression (Figures 5B and 5C) whereby L1 and L3 had the highest expression (P < 0.05) (Figure 6B). IL-6 was expressed primarily in the airway epithelium (data not shown). In contrast to the other proteins, there were no significant regional differences in IL-6 expression (P = 0.89; data not shown). The number of neutrophils was higher (P = 0.007) in the protective group (17 [2]/mm2) than in the injurious group (7 [2]/mm2), but there were no significant regional differences (P = 0.40; data not shown).
Figure 5. Qualitative regional expression of Ccl2 and p53. (A and B) Ccl2 staining was localized to the airway epithelium and inflammatory cells (macrophages; arrows) and was also expressed diffusely throughout the parenchyma. (C and D) The expression pattern of p53 was similar to that of Ccl2. There was regional variation (quantified in Figure 6) such that, for example, the expression levels of Ccl2 and p53 were higher in (A and C) the L1 region than in (B and D) the L2 region. Scale bars: 70 μm.
[More] [Minimize]
Figure 6. Regional expression of Ccl2 and p53 protein in the left lobe of the injurious ventilation group. The regional (L1–L6) proportion of positive staining tissue for (A) Ccl2 and (B) p53 protein (based on immunohistochemistry) per unit of tissue area after subtracting the background level of stain from the isotype control. *P < 0.05. Data are shown as mean (SD). n = 6 per group.
[More] [Minimize]The only genes that were significantly associated with any of imaging measures were IL-6, Ccl2, and Ang-2 (Table 2). Expression of IL-6 was positively associated with sVt and regional distention (P < 0.05) but not with sFRC. Similarly, the expression of Ccl2 was positively associated with sVt and regional distention (P < 0.01) but not with sFRC (Table 2 and Figure 7). Similarly, the expression of Ang-2 was positively associated only with sVt (P < 0.05) (Table 2 and Figure 7).
| Gene Name | sFRC | sVt | Distention (sFRC + sVt) | |||
|---|---|---|---|---|---|---|
| R2 | P Value | R2 | P Value | R2 | P Value | |
| Ccl2 | 0.002 | 0.76 | 0.157 | <0.01 | 0.121 | <0.01 |
| Cxcl2 | <0.001 | 0.92 | 0.038 | 0.14 | 0.033 | 0.17 |
| IL-1β | 0.016 | 0.34 | 0.048 | 0.09 | 0.058 | 0.06 |
| IL-6 | 0.009 | 0.47 | 0.063 | 0.05 | 0.068 | 0.04 |
| c-fos | <0.001 | 0.92 | 0.025 | 0.23 | 0.022 | 0.26 |
| Tnf-α | 0.015 | 0.35 | 0.032 | 0.17 | 0.042 | 0.12 |
| Wnt1 | 0.023 | 0.25 | 0.007 | 0.54 | 0.016 | 0.34 |
| Nfe2l2 | 0.036 | 0.15 | <0.001 | 0.98 | 0.04 | 0.64 |
| RAGE | 0.005 | 0.58 | 0.018 | 0.31 | 0.009 | 0.46 |
| Ang-2 | 0.01 | 0.45 | 0.063 | 0.05 | 0.038 | 0.14 |
Figure 7. Relationship between regional gene expression (IL-6, Ccl-2, and Ang-2 [angiopoietin 2]) and lung volumes. Scatterplots show the relationship between the fold change in regional gene expression of (A and B) IL-6, (C and D) Ccl2, and (E and F) Ang-2, and (A, C, and E) regional sFRC and (B, D, and F) regional sVt in the injurious group. Lines represent the predicted association based on linear regression analysis.
[More] [Minimize]In this study, we assessed the impact of mechanical ventilation on regional lung volumes and gene expression. We found 1) a heterogeneous response to mechanical ventilation whereby sFRC varied regionally, independent of ventilation strategy, whereas sVt and distention (sFRC + sVt) varied regionally, depending on the ventilation strategy used; 2) an overall reduction in sFRC in response to 2 hours of injurious ventilation; 3) variations in regional gene expression levels that, in some cases (IL-6, Ccl2), depended on the ventilation strategy used; and 4) positive associations between the expression levels of IL-6, Ccl2, and Ang-2 and regional stretch but not sFRC. Collectively, these data highlight the complex and heterogeneous response of lung tissue to mechanical ventilation and how some but not all of the inflammatory response is linked to regional variations in tidal stretch. The lack of association between sFRC and altered gene expression suggests that overstretch is more detrimental than atelectasis.
We found that regional lung volumes varied in response to mechanical ventilation. This variability is consistent with that described in previous reports using larger-animal models and static lung volume measures (22, 23). Of interest is the fact that the regional variation in sFRC we observed was consistent between the protective and injurious ventilation strategies; the more distal regions of the lung seemed to be more susceptible to underventilation (i.e., low sFRC), whereas they were protected from overstretch. There was minimal change in sFRC and sVt over time in the protective group. In contrast, the injurious group showed a consistent loss of sFRC and significant variations in the sVt response over time, suggesting that this ventilation strategy alters the mechanical properties of the lung. The fact that we observed regional variations in lung stretch within minutes of mechanically ventilating the mice (i.e., at H0), as well as the fact that these regional variations were associated with altered gene expression, suggests that local lung injury may develop very soon after the commencement of ventilation.
The regional variations we observed in sVt were associated with the altered expression of IL-6, Ccl2, and Ang-2. IL-6 is a key inflammatory cytokine (32) produced by epithelial cells and macrophages and is important in the progression of sepsis (17). Ccl2, or MCP-1, is responsible for the recruitment of macrophages to the site of inflammation (33, 34). The release of IL-6 and Ccl2 are closely linked (35), whereby IL-6 induces Ccl2 expression by peripheral blood mononuclear cells (36), whereas Ccl2 induces the release of IL-6 by human epithelial cells (37). Increasing concentrations of IL-6 have been demonstrated in response to injurious ventilation in clinical (20) and experimental (17) studies. Similarly, injurious mechanical ventilation is associated with increasing concentrations of Ccl2 in lung tissue (38), BAL, and plasma samples (7, 38). Ang-2 is involved in the regulation of vascular endothelial permeability and is strongly linked to outcome in mechanically ventilated patients, particularly in those with ARDS (39). The link between the expression of this mediator and regional stretch highlights the role of overstretch in contributing to ventilator-induced vascular permeability and, potentially, edema, although we were not able to quantify the latter directly. Although demonstration of upregulation of these genes in response to mechanical ventilation is not novel, overstretch as the key driver of the expression of these mediators is suggested by the strong positive association between regional IL-6, Ccl2, and Ang-2 expression; regional sVt and regional distention; and the absence of an association with sFRC. Given the importance of these mediators in multiorgan dysfunction (7, 40) and patient mortality (39), our data suggest that avoidance of regional overstretch during initiation of ventilation may be a critical determinant of patient outcome.
Although the expression levels of other genes (Cxcl2, TNF-α, c-fos, Nfe2l2, and Wnt1) were altered in response to the ventilation strategies used, their levels were not correlated with regional sFRC or sVt. TNF-α and Cxcl2 are proinflammatory cytokines that are elevated in response to mechanical ventilation (41). Nfe2l2, c-Fos, and Wnt1 are involved in transcription, cell signaling, and mesenchymal responses, respectively, and have previously been associated with the response to mechanical ventilation (17, 42, 43). In the case of TNF-α, c-fos, and Wnt1, the greatest expression was observed in the injurious group compared with the protective group, whereas the expression of Nfe2l2 was equivalent between the protective and injurious groups, and the expression of Cxcl2 was highest in the protective group compared with the injurious group. The expression pattern of Cxcl2 matched our neutrophil data whereby there were no regional differences in neutrophil numbers, but there was an association with ventilation such that the greatest neutrophilia was observed in the protective ventilation group. These observations are consistent with the notion that Cxcl2 is involved in the recruitment of neutrophils during the early stages of inflammation (44). These variations suggest that the pattern of expression is correlated with the ventilation strategy. However, none of these genes were associated with regional sFRC or sVt, suggesting that local factors may not be driving the altered expression of these pathways. Alternatively, upregulation of the expression of these genes may be binary and based on a low threshold of activation in response to mechanical stretch, although this does not explain the expression pattern in Cxcl2. Clearly, the link between regional overstretch and underventilation and the subsequent activation of biological pathways is complex. Although we have identified a clear link between overstretch and the expression of IL-6, Ccl2, and Ang-2, the mechanical and biological processes regulating the altered expression of the other genes warrant further investigation.
To gain further insight into the key processes, using immunohistochemistry, we quantified IL-6 and Ccl2 protein expression to determine whether altered gene expression translated into protein synthesis and p53 expression to gain further insight into the cellular response. We found that Ccl2 protein expression matched the gene data, whereas there was no difference in expression of IL-6 protein. It is unclear whether the latter was due to a lack of translation or whether the timing was such that protein translation had not yet occurred. The pattern of p53 expression matched Ccl2. We assessed p53 as a potential marker of apoptosis (45); however, p53 has multiple functions in cell regulation. In this context, p53 has been shown to regulate Ccl2 expression (46); thus, the association between the expression of these proteins is perhaps not surprising. This suggests that the stretch-induced production of Ccl2 may be regulated by p53; however, we are unable to rule out other roles for p53 in this context.
This study has several limitations. First, our injurious ventilation strategy comprised both high-pressure and zero PEEP, so we cannot separate the effect of each of these on the global expression levels of the genes we measured. However, our ability to calculate regional measures of sFRC and sVt means that we were able to identify how these factors influence regional gene expression levels. We were also limited by the fact that our observations were based primarily on correlation between gene expression and sVt, although the weight of evidence would suggest that the links we made were causal, and we were able to confirm increased Ccl2 gene expression by immunohistochemistry.
In summary, we have demonstrated regional variations in sFRC and sVt in response to mechanical ventilation. By measuring the expression levels of a suite of genes, we were also able to assess the link between the mechanical response of the lung and alterations in regional gene expression. Interestingly, no alterations in gene expression levels were associated with markers of atelectasis, whereas alterations in expression of IL-6 and Ccl2 were clearly linked to regional overstretch. There were a number of genes with altered expression levels with particular ventilation strategies, but not the regional mechanical response, which warrants further investigation. In the context of therapeutic approaches to improve outcomes in critically ill patients who are mechanically ventilated, our observations suggest that proinflammatory pathways are activated very early in response to mechanical stretch. What is unclear is whether this response resolves when stretch is subsequently reduced. Our data also indicate that IL-6, Ccl2, and Ang-2 are the most sensitive to regional variation in stretch. However, the relative pathological significance of these mediators is unclear, with studies suggesting that upregulation of IL-6 may be protective (47), whereas upregulation of Ang-2 is detrimental (48); thus, the net effect may depend on the relative expression of these pathways. Further exploration of this interaction is necessary to further understand the pathogenesis of VILI. Our study clearly highlights the complexity of the link between mechanical ventilation and biotrauma.
| 1. | Phua J, Badia JR, Adhikari NKJ, Friedrich JO, Fowler RA, Singh JM, et al. Has mortality from acute respiratory distress syndrome decreased over time? a systematic review. Am J Respir Crit Care Med 2009;179:220–227. |
| 2. | Villar J, Blanco J, Kacmarek RM. Current incidence and outcome of the acute respiratory distress syndrome. Curr Opin Crit Care 2016;22:1–6. |
| 3. | Bellani G, Laffey JG, Pham T, Fan E, Brochard L, Esteban A, et al.; LUNG SAFE Investigators; ESICM Trials Group. Epidemiology, patterns of care, and mortality for patients with acute respiratory distress syndrome in intensive care units in 50 countries. JAMA 2016;315:788–800. |
| 4. | Ranieri VM, Rubenfeld GD, Thompson BT, Ferguson ND, Caldwell E, Fan E, et al.; ARDS Definition Task Force. Acute respiratory distress syndrome: the Berlin Definition. JAMA 2012;307:2526–2533. |
| 5. | Montgomery AB, Stager MA, Carrico CJ, Hudson LD. Causes of mortality in patients with the adult respiratory distress syndrome. Am Rev Respir Dis 1985;132:485–489. |
| 6. | Slutsky AS. History of mechanical ventilation: from Vesalius to ventilator-induced lung injury. Am J Respir Crit Care Med 2015;191:1106–1115. |
| 7. | Imai Y, Parodo J, Kajikawa O, de Perrot M, Fischer S, Edwards V, et al. Injurious mechanical ventilation and end-organ epithelial cell apoptosis and organ dysfunction in an experimental model of acute respiratory distress syndrome. JAMA 2003;289:2104–2112. |
| 8. | Carrasco Loza R, Villamizar Rodríguez G, Medel Fernández N. Ventilator-induced lung injury (VILI) in acute respiratory distress syndrome (ARDS): volutrauma and molecular effects. Open Respir Med J 2015;9:112–119. |
| 9. | Bailey TC, Maruscak AA, Martin EL, Forbes AR, Petersen A, McCaig LA, et al. The effects of long-term conventional mechanical ventilation on the lungs of adult rats. Crit Care Med 2008;36:2381–2387. |
| 10. | Dreyfuss D, Saumon G. Ventilator-induced lung injury: lessons from experimental studies. Am J Respir Crit Care Med 1998;157:294–323. |
| 11. | Brower RG, Matthay MA, Morris A, Schoenfeld D, Thompson BT, Wheeler A; 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. |
| 12. | Guérin C, Reignier J, Richard JC, Beuret P, Gacouin A, Boulain T, et al.; PROSEVA Study Group. Prone positioning in severe acute respiratory distress syndrome. N Engl J Med 2013;368:2159–2168. |
| 13. | Cavalcanti AB, Suzumura EA, Laranjeira LN, Paisani DM, Damiani LP, Guimarães HP, et al.; Writing Group for the Alveolar Recruitment for Acute Respiratory Distress Syndrome Trial (ART) Investigators. Effect of lung recruitment and titrated positive end-expiratory pressure (PEEP) vs low PEEP on mortality in patients with acute respiratory distress syndrome: a randomized clinical trial. JAMA 2017;318:1335–1345. |
| 14. | Gattinoni L, Marini JJ, Pesenti A, Quintel M, Mancebo J, Brochard L. The “baby lung” became an adult. Intensive Care Med 2016;42:663–673. |
| 15. | Cressoni M, Chiurazzi C, Gotti M, Amini M, Brioni M, Algieri I, et al. Lung inhomogeneities and time course of ventilator-induced mechanical injuries. Anesthesiology 2015;123:618–627. |
| 16. | Dargaville PA, Rimensberger PC, Frerichs I. Regional tidal ventilation and compliance during a stepwise vital capacity manoeuvre. Intensive Care Med 2010;36:1953–1961. |
| 17. | 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. |
| 18. | Pugin J. Molecular mechanisms of lung cell activation induced by cyclic stretch. Crit Care Med 2003;31(4, Suppl):S200–S206. |
| 19. | 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. |
| 20. | Stüber F, Wrigge H, Schroeder S, Wetegrove S, Zinserling J, Hoeft A, et al. Kinetic and reversibility of mechanical ventilation-associated pulmonary and systemic inflammatory response in patients with acute lung injury. Intensive Care Med 2002;28:834–841. |
| 21. | Cannizzaro V, Hantos Z, Sly PD, Zosky GR. Linking lung function and inflammatory responses in ventilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol 2011;300:L112–L120. |
| 22. | Wellman TJ, Winkler T, Costa EL, Musch G, Harris RS, Zheng H, et al. Effect of local tidal lung strain on inflammation in normal and lipopolysaccharide-exposed sheep. Crit Care Med 2014;42:e491–e500. |
| 23. | Paula LF, Wellman TJ, Winkler T, Spieth PM, Güldner A, Venegas JG, et al. Regional tidal lung strain in mechanically ventilated normal lungs. J Appl Physiol (1985) 2016;121:1335–1347. |
| 24. | Dubsky S, Hooper SB, Siu KKW, Fouras A. Synchrotron-based dynamic computed tomography of tissue motion for regional lung function measurement. J R Soc Interface 2012;9:2213–2224. |
| 25. | Zosky GR, Cannizzaro V, Hantos Z, Sly PD. Protective mechanical ventilation does not exacerbate lung function impairment or lung inflammation following influenza A infection. J Appl Physiol (1985) 2009;107:1472–1478. |
| 26. | Larsson DH, Takman PA, Lundström U, Burvall A, Hertz HMA. A 24 keV liquid-metal-jet x-ray source for biomedical applications. Rev Sci Instrum 2011;82:123701. |
| 27. | Samarage CR, Carnibella R, Preissner M, Jones HD, Pearson JT, Fouras A, et al. Technical note: contrast free angiography of the pulmonary vasculature in live mice using a laboratory x-ray source. Med Phys 2016;43:6017. |
| 28. | Kim EH, Preissner M, Carnibella RP, Samarage CR, Bennett E, Diniz MA, et al. Novel analysis of 4DCT imaging quantifies progressive increases in anatomic dead space during mechanical ventilation in mice. J Appl Physiol (1985) 2017;123:578–584. |
| 29. | Stahr CS, Samarage CR, Donnelley M, Farrow N, Morgan KS, Zosky G, et al. Quantification of heterogeneity in lung disease with image-based pulmonary function testing. Sci Rep 2016;6:29438. |
| 30. | Dubsky S, Zosky GR, Perks K, Samarage CR, Henon Y, Hooper SB, et al. Assessment of airway response distribution and paradoxical airway dilation in mice during methacholine challenge. J Appl Physiol (1985) 2017;122:503–510. |
| 31. | Frangi AF, Niessen WJ, Vincken KL, Viergever MA. Multiscale vessel enhancement filtering. In: Wells WM, Colchester A, Delp S, editors. Medical image computing and computer-assisted intervention — MICCAI’98: first international conference. MICCAI 1998. Lecture Notes in Computer Science, Vol. 1496. Berlin, Heidelberg: Springer; 1998. pp. 130–137. |
| 32. | Xing Z, Gauldie J, Cox G, Baumann H, Jordana M, Lei XF, et al. IL-6 is an antiinflammatory cytokine required for controlling local or systemic acute inflammatory responses. J Clin Invest 1998;101:311–320. |
| 33. | van Kooten C, van der Linde X, Woltman AM, van Es LA, Daha MR. Synergistic effect of interleukin-1 and CD40L on the activation of human renal tubular epithelial cells. Kidney Int 1999;56:41–51. |
| 34. | Prodjosudjadi W, Gerritsma JS, Klar-Mohamad N, Gerritsen AF, Bruijn JA, Daha MR, et al. Production and cytokine-mediated regulation of monocyte chemoattractant protein-1 by human proximal tubular epithelial cells. Kidney Int 1995;48:1477–1486. |
| 35. | Hosaka K, Rojas K, Fazal HZ, Schneider MB, Shores J, Federico V, et al. Monocyte chemotactic protein-1-interleukin-6-osteopontin pathway of intra-aneurysmal tissue healing. Stroke 2017;48:1052–1060. |
| 36. | Biswas P, Delfanti F, Bernasconi S, Mengozzi M, Cota M, Polentarutti N, et al. Interleukin-6 induces monocyte chemotactic protein-1 in peripheral blood mononuclear cells and in the U937 cell line. Blood 1998;91:258–265. |
| 37. | Viedt C, Dechend R, Fei J, Hänsch GM, Kreuzer J, Orth SR. MCP-1 induces inflammatory activation of human tubular epithelial cells: involvement of the transcription factors, nuclear factor-κB and activating protein-1. J Am Soc Nephrol 2002;13:1534–1547. |
| 38. | Altemeier WA, Matute-Bello G, Frevert CW, Kawata Y, Kajikawa O, Martin TR, et al. 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. |
| 39. | Zinter MS, Spicer A, Orwoll BO, Alkhouli M, Dvorak CC, Calfee CS, et al. Plasma angiopoietin-2 outperforms other markers of endothelial injury in prognosticating pediatric ARDS mortality. Am J Physiol Lung Cell Mol Physiol 2016;310:L224–L231. |
| 40. | Tesch GH, Schwarting A, Kinoshita K, Lan HY, Rollins BJ, Kelley VR. Monocyte chemoattractant protein-1 promotes macrophage-mediated tubular injury, but not glomerular injury, in nephrotoxic serum nephritis. J Clin Invest 1999;103:73–80. |
| 41. | Shosholcheva M. Jankulovski N, Kartalov A, Kuzmanovska B, Miladinova D. Synergistic effect of hyperoxia and biotrauma on ventilator-induced lung injury. Pril (Makedon Akad Nauk Umet Odd Med Nauki) 2017;38:91–96. |
| 42. | Shan Y, Akram A, Amatullah H, Zhou DY, Gali PL, Maron-Gutierrez T, et al. ATF3 protects pulmonary resident cells from acute and ventilator-induced lung injury by preventing Nrf2 degradation. Antioxid Redox Signal 2015;22:651–668. |
| 43. | Silva PL, Negrini D, Rocco PR. Mechanisms of ventilator-induced lung injury in healthy lungs. Best Pract Res Clin Anaesthesiol 2015;29:301–313. |
| 44. | De Filippo K, Dudeck A, Hasenberg M, Nye E, van Rooijen N, Hartmann K, et al. Mast cell and macrophage chemokines CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue inflammation. Blood 2013;121:4930–4937. |
| 45. | Ozaki T, Nakagawara A. Role of p53 in cell death and human cancers. Cancers (Basel) 2011;3:994–1013. |
| 46. | Tang X, Asano M, O’Reilly A, Farquhar A, Yang Y, Amar S. p53 is an important regulator of CCL2 gene expression. Curr Mol Med 2012;12:929–943. |
| 47. | Wolters PJ, Wray C, Sutherland RE, Kim SS, Koff J, Mao Y, et al. Neutrophil-derived IL-6 limits alveolar barrier disruption in experimental ventilator-induced lung injury. J Immunol 2009;182:8056–8062. |
| 48. | Bhandari V, Choo-Wing R, Lee CG, Zhu Z, Nedrelow JH, Chupp GL, et al. Hyperoxia causes angiopoietin 2-mediated acute lung injury and necrotic cell death. Nat Med 2006;12:1286–1293. |
* These authors contributed equally to this work.
Supported by the National Health and Medical Research Council (1077905) and the Royal Hobart Hospital Research Foundation (15-027).
Author Contributions: S.Y., M.P., E.B., and G.R.Z.: conducted the experiments; S.Y., M.P., S.D., R.C., R.O’T., L.R., H.J., P.A.D., A.F., and G.R.Z.: analyzed the data generated; S.D., R.C., R.O’T., L.R., H.J., P.A.D., A.F., and G.R.Z.: conceptualized the study; and S.Y., M.P., E.B., S.D., R.C., R.O’T., L.R., H.J., P.A.D., A.F., and G.R.Z.: drafted the manuscript and approved the final version.
Originally Published in Press as DOI: 10.1165/rcmb.2018-0143OC on November 14, 2018
Author disclosures are available with the text of this article at www.atsjournals.org.
