Conflicting physiological and genomic cardiopulmonary effects of recruitment maneuvers in murine acute lung injury

Abstract

Low tidal volume ventilation, although promoting atelectasis, is a protective strategy against ventilator-induced lung injury. Deep inflation (DI) recruitment maneuvers restore lung volumes, but potentially compromise lung parenchymal and vascular function via repetitive overdistention. Our objective was to examine cardiopulmonary physiological and transcriptional consequences of recruitment maneuvers. C57/BL6 mice challenged with either PBS or LPS via aspiration were placed on mechanical ventilation (5 h) using low tidal volume inflation (TI; 8 μl/g) alone or in combination with intermittent DIs (0.75 ml twice/min). Lung mechanics during TI ventilation significantly deteriorated, as assessed by forced oscillation technique and pressure-volume curves. DI mitigated the TI-induced alterations in lung mechanics, but induced a significant rise in right ventricle systolic pressures and pulmonary vascular resistances, especially in LPS-challenged animals. In addition, DI exacerbated the LPS-induced genome-wide lung inflammatory transcriptome, with prominent dysregulation of a gene cluster involving vascular processes, as well as increases in cytokine concentrations in bronchoalveolar lavage fluid and plasma. Gene ontology analyses of right ventricular tissue expression profiles also identified inflammatory signatures, as well as apoptosis and membrane organization ontologies, as potential elements in the response to acute pressure overload. Our results, although confirming the improvement in lung mechanics offered by DI, highlight a detrimental impact in sustaining inflammatory response and exacerbating lung vascular dysfunction, events contributing to increases in right ventricle afterload. These novel insights should be integrated into the clinical assessment of the risk/benefit of recruitment maneuver strategies.

Figure 1.

Airway pressures monitoring and single-frequency forced oscillation technique (FOT) results during 5 hours of mechanical ventilation using tidal inflations (TIs) or deep inflations (DIs) in mice after PBS or LPS aspiration. The depicted nomenclature of a, b, c, d, e, and f denote a Bonferroni-corrected P value less than 0.05 for the Mann-Whitney pairwise comparisons (after Kruskal Wallis test): PBS + TI versus PBS + DI, PBS + TI versus LPS + TI, PBS + TI versus LPS + DI, PBS + DI versus LPS + TI, PBS + DI versus LPD + DI, and LPS + TI versus LPS + DI, respectively. Triangles and inverted triangles denote significant increase and decrease, respectively (as compared with H0_after volume history standardization) with Bonferroni-corrected P value less than 0.05 for pairwise Wilcoxon (after Friedman's test) (n = 6–9 animals per group). H0_before VHS denotes experiment start before volume history standardization. H0_after VHS denotes experiment start after volume history standardization.

Figure 2.

Low-frequency FOT results and pressure–volume curve data before and during 5 hours of mechanical ventilation using TIs or DIs in mice after PBS or LPS aspiration. a, b, c, d, e, and f denote a Bonferroni-corrected P value less than 0.05 for the Mann-Whitney pairwise comparisons (after Kruskal Wallis test): PBS + TI versus PBS + DI, PBS + TI versus LPS + TI, PBS + TI versus LPS + DI, PBS + DI versus LPS + TI, PBS + DI versus LPD + DI, and LPS + TI versus LPS + DI, respectively. Triangles and inverted triangles denote significant increase and decrease, respectively (as compared with H0_after volume history standardization) with Bonferroni-corrected P value less than 0.05 for pairwise Wilcoxon (after Friedman's test) (n = 6–9 animals per group). H0_before VHS denotes experiment start before volume history standardization. H0_after VHS denotes experiment start after volume history standardization.

Figure 3.

Expression pattern of dysregulated lung genes in mice exposed to mechanical ventilation using TIs or DIs after PBS or LPS aspiration. The expression levels of dysregulated lung genes identified using significance analysis of microarrays for LPS + DI versus PBS + TI comparison (A) and for LPS + TI versus PBS + TI comparison (B) are displayed by hierarchical clustering (using Bioconductor). Sample clustering is displayed at the top; gene clusters are displayed on the left; blue, white, and red represent expression levels below, at, and above mean level, respectively.

Figure 4.

Top 10 clusters of gene ontology categories and pathways enriched by genes dysregulated by LPS + DI (using PBS + TI as the reference group) in lungs. Groups were compared using significance analysis of microarray (LPS + DI versus PBS + TI and LPS + TI versus PBS + TI), and the lists of dysregulated genes were explored by Database for Annotation, Visualization and Integrated Discovery functional annotation clustering to assess significance of gene-term enrichment. Bars represent the log-transformed enrichment P value (modified Fischer's exact test) of each category for the LPS + DI versus PBS + TI comparison (dark gray) and for the LPS + TI versus PBS + TI comparison (light gray). The top 10 clusters are those with the highest enrichment score (calculated as the geometric mean of the enrichment P values of all categories within the cluster) for the LPS + DI versus PBS + TI comparison. The vertical dashed line represents the 0.05 significance threshold. TI, ventilation with TIs only.

Figure 5.

Fold changes in lung microarray expression of selected genes involved in inflammation and vascular dysfunction. Comparisons of gene expression after LPS + DI versus PBS + TI (light gray) and LPS + TI versus PBS + TI (dark gray) (A) and their correlation with RT-PCR validation assays (B). (C) The ratio of angiopoietin-2 to angiopoietin-1 array expression level (mean and SEM) in each group. LPS, LPS aspiration; qPCR, quantitative PCR; TI, ventilation with TIs only; Scube2, signal peptide CUB domain epidermal growth factor–like 2.

Figure 6.

Fold changes in concentrations of various cytokines, chemokines, and endothelium markers in bronchoalveolar lavage (A) and plasma (B) by multianalyte assay for LPS + DI versus PBS + TI (dark gray) and LPS + TI versus PBS + TI (light gray) comparisons. *P < 0.05 (Mann-Withney test after Kruskal-Wallis test) as compared with PBS + TI; ^#P < 0.05 (Mann-Whitney test after Kruskal-Wallis test) as compared with LPS + TI. G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte/macrophage colony–stimulating factor; KC, keratinocyte-derived cytokine; LIF, leukemia inhibitory factor; M-CSF, macrophage colony-stimulating factor; MCP, monocyte chemoattractant protein; MIG, monokine induced by interferon-γ; MIP, macrophage inflammatory protein; PAI, plasminogen activator inhibitor; RANTES, regulated upon activation, normal T cell expressed and secreted; sICAM, soluble intercellular adhesion molecule; sVCAM, soluble vascular cell adhesion molecule; Th, T helper cell; VEGF, vascular endothelial growth factor.