Genetic Targets of Hydrogen Sulfide in Ventilator-Induced Lung Injury – A Microarray Study

<div><p>Recently, we have shown that inhalation of hydrogen sulfide (H₂S) protects against ventilator-induced lung injury (VILI). In the present study, we aimed to determine the underlying molecular mechanisms of H₂S-dependent lung protection by analyzing gene expression profiles in mice. C57BL/6 mice were subjected to spontaneous breathing or mechanical ventilation in the absence or presence of H₂S (80 parts per million). Gene expression profiles were determined by microarray, sqRT-PCR and Western Blot analyses. The association of Atf3 in protection against VILI was confirmed with a Vivo-Morpholino knockout model. Mechanical ventilation caused a significant lung inflammation and damage that was prevented in the presence of H₂S. Mechanical ventilation favoured the expression of genes involved in inflammation, leukocyte activation and chemotaxis. In contrast, ventilation with H₂S activated genes involved in extracellular matrix remodelling, angiogenesis, inhibition of apoptosis, and inflammation. Amongst others, H₂S administration induced Atf3, an anti-inflammatory and anti-apoptotic regulator. Morpholino mediated reduction of Atf3 resulted in elevated lung injury despite the presence of H₂S. In conclusion, lung protection by H₂S during mechanical ventilation is associated with down-regulation of genes related to oxidative stress and inflammation and up-regulation of anti-apoptotic and anti-inflammatory genes. Here we show that Atf3 is clearly involved in H₂S mediated protection.</p></div

Venn diagram (A) showing the intersections between the lists of differentially regulated genes in response to 6 h mechanical ventilation (effect of ventilation, S1), mechanical ventilation with supplemented H₂S (effect of H₂S ventilation, S2) and, genes found differentially regulated in both groups (S3).

<p>The lists of effect-specific and intersection genes are shown in supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102401#pone.0102401.s001" target="_blank">tables S1</a> to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102401#pone.0102401.s003" target="_blank">S3</a>. Only genes with statistical relevance after unpaired Bayer T-test p≤0.005, false discovery rate (FDR) ≤10% and effect size ≥1.7 were included in the diagram (created using Genedata Analyst 2.2.6b software). Gene enrichment analysis (GEA) (B+C) of significant genes induced during mechanical ventilation (S1) and mechanical ventilation with H₂S (S2). Effect size was transformed to log2 ratio prior GEA performed with the MetaCore software. Only significant events, p<0.02, FDR<15% and including minimum of 5 genes GO processes (B) and GO processes networks (C) are shown. Shades of red (up-regulated) and blue (down-regulated) coded the degree of significance of the corresponding annotation.</p

Effect of reduced Atf3 protein synthesis on VILI.

<p>(A) Representative pictures of H&E stained lung tissue from control animals and animals mechanically ventilated with synthetic air in the presence or absence of H₂S. Control-Morpholino or Atf3-Morpholino treated mice were ventilated with supplemented H₂S. (B) Ventilator-induced lung injury (VILI) score was measured from the histology samples and (C) the relative amount of neutrophils was determined by cytospin analysis of BALF. Data represent means ±SEM for n = 4/group. Analysis of variance (Student–Newman–Keuls post hoc test), *P<0.05 vs. control; ^#P<0.05 vs. H₂S control-Morpfolino; ^§P<0.05 vs. air-ventilated group.</p

Effect of ventilation and hydrogen sulfide (H₂S) treatment on lung injury.

<p>(A) Representative pictures of H&E stained lung tissue from animals mechanically ventilated (vent)(12 ml/kg, 6 h) in the absence or presence of supplemental H₂S (80 ppm) and their corresponding non-ventilated controls. (B) Ventilator-induced lung injury (VILI) score was measured from the histology samples and (C) the relative amount of neutrophils was determined by cytospin analysis of BALF. Data represent means ±SEM for n = 5/group. Analysis of variance (Student–Newman–Keuls post hoc test), *P<0.05 vs. control; ^#P<0.05 vs. H₂S control; ^§P<0.05 vs. H₂S-ventilated group.</p

Verification of the microarray expression profiles of Socs3, Atf3 and Gadd45a by semi-quantitative RT-PCR.

<p>(A) Microarray expression levels of the control group (spontaneously air breathing animals) were used to calculate the effect of mechanical ventilation, spontaneous inhalation of H₂S, and ventilation in the presence of H₂S. (B) RT-PCR expression values for each gene were normalized to GAPDH. Data represent median of fold change ±SEM for n = 5/group. Analysis of variance (Student–Newman–Keuls post hoc test), *P<0.05 vs. control; ^#P<0.05 vs. H₂S control; ^§P<0.05 vs. air-ventilated group.</p