Use of consomic rats for genomic insights into ventilator-associated lung injury

Abstract

Increasing evidence supports the contribution of genetic influences on susceptibility/severity in acute lung injury (ALI), a devastating syndrome requiring mechanical ventilation with subsequent risk for ventilator-associated lung injury (VALI). To identify VALI candidate genes, we determined that Brown Norway (BN) and Dahl salt-sensitive (SS) rat strains were differentially sensitive to VALI (tidal volume of 20 ml/kg, 85 breaths/min, 2 h) defined by bronchoalveolar lavage (BAL) protein and leukocytes. We next exploited differential sensitivities and phenotyped both the VALI-sensitive BN and the VALI-resistant SS rat strains by expression profiling coupled to a bioinformatic-intense candidate gene approach (Significance Analysis of Microarrays, i.e., SAM). We identified 106 differentially expressed VALI genes representing gene ontologies such as "transcription" and "chemotaxis/cell motility." We mapped the chromosomal location of the differentially expressed probe sets and selected consomic SS rats with single BN introgressions of chromosomes 2, 13, and 16 (based on the highest density of probe sets) while also choosing chromosome 20 (low probe sets density). VALI exposure of consomic rats with introgressions of BN chromosomes 13 and 16 resulted in significant increases in both BAL cells and protein (compared to parental SS strain), whereas introgression of BN chromosome 2 displayed a large increase only in BAL protein. Introgression of BN chromosome 20 had a minimal effect. These results suggest that genes residing on BN chromosomes 2, 13, and 16 confer increased sensitivity to high tidal volume ventilation. We speculate that the consomic-microarray-SAM approach is a time- and resource-efficient tool for the genetic dissection of complex diseases including VALI.

Fig. 1

Survey of specific rodent strains for sensitivity to mechanical ventilatory stress: bronchoalveolar lavage (BAL) cell counts and protein levels. BAL inflammatory cells (A) and protein content (B) were used to assess lung injury induced by high tidal volume (HTV) mechanical ventilation in adult male Sprague-Dawley (SD), Dahl salt-sensitive (SS), and Brown Norway (BN) rats exposed to HTV ventilation for 2 h. HTV mechanical ventilation (20 ml/kg, see MATERIALS AND METHODS) induced a significant increase in BAL cells (A) and protein (B) in the BN strain. There were no significant changes in BAL cells or protein in either the SS or SD strains following HTV challenge. Inset: the percent (%) increase in BAL cell counts (A) or protein level (B) by strain compared with controls (i.e., without HTV). **P < 0.01; n = number of animals per condition.

Fig. 2

Survey of specific rodent strains for sensitivity to mechanical ventilatory stress: Evans blue dye (EBD) vascular leakage. EBD content was quantified to assess albumin leak across the lung vascular bed in rodents injured with HTV mechanical ventilation (20 ml/kg, 2 h). SD, Dahl SS, and BN male rats challenged in this manner revealed significant increases in EBD deposition noted only in the BN rat strain. There were no significant changes in EBD content in either the SS or SD strains. Inset: the percent increase in EBD content by strain compared with controls (i.e., without HTV). *P < 0.05. **P < 0.01.

Fig. 3

Gene ontologies (GO) involved in rodent lung responses to mechanical ventilation-induced mechanical stress. We employed OntoExpress, a program that uses a relational database to link genes in a given data set, as another level of filtering of the genomic data derived by microarray analyses. Ontology analysis of the 106 genes in our mechanical ventilation array data set shows biological relationships between genes and gene products, giving experimental gene array expression data a biological context. The “protein binding” ontology was identified as the major mechanical stretch-related acute lung injury (ALI) biological process with the highest percentage of genes per total number of identified genes (106). Significant representation was also identified in the signal transduction, DNA binding, cell proliferation, cell motility/chemotaxis, cell adhesion, and inflammation ontologies. Parenthetical annotation reflects the number of genes that mapped to the indicated gene Ontology. All eight ontologies had were significant at P < 0.001.

Fig. 4

Chromosomal distribution of differentially expressed probe set-defined transcripts from rodent lungs: ratio of the observed chromosomal distribution of differentially expressed rodent lung probe sets evoked by HTV mechanical ventilation over that of the predicted distribution (352). Predicted distribution, in this case, equates to an equal distribution of the HTV-driven probe sets (352) between all chromosomes normalized by the individual chromosomal probe representation on the microarray chip. With the goal of accentuating the greatest probe set-dense chromosomes for consomic selection, a chromosome with a ratio greater than 1 (where observed equals predicted) represents a significant trend in surpassing the chromosome's expected probe set density over other chromosomes and, hence, an increase in its potential contribution to the ventilator-associated lung injury (VALI) phenotype. As shown, chromosomes 2, 13, 16, and 17 (as highlighted in black) demonstrated the greatest density of probe sets (over a ratio of 1.2, thick black line), and since the SS:BN17 strain was not available, we studied strains SS:BN13, 16, and 2. We also studied the SS:BN20 strain (shown in striped bar) to study the trend for a chromosome with a low probe set density given the ratio attributed to its chromosome to be lower than 1. Parenthetical annotation reflects the number of unique genes that mapped to the indicated chromosome.

Fig. 5

Effect of BN chromosome introgression (chromosomes 2, 13, and 16) into the Dahl SS background on VALI phenotype. SS rats with introgression of BN chromosome 16 into the resistant SS background (SS:BN16) and exposed to HTV mechanical ventilation exhibited increased BAL cell counts (A) and protein level (B). Similar increases were observed with chromosome 13 introgression (SS:BN13) (BAL cell contents, C and D) while chromo-some 2 introgression (SS:BN2) showed divergent responses in BAL cell counts (E) vs. BAL protein level (F) compared with the SS parent. These data suggest that BN chromosomes 13 and 16 conferred increased sensitivity to HTV, and chromosome 2 showed an intermediate phenotype. SS rats with introgression of BN chromosome 20 into the resistant SS background (SS:BN20) and exposed to HTV mechanical ventilation did not exhibit changes in BAL cell counts (G) and a mild nonsignificant increase (P < 0.05) in protein level (H) compared with the SS strain. These data suggest that BN chromosome 20 did not confer increased sensitivity to HTV, unlike chromosomes 2, 13, and 16. *P < 0.05. **P < 0.01.