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. 2009 Jun;296(6):L888-900.
doi: 10.1152/ajplung.90369.2008. Epub 2009 Mar 13.

Cigarette smoke-induced emphysema in A/J mice is associated with pulmonary oxidative stress, apoptosis of lung cells, and global alterations in gene expression

Tirumalai Rangasamy  1 , Vikas Misra, Lijie Zhen, Clarke G Tankersley, Rubin M Tuder, Shyam Biswal
Affiliations

Cigarette smoke-induced emphysema in A/J mice is associated with pulmonary oxidative stress, apoptosis of lung cells, and global alterations in gene expression

Tirumalai Rangasamy et al. Am J Physiol Lung Cell Mol Physiol. 2009 Jun.

Abstract

Cigarette smoking is the major risk factor for developing chronic obstructive pulmonary disease, the fourth leading cause of deaths in the United States. Despite recent advances, the molecular mechanisms involved in the initiation and progression of this disease remain elusive. We used Affymetrix Gene Chip arrays to determine the temporal alterations in global gene expression during the progression of pulmonary emphysema in A/J mice. Chronic cigarette smoke (CS) exposure caused pulmonary emphysema in A/J mice, which was associated with pronounced bronchoalveolar inflammation, enhanced oxidative stress, and increased apoptosis of alveolar septal cells. Microarray analysis revealed the upregulation of 1,190, 715, 260, and 246 genes and the downregulation of 1,840, 730, 442, and 236 genes in the lungs of mice exposed to CS for 5 h, 8 days, and 1.5 and 6 mo, respectively. Most of the genes belong to the functional categories of phase I genes, Nrf2-regulated antioxidant and phase II genes, phase III detoxification genes, and others including immune/inflammatory response genes. Induction of the genes encoding multiple phase I enzymes was markedly higher in the emphysematous lungs, whereas reduced expression of various cytoprotective genes constituting ubiquitin-proteasome complex, cell survival pathways, solute carriers and transporters, transcription factors, and Nrf2-regulated antioxidant and phase II-responsive genes was noted. Our data indicate that the progression of CS-induced emphysema is associated with a steady decline in the expression of various genes involved in multiple pathways in the lungs of A/J mice. Many of the genes discovered in this study could rationally play an important role in the susceptibility to CS-induced emphysema.

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Figures

Fig. 1.
Fig. 1.
Increased air space enlargement in A/J mice exposed to chronic cigarette smoke (CS). A: H&E-stained lung sections from A/J mice exposed to room air or CS at the indicated time points. Lung sections from the 6-mo CS-exposed mice show increased alveolar destruction and air space enlargement compared with the lung sections from age-matched air-exposed A/J mice. Sections from the age-matched air-exposed mice show normal alveolar structure (n = 5 mice/group); original magnification, ×20. The images (15 fields/slide) of the H&E-stained lung sections from the air- and CS-exposed mice were acquired with a Nikon E800 microscope, and alveolar diameter (B) and mean linear intercepts (C) were determined by computer-assisted morphometry with the Image Pro Plus software. Six-month CS-exposed A/J mice show a significantly increased alveolar diameter (B) and mean linear intercept (C) compared with 1.5-mo CS-exposed mice. Data are means ± SE. *P ≤ 0.05.
Fig. 2.
Fig. 2.
Increased oxidative stress in the lungs of chronic CS-exposed A/J mice. A: the occurrence of oxidative stress in the lungs of 6-mo CS-exposed mice was determined by immunohistochemistry with anti-8-oxo-dG antibody. Lung sections from the CS-exposed A/J mice show increased staining for 8-oxo-dG (indicated by arrows) compared with lung sections from the age-matched air-exposed control mice. Normal mouse IgG1 was used as a negative control (magnification, ×20). B: quantification of 8-oxo-dG-positive cells in lungs after 6 mo of CS exposure. Increased number (28.3 anti-8-oxo-dG-positive cells/10 fields) of 8-oxo-dG-positive cells was detected in the lungs of CS-exposed mice. Few (4.2 anti-8-oxo-dG-positive cells/10 fields) 8-oxo-dG-positive cells were detected in the lung tissues of air-exposed mice (n = 5 mice/group). Values (positive cells/10 fields) are represented as means ± SE, *P ≤ 0.05 vs. air-exposed A/J mice.
Fig. 3.
Fig. 3.
Chronic CS exposure causes alveolar cell apoptosis in A/J mice. A: lung sections (n = 5) from air- or CS-exposed (6-mo) mice were subjected to TUNEL (middle) and DAPI (left) staining. Merged images are shown at right. Overlapping DAPI in blue and FITC in green create a magenta, apoptotic-positive signal. B: CS-exposed mice (6-mo) showed abundant TUNEL-positive alveolar septal cells compared with air-exposed mice (n = 5 mice/group). Values are represented as means ± SE. P ≤ 0.05. C: identification of apoptotic type II epithelial cells (top) and endothelial cells (bottom) in the lungs of CS-exposed mice (6-mo). Type II epithelial cells and endothelial cells were detected using anti-SPC and anti-CD34 antibodies, respectively. Nuclei were detected with DAPI (blue). The merged images with colocalization of cell-specific markers (cytoplasmic red signal) with apoptosis (green FITC + blue DAPI) signal, resulting in lavender-like signal (yellow arrows), are shown. D: active caspase-3 expression in lung sections from chronic CS-exposed (6-mo) mice. CS-exposed A/J mice show increased numbers of caspase-3-positive cells (indicated by arrows) in the lungs (n = 5/group) (magnification, ×20). E: number of caspase-3-positive cells in the lungs of age-matched air- or CS-exposed mice. Caspase-3-positive cells were significantly (*P ≤ 0.05) higher in the lungs of 6-mo CS-exposed mice than the air-exposed mice. Values are represented as means ± SE.
Fig. 4.
Fig. 4.
Graph depicting the numbers of differentially expressed genes (upregulated or downregulated) at each exposure time point. Solid black bars above the horizontal line represent the number of upregulated genes at each time point. Similarly, white bars below the horizontal line represent the number of downregulated genes at the same time points.
Fig. 5.
Fig. 5.
Venn diagrams depicting the extent of overlap between the gene expression profiles from the lungs of mice exposed to CS for various time periods. Groups were compared, and genes that satisfied the criteria of fold change ≥1.5 and P ≤ 0.05 were designated as significantly changed genes. The number of genes summarized in the Venn plot are as follows: circles shaded red, green, blue, and gray represent the total numbers of genes upregulated (A) or downregulated (B) with CS exposure at 5 h, 8 days, 1.5 mo, and 6 mo, respectively.
Fig. 6.
Fig. 6.
Pulmonary gene expression profiles of A/J mice exposed to CS. Cluster analysis shows the expression of phase I genes, antioxidant and phase II detoxification genes, phase III genes, growth factors, genes constituting the ubiquitin-proteasome complex, and genes involved in cell survival, apoptosis, and extracellular matrix maintenance. The genes are visualized by Treeview. Green indicates upregulated genes, and black represents no significant change in expression. Gene symbols are provided.
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