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, 68 (12), 1579-88

Endothelin-1 Directs Airway Remodeling and Hyper-Reactivity in a Murine Asthma Model

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Endothelin-1 Directs Airway Remodeling and Hyper-Reactivity in a Murine Asthma Model

L G Gregory et al. Allergy.

Abstract

Background: The current paradigm describing asthma pathogenesis recognizes the central role of abnormal epithelial function in the generation and maintenance of the disease. However, the mechanisms responsible for the initiation of airway remodeling, which contributes to decreased lung function, remain elusive. Therefore, we aimed to determine the role of altered pulmonary gene expression in disease inception and identify proremodeling mediators.

Methods: Using an adenoviral vector, we generated mice overexpressing smad2, a TGF-β and activin A signaling molecule, in the lung. Animals were exposed to intranasal ovalbumin (OVA) without systemic sensitization.

Results: Control mice exposed to inhaled OVA showed no evidence of pulmonary inflammation, indices of remodeling, or airway hyper-reactivity. In contrast, local smad2 overexpression provoked airway hyper-reactivity in OVA-treated mice, concomitant with increased airway smooth muscle mass and peribronchial collagen deposition. Pulmonary eosinophilic inflammation was not evident, and there was no change in serum IgE or IgG1 levels. The profound remodeling changes were not mediated by classical pro-inflammatory Th2 cytokines. However, uric acid and interleukin-1β levels in the lung were increased. Epithelial-derived endothelin-1 and fibroblast growth factor were also augmented in smad2-expressing mice. Blocking endothelin-1 prevented these phenotypic changes.

Conclusions: Innate epithelial-derived mediators are sufficient to drive airway hyper-reactivity and remodeling in response to environmental insults in the absence of overt Th2-type inflammation in a model of noneosinophilic, noninflammed types of asthma. Targeting potential asthma therapies to epithelial cell function and modulation of locally released mediators may represent an effective avenue for therapeutic design.

Keywords: animal models; asthma; epithelium; innate immunity; remodeling.

Figures

Figure 1
Figure 1
Ovalbumin (OVA) exposure results in airway hyper-responsiveness (AHR) in the absence of inflammation in mice overexpressing smad2 in the airway epithelium. (A) Resistance and (B) compliance measured in tracheotomized animals in response to increasing doses of methacholine. (C) Lung sections stained with H&E. Original magnification ×40. Scale bar = 50 μm. Representative photomicrographs are shown. (D) Total cells recovered from lung tissue. (E) Eosinophils determined by differential counting of cytospins prepared from the lung digest. (F) Eotaxin levels in the lung as determined by ELISA (assay sensitivity 3 pg/ml). (G) CD4+T1ST2+ Th2 cells recovered from the lung and quantified by flow cytometry. (H) Serum total IgE and IgG1 levels measured by ELISA (assay sensitivity 2 ng/ml and 80 pg/ml, respectively). Data shown represent means ± SEM (N = 6–18) of at least two independent experiments. *P < 0.05 compared with PBS-treated groups or OVA-exposed AdC mice.
Figure 2
Figure 2
Airway remodeling induced by ovalbumin (OVA) in epithelial smad2-overexpressing mice. (A) Lung sections stained with periodic acid-Schiff (PAS) to identify mucin-containing (purple) cells. (B) Lung sections stained with antibody to proliferating cell nuclear antigen (PCNA, brown staining). (C) Quantitation of PCNA+ airway epithelial cells. (D) Sirius Red staining of lung sections depicts peribronchiolar and perivascular collagen (red). (E) Recently synthesized total lung collagen was quantified by a biochemical (Sircol) assay. (F) Quantitative image analysis of subepithelial peribronchiolar collagen density determined by measuring Sirius Red-stained collagen in lung sections under polarized light. Original magnification ×40. Scale bar = 50 μm. Representative photomicrographs are shown. Data shown represent means ± SEM (N = 6–12) of two independent experiments. *P < 0.05 compared with PBS-treated groups or OVA-exposed AdC mice.
Figure 3
Figure 3
Airway smooth muscle changes induced by ovalbumin (OVA) in AdS mice. (A) Lung sections stained with an antibody against α-smooth muscle actin (α-SMA, brown staining), depicting myofibroblasts and smooth muscle cells. (B) Quantification of α-SMA+ peribronchiolar area. (C) Lung sections stained with an antibody against proliferating cell nuclear antigen (PCNA, brown staining). (D) Quantitation of PCNA+ peribronchiolar mesenchymal cells. Original magnification ×40. Scale bar = 50 μm. Representative photomicrographs are shown. (E) Airway resistance and (F) compliance measured in tracheotomized animals in response to increasing doses of methacholine. Data shown represent means ± SEM (N = 4–12) of two independent experiments. *P < 0.05 compared with PBS-treated groups or OVA-exposed AdC mice. †P < 0.05 comparing AdS OVA-challenged mice treated with salbutamol or vehicle, PBS.
Figure 4
Figure 4
Ovalbumin (OVA) increases mediators of airway remodeling and airway hyper-responsiveness (AHR) in mice overexpressing smad2 in the airway epithelium. (A) Endothelin-1 expression (red-stained) in airway epithelial cells correlates with (B) endothelin-1 levels in the BALF (assay sensitivity 0.41 pg/ml). (C) Lung sections stained with an antibody against fibroblast growth factor (FGF, brown staining). (D) Pulmonary FGF-2 levels. (E) Pulmonary IL-1β (assay sensitivity 8 pg/ml) and (F) uric acid levels (assay sensitivity 100 nM). Representative photomicrographs are shown. Original magnification ×40. Scale bar = 50 μm. Data shown represent means ± SEM (N = 6–18) of at least two independent experiments. *P < 0.05 compared with PBS-treated groups or OVA-exposed AdC mice.
Figure 5
Figure 5
Airway remodeling and airway hyper-responsiveness (AHR) are driven by endothelin-1 and uric acid. (A) Resistance and (B) compliance measured in tracheotomised animals in response to increasing doses of methacholine. (C) Quantitative image analysis of subepithelial peribronchiolar collagen density determined by measuring Sirius Red-stained collagen in lung sections under polarized light. (D) Recently synthesized total lung collagen was quantified by a biochemical (Sircol) assay. (E) Quantification of α-smooth muscle actin (α-SMA)+ peribronchiolar area. (F) Quantitation of proliferating cell nuclear antigen (PCNA)+ peribronchiolar mesenchymal cells. (G) Pulmonary endothelin-1, (H) uric acid, (I) IL-1β, and (J) fibroblast growth factor-2 (FGF-2) levels. Data shown represent means ± SEM (N = 6–12) of two independent experiments. *P < 0.05 compared with PBS-treated groups or ovalbumin (OVA)-exposed AdC mice. †P < 0.05 comparing AdS OVA-challenged mice treated with PD142893 to AdS OVA PBS.

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