Increased nicotinamide adenine dinucleotide phosphate oxidase 4 expression mediates intrinsic airway smooth muscle hypercontractility in asthma

Abstract

Rationale: Asthma is characterized by disordered airway physiology as a consequence of increased airway smooth muscle contractility. The underlying cause of this hypercontractility is poorly understood.

Objectives: We sought to investigate whether the burden of oxidative stress in airway smooth muscle in asthma is heightened and mediated by an intrinsic abnormality promoting hypercontractility.

Methods: We examined the oxidative stress burden of airway smooth muscle in bronchial biopsies and primary cells from subjects with asthma and healthy controls. We determined the expression of targets implicated in the control of oxidative stress in airway smooth muscle and their role in contractility.

Measurements and main results: We found that the oxidative stress burden in the airway smooth muscle in individuals with asthma is heightened and related to the degree of airflow obstruction and airway hyperresponsiveness. This was independent of the asthmatic environment as in vitro primary airway smooth muscle from individuals with asthma compared with healthy controls demonstrated increased oxidative stress-induced DNA damage together with an increased production of reactive oxygen species. Genome-wide microarray of primary airway smooth muscle identified increased messenger RNA expression in asthma of NADPH oxidase (NOX) subtype 4. This NOX4 overexpression in asthma was supported by quantitative polymerase chain reaction, confirmed at the protein level. Airway smooth muscle from individuals with asthma exhibited increased agonist-induced contraction. This was abrogated by NOX4 small interfering RNA knockdown and the pharmacological inhibitors diphenyleneiodonium and apocynin.

Conclusions: Our findings support a critical role for NOX4 overexpression in asthma in the promotion of oxidative stress and consequent airway smooth muscle hypercontractility. This implicates NOX4 as a potential novel target for asthma therapy.

Figure 1.

Representative photomicrographs of bronchial biopsies illustrating 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) immunostaining in (A) an isotype control, (B) a subject with severe asthma, (C) the airway smooth muscle (ASM) bundle of a healthy control, and (D) the ASM bundle in a subject with severe asthma. Scale bars, 100 μm (A and B) and 25 μm (C and D). (E) Semiquantitative intensity score of 8-oxodG in ASM cells in subjects with asthma compared with healthy subjects. Data are shown as mean ± SEM; n = 37; comparison across groups Kruskal-Wallis P = 0.022 and between groups post hoc Dunn's pairwise comparison P values as shown. (F) 8-oxodG staining intensity was correlated negatively with airflow obstruction (FEV₁/FVC %) (Spearman's rank correlation coefficient).

Figure 2.

(A) Detection of DNA damage induced by hydrogen peroxide in airway smooth muscle (ASM) cells using the human 8-oxoguanine glycosylase 1-modified alkaline comet assay. Reactive oxygen species (ROS) promotes formation of 8-oxo-7,8-dihydroguanine in DNA. Human 8-oxoguanine glycosylase 1 is an 8-oxoguanine DNA glycosylase that recognizes and removes the damaged DNA caused by ROS. Tail moment is defined as the product of the tail length and the total DNA in the tail. It incorporates the smallest detectable size of migrating DNA (comet tail length) and the fragments (represented by the intensity of DNA in the tail). Data are shown as mean ± SEM (n = 9). (B) Detection of intracellular ROS, induced by hydrogen peroxide in ASM cells after 1 hour using 2′,7′-dichlorofluorescin diacetate, which detects hydrogen peroxide, peroxyl radicals, and peroxynitrite anions (mean ± SEM; n = 8). (C) Intracellular flow cytometric data shown as a scatter plot (mean ± SEM). (D) Representative flow cytometric analysis of NOX4 cell surface expression and intracellular NOX4 expression. Gray line corresponds to IgG isotype control, black line NOX4⁺. (E) Quantitative reverse transcription–polymerase chain reaction analysis of NOX4 mRNA in normal and asthmatic ASM cells (mean ± SEM; n = 23). (F) NOX4 mRNA expression correlated negatively with airflow obstruction. Comparisons were made across groups by Kruskal-Wallis test with post hoc Dunn's pairwise comparison for nonparametric data and by paired and unpaired t tests as appropriate for parametric data. Correlations were assessed by the Spearman rank test.

Figure 3.

(A) Percentage contraction of collagen gels impregnated with airway smooth muscle from donors with asthma (n = 19) versus healthy control donors (n = 8) over 1 hour following stimulation with 1 nM bradykinin, (B) area under the curve gel contraction (mean ± SEM), and (C) representative gel photographs taken at 0 hour and 1 hour time points. The comparison was made by unpaired t test. *P < 0.05.

Figure 4.

(A) Percentage contraction of collagen gels, following stimulation with 1 nM bradykinin, impregnated with airway smooth muscle from donors with asthma versus healthy control donors untransfected or transfected with negative control small interfering RNA (siRNA) or NOX4 siRNA, (B) representative gel photographs taken at 0 hour and 1 hour time points, and (C) area under the curve (AUC) gel contraction (mean ± SEM, n = 6 asthma, n = 4 healthy control). Between group comparisons were made by paired t tests, *P < 0.05, **P < 0.01. (D) Comparison of the difference in AUC between negative control siRNA and NOX4 siRNA in asthma versus healthy control. (E) Percentage contraction of collagen gels impregnated with airway smooth muscle from donors with asthma versus healthy control donors following stimulation with 1 nM bradykinin in the presence and absence of diphenyleneiodonium (1–5 μg/ml) (n = 4). (F) AUC gel contraction (mean ± SEM, n = 4). Across-group comparisons were made by linear regression.

Figure 5.

(A) Percentage contraction of collagen gels, following stimulation with 1 nM bradykinin, impregnated with airway smooth muscle from donors with asthma versus healthy control donors untransfected or transfected with negative control small interfering RNA (siRNA) or superoxide dismutase 2 siRNA, (B) representative gel photographs taken at 0 hour and 1 hour time points, and (C) AUC gel contraction (mean ± SEM, n = 8). Between-group comparisons were made by paired t tests. (D) Comparison of the difference in AUC between negative control siRNA and superoxide dismutase 2 siRNA in asthma versus healthy control. (E) Percentage contraction of collagen gels impregnated with airway smooth muscle from asthmatic versus healthy control donors following stimulation with 1 nM bradykinin in the presence and absence of manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (10–100 mg/ml) (n = 6). (F) AUC gel contraction (mean ± SEM, n = 6). Across-group comparisons were made by linear regression.