Airway smooth muscle NOX4 is upregulated and modulates ROS generation in COPD

Abstract

The burden of oxidative stress is increased in chronic obstructive pulmonary disease (COPD). However, whether the intra-cellular mechanisms controlling the oxidant/anti-oxidant balance in structural airway cells such as airway smooth muscle in COPD is altered is unclear. We sought to determine whether the expression of the NADPH oxidase (NOX)-4 is increased in airway smooth muscle in COPD both in vivo and primary cells in vitro and its role in hydrogen peroxide-induced reactive oxygen species generation. We found that in vivo NOX4 expression was up-regulated in the airway smooth muscle bundle in COPD (n = 9) and healthy controls with >20 pack year history (n = 4) compared to control subjects without a significant smoking history (n = 6). In vitro NOX4 expression was increased in airway smooth muscle cells from subjects with COPD (n = 5) compared to asthma (n = 7) and upregulated following TNF-α stimulation. Hydrogen peroxide-induced reactive oxygen species generation by airway smooth muscle cells in COPD (n = 5) was comparable to healthy controls (n = 9) but lower than asthma (n = 5); and was markedly attenuated by NOX4 inhibition. Our findings demonstrate that NOX4 expression is increased in vivo and in vitro in COPD and although we did not observe an intrinsic increase in oxidant-induced reactive oxygen species generation in COPD, it was reduced markedly by NOX4 inhibition supporting a potential therapeutic role for NOX4 in COPD.

Keywords: Airway smooth muscle; COPD; NOX4; Oxidative stress; Reactive oxygen species.

Fig. 1

a Example photomicrographs of ASM stained with (i) isotype control or (ii-iv) NOX4 in a healthy control with a less than or greater than 20 pack year history and subject with COPD respectively and (v) an example of a COPD subject with more intense staining at higher power. b NOX4 % positive staining assessed by thresholding (mean [SEM]). n = 6 (<20 pack years healthy controls), n = 4 (>20 pack years healthy controls), n = 9 (COPD). c Correlation between smoking history and NOX4 ASM expression. d NOX4 mRNA (measured in triplicate) in ASM from COPD versus healthy controls. n = 11 (healthy controls), n = 9 (COPD). e Percentage of cells expressing NOX4 in ASM from COPD and asthma subjects assessed by flow cytometry. n = 7 (asthma), n = 5 (COPD). f Geometric mean fluorescence intensity of NOX4 expression following incubation with control media or 50 ng/mL TNFα in ASM from subjects with COPD or asthma. n = 7 (asthma), n = 5 (COPD). g Detection of intracellular ROS (measured in triplicate; relative fluorescent units [RFU]) in ASM induced by hydrogen peroxide. n = 9 (healthy controls), n = 5 (asthma), n = 5 (COPD). h Detection of hydrogen peroxide-induced intracellular ROS following pre-incubation with NOX4 inhibitor. n = 10. Each point represents an individual donor: circles-healthy, square-COPD and triangle-asthma subjects