Asthma is a chronic inflammatory disease characterized by airway hyperresponsiveness and variable airflow obstruction. Despite the significant improvement of our understanding, the diagnosis and management of asthma are still significant challenges to physicians. Nitric oxide (NO), a free radical molecule detectable in exhaled breath, plays an important role in physiological function of airway regulation. Exhaled NO can be elevated in asthma and has been regarded as a noninvasive marker of airway inflammation and thus potentially useful to diagnose and monitor asthma. Current clinical application of exhaled NO as a biomarker has been greatly limited due to the significant inter-subject variability. The determinants of exhaled NO signal needs to be elucidated.
Inflammation in asthma occurs throughout the airway tree. Increasing evidence demonstrates that inflammation of smaller airways (diameter < 2 mm) is significant. Due to relative inaccessibility of small airways, investigation of the inflammatory process and NO release from smaller airways is greatly limited.
Increased inducible nitric oxide synthase (iNOS) expression, induced by inflammatory stimuli, in airway epithelial cells has been proposed to be the main source of exhaled NO. On the other hand, arginase expression and activity in asthmatic airways is also increased, may contribute to subepithelial fibrosis, and may impact NO production from airway epithelium by competing with NOS for the common substrate (L-arginine). We hypothesized that variability in exhaled NO in asthmatics is due, in part, to a balance of NOS (inflammation) and arginase (fibrosis) pathways in the airway epithelium. Both primary airway epithelial cells and an alveolar type II cell line (A549) are utilized to characterize the impact of the inflammatory cytokines and mediators (e.g. IL-13, IL-1β, TNF-α, IFN-γ and TGF-β2) on gas phase nitric oxide release by altering NOS and arginase activity in airway epithelial cells. Our results suggest that small airway epithelial cells are likely to be the primary source of exhaled NO in breath. IL-13 and cytomix (combination of IL-1β, TNF-α, IFN-γ) induce distinct NO release patterns in epithelial cells from different origins. TGF-β2 impacts cytomix-induced NO production in airway epithelial cells by reducing iNOS mRNA and protein levels, and may contribute to the intersubject variability of exhaled NO in clinically similar subjects with asthma.
In this dissertation, we also developed a steady state model of L-arginine transmembrane transport, NO production, diffusion, and gas phase NO release from lung epithelial cells. Our model predicts intracellular L-arginine and gas phase NO release over a wide range of initial extracellular L-arginine concentrations following stimulation with cytomix (TNF-α, IL-1β, and INF-γ). Relative sensitivity analysis demonstrates that enhanced arginase activity has little impact on L-arginine bioavailability for NOS. In addition, NOS activity is the dominant variable which impacts gas phase NO release.
In summary, we conclude that small airway epithelial cells are the probable source of NO present in exhaled breath, and iNOS expression is the primary determinant. Airway inflammation pattern and location, and the balance between fibrotic pathways (i.e., TGF-β, arginase) and inflammatory pathways (i.e., IL-13, iNOS) contribute to the variability of exhaled NO in asthmatic patients.
|Advisor:||George, Steven C.|
|Commitee:||Berns, Michael, Brenner, Matthew|
|School:||University of California, Irvine|
|Department:||Biomedical Engineering - Ph.D.|
|School Location:||United States -- California|
|Source:||DAI-B 72/04, Dissertation Abstracts International|
|Subjects:||Cellular biology, Biomedical engineering|
|Keywords:||Airway, Epithelial cells, Gas phase release, Nitric oxide|
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