Effects of smoking and oxidative stress on airway remodeling and phenotypic changes of the airway epithelium in asthma and COPD: strategies to restore the epithelial barrier, repair and steroid sensitivity

Asthma and COPD are chronic inflammatory airway diseases affecting millions of
people worldwide. Inhaled corticosteroids (ICS) are by far the most effective
treatment with a broad anti-inflammatory spectrum. Nevertheless, most COPD
patients and a proportion of severe asthma patients are
corticosteroid-resistant (CR) and to fail to respond to ICS even when higher
doses are given. These corticosteroid-resistant patients suffer from persistent
symptoms and repeated asthma exacerbations. It has been suggested that smoking
and oxidative stress may induce corticosteroid-resistance. The reactive oxygen
species (ROS) responsible for oxidative stress can be generated exogenously
(air pollutants, cigarette smoke) and endogenously by metabolic reactions.
After inhaling air pollutants or cigarette smoke, the bronchial epithelium is
exposed. Preliminary data from our own lab suggest that smoking and oxidative
stress may decrease epithelial cell-cell contact formation. This results not
only in a decreased barrier function, but also in an increased production of
pro-inflammatory mediators.

With this research project, we will investigate the effects of smoking and
oxidative stress on epithelial barrier function in patients with asthma and
COPD.

The most important research questions are:

1) Does exposure of cultured human epithelial cells to cigarette smoke or
oxidative stress decrease cell-cell contact formation and reduce the level of
caveolin-1?
2) Does exposure to cigarette smoke or oxidative stress render human cultured
bronchial epithelial cells less sensitive to the positive effects of
corticosteroids ?
3) Can the effects of smoking/oxidative stress on cultured human bronchial
epithelial cells be counteracted by (combinations of) corticosteroids, *2-
agonists, heme oxygenase-1, exposure to carbon monoxide, or vitamin D3? Is this
associated with an increase in caveolin-1?
4) What are the effects of modulation of caveolin-1 on cell-cell contact
formation?
5) Are there differences with respect to the above between patients with
allergic or non-allergic asthma, COPD, or healthy subjects?
6) Are there differences between cultured epithelial cells derived from
bronchial brushing and epithelial cells derived from nasal brushing?

This is an observational study. We will include a total of 60 well
characterized (lung function, PC20 methacholine, skin prick test) patients with
asthma and COPD. We will perform nasal and bronchial brushings. In addition,
bronchial biopsies are taken to investigate the level of epithelial shedding
and markers of epithelial integrity such as E-cadherin and caveolin-1. In
addition, we will investigate the expression of heme oxygenase 1.

Cultured epithelial cells will be stimulated with relevant inflammatory
mediators (e.g. TNF-*., IL-13) and the production of pro-inflammatory mediators
TARC, TSLP, IL-6, GM-CSF will be assessed in the presence and absence of
budesonide. Next, oxidative stress will applied by maintaining subconfluent
cells for 24 (or 48 hours) under 5% CO2/95% O2 which results in the production
of ROS. This allows us to investigate the effects of oxidative stress on
development corticosteroid resistance and the underlying mechanisms involved
(i.e. effects on the level of NF-*B (both cytoplasma and nucleus),
glucocorticoid receptor (GR)* and GR*, glucocorticoid responsive element (GRE),
HDAC-2, and p38 MAPK). Finally, we will assess the effects of treatment aimed
to protect against oxidative stress (e.g. N-acetylcysteine, upregulation of
HO-1 (protoporphyrin, adenoviral constructs), CO, curcumin, and resveratrol).
Finally, we will investigate the effects of treatment aimed to improve
corticosteroid responsiveness (e.g. *2-agonists, theophylline).

This study is not associated with large risks. Possible adverse reactions may
be:

- Blood collection may be painful and cause skin bruising.
- The provocation test with metahcholine may cause temporary dyspnea.
- The bronchoscopy may cause irritation of the airways and the bronchial biopsy
may cause local bleeding.
- The nasal brush may cause irritation of the nasal mucosa and may cause local
bleeding.