Abstracts

#51 Oxidized phosphotidylcholine, a bioactive product of oxidative damage, causes airway epithelial barrier impairment


Christopher Daniel Pascoe, CHRIM; Neilloy Roy, CHRIM; Emily Turner-Brannen, CHRIM; Aruni Jha, CHRIM; Andrew J Halayko, CHRIM; Adrian R West, CHRIM


Introduction

Impaired epithelial barrier function plays a key role in the pathophysiology of asthma. Diminished tight junction integrity, including loss of E-cadherin and zonula occludens-1 (ZO-1), and decreased wound healing capacity increases the permeability of the epithelial layer to irritants. A phosphatidylcholine (PC)-rich lung lining fluid is susceptible to oxidative damage that results in peroxidation of unsaturated phospholipid chains, generating bioactive oxidized 2-arachidonoyl-1-palmitoyl-sn-glycero-3-phosphocholines (OxPAPCs). Dysregulated redox balance in the asthmatic lung increases the potential for excessive accumulation of OxPAPCs. Though OxPAPCs can promote inflammation and tissue damage, their effects in promoting epithelial barrier dysfunction are unknown. Hypothesis: Oxidized phosphatidylcholine exposure impairs of airway epithelial barrier function by altering cellular bioenergetics.


Methods

The human airway epithelial cell line, Calu3, was grown to confluence, serum deprived and incubated with OxPAPCs or PSPC, a non-oxidizable negative control. After 24-hours cytotoxicity, cell stress, epithelial barrier function and permeability, and wound reapair was assessed. Cellular reactive oxygen species (ROS) and mitochondrial membrane potential was assessed with fluorescent dies 6 hours after OxPAPC exposure. Experiments were completed in triplicate. Values are expressed as mean ±SEM, significance set at p<0.05.


Results

LDH release was evident only after 24hrs at the highest concentration of OxPAPC tested (vs. PSPC). Compared to PSPC, OxPAPC-treated cultures exhibited greatly reduced TEER by ~6-fold, and increased trans-epithelial permeability by ~20-fold. High concentrations of OxPAPC (160 µg/mL) virtually abolished wound repair while lower concentrations (40-80 µg/mL) delayed epithelium repair and barrier function restoration by ≥24 hours. This was mirrored by a loss of DNA synthesis capacity in wounded epithelial cells exposed to OxPAPCs. OxPAPC also dose dependently altered cellular bioenergetics causing increased cellular ROS and mitochondrial membrane potential.


Conclusion

OxPAPC promote epithelial barrier and wound healing impairment by interfering with cellular redox and mitochondrial bioenergetics, which may contribute to asthma pathogenesis and symptoms.