RATIONALE:  Nitrogen mustard (NM) is a highly toxic alkylating agent.  It is classified as chemical threat due to its potential use as a warfare agent.  When inhaled, mustard exposure can cause both acute and chronic lung injury.  The aims of this study are to develop an in vitro culture system for modeling mustard-induced airway injury and to identify growth factors contributing to airway pathology.

METHODS:  Primary human bronchial epithelial cells co-cultured with primary human pulmonary endothelial cells were exposed to NM (25, 50, 100, 250 or 500 µM) or PBS (control) for 1 hour.  Lactate dehydrogenase (LDH) and trans-epithelial electrical resistance (TEER) were measured prior to and 24 hours after NM exposure.  Endothelial cultures were then stained for live/dead staining.  Exposed airway media was also analyzed for growth factors using Luminex multiplex technology. 

RESULTS:  A one-hour NM vapor exposure increased supernatant LDH and decreased TEER at 24 hours for all cultures exposed to NM concentrations greater than or equal to 50 µM.  Evidence of endothelial cell death occurred at NM concentrations of 250 and 500 µM. Vascular endothelial growth factor (VEGF) signaling, specifically VEGF-A and placental growth factor (PlGF), increased in NM-exposed co-culture media compared to PBS controls.   

CONCLUSIONS:  NM vapor exposure causes significant airway epithelial and endothelial injury.  Increased VEGF signaling occurred in airway co-cultures exposed to higher NM concentrations with concurrent endothelial cell death.  Future studies are required to validate the role of VEGF signaling in both acute and chronic mustard-induced airway pathology.