Scientific Session 1
DEXAMETHASONE SYNCHRONIZES RESPIRATORY EPITHELIAL REPAIR IN VITRO
S. F. Iqbal1,2,3, A. Benton1, A. Watson1,3, M. Rose1,3, R. Freishtat1,2,3 1Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC, USA; 2Division of Emergency Medicine, Children's National Medical Center, Washington, DC, USA and 3George Washington University, Washignton, DC, USA.
Purpose of Study
Glucocorticoids (GC) are a mainstay of asthma therapy. The current paradigm is that they are anti-inflammatory; however an emerging concept is that GC resynchronize out-of-phase repair processes in damaged respiratory epithelium. Our hypothesis is that injured asthmatic respiratory epithelium is characterized by asynchronous regeneration unresponsive to in vitro conditions that mimic the diurnal levels of endogenous GC, whereas injured normal respiratory epithelium regeneration is characterized by rapid healing induced by GC-induced cell cycle pauses.
Primary differentiated human airway bronchial epithelial cells (Mattek, Ashland, MA) from normal and asthmatic donors were grown at an air-liquid interface on collagen-coated Transwell membranes. After a 24 hr incubation in steroid-free medium, confluent cultures were exposed to 20 nM dexamethasone (DEX) and were mechanically wounded. Half of the cultures were pulsed for 2 hours daily with 20 nM DEX. Cells were harvested 48 h after wounding. Cell cycle synchrony was evaluated by flow cytometry for DNA content (7-AAD) in proliferating (BrdU+) cells.
Summary of Results
Bright-field microscopy (16x) of DEX+ and DEX- normal culture wounds showed complete healing by 24 and 48 h, respectively. DEX treatment did not visibly alter similarly cultured asthmatic cells, which were still unhealed at 48 h. Normal DEX+ cultures showed significantly more BrdU+ cells in G2 (72 9 vs. 29 5%; P = 0.05) and less in G1 (17 6 vs. 63 4%; P = 0.02) than asthmatic cells.
Proliferating asthmatic cells were more evenly distributed among G1 and G2 than normal cells, consistent with asynchronous cell cycling. Although …