6740 Investigating the effect of caffeine on hyperoxia and stretch-induced activation of TGFb: implications for bronchopulmonary dysplasia in preterm neonates

Objectives Bronchopulmonary dysplasia (BPD) is a parenchymal fibrotic lung disease that occurs in approximately two thirds of preterm infants born between 22- and 26-weeks' gestation. Mechanical ventilation (MV) and hyperoxygenation resulting from neonatal intensive care treatment are thought to be crucial drivers. Caffeine Objective Investigate the effect of caffeine on Mechanical Ventilation and Hyperoxygenation-induced TGFβ activation and TGFβ isoform expression. Methods Immortalised human bronchial epithelial cells (iHBECs) were subjected to cyclical mechanical stretch (CMS) using the Flexcell tension system (1Hz, equivalent to 60 stretches per minute) or hyperoxygenation (95% O2) in the presence of 0 or 10uM caffeine. Total cell protein was subjected to western blotting to detect phosphorylated Smad2/3 and TGFb isoforms. In parallel experiments total cell RNA was subjected to quantitative RT-PCR to detect TGFB1, TGFB2 and TGFB3. ELISA was used to measure TGFβ isoform release in to cell supernatants. Results CMS caused an increase in TGFβ activation as measured by increased PSmad2/3. CMS caused an increase in TGFβ1 release while reducing TGFβ2 release. Caffeine inhibited CMS-induced TGFβ1 release and led to increased TGFβ2 expression above CMS alone. Caffeine caused an increase in CMS induced TGFB2 mRNA. Hyperoxygenation had no effect on expression of the TGFβ isoforms. Conclusion CMS has differential effects of TGFβ isoform expression, which may be important in the pathogenesis of BPD. Crucially, caffeine may exert its protective effects by reducing TGFβ1 and increasing TGFβ2 expression.