246 - Amphiregulin Exerts Proangiogenic Effects in the Developing Murine Lungs

To test the hypothesis that Areg promotes the proangiogenic ability of the endothelial cells in the developing murine lungs exposed to hyperoxia.

Design/Methods:

In vivo studies: One-day-old C57BL/6J were exposed to normoxia (21% O₂) or hyperoxia (70% O₂) from postnatal day (P) 1 to P14 and the lung tissue was harvested on P14 to determine Areg expression. In vitro studies: To determine if Areg influences angiogenesis in the developing lungs, we performed: (1) loss-of-function studies by transfecting fetal murine lung endothelial cells with 50 nM control or Areg siRNA and (2) gain-of-function studies by treating these cells with PBS or 100 nM recombinant Areg protein. The transfected and treated cells were then exposed to normoxia (21% O₂ + 5% CO₂) or hyperoxia (70% O₂ + 5% CO₂) for up to 72 h to determine Areg expression and quantity their tubule formation ability. Statistical significance was determined using t-tests and ANOVA. Both in vivo and in vitro studies were repeated greater than three times with n = greater than five per group during each study. A p-value < 0.05 was considered significant.

Results: Hyperoxia exposure increased Areg mRNA levels by 1.5-fold in whole lungs (p < 0.05) and by 1.8-fold in fetal murine lung endothelial cells (p < 0.01). Areg siRNA efficiently decreased Areg expression by greater than 3-fold in fetal murine endothelial cells (p < 0.001) and decreased their ability to form tubules at baseline ( p < 0.01) and potentiated hyperoxia-mediated anti-angiogenic effects ( p < 0.05) . By contrast, Areg treatment exerted proangiogenic effects and reduced hyperoxia-mediated anti-angiogenic effects ( p < 0.01).

Conclusion(s):

Areg promotes endothelial cell tubule formation in the developing murine lungs exposed to hyperoxia. Our results indicate that Areg is a potential therapeutic target for experimental and clinical BPD with a predominant vascular phenotype.