QS144. Endotoxin Alters Branching Morphogenesis and Matrix Deposition of The Fetal Lung

Abstract

Background: The effects of immaturity and hypoplasia of the premature lung can be affected by proinflammatory stimuli in late gestation or the postnatal period from acute lung injury secondary to intensive ventilatory management or the metabolic consequences of surgery. These stimuli alter alveolarization and contribute to bronchopulmonary dysplasia. While prior research has focused primarily on late gestational effects of inflammation on alveolar development, we sought to study whether early gestational exposure to endotoxin affects branching morphogenesis, during the critical pseudoglandular stage of lung development. Methods: Gestational day 15 (E15) fetal rat lung explants (term=22 d) at the pseudoglandular stage were incubated at 37°C in 95% air/5% CO2 as submersion cultures on inserts in serum-free BGJb media supplemented with antibiotics and ascorbic acid. Explants were treated with either 200 ng/mL or 2 μg/mL LPS with controls and examined daily by phase microscopy. After 5 days, explants were fixed in 4% formaldehyde, paraffin embedded and sectioned at 5 μm in the coronal plane. Immunohistochemical analysis was performed with PECAM to define endothelial cells, VEGF to examine endothelial mitogenesis and COX-2 antibodies as a marker for prostaglandin synthesis. Air space fraction was analyzed with Image J software. Results: Phase contrast microscopy and hematoxylin-eosin histology revealed progressive, dose-related changes in air sac contraction and interstitial thickening. Compared with control E15 explants, day 5 explants incubated with high dose LPS demonstrated thickened and shrunken airway sacs with stunted branching and increased matrix deposition in interstitial areas (graph). By immunohistochemical staining, COX-2 was quantitatively increased after high dose LPS exposure, while PECAM was reduced. VGEF expression was unaltered. Conclusions: This is the first evidence of an inflammatory effect of LPS on the early phase of lung development in the fetal rat, affecting branching morphogenesis during the pseudoglandular phase. Fetal endothelial cells are clearly affected, while COX-2 elevation suggests activation of an as yet undefined fetal pulmonary inflammatory cascade. We speculate that the proinflammatory stimuli that may ultimately lead to abnormal pulmonary development affect epithelial-mesenchymal interaction and differentiation at a much earlier gestational age than was previously recognized.