O2159: Impact of Mechanical Compression on Retinoic Acid Pathway Signaling in an Ex vivo Fetal Lung Model of Congenital Diaphragmatic Hernia

Background: The retinoic acid (RA) signaling pathway is a key regulator of fetal lung specification, branching morphogenesis, and alveolarization. Although RA has been implicated in the pathogenesis of pulmonary hypoplasia in rodent and human congenital diaphragmatic hernia (CDH), the mechanistic link between compression forces produced by the herniated abdominal viscera and RA dysregulation has not been well explored in the fetal lung. The purpose of this study was to determine the impact of mechanical compression on canalicular stage retinoic acid signaling using a novel ex vivo lung modeling device.

Methods: After animal welfare approval (MO20M15), timed pregnant CD1 mice were sacrificed for whole fetal lung harvesting at E15.5 (canalicular stage, E16=physiologic diaphragm closure). To mimic the in utero mechanical forces encountered in CDH, fetal lungs (n=28) were cultured ex vivo for 48 hours between pre-formed 2% agarose discs within microdevices (Figure 1A) under a static force of 3.3 kPa (Compression Plus FX-5000C, Burlington, NC). Lungs were compared to ex vivo lungs without compression (controls, n=28) by whole mount immunofluorescence staining and by quantitative gene expression (p≤0.05).

Results: Whole mount immunofluorescence with lung epithelial progenitor NKX2.1 demonstrated less complex branching patterns among compressed lungs when compared to control lungs (Figure 1B). Compressed lungs expressed a more immature phenotype than control lungs, as demonstrated by increased expression of Nkx2.1 (compression: 1.94+/-0.60, control 1.09+/-0.45, p=0.03), and trend to increased expression of proximal airway Sox2 (compression: 1.71+/-0.48, control 1.08+/-0.49, p=0.073) and distal airway Sox9 (compression: 1.68+/-0.46, control 1.06+/-0.41, p=0.076). Compressed lungs had significantly reduced expression of mesenchymal Sma (compression: 0.52+/-0.17, control 1.01+/-0.19, p=xx), AT1 epithelial Hopx (compression: 0.51+/-0.13, control 1.01+/-0.11, p< 0.001), AT1 epithelial Pdpn (compression: 0.63+/-0.23, control 0.97+/-0.22, p=0.025), and AT2 epithelial Sftpb (compression: 0.42+/-0.16, control 1.02+/-0.23, p=0.002). Although there were no differences in extracellular retinol binding protein (Rbp4) levels, expression of the cell membrane receptor Stra6 (compression: 0.64+/-0.15, control 1.03+/-0.15, p=0.002), enzymes along the RA pathway (Adh1, Aldh1), and cytoplasmic retinoic acid binding protein Crabp1 (compression: 0.44+/-0.24, control 1.07+/-0.23, p=0.002) were significantly decreased in the compression group (Figure 2).

Conclusion: This novel ex vivo compression modeling platform recapitulated many of the pathologic features of the CDH lung, including reduced distal airway branching and a more immature pulmonary phenotype as revealed by impaired epithelial-mesenchymal differentiation. Furthermore, isolated compression produced profound impairments at multiple stages along the RA biosynthesis pathway. These results highlight the mechanoresponsive nature of RA in the pathogenesis of pulmonary hypoplasia and suggest the possibility for novel molecular targets aimed to reversing abnormal lung development in patients with severe CDH.