O2167: Tracheal Ligation of Hypoplastic Fetal Lungs Induces Nuclear Yap Activation in Airway Epithelium

Background: Tracheal occlusion to induce canalicular stage lung growth has been shown to improve survival in a recent multicenter trial of fetuses with severe congenital diaphragmatic hernia (CDH). However, the underlying mechanisms that induce pulmonary growth in this setting remain essentially unknown. Our group hypothesized that the proximal-distal fetal lung abnormalities observed in the CDH lung may be reversed by activation of nuclear Yap (nYap), a mechanosensor protein that is an important regulator for controlling organ size and development. In this study, we sought to develop an ex-vivo hypoplastic lung model of tracheal ligation (TL) to determine whether pulmonary growth induction was associated with nYap activation.

Methods: After animal welfare approval (MO21M344), time-mated CD1 pregnant mice were gavaged nitrofen/4-biphenyl carboxylic acid (BPCA) at E8.5 to induce CDH fetal lung hypoplasia. At E16.5, control and nitrofen/BPCA mice were euthanized, and fetuses were harvested by laparotomy (Figure 1). In the TL group, a Hamilton 31-G/10 uL syringe (Model#7625-01/7762-04) was used to instill 2:1 DMEM/20% fetal bovine serum:trypan blue prior to tracheal occlusion with a fine vicryl suture (Figure 1). TL and non-ligated (no TL) lungs were cultured for 72 hours between pre-formed 2% agarose discs prior to quantitative gene expression and immunohistochemistry (IHC) studies. Data were expressed as mean+/-SD with statistical significance at p< 0.05.

Results: Control TL lungs (n=36) demonstrated increased expression of nYap-associated genes, as demonstrated by increased Ctgf (TL: 1.37+/-0.30 vs. no TL: 1.02+/-0.20, p=0.004) and Cyr61 (TL: 1.79+/-0.38 vs. no TL: 1.04+/-0.31, p< 0.001) compared to control, no TL lungs (n=42). Increased nYap expression after TL was also identified in nitrofen/BPCA lungs (n=28) when compared to no TL lungs (n=32) (Ctgf: 1.57+/-0.43 vs. 1.02+/-0.23, respectively; p=0.02) (Cyr61: 1.60+/-0.34 vs. 1.01+/-0.17, respectively; p=0.003) (Figure 2). Following TL, there was evidence of stretch within the airways, with distended bronchi on IHC, as well as upregulation of nYap in epithelial cells lining the proximal and distal bronchi (Figure 2). Fgf10 was also upregulated following TL in control (1.79+/-0.38 vs. 1.04+/-0.31, p< 0.001) and nitrofen/BPCA (2.25+/-0.72 vs. 0.98+/-0.31, p=0.003) lungs. Markers of AT1 cells were significantly upregulated after TL in control lungs, including Hopx (TL: 1.26+/-0.28 vs. no TL: 1.01+/-0.15, p=0.03) and Podoplanin (Pdpn) (TL: 2.62+/-1.63 vs. no TL: 1.06+/-0.41, p=0.02). TL in the nitrofen/BPCA model led to an increased trend in Hopx (1.46+/-0.63 vs. 1.01+/-0.14, p=0.11) and a significant increase in Pdpn (1.61+/-0.49 vs. 0.98+/-0.45, p=0.03).

Conclusion: This novel ex-vivo TL model of CDH lung hypoplasia revealed upregulation of Fgf10 and nYap in both control and hypoplastic lungs during the canalicular stage of development. These results suggest a critical role of nYap signaling in reversing lung hypoplasia and suggest the development of novel molecular targets aimed at manipulating mechanotransduction pathways within the CDH lung.