B75-22 Differential Expression of Wnt3a on Extracellular Vesicles Over Time in Lung Development and Injury

Abstract Background EVs are increasingly recognized as modulators of cell-cell interactions in the lung, and we recently reported changes in EV surface-profiles across human lung development. Lung injury after preterm birth remains a leading complication of prematurity and associates with dysregulated Wnt signaling, an essential pathway for lung development and repair. Building on the discovery that all Wnt ligands are expressed on EVs, we investigated the Wnt signaling profile across human development and in models of reversible and irreversible neonatal lung injury, the latter of which phenocopies bronchopulmonary dysplasia. Methods For murine studies, we used two established neonatal injury models: 1) Permanent injury: Hyperoxia (70% O2) from P1 to P5 and intranasal LPS on P3 and P4; 2) Reversible injury: Intranasal LPS on P3 and P4. We collected bronchoalveolar lavage fluid (BALF) from injured and noninjured mice at P7 and P14. EVs were isolated from BALF, and interrogated Wnt3a by ELISA. For human studies, we collected tracheal aspirates (TAs) from preterm infants 24-34 weeks gestation and analyzed samples with exosome and Wnt3A ELISAs. We performed single-cell transcriptomics from non-injured, reversible injury, and permanent injury model mice and compared expression of Wnt3A and EV hallmark genes, and spatially and temporally validated these findings in FFPE lung tissue blocks from both mouse models and our Human Infant Lung Repository. Results We consistently detected EVs in murine BALF and TAs from preterm infants. Murine BALF demonstrated differential detection of Wnt3a at P7, with decreased Wnt3A in the permanent injury model relative to non-injured and reversibly injured mice, with differential expression absent by P14. These differences are consistent with decreased Wnt3A expression by alveolar type 1 cells at P7 in our permanent injury model. This finding is replicated in preterm infant TAs in the late canalicular to saccular stage of lung development and lung blocks from preterm infants with evolving BPD relative to non-injured preterm controls. Conclusions In mice with severe lung injury, we note deficits in Wnt3A on EVs during recovery and similar stage-specific trajectory in human TAs and lung tissue. As Wnt3A is expressed transcriptionally during this period of surface area expansion, we speculate a loss of Wnt3A directly contributes to failure of regeneration during severe neonatal lung injury. Future work will explore a possible causal role for deficits in Wnt3A (and other Wnt ligands) in the development of BPD and identify an optimal therapeutic window for restoration of Wnt signaling to promote lung regeneration. This abstract is funded by: Vanderbilt Department of Pediatrics Turner Hazinski Award