Abstract Rationale Bronchopulmonary Dysplasia (BPD) is a consequence of disrupted lung development after preterm birth, with structural deficits at every level of the respiratory axis. BPD with lower airway disease is a clinically significant phenotype with increased mortality. Molecular mechanisms whereby preterm birth alters the trajectory of normal airway development are a knowledge gap. Objectives To characterize a cellular and molecular endotype of evolving BPD (eBPD, defined as lung disease in premature infants 30 days of age) with lower airway disease. Methods We used orthogonal methods, including ex vivo organotypic culture and bulk RNA sequencing of airway epithelial cells (AECs) from eBPD patients with and without exposure to hyperoxic injury as well as single cell RNA sequencing, histologic analysis and spatial transcriptomics in well-characterized pathologic samples from patients with evolving and established BPD. Measurements and Main Results In our ex vivo organotypic AEC model, we identified structural defects in AECs from patients with eBPD including loss of cilia. In bulk RNA sequencing studies, we saw loss of ciliated cell programming in eBPD AECs, including decreased TP73 (67-fold), FoxJ1 (387-fold), MCIDAS (13-fold), MYB (62-fold), and CCNO (19-fold), as well as decreased SOX2 (28-fold) expression. AECs cultured from patients with eBPD had increased VIM (22-fold) expression compared with AECs from healthy pediatric controls, and this increase in VIM coincided with squamous morphological change after hyperoxia exposure. These ex vivo findings were recapitulated in pathological samples from premature infants with eBPD, where we observed an expansion of vimentin-expressing structural plastic airway cells (VESPAs), defined as VIM+KRT5+TP63+ cells proximally and VIM+KRT5+SCGB3A2+ cells distally, in eBPD vs premature comparators (Figure 1). VESPAs were further characterized at a single-cell level in distal airways from patients with established BPD: VIM was expressed in secretory AECs (RASCs and SCGB3A1+/SCGB3A2+ cells). Finally, by spatial transcriptomics we found a correlation between decreased SOX2 levels with increasing days of lung injury in the proximal airway epithelium that mirrors this molecular change in eBPD AEC cultures. Conclusions Infants with eBPD have impaired AEC differentiation with an expansion of VESPAs and concomitant loss of cilia after hyperoxic injury, findings that mimic the effects of prematurity on injured airway cells in human patients. Loss of SOX2 that drives plasticity may be one mechanism for changes of airway epithelial cell fate. A VIM-high SOX2-low AEC endotype may identify airway disease in eBPD, with future experiments planned to explore the roles of these cells across time and disease state. This abstract is funded by: NIH
Eldredge et al. (Fri,) studied this question.