New Insights into the Natural History of Bronchopulmonary Dysplasia from Proteomics and Multiplexed Immunohistochemistry

Bronchopulmonary dysplasia (BPD) is a disease of prematurity related to the arrest of normal lung development. The objective of this study was to better understand how proteome modulation and cell-type shifts are noted in BPD pathology. Pediatric human donors aged 1-3 yr were classified based on history of prematurity and histopathology consistent with "healed" BPD (hBPD, = 3) and "established" BPD (eBPD, = 3) compared with respective full-term born ( = 6) age-matched term controls. Proteins were quantified by tandem mass spectroscopy with selected Western blot validations. Multiplexed immunofluorescence (MxIF) microscopy was performed on lung sections to enumerate cell types. Protein abundances and MxIF cell frequencies were compared among groups using ANOVA. Cell type and ontology enrichment were performed using an in-house tool and/or EnrichR. Proteomics detected 5,746 unique proteins, 186 upregulated and 534 downregulated, in eBPD versus control with fewer proteins differentially abundant in hBPD as compared with age-matched term controls. Cell-type enrichment suggested a loss of alveolar type I, alveolar type II, endothelial/capillary, and lymphatics, and an increase in smooth muscle and fibroblasts consistent with MxIF. Histochemistry and Western analysis also supported predictions of upregulated ferroptosis in eBPD versus control. Finally, several extracellular matrix components mapping to angiogenesis signaling pathways were altered in eBPD. Despite clear parsing by protein abundance, comparative MxIF analysis confirms phenotypic variability in BPD. This work provides the first demonstration of tandem mass spectrometry and multiplexed molecular analysis of human lung tissue for critical elucidation of BPD trajectory-defining factors into early childhood. We provide new insights into the natural history of bronchopulmonary dysplasia in donor human lungs after the neonatal intensive care unit hospitalization. This study provides new insights into how the proteome and histopathology of BPD changes in early childhood, uncovering novel pathways for future study.