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 contribute to BPD pathology. Pediatric human donors aged 1-3 years were classified based on history of prematurity and histopathology consistent with "healed" BPD (hBPD, n=3) and "established" BPD (eBPD, n=3) compared with respective full term born (n=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 was performed using an in-house tool and/or EnrichR. Proteomics detected 5,746 unique proteins, 186 upregulated and 534 downregulated, in eBPD vs. Control with fewer proteins differentially expressed in hBPD as compared to 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 vs. Control. Finally, several extracellular matrix components mapping to angiogenesis signaling pathways were altered in eBPD. Despite clear parsing by protein expression, comparative MxIF analysis confirms phenotypic variability in BPD. This work provides a 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.