214 - New Insights into Bronchopulmonary Dysplasia using Region Specific Proteomics

Publication Number: 214.5962

Andrew M. Dylag, University of Rochester, Rochester, NY, United States; Gail H.. Deutsch, University of Washington School of Medicine, Seattle, WA 98105, WA, United States; Jeffrey M. Purkerson, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States; Ravi S. Misra, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States; Matthew Jehrio, University of Rochester School of Medicine and Dentistry, rochester, NY, United States; Heidie L. Huyck, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States; Thomas Mariani, University of Rochester School of Medicine and Dentistry, Rochester, NY, United States; Geremy Clair, Pacific Northwest National Laboratory, Richland, WA, United States; Gloria S. Pryhuber, Golisano Children's Hospital at The University of Rochester Medical Center, Rochester, NY, United States

Background: Bronchopulmonary Dysplasia (BPD) is a disease of prematurity related to an arrest of normal lung development characterized by both airway and parenchymal disease.

Objective: To apply laser captured microscopy (LCM) to identify region-specific proteins and pathways differentially expressed in post-hospitalization BPD and to determine the precise location of observed changes in the proteome using highly multiplexed immunofluorescence (MxIF).

Design/Methods: Pediatric human donors aged 1.3 to 3 years were classified based on history of prematurity and histopathologic severity of BPD (N=3 “Recovered” BPD [rBPD] and N=3 “Established” BPD [eBPD]) with respective full term (N=6) age-matched controls. Four distinct tissue regions were isolated by LCM: large airway (AW), membranous bronchiole (MB), respiratory bronchiole (RB) and alveolar (ALV). Proteins were identified by >2 unique peptide fragments by tandem mass spectrometry. MxIF microscopy (30-40 target antibodies) was performed on lung tissue microarrays enriched for the 4 regions. Protein abundances and MxIF cell frequencies were compared using ANOVA, paired t-test, and presence-absence glm-binomial tests. Cell segmentation, cell typing, protein localization, quantification and visualization were performed using DeepCell, scipy, scanpy, squidpy, and Napari.

Results: Proteomics detected 3261 unique proteins, 2149 differentially quantifiable in the alveolar region and 2396 in the combined airway regions. Several key proteins were enriched in the ALV region (SCEL, AGER, ABCA3) and AW region (COL1A2, SCGB1A1, DES) validating the LCM-mediated regional enrichment. Tight junction proteins were increased in ALV while desmosomes were predominant in AW. Infant lung with eBPD showed upregulated apoptosis and collagen (COL14A1), with downregulated neutrophil-related pathways in the alveolar region. Large airway (AW) in eBPD showed upregulated lysosomal enzymes HEXA and HEXB, and downregulated ICAM1. Bronchioles demonstrated increased annexins (ANXA1, ANXA4, ANXA5) and decreased collagens (COL1A1, COL6A6), neutrophil (S100A8), and complement (C4B) proteins. Comparative MxIF analysis (Figure 1) demonstrated heterogeneity in BPD but confirmed major findings.

Conclusion(s): Region-specific, protein, and functional pathway dysregulation was detected in eBPD, with fewer differentially expressed proteins in rBPD. Region- and neighborhood-specific multi-modal human tissue analysis provides cell and extracellular matrix insights into BPD pathogenic mechanisms that could inform prevention and treatment.

Region Specific Multiplexed Immunofluorescence Microscopy in BPD.
Representative Airway (top) and Alveolar (bottom) sections for the Control (left), rBPD (middle), and eBPD (right) lung sections. Selected proteins show localization to lung regions, and differential expression based on BPD classification. Scale Bar = 500µm.