470 - Single Cell Transcriptomic Analysis of Pulmonary Adventitial Fibroblasts Reveals Unique Roles in Lung Development and in Hyperoxic Lung Injury

Poster Number: 470
Publication Number: 470.432

Nina E. Suresh, Stanford University School of Medicine, Redwood City, CA, United States; Fabio Zanini, UNSW Sydney, Sydney, New South Wales, Australia; Carsten Knutsen, Lucile Packard Children's Hospital Stanford, Palo Alto, CA, United States; Xibing Che, Stanford University School of Medicine, Palo Alto, CA, United States; Min Liu, Stanford University School of Medicine, Stanford, CA, United States; David N. Cornfield, Stanford University, Stanford, CA, United States; Cristina M. Alvira, Stanford University School of Medicine, Stanford, CA, United States

Background: The lung mesenchyme is comprised of many distinct cell types that are essential for development, including adventitial fibroblasts (AF). AF mediate pathologic pulmonary vascular remodeling in the response to chronic hypoxia, but their role in normal and aberrant postnatal lung development is not known.

Objective: We hypothesized that single cell RNA sequencing would identify distinct alterations in the AF transcriptome across development and in response to lung injury.

Design/Methods: Over 3000 single lung mesenchymal cells from mice at E18.5, P1, P7, and P21, and from P7 mice exposed to hyperoxia from birth, were sequenced at a depth of 1 million reads per cell. Mapped reads were quantified and analyzed using principal component analysis, determination of differentially expressed genes, and receptor-ligand profiling. AF and select genes were validated with multiplexed fluorescent in situ hybridization.

Results: Unsupervised clustering identified 2 transcriptionally distinct AF clusters; an early cluster comprised of cells from P1 and P7, and a late cluster of cells from P21. Detection of the highly discriminating gene Serpinf1, in situ, identified AF both within perivascular cuffs and lining conducting airways in close proximity to airway smooth muscle cells (ASM). Across postnatal development, AF pathways promoting extracellular matrix organization were upregulated, while proliferation, angiogenesis, and metabolic pathways were downregulated. Hyperoxia increased AF abundance more than 2-fold, and in situ quantification showed that hyperoxia increased AF abundance specifically around airways (1.72-fold, p< 0.001), but not in perivascular cuffs. Pathway analysis of genes dysregulated by hyperoxia found enrichment in cytoskeletal reorganization (p < 0.0001), including genes that regulate cell motility (e.g. Actr3, Myh9, and Twf1). Receptor-ligand profiling between AF and ASM showed significant increases in Jag1 (p < 0.0001) in AF and Notch2 in both AF (p < 0.0001) and ASM (p < 0.0001). ASM highly expressed Wnt5a in both normoxia and hyperoxia, but hyperoxia significantly increased AF expression of Fzd2 (p < 0.0001), a receptor for canonical and non-canonical Wnt signaling with key roles in proliferation and calcium mobilization.Conclusion(s): These data demonstrate dynamic AF diversity in lung development and highlight distinct interactions between AF and ASM, including putative enhancement of Notch and Wnt signaling in hyperoxia. We speculate that AF proliferation around airways in hyperoxia may contribute to pathologic airway remodeling in disorders such as bronchopulmonary dysplasia and asthma.