Maladaptive functional changes in alveolar fibroblasts due to perinatal hyperoxia impair epithelial differentiation

Abstract

Infants born prematurely worldwide have up to a 50% chance of developing bronchopulmonary dysplasia (BPD), a clinical morbidity characterized by dysregulated lung alveolarization and microvascular development. It is known that PDGFR alpha–positive (PDGFRA + ) fibroblasts are critical for alveolarization and that PDGFRA + fibroblasts are reduced in BPD. A better understanding of fibroblast heterogeneity and functional activation status during pathogenesis is required to develop mesenchymal population–targeted therapies for BPD. In this study, we utilized a neonatal hyperoxia mouse model (90% O 2 postnatal days 0–7, PN0–PN7) and performed studies on sorted PDGFRA + cells during injury and room air recovery. After hyperoxia injury, PDGFRA + matrix and myofibroblasts decreased and PDGFRA + lipofibroblasts increased by transcriptional signature and population size. PDGFRA + matrix and myofibroblasts recovered during repair (PN10). After 7 days of in vivo hyperoxia, PDGFRA + sorted fibroblasts had reduced contractility in vitro, reflecting loss of myofibroblast commitment. Organoids made with PN7 PDGFRA + fibroblasts from hyperoxia in mice exhibited reduced alveolar type 1 cell differentiation, suggesting reduced alveolar niche-supporting PDGFRA + matrix fibroblast function. Pathway analysis predicted reduced WNT signaling in hyperoxia fibroblasts. In alveolar organoids from hyperoxia-exposed fibroblasts, WNT activation by CHIR increased the size and number of alveolar organoids and enhanced alveolar type 2 cell differentiation.

Matrix fibroblast function during alveolarization is dependent on GATA6

“…Primary PDGFRA + fibroblasts support in vitro lung organoid formation and can be used to interrogate epithelial mesenchymal interactions that promote organoid formation, growth and AT2/AT1 differentiation (42, 43, 52). PDGFRA + cells from PN7 control and GATA6 PDGFRAτι/τι lungs were cultured with normal CD326 sorted epithelial cells from adult mouse lungs.…”

“…In the adult lung the AXIN2 cell-lineage derived PDGFRα positive MANCs support AT1 and AT2 differentiation and function (15). In vitro, PDGFRa + FBs support formation of alveolar organoids when cultured with primary AECs (42, 43, 45, 52). In the GATA6 PDGFRAΔ/Δ lungs we observed an increase in AT2 cells.…”

“…Loss of beneficial matrix-producing fibroblasts affects alveolar repair in aged and IPF lungs, suggesting the importance of fibroblast subpopulations in maintaining the normal epithelial cell niche (42). In a recent study of perinatal hyperoxia exposure we demonstrated trans differentiation of myo to lipofibroblasts which was associatedwith impaired AT2 to AT1 transition (43). In the adult lung the AXIN2 cell-lineage derived PDGFRα positive MANCs support AT1 and AT2 differentiation and function (15).…”

“…Perinatal hyperoxia exposure decreased matrix and increased lipo fibroblast function in PDGFRA + fibroblasts (43). Lipofibroblast differentiation was assessed by immunofluorescence staining for adipose differentiation-related protein (ADFP).…”

“…Recent studies demonstrate that loss of matrix fibroblast function with advanced age and in idiopathic pulmonary fibrosis results in impaired alveolar regeneration and impaired AT1 differentiation and is associated with fragmented ECM (42). In a perinatal hyperoxia model the number of PDGFRa + fibroblasts was reduced and PDGFRa + fibroblasts switched from a myo to a lipo phenotype in association with reduction in matrix organizing function and lack of AT1 differentiation (43). The molecular regulator controlling matrix fibroblasts and their specific role in alveolarization are unknown.…”