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

Matthew R Riccetti,Jason Woods,Jenna Green,John Snowball,Bonny Lami,Mehari Endale,Marion Waltamath,Sydney E Dautel,Shawn K Ahlfeld,Anne-Karina T Perl,Mereena George Ushakumary

doi:10.1172/jci.insight.152404

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% O2 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.

Highlights

Humans are born during the alveolar phase of lung development, when secondary crests form and newly forming alveolar walls extend into the airway lumen to increase surface area for gas exchange [1, 2]
Transcriptional profiling reveals dynamic changes in PDGFRA+ fibroblast proliferation and functional differentiation during and after neonatal hyperoxia exposure Neonatal pups were exposed to 90% O2 hyperoxia (O2) or room air (RA) starting at postnatal day 0 (PN0), and harvested during O2 exposure (PN4, PN7) or after recovery (PN10) (Figure 1 A)
While gene expression of Hopx and Pdgfa in PN7 magnetic-activated cell sorted (MACS)-isolated hyperoxia CD326+ epithelium transcripts were increased in RT-qPCR, suggesting that the changes are at a protein rather than RNA level (Supplemental Figure 1 F-Hf)

Summary

Introduction

Humans are born during the alveolar phase of lung development, when secondary crests form and newly forming alveolar walls extend into the airway lumen to increase surface area for gas exchange [1, 2]. Previous studies demonstrate that PDGFRA+ fibroblasts are reduced in BPD patient and BPD animal model lungs [38,39,40], and genetic ablation of PDGFRA+ fibroblasts during alveolarization cause persistent alveolar simplification [12]. It remains unknown what dynamic changes occur in the heterogenous mix of PDGFRA+ fibroblasts (matrix, myo-, and lipofibroblasts) during neonatal hyperoxia exposure and room air recovery, and how altered function of PDGFRA+ fibroblasts or changed contribution of PDGFRA+ fibroblast subpopulations result in impaired alveolar formation and alveolar epithelial differentiation

Methods

Results

Conclusion