Maladaptive functional changes in alveolar fibroblasts due to perinatal hyperoxia impair epithelial differentiation
Matthew R. Riccetti, Mereena George Ushakumary, Marion Waltamath, Jenna Green, John Snowball, Sydney E. Dautel, Mehari Endale, Bonny Lami, Jason Woods, Shawn K. Ahlfeld, Anne-Karina T. Perl
Matthew R. Riccetti, Mereena George Ushakumary, Marion Waltamath, Jenna Green, John Snowball, Sydney E. Dautel, Mehari Endale, Bonny Lami, Jason Woods, Shawn K. Ahlfeld, Anne-Karina T. Perl
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Research Article Development Pulmonology

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% 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.

Authors

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

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Figure 4

PDGFRA+ fibroblasts exposed to hyperoxia fail to support epithelial differentiation in organoid culture.

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PDGFRA+ fibroblasts exposed to hyperoxia fail to support epithelial diff...
(A) Organoids were made by coculturing fibroblasts from PN7 RA or O2-exposed (PN0–PN7, 90% O2) with adult RA epithelium, cultured for 3 weeks, and bright-field images were taken on fixed samples. Scale bar =1 mm. (B) Alveolar organoids were quantified on Nikon Elements by diameter, revealing reduced small colonies in hyperoxia. n = 8–10, control (RA) and experimental (O2) organoid transwells (replicates) were used. A 2-tailed Student’s t test was used, ****P < 0.0001. Error bars ± SD. (C, E, G, and I) Immunofluorescence on paraffin-embedded sections for markers of epithelial differentiation. Scale bars = 100 μm. (D, F, H, and J) Organoids were quantified using Nikon elements. Nuclear antibody stain HOPX was quantified by taking total antibody over DAPI, and cytoplasmic and membrane stains (SPC, AGER, CCSP) were quantified as % area over DAPI area. n = 8–10, control (RA) and experimental (O2) organoid transwells (replicates) were used. A 2-tailed Student’s t test was used, *P < 0.05; ****P < 0.0001. Data displayed as box-and-whisker plot. The box plots depict the minimum and maximum values (whiskers), the upper and lower quartiles, and the median. The length of the box represents the interquartile range. (K) Collagen contraction assay on PN7 PDGFRA+ RA and O2 fibroblasts was performed, then imaged after 3 days in culture. (L) Quantification of collagen assay average diameter in mm. Scale bar = 3.61 mm. Average diameter calculated, n = 5, RA and O2 collagen pellets were used. A 2-tailed Student’s t test was used, **P < 0.01. Error bars show mean ± SD. (M) Schematic showing how PN7 O2 PDGFRA+ fibroblasts fail to contract or support alveolar organoids in vitro.