184 - TGFβ regulates type 1 pulmonary epithelial cell plasticity and extracellular matrix gene transcription

Monday, May 1, 2023

9:30 AM – 11:30 AM ET

Poster Number: 184
Publication Number: 184.403

Danielle Callaway, Childrens Hospital of Philadelphia, Philadelphia, PA, United States; Ian J. Penkala, Toronto Animal Heath Partners, Toronto, ON, Canada; su zhou, Perelman School of Medicine at the University of Pennsylvania, Philadelphia,, PA, United States; Fabian Cardenas-Diaz, University of Pennsylvania, Philadelphia, PA, United States; Apoorva Babu, Perelman School of Medicine at the University of Pennsylvania, Bala Cynwyd, PA, United States; Michael Morley, Perelman School of Medicine at the University of Pennsylvania, Philadephia, PA, United States; Edward Morrisey, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States

Presenting Author(s)

Danielle Callaway, MD, PhD (she/her/hers)

T32 Research Fellow
Childrens Hospital of Philadelphia
Philadelphia, Pennsylvania, United States

Background: The TGFβ superfamily is a suspected contributor to bronchopulmonary dysplasia (BPD) although the mechanism is unclear. Prior work indicates that both TGFβ overexpression and attenuation in pulmonary epithelial cells result in arrested alveolarization in mice, a hallmark of BPD. In type 1 pulmonary alveolar (AT1) cells, TGFβ is enriched and involved in in vitro cellular spreading. Furthermore, recent evidence points to a role for AT1 cells as regulators of the extracellular (ECM) matrix, which, in other cell types, is controlled by TGFβ.

Objective: To determine the role of TGFβ in AT1 cells at homeostasis and after hyperoxic lung injury

Design/Methods: Mice lacking the main TGFβ receptor, Tgfbr2, in AT1 cells under a tamoxifen-inducible reporter were generated (Hopx^CreERT2:Tgfbr2:R26R^EYFP). AT1-AT2 cell reprogramming, histology, and other analyses were evaluated following injection at P0 and assessment at P5 and P42. Hyperoxia ( >95% O2) was performed at P0-P5 with analysis at P5 and P42 including measures of mean linear intercept (MLI) and mean septal thickness (MST). Bulk RNA sequencing was done on sorted AT1 cells at P5 with qRT-PCR at P5 and P42. In vitro experiments of sorted AT1 cells were performed on fibronectin-coated plates with addition of TGFβ inhibitor.

Results: Loss of Tgfbr2 resulted in increased AT1 to AT2 cell reprogramming with no difference in proliferation, apoptosis, or total AT2 numbers. On histology, loss of Tgfbr2 led to increased MLI and MST. Neonatal hyperoxia yielded a significant increase in lineage-traced AT2 cells and MLI over normoxia in both groups. MST after hyperoxia was elevated in Tgfrb2^flfl lungs at P5 compared to the control and normoxia but returned to baseline at P42. Bulk RNA sequencing and qRT-PCR showed a significant decrease in several ECM-related genes including glycoproteins, AT1-specific collagen IV subtypes, and ECM regulators with loss of Tgfbr2. Chemical inhibition of TGFβ in vitro also attenuated ECM gene transcription and reduced AT1 cell spreading.

Conclusion(s): Loss of Tgfbr2 resulted in increased AT1 to AT2 cell reprogramming as well as perturbations in the developing pulmonary architecture that were exacerbated with exposure to hyperoxia, indicating that hyperoxia-induced changes in AT1 cells are not mediated by TGFβ. RNA sequencing and qRT-PCR indicated that loss of Tgfbr2 in vivo and in vitro resulted in decreased transcription of several genes of the ECM involved in AT1 basement membrane structure and remodeling. Together, this points to a unique role of TGFβ to regulate ECM-related gene transcription in AT1 cells to impact the developing lung.