Oxygen Therapy as a Driver of Endothelial-to-Mesenchymal Transition via p53-Signaling in Lung Fibrosis

The effect of oxygen therapy in pulmonary fibrosis is understudied and it remains unknown whether even moderate oxygen concentrations (MOC) contribute to the remodelling of the gas-exchange area. We investigate the effect of MOC on endothelial cell (EC) injury and endothelial-to-mesenchymal transition (EndMt) with a focus on p53 signalling. To this end, we exposed ECs to MOC (FiO2=0.4) or room air (RA, FiO2=0.21) for 8hrs and characterized markers of EC injury and EndMT, p53 signalling, cell cycle regulation, and EC function, complemented by secretome analysis. Chip-based studies (OrganoPlate® Graft, Mimetas) allow us to investigate the impact of oxygen-induced EC injury on hPSC-derived alveolar organoids. MOC exposure in HUVECs and HPMECs resulted in a decrease of EC (CD31, VE-Cad, vWF) and an increase in mesenchymal markers (aSMA, FSP-1), accompanied by an increase in IL6 release (HPMEC-1.83, HUVEC-2.02 (fold change (FC) each)) as well as a decrease in cell viability (Wst-1) (HPMECs-0.38, HUVECs-0.55, FC each) and tube formation. In contrast, Eahy926, characterized by endogenous p53 upregulation, showed an ameliorated MOC response. Markers of cell cycle regulation (CDK1, CCNA2) were downregulated in ECs after MOC exposure, supporting the relevance of MOC-induced effects on p53 signaling. Ongoing studies establish the engraftment of alveolar organoids onto a chip-based, perfusable vascular bed derived from MOC or RA exposed ECs. Exposure to MOC efficiently induced EndMT, and reduced EC functionality associated with altered p53 signalling. Next to on-chip experiments that determine the effects on the alveolar epithelium, p53 restoration by Nutlins will establish a causal relation.