Identification of Ephrin type-B receptor 4 (EphB4) as a critical mediator of tissue fibrosis [RNA-seq]

Pulmonary fibrosis (PF) is a pathology associated with interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF). Fibrosis promotes continual secretion of extracellular matrix (ECM), producing non-functional scar tissue and causing organ failure. This study investigated the tyrosine kinase receptor Ephrin type-B receptor 4 (EphB4) as a mediator of PF. To this end, we generated mice with conditional Col1a2-driven deletion of Ephb4 and used a preclinical mouse model of PF, total and single nuclei RNA (snRNA) sequencing, NanoString, previously published single cell data, computational analysis, and functional assays of mouse and human healthy control and IPF lung fibroblasts. Col1a2-CreERT-driven Ephb4 deletion, or EphB4 inhibition via NVP-BHG712, markedly protected against bleomycin-induced PF. Total RNA-sequencing of fibroblasts isolated from Ephb4-deficient fibrotic mouse lungs exhibited reduced expression of ECM, ER Cargo, and protein trafficking-related genes. NVP-BHG712 reduced expression of these identified genes in mouse lung fibroblasts under fibrotic conditions in vitro. SnRNA sequencing of mouse lungs treated with NVP-BHG712 identified transcriptomic changes of ECM genes in specific fibroblast subpopulations. RNA sequencing, computational, and functional assays using mouse and human IPF fibroblasts identified elastin as a key mediator involved in EphB4 signaling. Combined, our data show that EphB4 is a crucial mediator of PF. Primary human IPF and control donor lung fibroblasts (HLF; from control donor lungs) were treated for 24 h with either 250 nM of the Ephb4 inhibitor NVP-BHG712 or 0.1% DMSO (as with control donors). Total RNA was then extracted, and sequencing libraries were prepared using Illumina's TruSEQ stranded total RNA library preparation kit and sequenced on Illumina's NextSeq550. Differential expression analysis and subsequent pathway analyses were performed. Following are the groupings for 16 samples: 0.1% DMSO treatment - 4 IPF samples - IPF1C, IPF2C, IPF3C, IPF4C 250 nM NVP-BHG712 treatment - 4 IPF samples - IPF1I, IPF2I, IPF3I, IPF4I 0.1% DMSO treatment - 4 HLF samples - HLF1_Vehicle, HLF2_Vehicle, HLF3_Vehicle, HLF5_Vehicle 250 nM NVP-BHG712 treatment - 4 HLF samples - HLF1_NVP-BHG712, HLF2_NVP-BHG712, HLF3_NVP-BHG712, HLF5_NVP-BHG712