Identification of Ephrin type-B receptor 4 as a critical mediator of tissue fibrosis
Brian Wu, Starlee S. Lively, Shabana Vohra, Noah Fine, Chiara Pastrello, Anca Maglaviceanu, Osvaldo Espin-Garcia, Evan Pollock-Tahiri, Sayaka Nakamura, Paramvir Kaur, Keemo Delos Santos, Jason S. Rockel, Pratibha Potla, Himanshi Gupta, Poulami Datta, Laura Tang, Jacob Kwon, Akihiro Nakamura, Matthew B. Buechler, Rajiv Gandhi, Jiangping Wu, Boris Hinz, Igor Jurisica, Mohit Kapoor
Brian Wu, Starlee S. Lively, Shabana Vohra, Noah Fine, Chiara Pastrello, Anca Maglaviceanu, Osvaldo Espin-Garcia, Evan Pollock-Tahiri, Sayaka Nakamura, Paramvir Kaur, Keemo Delos Santos, Jason S. Rockel, Pratibha Potla, Himanshi Gupta, Poulami Datta, Laura Tang, Jacob Kwon, Akihiro Nakamura, Matthew B. Buechler, Rajiv Gandhi, Jiangping Wu, Boris Hinz, Igor Jurisica, Mohit Kapoor
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Research Article Cell biology Pulmonology

Identification of Ephrin type-B receptor 4 as a critical mediator of tissue fibrosis

Abstract

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 nonfunctional 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-Seq 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-Seq of mouse lungs treated with NVP-BHG712 identified transcriptomic changes of ECM genes in specific fibroblast subpopulations. RNA-Seq, 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.

Authors

Brian Wu, Starlee S. Lively, Shabana Vohra, Noah Fine, Chiara Pastrello, Anca Maglaviceanu, Osvaldo Espin-Garcia, Evan Pollock-Tahiri, Sayaka Nakamura, Paramvir Kaur, Keemo Delos Santos, Jason S. Rockel, Pratibha Potla, Himanshi Gupta, Poulami Datta, Laura Tang, Jacob Kwon, Akihiro Nakamura, Matthew B. Buechler, Rajiv Gandhi, Jiangping Wu, Boris Hinz, Igor Jurisica, Mohit Kapoor

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Figure 8

EphB4 signaling in PF.

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EphB4 signaling in PF. (A and B) Human IPF lung fibroblasts were treated...
(A and B) Human IPF lung fibroblasts were treated with TGF-β1 or PBS for 24 hours. Cells (total 16 samples; n = 4 patients/group) were then treated with either NVP-BHG712 (250 nM) (A) or Ephb4 siRNA (B), or their respective controls, for an additional 24 hours. Protein lysates were collected, and Western blotting was performed to detect ELN protein levels. ImageJ densitometry was performed and normalized to GAPDH (mean ± SD). *P < 0.05; **P < 0.01, as determined by paired 2-way ANOVA followed by Tukey’s post hoc test adjusted for multiple comparisons. (C) Western blotting of siRNA treated IPF cells to detect P-SMAD 2/3 and total SMAD 2/3 (n = 4/group). (D and E) Verhoeff Van Gieson staining was performed on lung sections from mice with Ephb4 conditional knockout or respective control mice to visualize ELN (n = 5/group) following bleomycin or PBS instillation (D), or mice with bleomycin or PBS instillation treated with EphB4 inhibitor NVP-BHG712 (10 mg/kg) or vehicle (n = 5/group) (E). ELN staining was semiquantitatively scored (scores 0–3). ****P < 0.0001 as determined by 2-way ANOVA followed by Tukey’s post hoc tests adjusted for multiple comparisons. (F) Schematic depicting the role of EphB4 signaling in PF.