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 3

Ephb4-CKO fibrotic mouse lung fibroblasts exhibit unique transcriptomic profiles.

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Ephb4-CKO fibrotic mouse lung fibroblasts exhibit unique transcriptomic...
(A) Ephb4f/f;Col1a2-CreERT mice were treated with 4-hydroxytamoxifen (1 mg/day for 5 days) or corn oil control at 4 weeks of age and were then intratracheally instilled with PBS or bleomycin (1.2 U/kg) at 6 weeks of age. Mice were sacrificed 14 days after bleomycin or PBS challenge, and lung fibroblasts were isolated and cultured (n = 4 mice per group). Fibroblasts were enriched with a single passage followed by RNA extraction for total RNA-Seq. (B) Pairwise comparisons were made between bleomycin and PBS-treated Ephb4 control mice, or between Ephb4-CKO and control mice under bleomycin condition (n = 4 mice/group). Differentially expressed genes (DEGs) were determined using an FDR cutoff of q < 0.05 (red). In the bleomycin versus PBS comparison, 257 DEGs were identified in fibroblasts, including 155 upregulated and 102 downregulated DEGs. In the Ephb4-CKO versus control comparison, 317 DEGs were identified in fibroblasts, including 103 upregulated and 214 downregulated DEGs. (C) DEGs from both comparisons were visualized using heatmaps. (D) Lists of both upregulated and downregulated DEGs in the RNA-Seq dataset of Ephb4 CKO (n = 4) versus Ephb4 control mice (n = 4) treated with bleomycin were analyzed separately using pathDIP to identify subsets of genes enriched for unique pathways in the Reactome database. Gene-pathway associations were visualized in NAViGaTOR v.3.0.16. (E) Unique gene-pathway enrichments from the downregulated gene list visualized by dot plot, with significance determined using the Benjamini and Hochberg method (with cutoff of q < 0.01). (F) The expression of downregulated DEGs associated with ECM organization, protein transport, and ER cargo concentration visualized by Wideplot.