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
View: Text | PDF
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

×

Figure 2

Ephb4 CKO mice exhibit a protective phenotype against the development of pulmonary fibrosis.

Options: View larger image (or click on image) Download as PowerPoint
Ephb4 CKO mice exhibit a protective phenotype against the development o...
Ephb4f/f mice were crossed with Col1a2-CreERT mice to generate a line with conditional Col1a2-driven deletion of Ephb4 (Ephb4 CKO). (A) Genotyping was performed to confirm the presence of homozygous floxed Ephb4 alleles and Cre. (B) Mice treated with 4-hydroxytamoxifen to induce the CKO were sacrificed and primary lung fibroblasts were cultured to first passage. RNA was collected and Ephb4 was quantified by qPCR (n = 6–7). Protein lysates were collected from cell cultures and EphB4 protein was quantified by Western blotting (n = 5). (C) A mouse model for inducing fibrosis in Ephb4-CKO mice was performed using intratracheal bleomycin challenge or PBS (control) following 4-hydroxytamoxifen-induced deletion. (D) Mouse lungs were collected at end point (9 weeks of age) and stained using Gomori’s One Step collagen stain (blue). Representative histology images from 2 separate animals/group are shown. Fibrosis was quantified either by modified Ashcroft scoring or with ImageJ total lung collagen content quantification (n = 8 mice/group). (E) αSMA immunostaining was performed in lung tissue sections to assess fibrotic activity in Ephb4-CKO and control mice with and without bleomycin challenge (n = 5). Semiquantitative scoring (scores 0–3) of the staining intensity was assigned to each mouse lung. Scale bar: 400 µM; original magnification, x 10. (F) Primary lung fibroblasts were isolated from Ephb4 Col1a2-CreERT mice treated with 4-hydroxytamoxifen or corn oil and then bleomycin. RNA was isolated from fibroblasts, and qPCR was performed to detect Col1a1 and Acta2 (n = 4). For qPCR (B and F), 2–ΔCT values were used to represent relative expression of the gene of interest normalized to Gapdh. For all graphs, data are expressed as mean ± SD where differences (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) were identified using either 2-tailed Student’s t test (B) or 2-way ANOVA with Tukey’s post hoc tests (D–F).