Transplantation of endothelial progenitor cells attenuates pulmonary fibrosis via IFITM3-containing exosomes
Ying-Wei Lan, Jonathan Do, Wen Gao, Zicheng Deng, Xiaomei Xia, Enhong Li, Jillian Kash, Gautam Verma, Angara Sureshbabu, Kenneth S. Knox, Ross M. Bremner, Nicholas E. Banovich, Vladimir V. Kalinichenko, Tanya V. Kalin
Ying-Wei Lan, Jonathan Do, Wen Gao, Zicheng Deng, Xiaomei Xia, Enhong Li, Jillian Kash, Gautam Verma, Angara Sureshbabu, Kenneth S. Knox, Ross M. Bremner, Nicholas E. Banovich, Vladimir V. Kalinichenko, Tanya V. Kalin
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Research Article Pulmonology Vascular biology

Transplantation of endothelial progenitor cells attenuates pulmonary fibrosis via IFITM3-containing exosomes

Abstract

Endothelial progenitor cells (EPCs) are critical for vascular regeneration after injury. However, their role in pulmonary fibrosis (PF) remains unclear. Here, we identified a previously unrecognized population of lung capillary EPCs coexpressing c-KIT and FOXF1 (capEPCs) in adult human and mouse lungs. capEPCs were significantly reduced in lungs from patients with PF and in bleomycin-injured mice, implicating loss of this regenerative endothelial population in disease pathogenesis. Transplantation of donor capEPCs attenuated experimental PF, improved survival, reduced collagen deposition, and restored lung function. Donor capEPCs engrafted into the lung microcirculation of bleomycin-injured mice. Single-cell RNA sequencing identified interferon-induced transmembrane protein 3 as a highly enriched transcript in capEPCs. Conditioned media from IFITM3-overexpressing EPCs or recombinant IFITM3 enhanced endothelial proliferation and angiogenesis while suppressing TGF-β1–induced fibroblast activation in vitro. IFITM3 was found in capEPC-derived exosomes. Treatment with IFITM3-containing exosomes recapitulated the therapeutic effects of capEPC transplantation by improving endothelial function, inhibiting fibroblast activation, increasing animal survival, reducing lung fibrosis, and restoring lung function. Together these findings identified capEPC deficiency as a feature of PF and demonstrated that IFITM3-containing exosomes promoted vascular repair. Restoration of capEPC regenerative function or delivery of IFITM3-enriched exosomes may represent a promising therapeutic strategy for human PF.

Authors

Ying-Wei Lan, Jonathan Do, Wen Gao, Zicheng Deng, Xiaomei Xia, Enhong Li, Jillian Kash, Gautam Verma, Angara Sureshbabu, Kenneth S. Knox, Ross M. Bremner, Nicholas E. Banovich, Vladimir V. Kalinichenko, Tanya V. Kalin

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Figure 2

Mouse lung capEPCs localize within CAP1 and are decreased in lungs of bleomycin-treated mice.

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Mouse lung capEPCs localize within CAP1 and are decreased in lungs of bl...
(A) UMAP visualization of lung EC subclusters from control and bleomycin-treated mouse lungs (GSE264151 and GSE264162), highlighting capEPCs (FOXF1+KIT+) in red. (B) Quantification of FOXF1+KIT+ capEPCs across endothelial subtypes in control and bleomycin-treated lungs. Data are shown as the percentage of FOXF1+KIT+ cells among total ECs within each subtype, calculated separately for each mouse lung. Each dot represents an individual mouse. Horizontal lines indicate the mean. Statistical significance was assessed using the Wilcoxon’s rank-sum test. (C) Reduced abundance of capEPCs following bleomycin injury. Left: UMAP visualization of FOXF1+KIT+ capEPCs (red dots) in control and bleomycin-treated lungs. Dashed outlines indicate the capEPC-enriched region. Right: Percentage of capEPCs among total ECs, calculated per mouse lung (control, n = 4; bleomycin, n = 3). Statistical significance was assessed using a linear model with dataset covariate adjustment. (D) Pathway enrichment analysis of capEPCs under fibrotic conditions, showing signaling pathways up- or downregulated following bleomycin treatment. (E) Top: Immunofluorescent staining of lung sections from control (n = 6) and bleomycin-treated mice (n = 8) for CD31 (green), FOXF1 (white), KIT (red), and DAPI (blue). Bottom: Quantification of CD31+, FOXF1+, and KIT+ cells expressed as a percentage of total DAPI+ nuclei, and FOXF1+KIT+ capEPCs expressed as a percentage of total CD31+ ECs, calculated per lung. Representative images from independent lungs are shown. Scale bars: 100 μm. (F) Flow cytometry analysis using Foxf1-GFP reporter mice (experimental design shown above) shows decreased percentage of capEPCs (CD117+Foxf1+ ECs) in bleomycin-treated lungs (n = 5 mice per group). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ****P < 0.0001. For E and F, unpaired 2-tailed t tests were used.