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 8

Treatment with IFITM3-containing exosomes improves EC functions but inhibits fibroblast activation.

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Treatment with IFITM3-containing exosomes improves EC functions but inhi...
(A) Schematic diagram illustrating generation of ECs expressing exogenous IFITM3 fused to a HiBiT tag under doxycycline-inducible control. FOXF1 siRNA was used to suppress endogenous IFITM3 expression. Doxycycline (Dox) treatment induced HiBiT-IFITM3 expression, and conditioned media were collected for exosome isolation. Isolated exosomes were subsequently applied to endothelial cells (HPAECs) or fibroblasts (CCD-19Lu). (B) Size distribution of isolated exosomes measured by dynamic light scattering, showing similar particle profiles across groups (Exo Control, n = 3; IFITM3lo exosomes, n = 8; IFITM3hi exosomes, n = 5 independent exosome preparations). (C) Nano-Glo HiBiT extracellular detection assay shows increased HiBiT-IFITM3 signal in exosomes from Dox-induced cells (IFITM3hi exosomes; n = 4) compared with non-induced controls (IFITM3lo exosomes; n = 3). (D) Left: Endothelial tube formation assay in HPAECs shows impaired angiogenesis following treatment with IFITM3lo exosomes, while IFITM3hi exosomes restore tube-forming ability. Scale bars: 100 μm. Right: Quantification of vessel length (n = 12). Boxes indicate the 25th to 75th percentiles, center lines indicate the medians, and whiskers indicate the minimum and maximum values. (E) Cell proliferation assay using CCD-19Lu fibroblasts shows that IFITM3hi exosomes suppress TGF-β1–induced fibroblast proliferation at 24 and 48 hours, compared with IFITM3lo exosomes (n = 3). (F) qRT-PCR analysis demonstrates that treatment with IFITM3hi exosomes suppresses TGF-β1–induced expression of the profibrotic genes FN1, VIM, and COL3A1 in CCD-19Lu fibroblasts (control, n = 5; Exo Control + TGF-β1, n = 3; IFITM3lo exosomes + TGF-β1, n = 3; IFITM3hi exosomes + TGF-β1, n = 4 for each gene). Data are presented as mean ± SD. One-way ANOVA with Fisher’s LSD test was used for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.