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. 2019 Feb 13;23(1):44.
doi: 10.1186/s13054-019-2339-3.

Exosomes from endothelial progenitor cells improve outcomes of the lipopolysaccharide-induced acute lung injury

Yue Zhou  1   2 , Pengfei Li  1 , Andrew J Goodwin  3 , James A Cook  4 , Perry V Halushka  5   6 , Eugene Chang  7 , Basilia Zingarelli  8 , Hongkuan Fan  9   10
Affiliations

Exosomes from endothelial progenitor cells improve outcomes of the lipopolysaccharide-induced acute lung injury

Yue Zhou et al. Crit Care. .

Abstract

Background: The acute respiratory distress syndrome (ARDS) is characterized by disruption of the alveolar-capillary barrier resulting in accumulation of proteinaceous edema and increased inflammatory cells in the alveolar space. We previously found that endothelial progenitor cell (EPC) exosomes prevent endothelial dysfunction and lung injury in sepsis in part due to their encapsulation of miRNA-126. However, the effects of EPC exosomes in acute lung injury (ALI) remain unknown.

Methods: To determine if EPC exosomes would have beneficial effects in ALI, intratracheal administration of lipopolysaccharide (LPS) was used to induce ALI in mice. Lung permeability, inflammation, and the role of miRNA-126 in the alveolar-epithelial barrier function were examined.

Results: The intratracheal administration of EPC exosomes reduced lung injury following LPS-induced ALI at 24 and 48 h. Compared to placebo, intratracheal administration of EPC exosomes significantly reduced the cell number, protein concentration, and cytokines/chemokines in the bronchoalveolar lavage fluid (BALF), indicating a reduction in permeability and inflammation. Further, EPC exosomes reduced myeloperoxidase (MPO) activity, lung injury score, and pulmonary edema, demonstrating protection against lung injury. Murine fibroblast (NIH3T3) exosomes, which do not contain abundant miRNA-126, did not provide these beneficial effects. In human small airway epithelial cells (SAECs), we found that overexpression of miRNA-126-3p can target phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2), while overexpression of miRNA-126-5p inhibits the inflammatory alarmin HMGB1 and permeability factor VEGFα. Interestingly, both miR-126-3p and 5p increase the expression of tight junction proteins suggesting a potential mechanism by which miRNA-126 may mitigate LPS-induced lung injury.

Conclusions: Our data demonstrated that human EPC exosomes are beneficial in LPS-induced ALI mice, in part through the delivery of miRNA-126 into the injured alveolus.

Keywords: Acute lung injury; Exosomes; Tight junction protein; miR-126.

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Conflict of interest statement

Ethics approval and consent to participate

This study was approved by the Institutional Review Board for Human Research at the Medical University of South Carolina. Informed consent was obtained from the mother for all cord blood collections.

Investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the NIH and were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina.

Consent for publication

All listed authors consent to the submission and all data are used with the consent of the person generating the data.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Characterization of exosomes derived from human EPCs and NIH3T3 fibroblasts. a, b Exosome size distribution and concentration were measured by nanoparticle tracking analysis (NTA) with ZetaView. c Detection of exosome markers including CD9, CD63, and CD81 in EPC exosomes and NIH3T3 exosomes by western blot
Fig. 2
Fig. 2
Therapeutic effects of EPC exosomes on bronchoalveolar lavage fluid (BALF) cell counts and protein concentration in LPS-induced acute lung injury. Mice were subjected to acute lung injury (ALI) by LPS instillation and treated with either EPC exosomes or NIH3T3 exosomes or PBS at 4 h after injury. BALF total cell counts (a) and BALF protein concentration (b) were determined at 24 h after injury. *p < 0.05 compared with the PBS group; #p < 0.05 compared with the LPS group. n = 6–7 mice per group. Results are represented as mean ± SE
Fig. 3
Fig. 3
Therapeutic effects of EPC exosomes on BALF cytokines and chemokines in LPS-induced acute lung injury. BALF cytokines TNF-α (a), IL-6 (b), IL-1β (c), and IFNγ (d) and chemokines MIP-1 (e), MIP-2 (f), MIG (g), and IP-10 (h) were determined by mouse cytokine and chemokine array at 24 h after LPS installation. *p < 0.05 compared with the PBS group; #p < 0.05 compared with the LPS group. ##p < 0.05 compared with the LPS+EPC exosomes group. n = 4 mice per group. Results are represented as mean ± SE
Fig. 4
Fig. 4
Therapeutic effects of EPC exosomes on alveolar edema in LPS-induced acute lung injury. Lung water content was calculated as the ratio of wet weight to dry weight (a), and vascular leakage in lung tissue was measured via injecting Evans blue dye at 24 h after LPS instillation (b). *p < 0.05 compared with the PBS group; #p < 0.05 compared with the LPS group. n = 4–6 mice per group. Results are represented as mean ± SE
Fig. 5
Fig. 5
Therapeutic effects of EPC exosomes on LPS-induced acute lung injury by histological analysis and myeloperoxidase (MPO) activity. Lung (a) sections were stained with H&E and examined histologically at 48 h after LPS instillation. The representative sections are shown at × 400 original magnification, and scale bars are 20 mm. The PBS group showed normal lung tissue including thin alveolar walls and few alveolar macrophages. Yellow arrows indicate neutrophils in the alveolar space, green arrows indicate neutrophils in the interstitial space, blue arrows indicates hyaline membranes, and black arrows indicate thickening of the alveolar walls. Lung injury scores (b) were assessed. *p < 0.05 compared with the PBS group; #p < 0.05 compared with the LPS group. n = 4 mice per group. Results are represented as mean ± SE. MPO activity (c) in the lung tissue were measured at 24 h after LPS instillation. *p < 0.05 compared with the PBS group; #p < 0.05 compared with the LPS group. n = 3–6 mice per group. Results are represented as mean ± SE
Fig. 6
Fig. 6
miRNA-126-3p and miRNA-126-5p target PIK3R2, HMGB1, and VEGFα and regulate tight junction protein expression levels in human small airway epithelial cells (SAECs). To verify RNA sequencing data (Tables 1 and 2), SAECs were transfected with either miR-126-3p mimic, miR-126-5p mimic, or control miRNA for 48 h. PIK3R2, the target of miR-126-3p (a), epithelial tight junction claudin1 (b), claudin4 (c), and occludin (d) mRNA levels were measured by RT-qPCR. HMGB1 (e) and VEGFα (f), the targets of miR-126-5p; epithelial tight junction claudin1 (g); and claudin4 (h) mRNA levels were determined by RT-qPCR. GAPDH served as an internal control. *p < 0.05 compared with the control group; the experiments were performed at least three independent times. Results are represented as mean ± SE
Fig. 7
Fig. 7
miRNA-126-3p and miRNA-126-5p regulate the expression levels of cell tight junction proteins in LPS-stimulated SAECs. SAECs were transfected with either miR-126-3p mimic, miR-126-5p mimic, or control miRNA for 48 h and stimulated with LPS (100 ng/ml) for 24 h. mRNA levels of claudin1 (a, d), claudin4 (b, e), and occludin (c) in SAECs were measured by RT-qPCR. GAPDH served as an internal control. *p < 0.05 compared with the control group; #p < 0.05 compared with the LPS group. The experiments were performed at least three independent times. Results are represented as mean ± SE
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