Adipose stem cell-derived nanovesicles inhibit emphysema primarily via an FGF2-dependent pathway

Abstract

Cell therapy using stem cells has produced therapeutic benefits in animal models of COPD. Secretory mediators are proposed as one mechanism for stem cell effects because very few stem cells engraft after injection into recipient animals. Recently, nanovesicles that overcome the disadvantages of natural exosomes have been generated artificially from cells. We generated artificial nanovesicles from adipose-derived stem cells (ASCs) using sequential penetration through polycarbonate membranes. ASC-derived artificial nanovesicles displayed a 100 nm-sized spherical shape similar to ASC-derived natural exosomes and expressed both exosomal and stem cell markers. The proliferation rate of lung epithelial cells was increased in cells treated with ASC-derived artificial nanovesicles compared with cells treated with ASC-derived natural exosomes. The lower dose of ASC-derived artificial nanovesicles had similar regenerative capacity compared with a higher dose of ASCs and ASC-derived natural exosomes. In addition, FGF2 levels in the lungs of mice treated with ASC-derived artificial nanovesicles were increased. The uptake of ASC-derived artificial nanovesicles was inhibited by heparin, which is a competitive inhibitor of heparan sulfate proteoglycan that is associated with FGF2 signaling. Taken together, the data indicate that lower doses of ASC-derived artificial nanovesicles may have beneficial effects similar to higher doses of ASCs or ASC-derived natural exosomes in an animal model with emphysema, suggesting that artificial nanovesicles may have economic advantages that warrant future clinical studies.

Figure 1

Characterization of artificial nanovesicles generated from ASCs. (a) Transmission electron microscopy (TEM) image. (b) Nanoparticle size and number measured by nanoparticle tracking analysis. (c) Western blot analysis of artificial nanovesicles from ASCs using an exosomal marker. (d) Western blot analysis of artificial nanovesicles from ASCs using an ASC marker. NV: ASC-derived artificial nanovesicles, ASC: Adipose-derived stem cells. (e) ELISA measurements of the VEGF, FGF2 and HGF levels in ASC-derived natural exosomes and artificial nanovesicles.

Figure 2

Proliferative capacity of ASC-derived natural exosomes and artificial nanovesicles in alveolar epithelial cells. A CCK8 assay was used to evaluate the proliferation rate in the MLE12 cell line incubated with 0.1 or 1 μg ml⁻¹ of ASC-derived natural exosomes and artificial nanovesicles for 24 h. The data were significantly different compared with NoTx (untreated cells) (*P<0.05).

Figure 3

Regeneration effects of ASC-derived artificial nanovesicles on elastase-induced emphysema mice. C57BL/6 mice were intratracheally injected with elastase on day 0 and then intratracheally injected with ASC-derived artificial nanovesicles (Nano), natural exosomes (Exo) or ASCs on day 7. (a) Lung histology on day 14 after staining with hematoxylin and eosin. ((−) control: no elastase injection; Ela: 0.4 U of elastase injection; Ela+ASC: elastase+1 × 10⁵ ASCs; Ela+Nano (1/3 ×): elastase+1/3 × artificial nanovesicles; Ela+Nano (1 ×): elastase+1 × artificial nanovesicles; Ela+Exo (1 ×): elastase+1 × exosomes). (b) Morphometric analysis of the mean linear intercept (n=8–12). The data were significantly different (*P<0.05, **P=0.0089, ***P=0.0001) between the elastase group and the groups with injected ASCs or artificial nanovesicles.

Figure 4

ASCs-derived artificial nanovesicles enhance growth factor expression in recipient lungs. C57BL/6 mice were intratracheally injected with elastase on day 0 and then intratracheally or intravenously injected with 1/3 × of artificial nanovesicles (Nano), 1 × of exosome (Exo) or 1 × 10⁵ of ASCs on day 7. On day 14, mice were sacrificed, and the lungs were collected. ELISA were performed for mouse FGF2 (a), VEGF (b) and HGF (c) using lung protein lysate. (*P<0.05)

Figure 5

Fate of ASC-derived artificial nanovesicles after intratracheal injection into mice. Fluorescence images in the lung at 4, 24, 72 and 168 h after DiI-labeled artificial nanovesicles were intratracheally injected into mice with elastase-induced emphysema. Representative images are shown (n=2) ((−): No nanovesicle injection).

Figure 6

Internalization mechanisms of ASC-derived artificial nanovesicles. (a) Confocal microscopy of MLE12 cells incubated with/without a 10 μg ml⁻¹ dose of ASC-derived artificial nanovesicles from ASCs for 2 h on a 35 mm tissue culture dish. ((−) Ctrl: without artificial nanovesicles, (+) Ctrl: with nanovesicles, LY294002 (1 μM), cytochalasin D (0.2 μM), chloropromazine (1 μg ml⁻¹), or heparin (10 μg ml⁻¹): cells were treated with each drug for 30 min before artificial nanovesicle treatment). (b) Confocal microscopy of MLE12 cells incubated with a 10 μg ml⁻¹ dose of ASC-derived artificial nanovesicles from ASCs for 2 h on a 27 mm collagen coated tissue culture dish ((+) Ctrl: with nanovesicles, Heparin: heparin (10 μg ml⁻¹) treatment for 30 min before artificial nanovesicle treatment).