Exosomes Derived from AT2R-Overexpressing BMSC Prevent Restenosis After Carotid Artery Injury by Attenuating the Injury-Induced Neointimal Hyperplasia

Figures

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  Fig. 1. The identification of ART2-contained exosomes. A Representative transmission electron microscopy images of our isolated exosomes. B Nanoparticle tracking analysis of the size of the isolated particles. C Immunoblotting analysis of the expression of exosomal markers and AT2R in our isolated exosomes

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  Fig. 2. The effects of AT2R-EXO on vascular endothelial cells and smooth muscle cells under hypoxia. A VECs growth was analyzed by cck-8 after hypoxia induced and treated with different concentrations of the indicated exosomes for 24 h and 48 h. Data are the means ± SD from five independent experiments. *P < 0.05 and **P < 0.01 vs. hypoxia group; #P < 0.05 vs. vehicle-EXO group. B VSMCs growth was analyzed by cck-8 after hypoxia induced and treated with different concentration of the indicated exosomes for 24 h and 48 h. Data are the means ± SD from five independent experiments. *P < 0.05 and **P < 0.01 vs. hypoxia group; #P < 0.05 vs. vehicle-EXO group. C Representative images showed the result of TUNEL assay to detect the effect of exosomes on hypoxia-induced cell apoptosis. The apoptosis ratio is shown in histogram. Data are the means ± SD from three independent experiments. **P < 0.01 vs. hypoxia group; ##P < 0.01 vs. vehicle-EXO group. D Representative microphotographs showing the migration of VECs after hypoxia induced and treated with exosomes for 24 h. The migrated cell numbers are shown in histogram. Data are the means ± SD from three independent experiments. **P < 0.01 vs.…

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  Fig. 3. The Mechanism of AT2R-EXO in the inhibition of neointimal hyperplasia. A Immunoblotting analysis of the expression of eNOS and iNOS in VECs after hypoxia induced and treated with exosomes for 48 h. Relative levels were quantitatively analyzed using ImageJ (NIH). Results are the mean ± SD from three independent experiments. **P < 0.01 vs. untreated control group; ##P < 0.01 vs. hypoxia group; §P < 0.01 vs. vehicle-EXO group. B The secretion of IL-1β was detected by ELISA assay in VECs after hypoxia induced and treated with exosomes for 24 h and 48 h. C The secretion of TNF-α was detected by ELISA assay in VECs after hypoxia induced and treated with exosomes for 24 h and 48 h. Data are the means ± SD from nine independent experiments. *P < 0.05 and **P < 0.01 vs. hypoxia group; #P < 0.05 vs. vehicle-EXO group. D NO levels in VECs. Data are shown as mean ± SD of three independent experiments performed in duplicate. ##P < 0.01, hypoxia + AT2R-EXO treatment vs. hypoxia treatment only; **P < 0.01, hypoxia + AT2R-EXO + L-NAME treatment vs. hypoxia + AT2R-EXO treatment; E immunoblotting analysis of the expression of the indicated genes in VSMCs after hypoxia induced and treated with…

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  Fig. 4. The effect of AT2R-EXO on NIH after carotid artery injury in vivo. A Representative intravital images showing the aggregation of exosomes in the rat carotid artery injury animal models after 2 weeks of treatment by intravenous injection. The luminescence is shown in histogram. Data are the means ± SD from five independent experiments. **P < 0.01 vs. vehicle-EXO group. B Representative images showing the H&E and Masson staining analysis of the formation of neointimal hyperplasia at 2 weeks and 4 weeks (n = 5/group, magnifications, × 40). C Quantitative analysis of the intimal area /media area ratio, D the neointimal area, E the media area, and F the collagen area of the indicated groups. Data are the means ± SD from five independent experiments. **P < 0.01 vs. sham group; ##P < 0.01 vs. saline group; §P < 0.05 vs. vehicle-EXO group

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  Fig. 5. The effects of AT2R-EXO on the function of injured carotid artery. A Representative images showing the zymography analysis of the activity of MMP2 and MMP9 in the injured carotid artery tissue samples after treatment with exosomes for 2 weeks and 4 weeks. B The relative activity is shown in histogram. Data are the means ± SD from five independent experiments. **P < 0.01 vs. sham group; ##P < 0.01 vs. saline group; §P < 0.05 vs. vehicle-EXO group. C The spectrum Doppler detecting of the blood flow of the injured carotid artery site. Data are the means ± SD from five independent experiments. ##P < 0.01 vs. saline group; P < 0.05 vs. vehicle-EXO group. D Immunoblotting analysis of the expression of the indicated genes in the injured carotid artery tissue samples after treatment with exosomes for 2 weeks and 4 weeks. E Relative expression levels were quantitatively analyzed using ImageJ (NIH). Data are the means ± SD from three independent experiments. **P < 0.01 vs. sham group; ##P < 0.01 vs. saline group; §P < 0.05 vs. vehicle-EXO group