Engineered Mesenchymal Stem Cell–Derived Extracellular Vesicles Reverse Endothelial–Mesenchymal Transition in Atherosclerosis

FIGURE 1. Characteristics of EVs–SIRT1–VEGF. (A) Schematic representation of the collection and isolation of SIRT1 overexpressed extracellular vesicles coupled with Chol‐VEGF aptamers. (B–D) The mRNA and protein level of SIRT1 in BMSCs, BMSCs + Ad‐vector or BMSCs + Ad‐SIRT1 were detected by qRT‐PCR and Western Blot. (E) TEM images of EVs, EVs‐SIRT1 and EVs‐SIRT1‐VEGF. Scale bar, 100 nm. (F) Size distribution of EVs, EVs‐SIRT1 and EVs‐SIRT1‐VEGF captured by the NTA system. (G) The protein level of SIRT1 and marker proteins of extracellular vesicles, such as CD9, CD63 and TSG101 in BMSCs, EVs, EVs‐SIRT1 or EVs‐SIRT1‐VEGF was detected by WB. (H) The combination of EVs‐SIRT1 and Chol‐VEGF aptamer was confirmed by confocal microscopy. Scale bar, 100 and 10 µm. (I–L) The expression profiles of the proteome were analysed via a heatmap, GO analysis, volcano plot and KEGG pathway analysis. (M) The expression profiles of small RNA sequencing results were analysed by KEGG pathway analysis.

FIGURE 2. In vitro anti‐fibrotic effect of EVs‐SIRT1‐VEGF. (A) Schematic representation of EVs‐SIRT1 reversing EndMT in vitro. (B) CCK‐8 assay showed the role of TGF‐β in MAECs validity. (C–E) qRT‐PCR and WB analysis showed Vimentin and VE‐cadherin expression levels of MAECs at different concentrations of TGF‐β. (F, G) The tube formation of MAECs treated by PBS, TGF‐β, TGF‐β + EVs TGF‐β + EVs‐SIRT1 and TGF‐β + EVs‐SIRT1‐VEGF. Scale bar, 500 µm. (H–J) qRT‐PCR and WB analysis showed Vimentin and VE‐cadherin expression levels of MAECs treated by PBS, TGF‐β, TGF‐β + EVs, TGF‐β + EVs‐SIRT1 and TGF‐β + EVs‐SIRT1‐VEGF. (K, L) Immunofluorescence double staining analysis of Vimentin and VE‐cadherin expression of MAECs treated by PBS, TGF‐β, TGF‐β + EVs, TGF‐β + EVs‐SIRT1 and TGF‐β + EVs‐SIRT1‐VEGF. Scale bar, 10 µm.

FIGURE 3. Activation of Nrf2 by EVs‐SIRT1‐VEGF in MAECs. (A, B) qRT‐PCR and (C–F) WB analysis showed antioxidative stress (SIRT1, KEAP1, Nrf2, NQO‐1, HO1) inflammatory (TNF‐α, eNOS), autophagy (LC3, p62) pathways and β‐actin expression of MAECs treated by PBS, TGF‐β, TGF‐β + EVs, TGF‐β + EVs‐SIRT1 and TGF‐β + EVs‐SIRT1‐VEGF. (G, H) Immunofluorescence double staining analysis of Vimentin and Nrf2 expression of MAECs treated by PBS, TGF‐β, TGF‐β + EVs TGF‐β + EVs‐SIRT1 and TGF‐β + EVs‐SIRT1‐VEGF. Scale bar, 10 µm.

FIGURE 4. Therapeutic efficacy of EVs‐SIRT1‐VEGF in APOE−/− mice. (A) Therapeutic schematic diagram of EVs‐SIRT1‐VEGF for AS mice. (B) Recording of body weight of AS mice during treatment. (C) The blood lipid level of each group at the end of treatment. (D) The gross ORO staining in aortic of each group at the end of treatment. (E, F) The ORO, Movat and HE staining in aortic root sections at the end of treatment. ORO scale bar, 50 µm. Movat and HE scale bar, 200 µm. (G, H) Immunofluorescence double‐staining analysis of Vimentin and VE‐cadherin expression in aortic root sections at the end of treatment. Scale bar, 50 µm. (I, J) Immunofluorescence staining analysis of SIRT1 expression in aortic sections at the end of treatment. Scale bar, 20 µm.