Perivascular Adipose-Derived Exosomes Reduce Foam Cell Formation by Regulating Expression of Cholesterol Transporters

Figures

• 

  Figure 1. Characterisation of SCAT-EXOs and PVAT-EXOs. Exosomes were identified by transmission electron microscopy (A). Scale bars: 200 nm. Nanoparticle tracking analysis showed the particle size distribution of exosomes (B). The exosome-related protein markers CD9, CD63, and TSG101 were measured by Western blotting (C).

• 

  Figure 2. SCAT-EXOs and PVAT-EXOs were transferred into macrophages. Exosomes were labelled with PKH26 (red) and incubated with RAW264.7 cells for 12 h. The cells were then stained with phalloidin (green) and DAPI (blue). Fluorescence signals were analysed by laser confocal microscopy. Ctrl: control group. Scale bars: 75 μm.

• 

  Figure 3. PVAT-EXOs reduced macrophage foam cell formation. RAW264.7 cells were incubated with SCAT-EXOs, PVAT-EXOs or equal amounts of PBS and then incubated with or without ox-LDL. Oil Red O staining indicated that PVAT-EXOs reduced macrophage foam cell formation (A). Scale bars: 50 μm. Lipid accumulation in macrophages was confirmed by determining the levels of total cholesterol (B), cholesterol esters (C) and free cholesterol (D). Ctrl: control group. The ultrastructure of foam cells was detected with transmission electron microscopy (E). Black arrows indicate phagosomes, white arrows indicate lysosomes, and red arrows indicate lipid droplets. Scale bars: 2 μm. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the ox-LDL treated group.

• 

  Figure 4. Macrophage polarisation was not affected by SCAT-EXOs or PVAT-EXOs. RAW264.7 cells were incubated with SCAT-EXOs, PVAT-EXOs or equal amounts of PBS and then incubated with lipopolysaccharide (LPS). The phenotypes of cells were analysed by flow cytometry (A). There was no significant difference in the percentages of CD206+/F4/80+ cells or CD80+/F4/80+ cells between the groups (B,C). The production of TNF-a (D) or IL-6 (E) was not affected by EXOs.

• 

  Figure 5. PVAT-EXOs reduced Dil-oxLDL uptake and promote cholesterol efflux in macrophages. After treatment with SCAT-EXOs, PVAT-EXOs or PBS, Dil-oxLDL uptake by RAW264.7 cells was analysed by fluorescence microscopy (A). In addition, the fluorescence intensities were quantified by flow cytometry (B). HDL-mediated cholesterol efflux was significantly promoted by PVAT-EXOs (C). Ctrl: control group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the Dil-oxLDL group or control group.

• 

  Figure 6. The expression of cholesterol transporters in macrophages was regulated by SCAT-EXOs and PVAT-EXOs. RAW264.7 cells were incubated with SCAT-EXOs, PVAT-EXOs or equal amounts of PBS and then incubated with ox-LDL. Western blot analysis showed the expression of the cholesterol transport proteins SR-A, CD36, LDL-R, LOX-1, ABCA1, and ABCG1 and the upstream proteins LXRα and PPARγ (A). The expression levels were normalised to β-actin. The mRNA expression levels of SR-A, CD36, LDL-R, LOX-1, ABCA1 and ABCG1 were analysed by real-time PCR and normalised to GAPDH (B). Ctrl: control group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the ox-LDL group.