Probiotic membrane vesicles ameliorate atherosclerotic plaques by promoting lipid efflux and polarization of foamy macrophages

Figures

• 

  Fig. 1. Characterization of MVs. (A) Schematic representation of isolated MVs. (B) TEM images and hydrodynamic diameters of MVs. (C) The average hydrodynamic diameters of MVs in normal saline over a 7-day period (n = 3). (D) The average zeta potentials of MVs in normal saline over a 7-day period (n = 3). (E) Schematic illustration of MVs loaded with ICG. (F-I) Flow analysis of different concentrations of MVs@ICG incubated with THP-1 + PMA or THP-1 + PMA + oxLDL for varying times. (J) Confocal imaging of MVs@ICG or ICG incubated with THP-1 + PMA or THP-1 + PMA + oxLDL. Cell nuclei were stained with DAPI, and the cytoskeleton was stained with FITC-phalloidin. (n = 3, scale bar = 50 μm)

• 

  Fig. 2. Targeted delivery of MVs in atherosclerotic mice. (A) Schematic representation of the preparation and imaging of atherosclerotic mice. (B) Representative ex vivo NIRF images of various tissues at different time points after tail vein injection of ICG or MVs@ICG. (C) Representative ex vivo NIRF images and oil red O staining of aortic tissues at different time points after tail vein injection of ICG or MVs@ICG. (D) HE staining and oil red O staining of atherosclerotic lesions and fluorescence images showing the accumulation of ICG in the atherosclerotic plaques of ApoE−/− mice injected with ICG or MVs@ICG. (E) Immunofluorescence staining of atherosclerotic lesions after tail vein injection of MVs@ICG; nuclei and macrophages were stained with DAPI and FITC-labelled F4/80, respectively. (n = 3, scale bar = 200 μm)

• 

  Fig. 3. Therapeutic efficacy of MVs in atherosclerotic mice. (A) Schematic representation of the preparation and treatment of atherosclerotic mice. (B) Representative ex vivo NIRF images of aortic tissues after the treatment. (C) Quantitative analysis of ICG fluorescent signals in the aorta 2 h postinjection. (D) Representative photographs of oil red-stained aortas. (E-F) HE staining and oil red O staining of atherosclerotic lesions in the aortic sinus in ApoE−/− mice injected with the control treatment or MVs. (G) Quantitative analysis of atherosclerotic lesions. (H) Fluorescence images showing the accumulation of ICG in atherosclerotic plaques in the aortic root sections of ApoE-/- mice. (I) Masson staining of atherosclerotic lesions in the aortic sinus. (J) The levels of T-CHO, TG, HDL-C and LDL-C in mice after the various treatments. (n = 5, mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001)

• 

  Fig. 4. Bioinformatic analysis of the mechanisms underlying the protective effects of MVs on AS development. (A) Schematic diagram of RNA-seq analysis. (B) Analysis of the quality control of the RNA sequencing data. (C) Changes in the number of genes after MVs treatment. (D) Significant changes in the expression levels of genes. (E) Alterations in various biological processes following MVs treatment. (F) Modifications in biological processes, including the promotion of lipid efflux and the regulation of macrophages, after MVs treatment

• 

  Fig. 5. MVs promotes lipid efflux by altering the expression of ABCA1. (A) Significant changes in signalling pathways, including the lipid and atherosclerosis pathways, following MVs treatment. (B) Significant alterations in genes involved in the lipid and atherosclerosis pathways. (C) Differential genes in the “lipid and atherosclerosis” pathway that intersected with genes that regulate lipid efflux. (D) Representative microscopy images of oil red O-stained foamy macrophages treated with various concentrations of MVs (0, 10, 20, 50 µg/mL; TO, TV1, TV2, TV3). (E-F) Western blot and RT‒qPCR analysis of ABCA1 in foamy macrophages treated with various concentrations of MVs (0, 10, 20, 50 µg/mL). (G) Immunohistochemical staining of ABCA1 in aortic tissues after the various treatments (control or MVs). (n = 5, mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001)

• 

  Fig. 6. MVs increased the expression of ABCA1 by upregulating the transcription factor NR1H3. (A) Differentially expressed genes obtained by RNA-seq analysis that intersected with transcription factors regulating ABCA1. (B-C) Western blot and RT‒qPCR analysis of NR1H3 in MVs-treated foamy macrophages. (D-F) MVs treatment or NR1H3 overexpression increased ABCA1 protein levels. (G-I) NR1H3 knockdown was reversed by MVs treatment. (J) Schematic diagram illustrating the mechanism by which MVs promote lipid efflux. (n = 5, mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001)

• 

  Fig. 7. MVs promote foamy macrophage polarization. (A) Schematic diagram illustrating how MVs promote macrophage polarization towards the M2 phenotype. (B) The downregulation of M1 markers, including CD86, NOS2, and TLR4. (C) The upregulation of M2 markers, including CD209, IL1R2, and TGM2. (D-E) The expression of cytokines in foamy macrophages and MVs-treated foamy macrophages. (F) Representative microscopy images of foamy macrophages and MVs-treated foamy macrophages. (G) Western blot analysis of M1 (CD86) and M2 (CD206) macrophage markers in foamy macrophages with or without MVs treatment. (H) Confocal images showing the expression of M1 (CD86) and M2 (CD206) macrophage markers in foamy macrophages with or without MVs treatment. (I) Immunohistochemical analysis of macrophage (F4/80), M1 macrophage (CD86) and M2 macrophage (CD206) marker expression in aortic tissues after the various treatments (control or MVs). (n = 5, mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001)