Exosomes as a messager to regulate the crosstalk between macrophages and cardiomyocytes under hypoxia conditions

Figures

• 

  FIGURE 1. The characterization and uptake of Exo derived from H9c2 cell lines cultured in normoxic or hypoxic environment. (A) Morphological characterization of Nor‐Exo and Hypo‐Exo by transmission electron microscopy (scale bar = 100 nm). (B) The surface makers (Alix, HSP70 and CD63) of Nor‐Exo and Hypo‐Exo were detected by western blotting. (C) The size distribution was measured using NanoSight analysis, which indicated that the diameter of most of the particles was within the range of 40–160 nm. ‘ns’ indicates p ≥ 0.05. (D) A laser scanning confocal microscope captured the PKH26‐labelled Exos (red fluorescence) taken up by RAW264.7 cells stained by DAPI (blue fluorescence) (scale bar = 20 μm)

• 

  FIGURE 2. Nor‐Exo and Hypo‐Exo derived from H9c2‐polarized macrophages. (A) Quantitative analysis of hypoxia‐inducible factor 1 alpha expression in Nor‐Exo and Hypo‐Exo as detected by western blotting. (B) Quantitative analysis of transforming growth factor beta expression in Nor‐Exo and Hypo‐Exo as detected by western blotting. (C) M1‐macrophage marker iNOS and M2‐macrophage marker Arg‐1 were evaluated in RAW 264.7 cells cocultured with PBS, Nor‐Exo or Hypo‐Exo for 48 h and assessed by western blotting. (D) The mRNA expression levels of IL‐1β, IL‐6, iNOS, TNF‐α, MCP‐1, Arg‐1, TGF‐β, IL‐10, Ym‐1 and Fizz‐1 in RAW 264.7 cells were determined by qRT‐PCR. (E) Western blot analysis of Arg‐1 levels in RAW264.7 cells pretreated with or without Dynasore, followed by the stimulation with PBS, Nor‐Exo or Hypo‐Exo. At least three independent experiments were performed for each group. Data are expressed as the mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001; ‘ns’ indicates p ≥ 0.05

• 

  FIGURE 3. Circulating exosomes modulate macrophage polarization in vitro. (A) The protein profile of hNor‐Exo and hAMI‐Exo was evaluated by western blotting. (B) Morphological characterization of hNor‐Exo and hAMI‐Exo by transmission electron microscopy (scale bar = 50 nm). (C) The size distribution of hNor‐Exo and hAMI‐Exo was measured using Nanosight analysis. ‘ns’ indicates p > 0.05. (D) A laser scanning confocal microscope captured the PKH26‐labelled hNor‐Exo or hAMI‐Exo (red fluorescence) taken up by RAW264.7 cells stained by DAPI (blue fluorescence) (scale bar = 50 μm). (E) Western blot analysis of HIF‐1α expressed in hNor‐Exo or hAMI‐Exo (n = 3). (F) RAW 264.7 cells were stimulated with PBS, hNor‐Exo or hAMI‐Exo for 48 h, the M1‐macrophage marker iNOS and M2‐macrophage marker Arg‐1 were analysed by western blot (n = 3). Data are expressed as means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001; “ns” indicates p ≥ 0.05

• 

  FIGURE 4. Circulating exosomes modulate macrophage polarization in vivo. (A) Representative images of iNOS‐positive staining: DAPI, blue; iNOS, green (n = 6, scale bar = 20 μm). (B) Representative images of CD206‐positive staining: DAPI, blue; CD206, red (n = 6, scale bar = 20 μm). Data are expressed as means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001; ‘ns’ indicates p ≥ 0.05

• 

  FIGURE 5. The related profiles and functions of miRNAs in circulating exosomes. (A) The heat map of differential expression microRNAs in exosomes derived from hNor‐Exo (n = 3) and hAMI‐Exo (n = 6). The red dots: up‐regulated microRNAs. Green dots: down‐regulated microRNAs. (B) GO analyses for target genes of the differentially expressed miRNAs. (C) KEGG pathway analyses of the target genes of the differentially expressed miRNAs

• 

  FIGURE 6. M2 macrophages alleviate oxidative stress injury in H9c2 cells. (A) Optical image of RAW264.7 cells treated with PBS, LPS (100 ng/mL) or IL‐4 (40 ng/mL) for 24 h. (B) Western blot analysis of iNOS and Arg‐1 expressed in RAW264.7 cells treated with PBS, LPS (100 ng/mL) or IL‐4 (40 ng/mL) for 24 h (n = 3). (C) Schematic image of H9c2 cells (bottom chamber) cocultured with M1 macrophages [RAW264.7 cells treated with LPS] or M2 macrophages [RAW264.7 cells treated with IL‐4] (upper chamber) in Transwell systems. (D) Expression of apoptosis‐related proteins (Bax and Bcl‐2) were evaluated in H2O2‐induced H9c2 cells cocultured with M1 macrophages or M2 macrophages and assessed by western blotting (n = 3). (E) Representative TUNEL staining for apoptotic cells in H2O2‐treated H9c2 cells cocultured with M1 macrophages or M2 macrophages; the red fluorescence represents apoptotic cells (n = 4, scale bar = 100 μm). Data are expressed as means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001; ‘ns’ indicates p ≥ 0.05