TGF-β-Containing Small Extracellular Vesicles From PM2.5-Activated Macrophages Induces Cardiotoxicity

Figures

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  Figure 1. PM2.5 induced cardiac inflammation and fibrosis. (A) Representative images of hematoxylin-eosin (H&E) staining in heart issue, scale bars: 100 μm. The black arrow indicated the infiltration of inflammatory cells. (B) Representative images of Masson staining in heart issue, scale bars: 100 μm. (C) Quantitation of cardiac fibrosis area. (D) Relative mRNA expressions of α-SMA, mmp9, Col1α1, and Col3α1 in the hearts of mice were analyzed by qPCR. (E–H) Relative protein expressions of α-SMA, Col I and Col III in the hearts of mice were analyzed by Western blot. All the data were presented as mean ± SEM (t-test), n = 5, *indicates p < 0.05, **indicates p < 0.01.

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  Figure 2. Serum sEV from PM2.5-exposed mice increased the level of fibrosis-related proteins in cardiomyocytes. (A) Representative TEM images of isolated serum sEV, scale bars: 500 nm, 100 nm. (B) Representative results of NTA demonstrated the concentration and distribution in FA-sEV and PM2.5-sEV. (C) The average concentration in FA-sEV and PM2.5-sEV. (D) Mean size in FA-sEV and PM2.5-sEV. (E) Representative images of Western blot in macrophage marker (F4/80), epithelial cell marker (E-cadherin), and sEV marker (Alix, CD9, and CD63) in FA-sEV and PM2.5-sEV. (F–I) Relative protein expressions of α-SMA, Col I, and Col III in HL-1 cells treated with sEV analyzed by Western blot (n = 4). All the data were presented as mean ± SEM (t-test), n = 5, *indicates p < 0.05, **indicates p < 0.01. N.S indicates no significance.

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  Figure 3. PM2.5-treated macrophage-sEV increased collagen expressions in cardiomyocytes. (A) HL-1 cells cocultured with RAW264.7 and MLE-12 in the presence or absence of PM2.5 and GW4869 in a transwell (0.4 μm) plate. (B–E) Cocultured with RAW264.7 and PM2.5 promoted the protein levels of α-SMA, Col I, and Col III in HL-1 cells through transwell system. All the data were presented as mean ± SEM (t-test, one-way ANOVA), *indicates p < 0.05, **indicates p < 0.01.

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  Figure 4. Separation and characterization of sEV from RAW264.7 and MLE-12 after PM2.5 exposure. (A,B) Cell viability analyzed by MTT assay. The RAW264.7 and MLE-12 were treated with various concentrations of PM2.5 for 1, 12, 24, and 48 h (n = 5). (C) The results of NTA demonstrated the concentration and distribution in CN-sEV and PM2.5-sEV. (D) Representative TEM images of isolated sEV, scale bars: 200 nm, 50 nm. (E) Representative images of Western blot in sEV marker Alix, CD9 and CD63 in CN-sEV and PM2.5-sEV. All the data were presented as mean ± SEM (t-test), n = 3–4, *indicates p < 0.05, **indicates p < 0.01.

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  Figure 5. Release of key cytokines in sEV by RAW264.7 and MLE-12 after 1, 12, 24, 48 h of PM2.5 exposure. (A–C) ELISA for TNFα, TGF-β, and IL-6 in sEV from RAW264.7 cells after 1, 12, 24, and 48 h of PM2.5 exposure. (D–F) ELISA for TNFα, TGF-β, and IL-6 in sEV from MLE-12 cells at 1, 12, 24, and 48 h after PM2.5 exposure. All the data were presented as mean ± SEM (two-way ANOVA), n = 3. *indicates p < 0.05, **indicates p < 0.01.

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  Figure 6. TGF-β-containing sEV-induced production of collagen through TGF-β-Smad2/3 signaling pathway in cardiomyocytes. (A) Representative images of HL-1 cells that were incubated with the RAW264.7-sEV of PKH26-labeled sEV (red), cytoskeleton was stained with tubulin (green) and nuclei with DAPI (blue). Scale bar: 50 μm, 20 μm. (B) Relative mRNA expressions of α-SMA, mmp9, Col1α1, Col1α2, Col3α1, and TGF-β in HL-1 after being treated with RAW264.7-sEV analyzed by qRT-PCR. (C–G) Relative protein expressions of GF-β, α-SMA, Col I, and Col III in HL-1 were analyzed by Western blot. (H–J) Quantification of normalized p-Smad2/Smad2, p-Smad3/Smad3 in HL-1. All the data were presented as mean ± SEM (t-test), n = 3–4, *indicates p < 0.05, **indicates p < 0.01.