Exosomes derived from plasma: promising immunomodulatory agents for promoting angiogenesis to treat radiation-induced vascular dysfunction

Figures

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  Figure 1. Exosomes were derived from healthy rat plasma. RP-Exos stimulated inflammatory reactivity of macrophages, which further enhanced vascular formation of HUVEC cells by upregulating angiogenic factors. Afterward, RP-Exos were applied to in vivo experiments, where they promoted angiogenesis and tissue regeneration in both irradiated skin and bone defects.

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  Figure 2. Characterization and endocytosis of RP-Exos. (A) Morphology of RP-Exos observed by transmission electron microscope (TEM). Scale bar = 500 nm. (B) Particle diameter distribution of RP-Exos measured by nanoparticle tracking analysis (NTA). (C) Western blot analysis of CD63 and TSG101 on RP-Exos and supernatant. (D–G) Representative fluorescence and bright-field images of RP-Exos endocytosed by macrophages. Nuclei of macrophages were stained with DAPI (blue). RP-Exos were stained with Dil (red). Bars = 25 um.

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  Figure 3. RP-Exo reversed IR effects on cell viability and cell cycle distribution of macrophages. (A) Cell viability as assessed by CCK-8 assay. Ionizing radiation impaired proliferation of macrophages while RP-Exos rescued the adverse effect. (B) Quantitative analysis of cell cycle distribution, n = 3 per group. IR induced macrophage cell cycle to arrest in the G2/M phase while RP-Exos inhibited the adverse effect. E0R0: untreated macrophages. E0R1: macrophages receiving ironizing radiation. E1R1: macrophages receiving ironizing radiation and RP-Exo intervention. * = P < 0.05, ∗∗ = P < 0.01, ∗∗∗ = P < 0.001. (C–K) Cell cycle distribution of macrophages in different groups.

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  Figure 4. RP-Exos promoted macrophage inflammatory and pro-angiogenic functions, which were decreased by IR. (A–B) Differentially expressed genes shown in a volcano plot (E0R0 vs E0R1; E1R1 vs E0R1). (C, E) KEGG and GO analysis. The downregulated genes in the E0R1 group compared with the E0R0 group were enriched in inflammatory and pro-angiogenic pathways or GO terms. (D, F) KEGG and GO analysis. The upregulated genes in E1R1 group compared with E0R1 group were also enriched in inflammation and angiogenesis-related pathways or GO terms. (G) Heat map analysis of candidate genes. E0R0: normal macrophages. E0R1: macrophages receiving ironizing radiation. E1R1: macrophages receiving ironizing radiation and RP-Exo intervention.

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  Figure 5. RP-Exos boosted pro-angiogenic gene and protein expression of macrophages, which were downregulated by IR. (A–C) RP-Exos upregulated expression of pro-angiogenic genes (Vegfa, Ndrg1, and Adm), reversing the side effect induced by IR. n = 4 per group (D) The protein levels of VEGFA, NDRG1, and ADM were analyzed by western blot. (E) Quantification analysis of the blots shown in (D). (F) HUVECs were cultured with conditioned medium collected from macrophages in the E0R0, E0R1, and E1R1 groups. E0R1 conditioned medium impaired tube formation ability of HUVECs, while tube sprouting results in the E1R1 conditioned medium-treated HUVEC group were closer to the normal condition (E0R0 group). (G) The number of nodes, junctions, branches, and total tube length were quantified. n = 6 per group. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.0001.

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  Figure 6. RP-Exos stimulated inflammatory response in irradiated macrophages. Inflammatory gene and M1 marker expression of (A) Tnf, (B) Cd86, (C) Nos2, (D) Cxcl2, (E) Lcn2, (F) Bnip3, (G) H2-Oa, (H) Gbp8, (I) Ddit4 in E0R0, E0R1, and E1R1 groups. E0R1: macrophages receiving ironizing radiation. E1R1: macrophages receiving ionizing radiation and RP-Exo intervention. n = 3 per group. * = P < 0.05, ∗∗ = P < 0.01, ∗∗∗ = P < 0.001, ∗∗∗∗ = P < 0.0001.

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  Figure 7. RP-Exos favored angiogenesis and regeneration in the skin defect model. (A) HE staining and CD31 immunohistochemical staining of untreated skin tissue sections in irradiated (R+) or non-irradiated (R-) rats. IR reduced the number of cells and blood vessels in skin tissues. (B, C) CD31 immunohistochemical staining, HE staining and Masson’s Trichrome staining of regenerated skin tissue in rats in the E0R1 and E1R1 groups at 21 days after operation. Tissues in the E1R1 group healed better and exhibited more blood vessels than tissues in the E0R1 group. E0R1: rats treated with PBS. E1R1: irradiated rats treated with RP-Exos.

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  Figure 8. RP-Exos accelerated angiogenesis and regeneration in the bone defect model. (A) HE staining and CD31 immunohistochemical staining of untreated cranial bone in irradiated (R+) or non-irradiated (R-) rats. Cells and blood vessels were reduced by IR. (B) αSMA immunohistochemical and immunofluorescence staining of regenerated bone tissue in rats in the E0R1 and E1R1 groups. B: newly formed bone; V: blood vessels; C: connective tissues around newly formed bone. (C-I, II, III, IV) Representative micro-CT images of newly formed bone tissues. The red circle in (C-I, II) represents the defect ( five mm in diameter). Different colors in (C-III, IV) represent different bone densities, with red denoting denser and blue denoting sparser densities. (C-V, VI) Gross images of bone defect sites in the E0R1 and E1R1 groups. (D–F) Micro-CT quantitative analyses of new bone formation (mm 3), defined as bone within the red circle in (C-V,er); Tb.Sp (mm), trabecular separation; Tb.N (1/mm), trabecular number. n = 12 per group. * = P < 0.05, ** = P < 0.01.