The miR‐126‐5p and miR‐212‐3p in the extracellular vesicles activate monocytes in the early stage of radiation‐induced vascular inflammation implicated in atherosclerosis

Figures

• 

  FIGURE 1. Extracellular vesicles (EVs) are released from various cell types in response to radiation exposure in mouse model. The size of isolated EVs from irradiated mice (0, 0.1 and 1 Gy) were validated and characterised by (a) nanoparticle tracking analysis and (b) transmission electron microscopy (TEM). TEM images show the morphology characteristic of EVs both at a close‐up (100 nm) and at a wild‐filed (scale bar, 1 μm). (c) The EVs markers CD9, CD63 and CD81 were also detected by western blotting. Without EV refer to the remaining fraction of plasma after EVs separation. (d) Using the conventional flow cytometer, EVs were characterised by staining with Annexin V (FITC), which is specific for phosphatidylserine containing EVs. (e) Imaging flow cytometry (ImageStreamX) was also performed to visualise Annexin V (FITC)‐stained EVs. Total Annexin V positive events are determined in the gate. (f) Following irradiation with 0.1, 0.5, 1 and 5 Gy, mouse plasma EVs were stained for the surface markers CD31+/CD42‐, CD105+, CD144+ and CD146+ for endothelial cell‐derived EVs. Monocyte‐, platelet‐ and leukocyte‐derived EVs were identified using CD11b+, CD41+ and CD45+, respectively. The…

• 

  FIGURE 2. Extracellular vesicles (EVs) are released from various cell types in response to radiation exposure in human blood sample. (a) Radiation‐induced dicentric chromosomes in lymphocytes were evaluated using the DCScore program and presented as estimated doses (y‐axis) compared with the actual irradiated doses (x‐axis). Dicentric chromosomes (indicated by the arrow in the right panel) were specifically observed in irradiated cells. (b) Phosphorylated H2AX foci were analysed using ImageStreamX. Representative image of the foci (left panel) and graph showing the number of foci, with each bar representing the mean value ± SD (right panel). (c) EVs in the human PB samples were evaluated by flow cytometry as described in Section 2, and analysed using the linear regression model. Dashed lines indicate 95% confidence intervals for the regression line. Several cell‐specific markers were used including HLA‐DR+ for monocytes, CD45+ for leukocytes, CD41+ for platelet, and CD31+/CD42‐, CD105+, CD144+ and CD146+ for endothelial cells. All data are presented as mean ± SD. ***p < 0.001, **p < 0.01 and *p < 0.05 versus 0 Gy.

• 

  FIGURE 3. Endothelial and monocytic EVs are released at different time‐points following radiation exposure. Isolated EVs from co‐cultures of HUVECs and THP‐1 cells were analysed using cell‐type specific markers, (a) CD105+ and CD31+ for endothelial cells, and (b) CD11b+ and HLA‐DR+ for monocytes. The value at each time‐point was determined from five independent experiments and presented as relative fold‐change compared to non‐irradiated control cells. (c) Adhesion activity was examined using CFSE‐labelled THP‐1 cells (green) and PKH26‐labeled HUVECs (red). Adherent THP‐1 cells are indicated by arrows. Scale bar, 100 μm. The expression of cell surface adhesion molecules was determined at 4, 24 or 48 h after radiation exposure, using the following antibodies: (d) LFA1 and Mac1 for THP‐1 cells and (e) ICAM‐1 and ICAM‐2 for HUVECs. The mRNA levels of TNF‐α, IFN‐γ and IL‐13 were determined in co‐cultured (f) HUVECs and (g) THP‐1 cells after separation using qRT‐PCR. Data are represented as mean ± SD from three independent experiments with three replicates each. ***p < 0.001, **p < 0.01 and *p < 0.05 versus 0 Gy.

• 

  FIGURE 4. Endothelial EVs from irradiated co‐cultures induce activation of non‐irradiated monocytes in vitro. (a) CFSE‐labelled HUVECs were co‐cultured with PKH26‐labelled THP‐1 cells. Endothelial EVs (marked with CFSE) were increased in response to radiation exposure, however, no change in monocytic EVs (marked with PKH26) was observed. (b) CSFE‐labelled endothelial EVs (EEVs, green) were internalised by THP‐1 cells stained with DAPI (blue). Scale bar, 10 μm. Fluorescence intensity was quantified to determine EV uptake by THP‐1 cells. (c) Monocytic EVs were identified using the cell‐specific makers HLA‐DR+ and CD11b+, 24 h after treatment with endothelial EVs from the irradiated (0.1 and 1 Gy) HUVEC‐THP‐1 co‐cultures. CM refers to the conditioned media after isolation of endothelial EVs. (d) Surface expression of adhesion molecules in EEV‐treated or irradiated THP‐1 cells was examined using anti‐CD11a+/CD18+ (LFA1) and anti‐CD11b+/CD18+ (Mac1) antibodies. (e) Non‐irradiated THP‐1 cells were treated for 24 h with two concentrations (1× and 2×) of radiation‐induced (1 Gy) endothelial EVs. (f) Expression of genes related to monocyte adhesion in EEV‐treated non‐irradiated THP‐1 cells was…

• 

  FIGURE 5. Plasma EVs from irradiated mice induce immune response in non‐irradiated mice. (a) Illustration showing the experimental design and workflow for in vivo analyses. The transplanted EVs isolated from mice receiving different doses of radiation (0, 0.1 and 1 Gy) (n = 6 mice per group) were injected into non‐irradiated mice (n = 4 mice per group) through the tail vein. EVs were isolated from. (b) Using the conventional flow‐cytometry (c) and imaging flow‐cytometry, isolated EVs from the mice 24 h after radiation exposure were further analysed using cell‐type specific markers, CD31+/CD42‐ for endothelial cells and CD11b+ for monocytes. (d) The number of neutrophils (CD45+/CD11b+/Ly6G+) and macrophages (CD45+/CD11b+/F4/80+) was determined in the peripheral blood, spleen and bone marrow samples from EV‐recipient mice. (e) The levels of CD11a+/CD18+(LFA1) and CD11b+/CD18+(Mac1) were also determined in the above‐mentioned samples by flow cytometry. Data are represented as mean ± SD from three independent experiments with three replicates each. ***p < 0.001, **p < 0.01 and *p < 0.05 versus control. (f) Cytokine array was performed on leukocytes isolated from peripheral blood of…

• 

  FIGURE 6. EVs are released in the blood in response to radiation exposure. (a) Heatmap of 85 differentially abundant miRNAs in plasma EVs from non‐irradiated and irradiated (1 Gy) mice (Fold change: 1.5). Red colour indicates high expression level and green colour indicates low expression level. (b) Targeted pathways cluster of 10 miRNA candidates generated using the KEGG pathway analysis. Using qRT‐PCR, the levels of the 10 candidate miRNAs were analysed in (c) endothelial EVs from the co‐culture medium at 4 h after irradiation, (d) co‐cultured HUVECs at 4 h after irradiation and (e) THP‐1 cells at 24 h after treatment with endothelial EVs (isolated from the co‐culture medium at 4 h after radiation exposure). Data were derived from three biological replicates with three technical replicates each. Data are presented as mean ± SD. ***p < 0.001, **p < 0.01 and *p < 0.05 versus 0 Gy.

• 

  FIGURE 7. miR‐126‐5p and miR‐212‐3p mediates radiation‐induced damage signals from endothelial cells to monocytes. (a) Reduced expression of miR‐126‐5p and miR‐212‐3p was observed in HUVECs at 48 h after transfection with miRNA inhibitors. (b) At 24 h after radiation (1 Gy) exposure, adhesion molecules (LFA1 and Mac1) were examined in monocytes by flow‐cytometry following co‐culture with HUVECs transfected with miRNA inhibitors. (c) miRNA over‐expression using mimics in THP‐1 cells was verified by qRT‐PCR. (d) The expression of adhesion molecules in mimics‐transfected THP‐1 cells was compared to that in non‐transfected cells (control) and non‐transfected irradiated cells (irradiation). (e) Schematic representation of miR‐126‐5p and 212–3p constructs in dual‐luciferase assay is shown. Both pmirGLO empty vector and pmirGLO‐mismatch constructs was used as negative controls for antago‐miRNAs. Luciferase activity was measured using the transfected THP‐1 cells with pmirGLO‐miR‐126‐5p or 212–3p match/mismatch. (f) Expression of adhesion molecules was also analysed as described above. Data were derived from three biological replicates with three technical replicates each. Data are presented as…

• 

  FIGURE 8. MiR‐126‐5p and miR‐212‐3p promote macrophage differentiation and ox‐LDL uptake. (a) THP‐1 cells transfected with miRNA mimics were evaluated by flow cytometry for CD11b, CD14 and CD11c expression after treatment with 150 nM PMA. The histograms overlay depicts surface markers in transfected THP‐1 cells following PMA treatment (red), PMA treatment without transfection (blue) and control (light grey). (b) Foam cell formation of macrophages derived from mimics transfected THP‐1 cells was observed under microscope. Scale bar, 500 μm; magnification, 400×. (c) Lipid accumulation was assessed by extracting Oil‐red O from stained macrophages. (d) Total free cholesterol and (e) cholesteryl ester contents were determined in macrophages as described in Section 2. Data are represented as mean ± SD from three independent experiments with three replicates each. ***p < 0.001, **p < 0.01 and *p < 0.05 versus PMA.

• 

  FIGURE 9. Plasma endothelial EVs and their cargo are increased in radiation‐induced atherosclerotic Ldlr‐/‐ mice. (a) Representative images of atherosclerotic lesions in the aortic root at the level of the aortic valve shown at 100× magnification. Scale bar, 200 μm. Lesion area in the aortic sinus was quantified using the NIH ImageJ software. (b) Atherogenic index of plasma (AIP) were calculated with the levels of triglycerides and HDL‐cholesterol. (c) Plasma EVs in radiation‐induced atherosclerotic Ldlr‐/‐ mice were determined by flow cytometry as described previously. (d,e) The levels of miR‐126‐5p and miR‐212‐3p in plasma EVs of atherosclerotic mice were measured by qRT‐PCR. (f–h) Pearson's correlation analysis was performed to reveal the correlation between risks indices of atherosclerosis (AIP) and atherosclerotic plaques progress, EVs secretion, miR‐126‐5p expression, respectively. Data are represented as mean ± SD from three independent experiments with three replicates each. ***p < 0.001, **p < 0.01 and *p < 0.05 versus 0 Gy.

• 

  FIGURE 10. Schematic diagram depicting the initial stage of radiation‐induced vascular inflammation. Early responding endothelial EVs following radiation exposure stimulate monocytes leading to vascular inflammation. The levels of various miRNAs in endothelial EVs, including miR‐10a‐5p, miR‐126‐5p and miR‐212‐3p, are co‐ordinately regulated in response to radiation exposure.