Endothelial Microparticle-Mediated Transfer of microRNA-19b Inhibits the Function and Distribution of Lymphatic Vessels in Atherosclerotic Mice

Figures

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  FIGURE 1. Lymphatic vessel dysfunction occurs in the early stage of atherosclerosis. (A) Representative confocal image of FITC fluorescence intensity in popliteal lymph nodes from normal diet-fed and high-fat diet-fed ApoE-/- mice. (B) Quantitative analysis of the results shown in (A) for the normal control and atherosclerosis groups. *p < 0.05 compared with normal control group, n = 7 per group. (C) Representative Evans blue staining images of lymphatic vessels in legs of normal diet-fed and high-fat diet-fed ApoE-/- mice after 4 weeks of feeding. (D) Amount of Evans blue dye retained in the injected leg lymph node at 10 min after injection. (E) Amount of Evans blue dye retained in opposite leg lymph node at 10 min after injection. *p < 0.05 compared with normal control group, n = 7 per group. (F–G) RT-PCR results for the expression of miRNA-19b in blood and atherosclerotic artery tissue.

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  FIGURE 2. miR-19b levels in different tissues from mice in the three treatment groups after 4, 6, and 8 weeks of treatment. (A) Schematic of the experimental procedure for EMP or contrl treatment and different observation points. (B) After 4 and 6 weeks (C) and 8 weeks (D) of treatment, miR-19b levels in aortic tissue of mice injected with the indicated EMPs. *p < 0.05 compared with EMPmiR-19b group, n = 7–8 per group. (E) After 4 and 6 weeks (F) and 8 weeks (G) of treatment, miR-19b levels in lymph fluid of mice injected with the indicated EMPs. *p < 0.05 compared with EMPmiR-19b group, n = 7–8 per group.

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  FIGURE 3. EMPmiR-19b promotes atherosclerotic plaque formation in ApoE−/− mice. (A) Carotid ultrasound was used to assess arterial flow velocity (V) of carotid artery. The peak systolic velocity (PSV) and end-diastolic velocity (EDV) were measured and used to calculated the resistive index (RI) = (PSV–EDV)/PSV, as a flow parameter reflecting vascular resistance and vascular compliance. (B) Aortic diameter in systolic stage as a measure of artery size. (C) Gross pathological specimen from high-fat diet-fed ApoE-/- mice intravenously injected with PBS, EMPcontrol or EMPmiR-19b for 4, 6, or 8 weeks. Representative images are presented. (D) Representative images of H&E staining of aortic sections from high-fat diet-fed ApoE−/− mice intravenously injected with PBS, EMPcontrol, or EMPmiR-19b for 4, 6, or 8 weeks. (E) Quantitative morphometric analysis of lesion area in (D). *p < 0.05 compared with EMPmiR-19b group, n = 7–8 per group.

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  FIGURE 4. EMPmiR-19b limits lymphatic network distribution along atherosclerotic arteries. (A) Representative immunofluorescence staining images of atherosclerotic arteries from high-fat diet-fed ApoE-/- mice intravenously injected with PBS, EMPcontrol, or EMPmiR-19b for 8 weeks. In the images, green fluorescence represents LYVE1, a relatively specific marker of lymphatic vessels, while red fluorescence represents CD68. LYVE1+/CD68- tubular structures are identified as lymphatic vessels. (B) Quantitative analysis of the results shown in (A). *p < 0.05 compared with EMPmiR-19b group, n = 7–8 per group.

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  FIGURE 5. EMPmiR-19b induce lymphatic system dysfunction in ApoE-/- mice. (A) Representative confocal images of FITC fluorescence intensity in popliteal lymph nodes of high-fat diet-fed ApoE-/- mice intravenously injected with PBS, EMPcontrol, or EMPmiR-19b for 4, 6, or 8 weeks. (B–D) Quantitative analysis of the results shown in (A). *p < 0.05 compared with EMPmiR-19b group, n = 5–6 per group. (E) Representative images of Evans blue staining of lymphatic vessels in legs from high-fat diet-fed ApoE-/- mice intravenously injected with PBS, EMPcontrol, or EMPmiR-19b for 4, 6, or 8 weeks.

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  FIGURE 6. Effects of miR-19b on LEC migration and lymphangiogenesis. LECs were respectively transfected with miR-19b mimic (miR-19b mimic group) or a negative control fragment (negative control group), as well as miR-19b inhibitor (miR-19b inhibitor group) or a corresponding negative control (inhibitor negative control group). (A) Cell migration was assessed by Transwell migration assay and (B) quantified by measuring the number of migrated LECs; n = 3. (C) In vitro lymphangiogenesis was analyzed by tube formation assay and quantified by measuring the total number of (D) branching points and (E) loops; n = 3. Magnification, ×100. *p < 0.05 compared with negative control group, # p < 0.05 compared with the negative control group.

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  FIGURE 7. Regulatory effects of miR-19b on TGFβRII expression in LECs. (A) Schematic of miRNA-19b target prediction. (B) Schematic representation of the 3′ untranslated region of TGFβRII mRNA with the predicted target sites for miR-19b as well as miR-19b inhibitor (miR-19b inhibitor group) and a corresponding negative control (inhibitor negative control group). (C) LECs were transfected with miR-19b mimic at different concentrations or a negative control fragment, and miR-19b expression was measured by RT-PCR and normalized to RNU6B expression. *p < 0.05 compared with the respective control group. (D) LECs were transfected with miR-19b inhibitor at different concentrations and a corresponding negative control fragment, and miR-19b expression was measured by RT-PCR and normalized to RNU6B expression. # p < 0.05 compared with negative control. (E) LECs were transfected with miR-19b mimic (miR-19b mimic group) or a negative control fragment (negative control group) as well as miR-19b inhibitor (miR-19b inhibitor group) or a corresponding negative control (inhibitor negative control group), and TGFβRII mRNA expression was measured in the different groups. *p < 0.05, # p < 0.05 compared with…