Ultrasound Molecular Imaging of Atherosclerosis for Early Diagnosis and Therapeutic Evaluation through Leucocyte-like Multiple Targeted Microbubbles

Figures

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  Figure 1. Preparation and characterization of MBVIS. (A) Schematic diagram of targeted MBVIS. (B) Representative photomicrograph of MBVIS (scale bar = 10 μm); inset shows a bottle of MBVIS suspension. (C) The size distribution curve of MBVIS with an obvious peak at 1-3 μm.

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  Figure 2. Fluorescence microscopic examination of the fluorophore-labelled targeted MBs. Representative fluorescence micrographs of single-/double-/triple- targeted MBs (scale bar = 10 μm) indicated the successful conjugation of three kinds of ligands to the surface of corresponding MBs.

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  Figure 3. In vitro static adhesion of targeted MBs to inflammatory cells. (A-C) Correlation analysis between the concentrations of TNF-α and the expression levels of VCAM-1, ICAM-1 and P-selectin on bEnd.3 cells stimulated by TNF-α for 8 h. (D) Representative bright-field micrographs of targeted MBs and MBIgG bound to stimulated bEend.3 cells (40 ng/mL TNF-α; scale bar =10 μm). (E) Quantitative assay for the number of MBs adhered onto bEnd.3 cells. #: P < 0.05 and *: P < 0.01. (F) The number of MBVIS attached to cells stimulated with 0, 10, 20 or 40 ng/mL TNF-α. *: P < 0.05. (F) Correlation analysis between the number of attached MBVIS and concentrations of TNF-α used in the stimulation experiment.

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  Figure 4. Dynamic adhesion of MBs under the parallel plate flow chamber. (A) The number of MBs bound to the TNF-α-stimulated cells in different flow shear stresses after 4 min. (B) The number of MBs bound to the TNF-α-stimulated cells at different times under 4 dyn/cm2 flow shear stress.

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  Figure 5. In vivo ultrasound molecular imaging of the ascending aorta. (A) Representative color-coded ultrasound images after injection of various kinds of MBs at the 10 week feeding time, and (B) the quantitative analysis of ultrasound signal intensities. *: P < 0.05. (C) Representative color-coded images from four groups after injection of MBVIS, and (D) the quantitative analysis of ultrasound signal intensities. Stronger signal could be observed in the A-HD group than in the control groups at the same time. *: P < 0.05. Scale bar = 1 mm.

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  Figure 6. Progression of atherosclerotic plaques and expression of VCAM-1, ICAM-1 and P-selectin. (A) Representative micrographs of H&E-stained aorta sections from ApoE-/- mice fed HD for 6, 10, 14 or 20 weeks. (B) Quantitative analysis of intima-medial thickness. The intima-medial thickness increased with the extension of feeding time. *: P < 0.05. Scale bars in micrographs with low magnification are 200μm, and 100μm in micrographs with high magnification. (C) Representative micrographs of aorta sections stained with Oil Red O. An apparent atherosclerotic plaque could be observed at the 20-week HD feeding time. (D) Giant plaques in sections from mice fed 30 weeks occupied almost the whole lumen. (E) Representative micrographs of immunofluorescence staining for VCAM-1, ICAM-1 and P-selectin proteins on endothelium of ascending aorta from A-HD mice. VCAM-1, ICAM-1 and P-selectin were labelled as blue, red and green. Scale bar = 100 μm. (F) Western blotting analysis of the expression of VCAM-1, ICAM-1 and P-selectin from the aorta tissue of A-HD, A-RD, C-HD and C-RD mice fed for 6, 10 or 14 weeks. (G-I) Quantitative analysis of the expression levels of VCAM-1, ICAM-1 and P-selectin. The…

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  Figure 7. Ultrasound molecular imaging assessment of atorvastatin efficacy. (A) Representative H&E images of ascending aorta sections from ApoE-/- mice fed atorvastatin or placebo in their daily hypercholesterolemic diets for 8 weeks. Scale bar = 50 μm. (B) Quantitative analysis of intima-medial thickness, with reduced intima-medial thickness in the atorvastatin-treated A-HD mice. *: P < 0.05. (C) Representative color-coded images of ascending aorta generated from adherent MBVIS or MBIgG. Scale bar = 1 mm. (D) Quantitative analysis of signal intensities from (C). A-HD mice were raised with atorvastatin or placebo added into their daily hypercholesterolemic diet for 8 weeks. *: P < 0.05.

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  Figure 8. Expression detection of VCAM-1, ICAM-1 and P-selectin after atorvastatin treatment. (A) Representative micrographs of immunofluorescence staining for VCAM-1, ICAM-1 and P-selectin proteins on endothelium of ascending aorta from A-HD mice with atorvastatin or placebo treatment. Scale bar = 100 μm. (B) Western blotting analysis of the expression of VCAM-1, ICAM-1 and P-selectin from the aorta tissue of A-HD mice with atorvastatin or placebo treatment. Significantly reduced expression was found in the atorvastatin-treated animals. *: P < 0.05.