| Backgroud and ObjectiveAtherosclerotic vulnerable plaque (AVP) and its complication is the "number one killer" to human health. The unpredictable sudden breakage or erosion of vulnerable plaque, platelet activation and thrombosis could lead to the rapid occlusion of vascular, which is the most important pathogenesis of sudden fatal and nonfatal cardiovascular/cerebrovascular events such as myocardial infarction, cerebral thrombosis. And the rupture of vulnerable atherosclerotic plaques is regarded as the most important initiating factor. However, the occurrence and development of atherosclerotic plaque is a chronic insidious process, and most cardiovascular and cerebrovascular events have no signs before the onset. Hence, identifying high-risk atherosclerotic plaques is necessary for guiding the management and has important clinical significance in preventing the occurrence of severe cardiovascular events. But currently there are no effective methods of early identification and detection of AVP in clinic. With the emerging and developing of the targeted molecular imaging technology, a variety of noninvasive molecular imaging approaches, such as ultrasound, PET, MRI and CT and so on, can detect vulnerable plaque on a subcellular level with great enthusiasm.Among those molecular imaging methods, targeted ultrasound molecular imaging (UMI) is to connect specific ligand that targeting the lesion-specific molecular with ultrasound microbubble surface, to construct targeted ultrasound microbubbles, which will be considered as molecular probe. With combination of contrast enhanced ultrasound (CEU), the specific molecules adhered on the endothelial cells can be imaging on molecular level, further achieving the purpose of specific diagnosis of diseases at a molecular level. As compared with other traditional imaging techniques, UMI technology has the advantages of real time, high spatial and temporal resolution, no radiation pollution, portable and inexpensive features to evaluate atherosclerotic phenotypes. It is the ideal method for in vivo assessment of the temporal and spatial variation and characteristics of fixed molecular targets in blood circulation, and it is particularly suitable for in vivo localization, quantification and visualization of molecular targets expressed on the the surfure of endothelium and actived platelet under the condition of plaque inflammation and arterial thrombosis. Currently, UMI can be used to detect molecular targets on the surface of activated platelets and endothelial cells that are accessible to microbubbles, including activated von Willebrand factor (vWF) for platelet adhesion, vascular cell adhesion molecule-1 and P-selectin for inflammation, and vascular endothelial growth factor receptor-2 and interleukin-8 for neovascularization, all of which are expressed on the endothelial surface. To date, there are no reports of an atherosclerotic profile of activated platelets and thrombosis obtained by UMI. While these play an important role in the progression of atherosclerotic lesions and are also closely ralated with the plaque vulnerability; more often, atherothrombosis is asymptomatic and insidious, and can be a warning sign for high-risk plaques which can potentially be detected by UMI of activated platelets. Therefore, UMI of "active platelets on plaque endothelium" have provided a very valuable approach for the clinically detecting AVP and its risk stratification, futher guiding the treatment and preventing the occurrence of severe cardiovascular events.The glycoprotein (GP) Ⅱb/Ⅲa complex, also known as αⅡbβ3 integrin, is the major receptor expressed on the surface of activated platelets and also is a biomarker of platet activation; it could combine with binding sites on RGD sequences (arginine-glycine-aspartic acid, Arg-Gly-Asp) of many adhesion proteins such as the fibrinogen, and is essential for their interactions with other activated platelets and adjacent cells in atherothrombosis, and is the final common pathway of many factors induced platelet aggregation and thrombosis. Previous studies reports that platelet aggregation and GP Ⅱb/Ⅲa expression on the circulating platelet surface or atherosclerosis were found to be higher in acute coronary syndrome or unstable angina than in stable angina or healthy volunteers. Thus, the GP Ⅱb/Ⅲa receptor is a potential marker for imaging aggregated platelets in atherosclerotic plaques, which is considered as an important targeted molecular during detection of thrombi and anticoagulant therapy. However, data on the correlation between GP Ⅱb/Ⅲa on platelets in advanced atherosclerotic lesions and plaque severity are limited, and it is unclear whether GP Ⅱb/Ⅲa on activated platelets can serve as a biomarker for high-risk plaques, which is also the first problem to be solved in this study.Peptides containing the Arg-Gly-Asp (RGD) sequence are highly adhesive for GP Ⅱb/Ⅲa. Existing researches had connected RGD series linear oligopeptide on ultrasound microbubble surface, and successfully completed the targeted UMI of deep vein and atrial thrombus through the specific binding between targeted microbubbles and actived platelet GP Ⅱb/Ⅲa receptor in thrombosis. But because of high shear stress and "axial flow" characteristics in the artery, microbubbles with stronger targeted binding capacity are required to realize the targeted molecular imaging of arterial thrombus. Studies have shown that synthetic cyclic RGD peptide sequence could mimic human endogenous RGD binding sites, making it has 30-fold greater affinity for the GP Ⅱb/Ⅲa complex than the linear form, which is particularly advantageous for binding under the conditions of rapid blood flow that occur in atherosclerotic arteries. At present, our research group had successfully constructed cyclic RGD peptide modified targeted ultrasound microbubbles, and had verified their adhesive ability to GP Ⅱb/Ⅲa in vitro and in vivo, futher realize the UMI of large arterial thrombosis. But the large artery acute thrombosis models used in above studies were not close to pathological mechanism of in vivo atherosclerotic thrombosis, which could not reflect the occurrence, development and evolution of atherosclerotic plaque. Accordingly, in the study, we used apolipoprotein E knockout (ApoE-/-) mice to establish a real mice model of atherosclerotic plaques, providing a basis for identification of vulnerable plaque by UMI.Therefore, in this study, C57BL/6 wide type and ApoE-/- mice were used to establish mice models of different gradient atherosclerotic plaques. The current study examined whether GP Ⅱb/Ⅲa receptors on activated platelets that are adhered and aggregated on the endothelium can serve as a biomarker of atherosclerotic plaque instability by the methods of histopathology, scanning and transmission electron microscopy and immunofluorescence; and GP Ⅱb/Ⅲa can be quantified by contrast-enhanced ultrasound using MB-cRGDs to identify vulnerable plaque, providing a new noninvasive and effective method for identification and evaluation of vulnerable plaque.Methods1. Preparation of microbubbles and identification of the physicochemical propertiesA variety of related lipid material and synthetic phospholipid cyclic five peptide or the same type of nonspecific peptide with similar molecular weight and structure, were added in distilled water according to a certain proportion were mixed together and suspended in 20 ml purified water in 75℃ water bath, and then purged with perfluoropropane, followed by agitation with high shear machine to form milky white liquid, which is microbubbles modified with cyclic RGD (MB-cRGD) and isotype control non-targeted microbubbles (MB-CON), then stewing, washing and purified for 3 times. Prepared and purified microbubbles were reserved in the 4℃ of the refrigerator. The morphology of the microbubbles was observed by microscope, and the concentration and size distribution in each group were measured using a Coulter Counter device.2. Preparation of mice models of different gradient atherosclerotic plaques and its groupC57BL/6 wide type and ApoE-/- mice were used to establish mice models of different gradient atherosclerotic plaques and control models. C57BL/6 wide type and ApoE-/- mice were respectively given chow diet hypercholesterolemic diet (HCD), and maintained for 30 weeks starting at 6 weeks of age. Four groups of mice were used in this study:C57BL/6+ chow, C57BL/6+HCD, ApoE-/- +chow and ApoE-/- +HCD.3. Attachment of fluorescence-labeled platelets to the aorta were verified with fluorescent intrvital microscopyFreshly isolated platelets were obtained from whole blood collected from normal healthy C57BL/6 mice, and anticoagulated with 0.129M sodium citrate, followed extraction of platelets by centrifugation and preparation of platelet suspension. The platelets were incubated with calcein AM (300 ng/ml in PBS) for 15 min in the dark. Fluorescence-labeled platelets were injected into four different mouse groups through the tail vein (n=6 each); with an equivalent amount of calcein-AM without platelets injected into the negative control mice (n=6) and PBS without calcein-AM injected into the autofluorescence control ApoE-/-+HCD mice (n=6). Animals were sacrificed 15 min after injection, and the aorta was harvested and immediately frozen in optimum cutting temperature medium. The tissue was sectioned on a cryostat, and visualized the attachment of fluorescence-labeled platelets to the aorta at 480 nm under an epifluorescence microscope, and imaged with a C150L charge-coupled device camera.4. Detection of activated platelets and atherothrombus by electron microscopy (EM)After the image acquisition of targeted UMI,6 mice were randomly selected from each group, abdominal aorta tissue samples were obtained and fixed in situ with 2.5% glutaraldehyde for 4 h, and a subset of these was prepared for EM following a standard procedure. The activated platelets adhered on the luminal surface was observed by scanning EM operated at 20 kV. Platelets adhered to the surface of the vessel lumen were quantified by counting the average number of platelets over 10 optical fields (25.2×25.2 mm per field). Transmission EM was used at 80 kV to examine activated platelets in the abdominal aortic tissue of ApoE-/- +HCD mice.5. HE, masson, and immunohistochemistryAfter the image acquisition of targeted UMI, the remaining 6 mice in each group, abdominal aorta tissue samples were obtained and fixed, dehydration and paraffin embedding. HE, Masson staining and immunohistochemical staining were performed on paraffin-embedded serial sections cut at a thickness of 3μm.6. Plaque quantification based on histopathologic indicatorsAfter the serial sections were peformed on HE, Masson staining and immunohistochemistry, lipid deposition and collagen fiber content were measured by planimetry and expressed as a percentage of total plaque area. The area of positive immunoreactivity was quantified using Image-Pro Plus (Media Cybernetics, Rockville, MD, USA) and expressed as a percentage of the total area of the plaque or vessel wall. The SMC and macrophage contents of plaque were quantified as percentages of positive to total plaque area. The GP Ⅱb/Ⅲa content of plaques was measured by two methods:as percentages of GP Ⅱb/Ⅲa expression in the plaque and of GP Ⅱb/Ⅲa coverage of the endothelium.All measurements and analyses of UMI, scanning EM, histology, and immunolabeling data were performed by individuals who were blinded to the experimental design. The necrotic center/fiber cap (NC/FC) ratio was measured through the NC and FC size, and plaque vulnerability index was calculated using the formula:vulnerability index= (lipid deposit and macrophages)/(collagen fibers and SMCs).7. Construction of endothelial inflammatory model and isolation of human platelets and immune-fluorescence detectionHuman umbilical vein endothelial cell (HUVEC) was cultured in a confocal culture dish, and stimulated with various concentrations of recombinant tumor necrosis factor-α (TNF-α) recombinant protein for 1h, and vWF expression on HUVECs was measured by immunofluorescence labeling.20 ml human venous blood was obtained from healthy adult volunteers who did not take any drugs in the 10 days prior to blood collection. Blood was drawn into syringes containing 0.129M sodium citrate anticoagulant. The anti-coagulated blood was centrifuged at 200×g for 10 min to obtain platelet-rich plasma (PRP). Platelets were purified from PRP by centrifugation at 1000xg and washed twice. To selectively pellet erythrocytes, platelets were resuspended in the same buffer and centrifuged twice at 120xg for 3 min. The purified platelets were resuspended in the medium and incubated at 37℃ and 5% CO2. To measure the effect of vWF on GP Ⅱb/Ⅲa expression, platelets were then stimulated with various concentrations of recombinant vWF for 1h, and GP Ⅱb/Ⅲa expression on platelets was measured by immunofluorescence labeling.8. Targeted UMI of mouse abdominal aorta and its image analysisAfter preparation of animal models,12 mice were randomly selected from each group and equally divided into experimental group (n=24) and blocking group (n=24). The ultrasonic probe (15L7) were fixed on top of the mouse abdominal aorta; the position the probe was unchanged after adjusting the position to obtain good abdominal aortic long axis plane, and the parameters of the instrument were constant in the whole process of the experiment. CEU was performed with second harmonic imaging, and the probe transmitting and receiving frequency were 7 and 15MHz respectively and mechanical index (MI) of 0.18. The time interval of ultrasonic transmission is set to 10s. 1×106 MB-cRGD or MB-CON were intravenous bolus injected into each subgroup of mice in experimental group or blocking group in a total volume of 0.1 ml in a random order. The mice in the blocking were administrated 18ug/g eptifibatide through tail vein before injection of microbubble to saturated integrin GP Ⅱb/Ⅲa receptor. CEU images of the aorta were acquired at lOmin after injection of microbubbles, and then the MI was transiently increased to 1.0 for 3 s to destroy adhered MBs, and post-destruction images were acquired at an index of 0.18 to obtain the background signal, all the images were measured with video intensity (Ⅵ) and color-encoded tnrough MCE2.7 color-encoded software.9. Statistical analysisData were presented as the mean ± standard deviation and analyzed using SPSS v.13.0 (SPSS Inc., Chicago, IL, USA), A P value<0.05 (two-tail) was considered statistically significant. Comparisons between two groups were performed with the independent-samples t-test. Multi-group comparisons with only one grouping factor were performed with one-way analysis of variance with equal variances, A Bonferroni correction was calculated for multiple comparisons of continuous variables; while approximate F test Welch method for analysis of variance without equal variances, Dunnett’s T3 test was used for multiple comparisons. The variance analysis of factorial design was used to compare the multiple comparisons with 3 grouping factors. Spearman rank correlations were used to assess the linear correlation between selected variables.Results1. Microbubbles characterizationMB-cRGD and MB-CON prepared by agitation with high shear machine were transparent microbubbles, similar size and shape observed under microscope. The mean diameter of MB-cRGD and MB-CON were respectively approximately 2.52 ±0.28 μm and 2.54 ±0.30 μm, and the mean concentrations were respectively approximately (1.13±0.19) × 109 MBs per ml and (1.12±0.22)×109 MBs per ml. There were no significant differences in the mean diameter or concentration (p>0.05).2. Preparation of atherosclerotic plaqueThe results of oil red O staining from the four groups of mice showed that the most obvious atherosclerotic plaque was observed in the ApoE-/- +HCD group, followed by ApoE-/- +chow, C57BL/6+ HCD, and C57BL/6+ chow groups. In C57BL/6+ HCD group, only a small amount of abdominal aorta was stained red. No atherosclerotic plaque was found in the abdominal aorta of C57BL/6+ chow mice group.3. Fluorescent platelets attach to the endothelium of atherosclerotic lesions.Freshly isolated platelets were labeled with calcein-AM, and showed green fluorescence observed under fluorescence microscope. The fluorescent platelets were injected into the mice via tail vein, and frozen sections were prepared to observe the activated platelets adhering to the atherosclerotic lesions by epifluorescence microscopy; The highest numbers of fluorescence-labeled platelets were observed in the ApoE-y-+HCD group, followed by ApoE-/- +chow, C57BL/6+ HCD and C57BL/6+chow groups (p<0.05). There were no fluorescence-labeled platelets observed on the endothelium of the negative control group, or on atherosclerotic plaque in the autofluorescence control group.4. Activated-platelets adhere and aggregate on atherosclerotic plaque.The aggregation of platelets on the luminal surface of the endothelium was examined by scanning EM. In ApoE-/- +HCD group, much more platelets were recruited in the atherosclerotic lesions, while some of them aggregated, suggesting an adhering to each other. Similar to results obtained with the fluorescent probe, the highest number of platelets was observed in the ApoE-/- +HCD group, followed by ApoE-/- +chow, C57BL/6+HCD, and C57BL/6+chow groups (P<0.05). Because the size of platelet is variable (diameter may differ from 1-8μm), thus it is not reliable enough to identify platelet according to its size by using scanning EM. We further confirmed the structure of platelet by transmission EM. We noted that the platelets could be recruited in the atherosclerotic plaque or bind to endothelial cells, and the microparticles, a feature structure of platelet, could be clearly observed in the platelets. Furthermore, thrombi—which are enriched in platelets and contain a large number of red blood cells (RBCs) and an abundance of fibrin—were detected at the site of vascular injury in ApoE-/- +HCD group.5. HE, masson, and immunohistochemistry5.1 GP Ⅱb/Ⅲa immunohistochemistry:GP Ⅱb/Ⅲa expression in abdominal aortic plaques was assessed in the four experimental groups; GP Ⅱb/Ⅲa coverage of the endothelium was highest in the ApoE-/- + HCD group, followed by ApoE-/- +chow, C57BL/6+HCD, and C57BL/6+ chow groups (p<0.05), and a similar expression pattern was observed in plaques. Interestingly, GP Ⅱb/Ⅲa coverage of the endothelium was correlated with GP Ⅱb/Ⅲa expression in plaques (r=0.897, p<0.001), and GP Ⅱb/Ⅲa-rich thrombi were observed at the site of vascular injury in ApoE-/- +HCD mice group.5.2 HE, masson, and immunohistochemistry of CD68 and a-SMA:Hematoxylin and eosin and Masson’s trichrome staining, as well as a-SMA and CD68 immunolabeling of abdominal aortic plaques, revealed the highest percentage of macrophage-positive to total plaque area, NC/FC, and vulnerability index in ApoE-/- + HCD mice, followed by ApoE-/- +chow, C57BL/6+HCD, and C57BL/6+ chow mice (p<0.05). In contrast, the percentage of SMC to total plaque area showed the opposite trend. Notably, GP Ⅱb/Ⅲa coverage of the endothelium and expression in the whole plaque correlated with plaque indicators such as vulnerability index and necrotic center/fibrous cap (NC/FC).6. Cell/platelets immunofluorescenceTo test the effect of inflammatory stimulation on endothelia, HUVECs were stimulated with various concentrations of TNF-α and vWF expression was assessed by immuofluorescence. We found that TNF-α-induced vWF expression on HUVECs as well as vWF-induced GP Ⅱb/Ⅲa expression on platelets was dose-dependent. The specificity of the immunoreactivity was confirmed by the absence of fluorescence in HUVECs or platelets incubated with secondary antibodies alone. This further indicated that the inflammatory reaction lead to the increased expression of endothelial vWF and initiated platelet activation/aggregation, resulting in the high expression of GP Ⅱb/Ⅲa on activated and adhered platelets. Hence, GP Ⅱb/Ⅲa functions as a biomarker of vulnerable plaques as a result of inflammation and vWF activation, in accordance with previous findings.7. Targeted imaging of atherosclerotic plaques.Ten min after injection of MB-cRGD or MB-CON via tail vein. In testing group, CEU images for MB-cRGD showed that more obvious abdominal aortic ultrasound imaging can be seen in ApoE-/- +HCD mice group, followed by ApoE-/-+ chow, C57BL/6+HCD and C57BL/6+ chow groups. CEU for MB-CON showed no obvious abdominal aortic imaging enchancement in each animal group. In blocking group, CEU images of MB-cRGD and MB-CON also showed no obvious abdominal aortic imaging enchancement in each animal group.Quantitative analysis of Ⅵ value:Background-subtracted VI for MB-cRGD and MB-CON were comparable in both the testing and blocking groups in C57BL/6+ chow mice, but was significantly higher for MB-cRGD compared to MB-CON in the other three testing groups; The VI value of MB-cRGD in ApoE-/- +HCD was 20.30±3.10, and much higer than ApoE-/- +chow group (13.47±2.50, p<0.05) and C57BL/6+ HCD group (6.99 ±1.68, p<0.05). In blocking group, the VI value of MB-cRGD and MB-CON in each animal group had no significant difference (p>0.05), both of which are significantly lower than the VI value of ApoE"/- +HCD group (p<0.05) and ApoE-/- +chow group in experimental group (p<0.05), suggesting that the binding between MB-cRGD and GP Ⅱb/Ⅲa receptor was special and could be substantial inhibited by Eptifibatide.8. Correlation analysis between VI value of UMI and histopathological indexSpearman correlation analysis found that the VI of MB-cRGD in the testing group was correlated with GP Ⅱb/Ⅲa coverage of the endothelium and expression in plaques (r=0.877 and r=0.872, each p<0.001), as well as with vulnerability index and NC/FC (r=0.883 and r=0.875,each p<0.001).Conclusion1. GP Ⅱb/Ⅲa receptor of activated platelets on the endothelial surface was significantly related to plaque vulnerability, which could be served as a biomarker of vulnerable plaque;2. GP Ⅱb/Ⅲa receptor was highly expressed on the activated platelets, leading to the atherosclerotic plaque instability through the mechanism of "inflammatory stimulation-vWF activation on endothelial cells-platelet activation/aggregation".3. GP Ⅱb/Ⅲa receptor on activated platelets can be quantified by UMI using MB-cRGDs, whose VI value was significantly related to GP Ⅱb/Ⅲa receptor expression and plaque vulnerability, identifying vulnerable plaques and providing assist in the prevention of acute cardiovascular events. |
PDF Full Text Request