| BackgroundAtherosclerosis(AS)is the main cause of a series of acute cardiovascular events such as myocardial infarction,sudden cardiac deaths and stroke.AS plaque rupture and thrombosis are responsible.It is particularly important to predict the progression of AS prospectively and find an effective diagnosis and intervention strategy for AS vulnerable plaques.In recent years,molecular ultrasound imaging based on the targeted markers plays increasling important roles in disease diagnosis and treatment with a series of advantages such as sensitivity,specificity and accuracy.As the basis of ultrasonic molecular imaging technology,development of targeted molecular probes is critical.AS was considered to be a chronic inflammatory disease and the macrophages were the earliest inflammatory cells invaded to AS lesions.Macrophage apoptosis plays a critical role in the progression of AS lesions and vulnerable plaques formation,development,instability and rupture.When AS occurs,circulating monocytes migrate across the membrane to subintima and differentiate into macrophages,which absorb large amounts of lipid to form foam cells and eventually undergo apoptosis.Research has shown that the number of apoptotic macrophages was relatively less in the early stable plaque stage,but it was highly expressed in vulnerable AS plaques.This is expected to be a new target for the diagnosis and treatment of vulnerable plaques.Excessive apoptosis of macrophages can also accelerate the secretion of inflammatory factors and tissue factor(TF)expression in plaque.Phosphatidyl serine(PS)externalized on the surface of apoptotic cells is the most effective and main inducer of TF activation.In view of the important role of TF in AS lesions,it will be helpful to identify plaque vulnerability in early stage and improve the progression of AS lesions for development of a novel ultrasonic molecular probe through identification of plaque vulnerability and regulation of TF activation.This will provide a new strategy for reducing acute cardiovascular events.Methods1.Annexin V nanobubbles(NBav),an ultrasonic targeted molecular imaging probe,were prepared by "optimized thin-film hydration" and "biotin-avidin-biotin"methods,and then the characterization and echogenicity of NBav in vitro were measured and analyzed respectively.2.Establishment of the ApoE-/-mouse atherosclerotic plaque model and verification of model by high-frequency ultrasound imaging,anatomical observation and oil red O staining.3.Ultrasound molecular imaging was performed on AS plaques in ApoE-/-mice with NBAⅤ and control Nanobubbles(NBctrl).According to the imaging results,the plaques were incorprated into vulnerable plaque group and stable plaque group.4.After ultrasound molecular imaging,the value of NBAⅤ in the diagnosis of vulnerable plaques and progression of AS in mice were confirmed and analyzed by histological evaluation.5.In vitro,the targeting binding ability of NBav to ox-LDL-induced apoptotic macrophages was observed and the effect of NBAⅤ on TF level was analyzed.6.In vivo,the effect of NBAⅤ on TF expression in plaque of AS was evaluated and the possible mechanism of NBAⅤ intervention in vulnerable plaques was investigated.Results1.Preparation,characterization and echogenicity of NBAⅤ In vitro1)NBAⅤ presented monodisperse spherical particles with uniform size by Scanning electron microscopy.NBAⅤ that labeded by DiI were scattered red fluorescent spots with fluorescence microscope.2)The result of size analysis showed that NBAⅤ had a size distribution of 519.9±9.4nm with a PDI of 0.142± 0.038 and a negative zeta potential of-23.3±2.1mV.3)The bubble size of NBAⅤ remained relatively constant at 4℃,remaining approximately 610nm within the 48h after production.NBAⅤ remained within the nano size range(<650nm)for 120min in 37℃,indicating that NBAⅤ were relatively stable.In the following experiments,freshly made bubbles were used within 48h.4)The results of in vitro imaging showed that the echo intensity of NBAⅤ was significantly higher than that of the control saline(120.10± 2.13 vs 8.64± 0.25,P<0.001),indicating that NBAⅤ has a good developing effect and can be used for subsequent in vivo ultrasound molecular imaging studies.2.Establishment and verification of the ApoE-/-mouse atherosclerotic plaque modelApoE-/-mice were fed a high-fat a diet for more than 8 weeks,then the AS plaque was observed by high-frequency ultrasound.Anatomical observation and oil red O staining further confirmed the model of AS plaque.1)High frequency ultrasound could clearly showed that the thickened intima-media membrane and high or mixed echo plaques in the arteries of model mice.The arterial of control C57 mice showed smooth intima-media membrane without plaque formation.2)From the gross view of the aorta,a significant atherosclerosis plaques could be observed in ApoE-/-mice fed with high-fat diet for more than 10 weeks,but almost no atherosclerosis plaques formed in control mice.3)Furthermore,the oil-red O staining of arteries in vitro further confirmed the atherosclerotic plaques,which characterized by a large number of red-stained positive lipids.3.Value of NBAⅤ in diagnosis of vulnerable plaques and assessment of progression of AS in mice1)The echo of arteries lumen and atherosclerotic plaque in mice were observed enhanced at 10 seconds after NBAⅤ or NBCtrl injection intravenously,and the echo intensity gradually decreased with time.After 1-5min injection,the echo of NBAⅤ in the vessel lumen gradually decreased,but strong echo still existed in the plaque area.The echo of NBctrl in the lumen and plaque was decreased after injection.At different time points(1min,5min and l0min)after injection of NBAⅤ,the echo intensity of some plaques in the region of interest was significantly higher than that after injection of NBctrl,and the difference was statistically significant(lmin:106.33± 23.0 vs 62.33± 18.5,P<0.001;5min:101.33± 17.3 vs 41.67±10.3,P<0.01;10min:72.67± 10.2 vs 22.00± 5.2,P<0.01).2)According to the above experimental results,the plaques with and without differences between NBAⅤ and NBctrl imaging were analyzed for histopathology and immunology in vitro.The results showed that:A.HE staining:compared with normal vessels,atherosclerotic vessels presented significant morphological changes.The plaques protruding into the lumen had irregular and disordered structures and the ones which imaged different by NBav and NBctrl were rich in vacuolar adipose tissue.B.Masson and Oil red O staining:The results showed increment of atherosclerotic burden with abundant red stained lipid distribution in atherosclerotic arteries of differential imaging group(0.33 ± 0.03 vs 0.21 ± 0.01,P<0.05).Masson staining showed that the blue stained collagen fibers area were less in differential imaging group than those no differential imaging group(0.15 ± 0.07 vs 0.23 ± 0.01,P<0.01).C.CD68 and α-SMA staining:a large number of CD68 positive cells near the vascular lumen can be seen in plaque of differential imaging group with lessα-SMA positive cells.Further quantitative analysis revealed that the percentage of CD68 positive cells in the plaque of differential imaging group(14.73 ± 2.34%)was significantly higher than that in no differential imaging group(7.30 ± 0.81%,P<0.05),but the percentage of α-SMA positive cells were opposite between them(4.80 ± 0.40%vs 11.50 ± 1.50%,P<0.001).D.TUNEL staining:A large amount of red fluorescence was found in the plaques of the difference imaging group,which showed significant difference with undifferential imaging group(P<0.01).4.NBAⅤ targets apoptotic macrophages in vitro and inhibits TF expression in macrophages1)The ox-LDL treatment has been shown to markedly induce apoptosis of macrophage,with the apoptotic proportion of 28.22 ± 1.10%(50μg/ml),56.77 ±1.90%(75μg/ml)and 65.52±1.50%(100μg/ml),compared with 2.05±0.50%in the control group(P<0.05).The apoptosis rate of raw 264.7 cells induced by the 100μg/mL concentration of ox-LDL was slightly higher than that of 75μg/mL concentration group,no significant difference between them(P>0.05).Therefore,ox-LDL were used at 75μg/mL of concentration in the following experiments.2)ox-LDL exhibited an obvious effect on rise of TF concentration within 12h induction(532.7 ± 22.71pg/ml)and reached the highest value at 18h or 24h induction(18h:803.2±24.69pg/ml,24h:736.3±14.48pg/ml).3)Under the microscope,a large number of DiI-labeled NBAⅤ accumulated on the surface of apoptotic macrophages,while nothing was observed in the control group.4)NBAⅤ exhibited an obvious inhibiting effect on TF by ox-LDL inducement at 12h,as seen from the markedly decreased expression of TF expression(12h:532.7±22.71pg/ml vs 259.0±16.77pg/ml,,P<0.05;18h:803.2±24.69 pg/ml vs 247.1± 31.36pg/ml,P<0.01).5.Evaluation of intervention effect of NBAⅤ on TF in tissue of plaque1)The ApoE-/-mice were treated with NBAⅤ via tail vein for 8 weeks.The result showed that the positive TF staining areas of plaque began to decrease after 4w of NBAⅤ injection.With the increase of treatment,the TF positive staining area in plaque gradually decreased.Until 8th week of treatment,the TF positive area disappeared(0.065± 0.003),and there was no significant difference compared with the 6th week of treatment(0.067± 0.003,P>0.05).2)The level of TG,TC and LDL-C were significantly lower than that in the control group after NBAⅤ treatment for 6 weeks(P<0.05).The level of HDL-C was significantly higher than that of the untreated group(P<0.05).ConclusionsThis study mainly explored the feasibility of NBAⅤ in diagnosis of progression of AS lesions and vulnerable plaques in mice through ultrasonic targeted molecular imaging of ApoE-/-atherosclerotic plaques,and further analyzed the potential value of NBAⅤ in the intervention of vulnerable plaque lesions:1.The molecular imaging probe NBAⅤ was successfully prepared and characterized well,which can be further used in vivo ultrasound imaging.2.NBAⅤ has a good application value in the evaluation of AS lesion and the diagnosis of vulnerable plaques in high-fat fed mice.3.NBAⅤ may inhibit TF expression by targeting PS on the surface of apoptotic macrophages.NBAⅤ has a positive effect on improving lipid indexes of AS mice,suggesting that NBAⅤ can be used AS a molecular probe for anticoagulant therapy of AS.NBAⅤ can effectively intervene in the progression of AS lesions and reduce TF expression in plaques,which has potential effect on reducing thrombosis. |
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