| Background and ObjectivePlatelet activation occurred in many clinical diseases, such as ischemic cardiovascular and cerebrovascular diseases, thrombophilia diseases, immunity and metabolic diseases and so on. The activation of platelets and formation of thrombus or thromboembolism was the key to trigger the onset of catastrophic clinical events of myocardial infarction or stroke, which were direct reasons of death or disability, and the number of deaths accounted for more than half of the total, even far more than that caused by cancer, infectious diseases and respiratory diseases. Additionally, according to statistics, about70percent of fatal cardiovascular disease was caused by the rupture of atherosclerotic vulnerable plaque, which resulted in platelet activation, acute thrombus formation and thromboembolism. So platelet activation and atherosclerotic vulnerable plaques played pivotal roles in the occurence of serious clinical events. It had been demonstrated that platelet activation runs through the whole process of development and progression of atherosclerosis plaques, which not only induced thrombus formation but also released proinflammatory cytokines resulted in endothelial dysfunction and activation. Moreover, the net relationship between inflammation and the coagulation system would exacerbate the development of plaque instability/vulnerability and even rupture through multi-level mechanism, while plaque rupture, platelet activation and platelet-rich thrombus created a vicious cycle and jointly promote the onset of thrombotic events. Therefore, detecting activated platelets and thrombosis earlier would have great clinical significance to prevent and cure fatal clinical events, may reflect the process of inflammation and provide new ideas for the early identification of high-risk atherosclerotic plaques. However, at present, there had no effective means to diagnose arterial platelet thrombus in early stage. With the emerging and developing of targeted molecular imaging technique, existing non-invasive imaging modalities, such as ultrasound, MRI, CT, SPECT, had been positive explored to detect thrombus at molecular level.Among those molecular imaging modalities, targeted ultrasound molecular imaging involved the design and synthesis of targeted microbubbles that adhere to endothelium moleculars under disease-specific conditions. With the combination of contrast-enhanced ultrasound, the disease can be ultrasonically detected as a prolonged contrast effect that persisted in tissue beyond the normal washout time of nontargeted microbubbles. As compared with traditional imaging approaches characterized by detecting changes of diseases in physics or physiology, targeted contrast imaging could provide real-time, convenient, high spatial resolution and cheap capabilities for in vivo ultrasonic detection of phenotypic features of endothelium that predate clinical disease. Nowadays, some international researches had successfully applied targeted molecular image in thrombus of vein and arteriole, however, there was no report related to arterial thrombosis.The platelet glycoprotein GPIIb/IIIa receptors highly expressed on activated platelet surface were a sign of platelet activation, which can bind to the arginine-glycine-aspartic acid (RGD) binding site of fibrinogen and other adhesion proteins that was the final common pathway of platelet aggregation and thrombosis induced by a variety of factors. Thus, GPIIb/Ⅲa was an important target molecule in the thrombus detection and clinical anti-coagulant therapy. Several reasearches had showed that micobubbles combined with liner RGD could specifically adhere to GPIIb/IIIa receptors of activated platelet surface and then successfully implement ultrasound molecular imaging of deep vein and atrial thrombus. Due to the high-shear stress and "axial flow" phenomenon existed in large and middle-sized arteries, the molecular visualization of specific changes in arteries with targeted ultrasound needed more powerful targeted microbubbles. It had been found that the synthetic oligo-peptide sequence RGD loop which simulate the body’s natural RGD binding site, had a stronger capability to attach platelet glycoprotein GPⅡb/Ⅲa receptors than liner RGD. If the surface of general microbubbles was conjugated with oligo-peptide sequence RGD loop, the microbubbles will possess strong and perfect targeted efficacy that was expected to achieve early targeted ultrasound molecular imaging for activated platelets and thrombus in arteries in combination with contrast ultrasound.Presently, we had successfully constructed cyclic RGD-modified microbubbles targeted to GPIIb/IIIa receptors on abdominal arterial thrombus, however, thrombin induced thrombus, just as the methods (endothelium induried by Fecl3, endothelian mechanical damage, etc.) frequently used in the experimental study, was mixed thrombus. Because the procoagulant effect of inflammation and the vicious cycle caused by the ineraction of coagulation-inflammation network had become an important pathogenesis of platelet activation and thrombosis. It had been widely regarded as arterial thrombosis occurred in the earliest stages was mostly mediated by inflammation, which firstly activated platelets and thus formed platelet-rich thrombus. Therefore, inflammation-driven thrombosis in animal model would theoretically closer to the platelet activation and thrombosis in vivo. However, existing animal arterial thrombosis models mentioned above hadn’t related to the pathophysiological mechanisms of arterial thrombus formation. Then to build a kind of animal model that highly simulated arterial platelet thrombus formation induced by inflammation, will be beneficial to the research and clinical application of arterial thrombosis and inflammatory diseases. Arachidonic acid(AA) as an unsaturated fatty acid was widely present in the body, and had a pivotal regulatory role in inflammation and platelet activation by producing various biologically active substance, such as PGE2, PGI2, TXA2, LT, etc. At present, while researchers in home had successfully established human skin inflammation models by using AA in vitro, there had no studies to apply AA periadventially for simulating inflammation in vivo to induce arterial platelet thrombosis model.Therefore, in this study, microbubbles targeted to GPIIb/Ⅲa receptor(MBRGD) will be constructed by covalently combinding its specific ligands-cyclic Arg-Gly-Asp (RGD) peptide to the shell of general lipid microbubbles, and mouse carotid arterial platelet thrombosis will be established by applying AA periadventially. Respectively, the binding abilities of MBRGD to GPIIb/Ⅲa receptors immobilized on a culture dish under different shear stresses conditions were assessed in a parallel plate flow chamber, and platelet thrombus formation in carotid arteries were imaged and validatied with fluorescent intravital microscopy. Then, the mouse carotid thrombus were evaluated in vivo by the application of MBRGdD and contrast enhanced ultrasound (CEU). The aim of this study was to successfully construct targeted microbubbles with cyclic-RGD peptides and establish a kind of anmimal model of arterial inflammation-induced platlet thrombus, and then to explore the feasibility of targeted microbubble to evaluate activated platelets and thrombosis in arteries.Methods1. Preparation of microbubbles and determination of their biological propertiesCyclic pentapeptides and isotype nonspecific peptides were covalently attached to the lipid shell of general lipid microbubbles to prepare the targeted microbubbles (MBRGD) and isotype microbubbles (MBCON)-They were also washed by superpurified water three times to remove excess free unincorporated lipid. MBRGD and MBCON were storaged in refrigerator at4℃. The concentration and size distribution of these microbubbles were measured by a Coulter Multisizer counter.2. Evaluation the efficacy of MBRGD targeting to platelet glycoprotein IIb/IIIa(GPⅡb/Ⅲa) by using a parallel plate flow chamber2.1、 Attachment study:All culture dishes were equally divided into three groups, including dishes coated with platelet glycoprotein GPIIb/IIIa (1μg/ml), dishes coated with GPIIb/IIIa but blocked by a plenty of RGDfv, and dishes with the lack of GPIIb/IIIa (each n=3). MBRGD and MBCON (5x106/ml) were drawn through the parallel plate flow chamber at the different shear stress of0.5dyn/cm2,2.0dyn/cm2and4.0dyn/cm2, respectively. Quantitative analysis of microbubble accumulation was performed by counting the number of microbubbles adhered to chamber in the observed area under the microscope when the emergence of microbubbles for6min.2.2、 Detachment study:The parallel plate flow chambers were coated with GPIIb/IIIa (1μg/ml). MBrgd or MBcon (5×106/ml) were drawn into the flow chamber and allowed to interact with the target surface by flotation at zero flow for5min. Then0.9%sodium chloride solution was drawn through the flow chamber at a shear stress of0.2dyn/cm2to remove thoese stationary but not adhered microbubbles. The total number of microbubbles adhered was recorded and microbubble detachment was then assessed by increasing shear stress every30s until the microbubbles were completely detached. Recorded the full video and took photographys at a fixed field of vision (the objective lens was20times).3. Preparation of mouse model of carotid arterial platelet thrombus and imaging of thrombus formation with fluorescent intravital microscopyCarotid arteries of20KM mice were exposed and isolated from surrounding tissues by a piece of parafilm. The diameter of the carotid upstream was slightly reduced by loose permanent ligation.6μl AA at250mg/ml was randomly applied periadventially for30minutes in the one cartotid of mouse, while the contralateral carotid of each group was not handled but as its own controls.The whole blood of20KM mice were taken through heart and anticoagulanted with sodium citrate, followed by extracting platelet concentrate by centrifugation and preparing platelet suspensions. Platelets were incubated with1000ng/mL calcein-AM in PBS fir15minutes in the dark. Washed labeled platelets were injected in the tail vein of the receiving mice. The labeled platelets were observed and carotid thrombosis was recorded under a fluorescent microscope.4. Ultrasound molecular imagingAfter the preparation of animal models, mice were randomly divided into test group (n=10) and blocker group (n=10). Fixed ultrasound probe (15L8) at the diatal end of the common carotid artery, and justified the disposition of probe to obtain perfect transverse section of the both carotid arteries, then keeped still and all parameters remained constant. CEU molecular imaging were performed in second harmonic imaging mode with centerline frequency of7.0MHz and mechanical index (MI=0.18) for a duration of6minutes. MBCON and MBRGD were respectively and randomly injected via a catheter placed in the tail vein, and eptifibatide was injected in blocker group at18ug/g25to saturate the GPIIb/IIIa integrins before microbubbles adminstration. With the observation for6minutes, the microbubbles were destroyed by three seconds of continuous imaging with a high MI of1.9and the background subtracted VI of thrombus were measured.5. HE staining and immunohistochemistry:After CEU examination, all carotid arteries of all mice were harvested for the examination of pathology and immunohistochemisty.Results1. Results for microbubble preparation:The density of MBRGD and MBCON were about7.17-8.17×108/ml and7.05-8.58x×08/ml separately, the size distrubution for MBRGD and MBCON were about2.23-2.40μm,2.23-2.46μm respectively. There were no significant difference between both the concentrations and mean sizes (P>0.05).2. The efficacy of MBRGD targeted to GPIIb/IIIa in the parallel plate flow chamber studyThe parallel plate flow chamber attachment experiments:In the chambers coated with GPIIb/IIIa, it showed that the attachment number of MBRGD was significantly higher than that of MBCON at the same shear stress for6minutes (P <0.05); the attachment of both microbubbles to flow chamber decreased gradually as shear stress increased (P<0.05), but at high shear stress, MBRGD still had a certain attachment to GPIIb/IIIa. At0.5dyn/cm2shear stress, the adhesion number of MBRGD was (182.00±26.21), while at4.0dyn/cm2shear stress, the number was (37.67±11.24). However, there was no significant difference between microbubbles at the same shear stress in the group lack of GPIIb/IIIa or blocked with excess RGDfv against GPIIb/IIIa, even at the shear stress of0.5dyn/cm2, MBRGD rarely attached on the parallel plate. Additionally, untargeted microbubbles (MBCON) could not attach on the plate perfectly and sufficiently at all groups and all shear stresses. It showed that MBRGD possessed a good targeted ability to GPIIb/IIIa at a certain shear stress.The parallel plate flow chamber detachment experiments:The attachment number of MBRGD to chamber decreased gradually as shear stress increased. Half-maximal detachment was achieved at a shear stress of (26.09±2.93) dyn/cm2. when the MBGRD were completely detached, the shear stress was up to (98.28±2.86) dyn/cm2, while MBCON can not resist low shear stress, its half-maximal detachment and maximal detachment were only achieved at a shear stress of (5.14±1.40) dyn/cm2and (30.02±3.34) dyn/cm2.3. Imaging of thrombus formation with fluroscent intravital microscopyAfter the incubation of platelets suspensions with calcein-AM, platelets observed under a fluroscent microscope gave off green fluorescence. In the luminal of AA-applicated carotid, labeled platelets were gradually adhering to the vessel wall to from platelet thrombus. It had confirmed that AA surrounding the carotid could induce intra-arterial platelet-rich thrombosis.4. Ultrasound molecular imaging:After injection of MBRGD and MBcon via tail vein randomly and respectively for6minutes, MBRGD expressed stronger signal enhancement in AA-applied carotid, while no obvious contrast enhancement of both carotids can be observed when ultilizing the nonspecific MBCON-And in sham surgery group, there was also no enhanced signal in carotids with MBRGDdor MBCON-The VI of AA-applied carotid thrombus with MBRGd(21.24+3.67) was about6.43folds higher than that of thrombus with MBCON (3.30±1.51), and was higher than that of contralateral carotid (21.24±3.67vs.2.35±1.21, P＜0.05) with MBRGD-As respected, there had no greatly difference between the VI of AA-applied carotid and contralateral with MBcon, and the same result was observed in bolcker group with MBRGD and MBCON.5. HE staining and immunohistochemisty examination: A large number of platelets trabecular were observed in AA-applied carotids and GPIIb/IIIa receptors are expressed abundantly in the thrombus, while in contralateral carotid and sham surgery group, there were no substance formed and expressed in the lumen. It had been proved that the mousel model of carotid platlet thrombus was established successfully.Conclusions1. Microbubbles targeted to thrombus can be successfully constructed by covalently combinding oligopeptide RGD sequence to the shell of lipid microbubbles, and the targeted microbubbles possess strong ability to attach targets actively and resist a certain shear stress.2. Intravital fluorescent microscopy imaged that applying AA periadventially could induce intra-arterial platelet thrombosis in the mouse carotid, which can provide a new arterial thrombus model in which inflammation was predominant and was closer to thrombosis in vivo.3. In combination with CEU, ultrasound molecular imaging with MBRGD targeted to thrombus in vivo performed better than MBCON, which may be used to evaluate activated platelets and thrombosis in arteries effectively. |
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