Objective: Ultrasound molecular imaging (UMI) of glycoprotein (GP) IIb/IIIa receptor on activated platelets offers a unique means of identifying high-risk atherosclerosis. the potential mechanism of GP IIb/IIIa to serve as a biomarker of vulnerable plaques (see Supplemental Materials for details). Physique 1 Illustration of the experimental protocol. MB preparation The GP IIb/IIIa-targeted and unfavorable control MBs (MB-cRGD and MB-CON, respectively) had been generated by conjugating MBs to cyclic Arg-Gly-Asp-D-Phe-Cys (molecular pounds = 578.65, C24H34N8O7S) and cyclic Arg-Ala-Asp-D-Phe-Val (molecular weight = 588.67, C27H40N8O7) peptides, respectively, as described 24 previously. The binding characteristics from the MBs have already been reported 24 previously. MB-CON and MB-cRGD had been characterized using a Coulter counter-top (Beckman Coulter Inc., Brea, CA, USA) to look for the MB size and focus (discover Supplemental Components for information). After keeping track of, the MBs had been diluted with saline to a focus of 1107 and their half-life was assessed (discover Supplementary Components for information). Peptides had been synthesized by Peptides International Inc. (Louisville, KY, USA). Connection of fluorescence-labeled platelets towards the aorta Freshly isolated platelets had been obtained from entire blood gathered from normal healthful C57BL/6 mice and cleaned double with PBS. The platelets had been incubated with calcein AM (300 ng/ml in PBS; Invitrogen, Carlsbad, CA, USA) for 15 min at night as previously referred to 25. Fluorescence-labeled platelets had been injected into four different mouse groupings through the tail vein (n=6 each); with an equal quantity of calcein-AM without platelets injected in to the harmful control mice (n=6) and PBS without calcein-AM injected in to the autofluorescence Rabbit Polyclonal to BRF1 control ApoE-/-+HCD mice (n=6). Pets had been sacrificed 15 min after shot, as well as the aorta was gathered and instantly iced in ideal slicing temperatures moderate. The tissue was sectioned on a cryostat, visualized at 480 nm under an epifluorescence microscope, and imaged with a C150L charge-coupled device camera (Pixera, Santa Clara, CA, USA). Detection of activated platelets and atherothrombus by electron microscopy (EM) Abdominal aorta 527-95-7 manufacture tissue samples for the UMI study were fixed in situ with 2.5% glutaraldehyde, and 527-95-7 manufacture a subset of these was prepared for EM following a standard procedure. The luminal surface was observed by scanning EM (S-3000N; Hitachi, Tokyo, Japan) 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 (25.225.2 mm 527-95-7 manufacture per field). Transmission EM (Tecnai G2Spirit; Fei, Hillsboro, OR, USA) was used at 80 kV to examine activated platelets in the tissue of ApoE-/-+HCD mice. Histology and immunohistochemistry Samples of the abdominal aorta for histopathologic examination were obtained from all 24 mice used in the UMI study. Hematoxylin and eosin and Masson’s trichrome (MST-8003; Matxin Labs Pvt. Ltd., Bangalore, India) staining and immunohistochemistry were performed on paraffin-embedded serial sections cut at a thickness of 4 m. Immunohistochemistry was performed using a rabbit polyclonal primary antibody against mouse -easy muscle actin (-SMA), cluster of differentiation (CD)68, or GP IIb integrin (ab5694, ab125212, and ab63983, respectively; all from Abcam, Cambridge, MA, USA) to label easy muscle cells (SMCs), macrophages, and platelets, respectively. Plaque quantification based on histopathologic indicators Five representative serial sections each of the proximal, intermediate, and distal ends of the abdominal aorta were selected from each animal. Lipid deposition and collagen fiber content were measured by planimetry 27 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, as previously described 28. Histopathologic indicators (-SMA, CD68, and 527-95-7 manufacture GP IIb/IIIa expression) were quantified at three selected sites and averaged per section per site. All analyses and measurements of UMI, scanning EM, histology, and immunolabeling data had been performed by people who had been blinded towards the experimental style. The necrotic middle/fiber cover (NC/FC) proportion was assessed, and plaque vulnerability index was computed using 527-95-7 manufacture the formulation 29, 30: vulnerability index = (lipid deposit and macrophages).