title: In vivo ultrasound molecular imaging of inflammatory thrombosis in arteries with cyclic Arg-Gly-Asp-modified microbubbles targeted to glycoprotein IIb/IIIa.
abstract: OBJECTIVE
  Ultrasound molecular imaging has the potential to detect activated platelets, thus identifying atherosclerotic plaque instability before onset of serious clinical events. However, it has not been well defined in inflammatory arterial thrombosis. We hypothesized that microbubbles (MBs) target glycoprotein IIb/IIIa (GP IIb/IIIa) could achieve a noninvasive in vivo detection of inflammatory thrombosis in large arteries through contrast-enhanced ultrasound (CEU) imaging.
  
  
  METHODS
  Lipid shell-based gas-filled MBs were modified covalently with a cyclic Arg-Gly-Asp (RGD) peptide (MB-cRGD) targeted to activated GP IIb/IIIa or a negative control peptide (MB-CON) via thiol-maleimide coupling. Adherence of MB-cRGD and MB-CON to GP IIb/IIIa was determined in vitro by using a parallel plate flow chamber at variable shear stress (0.5-8 dynes/cm2). Inflammatory platelet thrombosis was induced by periadvential application of arachidonic acid (AA) to one of the bilateral carotids of C57BL/6 mice (n = 20) and confirmed through intravital fluorescence microscopy. Attachment of MBs was determined in vivo with CEU imaging of bilateral carotids in the AA application mice with (n = 10) or without (n = 10) pretreatment of GP IIb/IIIa antagonist. The expression of integrin GP IIb/IIIa was assessed through immunohistochemistry.
  
  
  RESULTS
  Microbubble-cRGD but not MB-CON had excellent affinity to GP IIb/IIIa under all shear stress conditions. Successful inflammatory platelet activation and thrombosis in AA application carotids were noted through intravital fluorescence microscopy. Contrast video intensity from adhered MB-cRGD in the thrombi was significantly higher than that from MB-CON (P < 0.05). Video intensity of MB-cRGD in the thrombi was suppressed significantly by preblocking with GP IIb/IIIa antagonist (P < 0.05) but not for MB-CON. Immunohistochemical finding demonstrates that expression of integrin GP IIb/IIIa in the thrombi was abundant; it was inhibited significantly through pretreatment with GP IIb/IIIa antagonist (P < 0.05).
  
  
  CONCLUSIONS
  Cyclic RGD-modified MBs targeted to GP IIb/IIIa with CEU are capable of detecting inflammation-activated platelets and thrombosis in large arteries, thus providing a potential tool for identification of vulnerable atherosclerotic plaques.
authors.affiliation: From the *Department of Cardiology, Nanfang Hospital, Southern Medical University; †Department of Cardiology, Panyu Central Hospital; ‡Key Lab for Organ Failure Research, Ministry of Education; §Department of Nephrology, Nanfang Hospital, Southern Medical University, Guangzhou; and ∥Institute of Ultrasonography, Chongqing Medical University, Chongqing, China.
authors.forename: Weilan
authors.initials: W
authors.lastname: Wu
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Shuxin
authors.initials: S
authors.lastname: Shen
authors.forename: Juefei
authors.initials: J
authors.lastname: Wu
authors.forename: Shengcun
authors.initials: S
authors.lastname: Guo
authors.forename: Liang
authors.initials: L
authors.lastname: Su
authors.forename: Fanfan
authors.initials: F
authors.lastname: Hou
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Wang
authors.forename: Yulin
authors.initials: Y
authors.lastname: Liao
authors.forename: Jianping
authors.initials: J
authors.lastname: Bin
mesh_terms: D000818:Animals; D001158:Arteries / Q000378:metabolism*; D003287:Contrast Media; D007124:Immunoenzyme Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D045423:Microbubbles; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D009842:Oligopeptides; D015539:Platelet Activation / Q000187:drug effects; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism*; D033081:Shear Strength; D013927:Thrombosis / Q000000981:diagnostic imaging* / Q000378:metabolism; D014463:Ultrasonography
chemical_list: D003287:Contrast Media; D009842:Oligopeptides; D010975:Platelet Aggregation Inhibitors; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C047981:arginyl-glycyl-aspartic acid