title: 64Cu-Labeled Divalent Cystine Knot Peptide for Imaging Carotid Atherosclerotic Plaques.
abstract: The rupture of vulnerable atherosclerotic plaques that lead to stroke and myocardial infarction may be induced by macrophage infiltration and augmented by the expression of integrin αvβ3. Indeed, atherosclerotic angiogenesis may be a promising marker of inflammation. In this study, an engineered integrin αvβ3-targeting PET probe, (64)Cu-NOTA-3-4A, derived from a divalent knottin miniprotein was evaluated in a mouse model for carotid atherosclerotic plaques.
  
  
  METHODS
  Atherosclerotic plaques in BALB/C mice, maintained on a high-fat diet, were induced with streptozotocin injection and carotid artery ligation and verified by MR imaging. Knottin 3-4A was synthesized by solid-phase peptide synthesis chemistry and coupled to 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) before radiolabeling with (64)Cu. PET probe stability in mouse serum was evaluated. Mice with carotid atherosclerotic plaques were injected via the tail vein with (64)Cu-NOTA-3-4A or (18)F-FDG, followed by small-animal PET/CT imaging at different time points. Receptor targeting specificity of the probe was verified by coinjection of c(RGDyK) administered in molar excess. Subsequently, carotid artery dissection and immunofluorescence staining were performed to evaluate target expression.
  
  
  RESULTS
  (64)Cu-NOTA-3-4A was synthesized in high radiochemical purity and yield and demonstrated molecular stability in both phosphate-buffered saline and mouse serum at 4 h. Small-animal PET/CT showed that (64)Cu-NOTA-3-4A accumulated at significantly higher levels in the neovasculature of carotid atherosclerotic plaques (7.41 ± 1.44 vs. 0.67 ± 0.23 percentage injected dose/gram, P < 0.05) than healthy or normal vessels at 1 h after injection. (18)F-FDG also accumulated in atherosclerotic lesions at 0.5 and 1 h after injection but at lower plaque-to-normal tissue ratios than (64)Cu-NOTA-3-4A. For example, plaque-to-normal carotid artery ratios for (18)F-FDG and (64)Cu-NOTA-3-4A at 1 h after injection were 3.75 and 14.71 (P < 0.05), respectively. Furthermore, uptake of (64)Cu-NOTA-3-4A in atherosclerotic plaques was effectively blocked (∼90% at 1 h after injection) by coinjection of c(RGDyK). Immunostaining confirmed integrin αvβ3 expression in both the infiltrating macrophages and the neovasculature of atherosclerotic plaques.
  
  
  CONCLUSIONS
  (64)Cu-NOTA-3-4A demonstrates specific accumulation in carotid atherosclerotic plaques in which macrophage infiltration and angiogenesis are responsible for elevated integrin αvβ3 levels. Therefore, (64)Cu-NOTA-3-4A may demonstrate clinical utility as a PET probe for atherosclerosis imaging or for the evaluation of therapies used to treat atherosclerosis.
authors.affiliation: Department of Nuclear Medicine, Zhongshan Hospital, Fudan University, Shanghai, China; and Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California.
authors.forename: Lei
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California.
authors.forename: Yingfeng
authors.initials: Y
authors.lastname: Tu
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California.
authors.forename: Richard H
authors.initials: RH
authors.lastname: Kimura
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California.
authors.forename: Frezghi
authors.initials: F
authors.lastname: Habte
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California zcheng@stanford.edu.
authors.forename: Hao
authors.initials: H
authors.lastname: Chen
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California.
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.affiliation: Department of Nuclear Medicine, Zhongshan Hospital, Fudan University, Shanghai, China; and.
authors.forename: Hongcheng
authors.initials: H
authors.lastname: Shi
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California.
authors.forename: Sanjiv Sam
authors.initials: SS
authors.lastname: Gambhir
authors.affiliation: Molecular Imaging Program at Stanford (MIPS), Department of Radiology and Bio-X Program, Canary Center at Stanford for Cancer Early Detection, Stanford University, Stanford, California zcheng@stanford.edu.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cheng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002339:Carotid Arteries / Q000000981:diagnostic imaging* / Q000473:pathology; D002340:Carotid Artery Diseases / Q000000981:diagnostic imaging*; D056831:Coordination Complexes / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D006571:Heterocyclic Compounds / Q000737:chemistry; D006573:Heterocyclic Compounds, 1-Ring; D007249:Inflammation; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D008279:Magnetic Resonance Imaging; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D058226:Plaque, Atherosclerotic / Q000000981:diagnostic imaging*; D049268:Positron-Emission Tomography; D012421:Rupture; D014057:Tomography, X-Ray Computed
chemical_list: C000600262:64Cu-NOTA-3-4A; D056831:Coordination Complexes; D006571:Heterocyclic Compounds; D006573:Heterocyclic Compounds, 1-Ring; D039302:Integrin alphaVbeta3; D010455:Peptides; C048993:1,4,7-triazacyclononane-N,N',N''-triacetic acid; D003545:Cysteine
keywords: 64Cu; PET/CT; atherosclerotic plaque; knottin; αvβ3 integrin