title: Dual-Modality Activity-Based Probes as Molecular Imaging Agents for Vascular Inflammation.
abstract: Macrophages are cellular mediators of vascular inflammation and are involved in the formation of atherosclerotic plaques. These immune cells secrete proteases such as matrix metalloproteinases and cathepsins that contribute to disease formation and progression. Here, we demonstrate that activity-based probes (ABPs) targeting cysteine cathepsins can be used in murine models of atherosclerosis to noninvasively image activated macrophage populations using both optical and PET/CT methods. The probes can also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations.
  
  
  METHODS
  Macrophage-rich carotid lesions were induced in FVB mice fed on a high-fat diet by streptozotocin injection followed by ligation of the left common carotid artery. Mice with carotid atherosclerotic plaques were injected with the optical or dual-modality probes BMV109 and BMV101, respectively, via the tail vein and noninvasively imaged by optical and small-animal PET/CT at different time points. After noninvasive imaging, the murine carotid arteries were imaged in situ and ex vivo, followed by immunofluorescence staining to confirm target labeling. Additionally, human carotid plaques were topically labeled with the probe and analyzed by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunofluorescence staining to confirm the primary targets of the probe.
  
  
  RESULTS
  Quantitative analysis of the signal intensity from both optical and PET/CT imaging showed significantly higher levels of accumulation of BMV109 and BMV101 (P < 0.005 and P < 0.05, respectively) in the ligated left carotid arteries than the right carotid or healthy arteries. Immunofluorescence staining for macrophages in cross-sectional slices of the murine artery demonstrated substantial infiltration of macrophages in the neointima and adventitia of the ligated left carotid arteries compared with the right. Analysis of the human plaque tissues by sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed that the primary targets of the probe were cathepsins X, B, S, and L. Immunofluorescence labeling of the human tissue with the probe demonstrated colocalization of the probe with CD68, elastin, and cathepsin S, similar to that observed in the experimental carotid inflammation murine model.
  
  
  CONCLUSIONS
  We demonstrate that ABPs targeting the cysteine cathepsins can be used in murine models of atherosclerosis to noninvasively image activated macrophage populations using both optical and PET/CT methods. The probes could also be used to topically label human carotid plaques demonstrating similar specific labeling of activated macrophage populations. Therefore, ABPs targeting the cysteine cathepsins are potentially valuable new reagents for rapid and noninvasive imaging of atherosclerotic disease progression and plaque vulnerability.
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California.
authors.forename: Nimali P
authors.initials: NP
authors.lastname: Withana
authors.affiliation: Department of Medicine (Cardiovascular), Stanford University School of Medicine, Stanford, California.
authors.forename: Toshinobu
authors.initials: T
authors.lastname: Saito
authors.affiliation: Department of Radiology, Stanford University School of Medicine, Stanford, California.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Ma
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California.
authors.forename: Megan
authors.initials: M
authors.lastname: Garland
authors.affiliation: Department of Radiology, Stanford University School of Medicine, Stanford, California.
authors.forename: Changhao
authors.initials: C
authors.lastname: Liu
authors.affiliation: Department of Medicine (Cardiovascular), Stanford University School of Medicine, Stanford, California.
authors.forename: Hisanori
authors.initials: H
authors.lastname: Kosuge
authors.affiliation: Department of Medicine (Cardiovascular), Stanford University School of Medicine, Stanford, California.
authors.forename: Myriam
authors.initials: M
authors.lastname: Amsallem
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California.
authors.forename: Martijn
authors.initials: M
authors.lastname: Verdoes
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California.
authors.forename: Leslie O
authors.initials: LO
authors.lastname: Ofori
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California; and.
authors.forename: Michael
authors.initials: M
authors.lastname: Fischbein
authors.affiliation: Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, California; and.
authors.forename: Mamoru
authors.initials: M
authors.lastname: Arakawa
authors.affiliation: Department of Radiology, Stanford University School of Medicine, Stanford, California mbogyo@stanford.edu zcheng@stanford.edu.
authors.forename: Zhen
authors.initials: Z
authors.lastname: Cheng
authors.affiliation: Department of Medicine (Cardiovascular), Stanford University School of Medicine, Stanford, California.
authors.forename: Michael V
authors.initials: MV
authors.lastname: McConnell
authors.affiliation: Department of Pathology, Stanford University School of Medicine, Stanford, California Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California mbogyo@stanford.edu zcheng@stanford.edu.
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
mesh_terms: D000818:Animals; D050197:Atherosclerosis / Q000000981:diagnostic imaging* / Q000378:metabolism*; D002339:Carotid Arteries / Q000000981:diagnostic imaging* / Q000378:metabolism*; D002403:Cathepsins / Q000378:metabolism; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D015335:Molecular Probes / Q000378:metabolism*; D049268:Positron-Emission Tomography / Q000379:methods*
chemical_list: D015335:Molecular Probes; D002403:Cathepsins
keywords: PET; PET/CT; activity-based probes; animal imaging; atherosclerosis; cathepsins; inflammation; macrophages; optical; vascular