title: Optical visualization of cathepsin K activity in atherosclerosis with a novel, protease-activatable fluorescence sensor.
abstract: BACKGROUND
  Cathepsin K (CatK), a potent elastinolytic and collagenolytic cysteine protease, likely participates in the evolution and destabilization of atherosclerotic plaques. To assess better the biology of CatK activity in vivo, we developed a novel near-infrared fluorescence (NIRF) probe for imaging of CatK and evaluated it in mouse and human atherosclerosis.
  
  
  RESULTS
  The NIRF imaging agent consists of the CatK peptide substrate GHPGGPQGKC-NH2 linked to an activatable fluorogenic polymer. In vitro, CatK produced a 2- to 14-fold activation of the agent over other cysteine and matrix metalloproteinases (P<0.0001), as well as a >8-fold activation over a control imaging agent (P<0.001). Optical imaging of atheroma revealed >100% NIRF signal increases in apolipoprotein E-/- mice in vivo (n=13; P<0.05, CatK imaging agent versus control agent) and in human carotid endarterectomy specimens ex vivo (n=14; P<0.05). Fluorescence microscopy of plaque sections demonstrated that enzymatically active CatK (positive NIRF signal) localized primarily in the vicinity of CatK-positive macrophages. Augmented NIRF signal (reflecting CatK activity) colocalized with disrupted elastin fibers within the media underlying plaques.
  
  
  CONCLUSIONS
  Use of this novel protease-activatable NIRF agent for optical imaging in vivo demonstrated preferential localization of enzymatically active CatK to macrophages, consistent with their known greater elastinolytic capabilities compared with smooth muscle cells. Augmented CatK proteolysis in atheromata further links CatK to vascular remodeling and plaque vulnerability.
authors.affiliation: Center for Molecular Imaging Research, Massachusetts General Hospital, Boston, MA 02129, USA. fjaffer@partners.org
authors.forename: Farouc A
authors.initials: FA
authors.lastname: Jaffer
authors.forename: Dong-Eog
authors.initials: DE
authors.lastname: Kim
authors.forename: Luisa
authors.initials: L
authors.lastname: Quinti
authors.forename: Ching-Hsuan
authors.initials: CH
authors.lastname: Tung
authors.forename: Elena
authors.initials: E
authors.lastname: Aikawa
authors.forename: Ashvin N
authors.initials: AN
authors.lastname: Pande
authors.forename: Rainer H
authors.initials: RH
authors.lastname: Kohler
authors.forename: Guo-Ping
authors.initials: GP
authors.lastname: Shi
authors.forename: Peter
authors.initials: P
authors.lastname: Libby
authors.forename: Ralph
authors.initials: R
authors.lastname: Weissleder
mesh_terms: D000818:Animals; D001057:Apolipoproteins E / Q000235:genetics; D002339:Carotid Arteries / Q000201:enzymology* / Q000473:pathology; D002340:Carotid Artery Diseases / Q000378:metabolism* / Q000473:pathology; D056657:Cathepsin K; D002403:Cathepsins / Q000378:metabolism*; D004549:Elastin / Q000378:metabolism; D004789:Enzyme Activation; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D007150:Immunohistochemistry; D008264:Macrophages / Q000201:enzymology / Q000473:pathology; D051379:Mice; D008817:Mice, Mutant Strains; D008856:Microscopy, Fluorescence / Q000379:methods*; D010447:Peptide Hydrolases; D019265:Spectroscopy, Near-Infrared / Q000379:methods*
chemical_list: D001057:Apolipoproteins E; D005456:Fluorescent Dyes; D004549:Elastin; D002403:Cathepsins; D010447:Peptide Hydrolases; C534130:CTSK protein, human; D056657:Cathepsin K; C534132:Ctsk protein, mouse; D016650:Fluorescein-5-isothiocyanate