Optical visualization of cathepsin K activity in atherosclerosis with a novel, protease-activatable fluorescence sensor.
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.
Jaffer
Farouc A
FA
Center for Molecular Imaging Research, Massachusetts General Hospital, Boston, MA 02129, USA. fjaffer@partners.org
Kim
Dong-Eog
DE
Quinti
Luisa
L
Tung
Ching-Hsuan
CH
Aikawa
Elena
E
Pande
Ashvin N
AN
Kohler
Rainer H
RH
Shi
Guo-Ping
GP
Libby
Peter
P
Weissleder
Ralph
R
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*
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