Objective-Proteases are emerging biomarkers of inflammatory diseases. In atherosclerosis, these enzymes are often secreted by inflammatory macrophages, digest the extracellular matrix of the fibrous cap, and destabilize atheromata. Protease function can be monitored with protease activatable imaging probes and quantitated in vivo by fluorescence molecular tomography (FMT). To address 2 major constraints currently associated with imaging of murine atherosclerosis (lack of highly sensitive probes and absence of anatomic information), we compared protease sensors (PS) of variable size and pharmacokinetics and coregistered FMT datasets with computed tomography (FMT-CT). Methods and Results-Coregistration of FMT and CT was achieved with a multimodal imaging cartridge containing fiducial markers detectable by both modalities. A high-resolution CT angiography protocol accurately localized fluorescence to the aortic root of atherosclerotic apoE Ϫ/Ϫ mice. To identify suitable sensors, we first modeled signal kinetics in-silico and then compared 3 probes with oligo-L-lysine cleavage sequences: PS-5, 5 nm in diameter containing 2 fluorochromes, PS-25, a 25-nm version with an elongated lysine chain and PS-40, a polymeric nanoparticle. Serial FMT-CT showed fastest kinetics for PS-5 but, surprisingly, highest fluorescence in lesions of the aortic root for PS-40. PS-40 robustly reported therapeutic effects of atorvastatin, corroborated by ex vivo imaging and qPCR for the model protease cathepsin B. Key Words: FMT-CT Ⅲ molecular imaging Ⅲ atherosclerosis Ⅲ protease activity Ⅲ inflammation A thin fibrous cap, presence of macrophages and foam cells, and increased activity of proteases are hallmarks of atherosclerotic lesions at risk for causing myocardial infarction and stroke. 1 Typically, the fibrous cap and the endothelial layer are damaged because of heightened inflammatory activity, thus exposing the thrombogenic plaque core to platelets and coagulation factors in the blood stream. This triggers thrombotic occlusion of the artery followed by ischemic injury of downstream tissue. 1 Positive remodeling of the artery may obscure detection of inflamed plaques as the vessel lumen can be virtually unchanged, while some protruding plaques that cause significant stenosis on angiography are fairly stable. 2 Hence, protease activity in a lesion may have a higher predictive value for ischemic events than reported for the anatomic degree of stenosis. 3 Protease presence in atherosclerotic lesions has been imaged with radiolabeled small molecule inhibitors 4,5 and more recently with a MRI detectable Gd chelate attached to an affinity peptide targeting MMPs. 6 Fluorescent protease sensors (PS), which are injected in an inactive form and only emit signal after cleavage of a peptide sequence specific to their target enzyme, have been used to image protease activity. 7 Cysteine proteases are mainly expressed by monocytes/macrophages, 8,9 cells which orchestrate atherosclerotic lesion development and destabilization. They promote digestio...

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