BACKGROUND: Protease-sensing molecular optical imaging can potentially identify vulnerable atherosclerotic plaques before cerebral infarcts occur. However, several translational issues need to be resolved before implementing human trials and clinical applications of the technique. We investigated whether molecular optical imaging reflects (1) atherosclerosis pathophysiology and (2) the atherosclerosis-modulating effect of a Western diet or atorvastatin treatment in ApoE-knockout mice. METHODS: We fed 21 8-week-old ApoE-knockout mice a normal chow diet (n=7), a Western diet (n=7), or a Western diet with atorvastatin (n=7) for 14 weeks. Blood was collected for measuring cholesterol levels at 24 hours after injecting the cathepsin-B (CatB) probe into the tail vein. Near-infrared fluorescence reflectance imaging of CatB activity was performed after excising the aorta. RESULTS: CatB molecular optical imaging precisely reflected the pathophysiologic alteration of atherosclerosis. The CatB activity signal was significantly stronger in the Western diet group (49.3+/-26.0 [mean+/-SD], arbitrary units) than in the atorvastatin group (25.3+/-8.7) and the normal chow diet group (20.6+/-21.1). The effect of the statin on CatB activity was independent of cholesterol lowering. CONCLUSIONS: CatB-sensing molecular optical imaging represents a valuable tool for evaluating atherosclerosis pathophysiology and for monitoring the effects of antiatherosclerotic therapeutic interventions.