Abstract 1725: Statin Therapy Reduces Cysteine Protease Activity in Atherosclerosis in vivo
Introduction: Recent investigations demonstrate that statins can suppress protease activity in atherosclerosis. Here we imaged statin-mediated reductions in cysteine proteolysis using a near infrared fluorescence (NIRF) imaging agent and in vivo laser scanning fluorescence microscopy (LSFM).
Methods: ApoE-/- mice (age 26 wks) were randomized to continue a high-cholesterol diet (HCD, n=6) or HCD with admixed statin (n=5, 0.01% w/w atorvastatin) for 8 weeks. Mice were then injected with a protease-activatable imaging agent (Prosense-680, 200 nmol/kg, VisEn Medical). After 24 hours, mice underwent intravital LSFM (6x6 micron resolution, Olympus, Japan) and injection of an intravascular agent (Angiosense-750). Z-stack projections underwent region-of-interest analysis, with inflamed pixels defined as >2000 fluorescence units. Resected aortas then underwent fluorescence reflectance imaging (FRI) and histopathological analysis.
Results: LSFM revealed focal NIRF protease signal in atheroma (Figure A⇓, green=protease activity, red=intravascular agent). In statin-treated animals, LSFM z-stacks revealed significantly less proteolytic plaque signal per pixel (p<0.05, Figure B⇓). Findings were confirmed on ex vivo FRI (47% signal reduction in the statin group, p<0.05). NIRF signal reductions in plaque sections correlated with decreases in the cysteine protease cathepsin B on immunohistochemistry and immunoblotting.
Conclusions: Statin treatment reduces cysteine protease activity in atheroma, and this effect can be imaged in vivo. These results show that protease imaging can serve as an in vivo biological readout of anti-atherosclerotic interventions.