Multispectral Near-Infrared Fluorescence Molecular Imaging of Matrix Metalloproteinases in a Human Carotid Plaque Using a Matrix-Degrading Metalloproteinase–Sensitive Activatable Fluorescent Probe

Abstract

Formation of unstable atherosclerotic plaque in the internal carotid artery carries a high risk for emboli and subsequent cerebral ischemic events. The fibrous cap of such a plaque may become thin and rupture as a result of the depletion of matrix components through the activation of proteolytic enzymes such as matrix-degrading proteinases. Enhanced matrix breakdown has been attributed primarily to a family of matrix-degrading metalloproteinases (MMPs) that are highly concentrated in atherosclerotic plaques by inflammatory cells (eg, macrophages, foam cells), smooth muscle cells and endothelial cells.1 Elevated serum MMP-9 concentration is associated with carotid plaque instability and the presence of infiltrated macrophages.2 Furthermore, analysis of the presence of MMP-9 protein by ELISA within excised carotid plaques revealed high MMP-9 protein mass in calcified segments at or near the carotid bifurcation and in segments with intraplaque hemorrhage. Gelatin zymography showed an increased gelatinase activity of MMP-9 in these segments.3 These data favor the important role of MMP-9 in the pathogenesis of plaque instability. We analyzed the topographic distribution of MMPs within an excised human carotid plaque by applying multispectral near-infrared fluorescence (NIRF) imaging (IVIS Spectrum, Caliper Life Sciences, Hopkinton, Mass). A surgical endarterectomy was performed on a 74-year-old women with a left-sided, symptomatic, >70% carotid stenosis. Immediately after endarterectomy, the plaque was placed in PBS and transported to the NIRF system. The plaque was then stretched out and fixed on a silicon plate with 25G needles. A PBS NIRF image was generated from both the intraluminal and extraluminal side of the plaque to determine the level of autofluorescence (background) (Figure 1). After incubation with …