18F-Fluoride Signal Amplification Identifies Microcalcifications Associated With Atherosclerotic Plaque Instability in Positron Emission Tomography/Computed Tomography Images

BACKGROUND: Microcalcifications in atherosclerotic plaques are destabilizing, predict adverse cardiovascular events, and are associated with increased morbidity and mortality. F-18-fluoride positron emission tomography (PET)/computed tomography (CT) imaging has demonstrated promise as a useful clinical diagnostic tool in identifying high-risk plaques; however, there is confusion as to the underlying mechanism of signal amplification seen in PET-positive, CT-negative image regions. This study tested the hypothesis that F-18-fluoride PET/CT can identify early microcalcifications. METHODS: F-18-fluoride signal amplification derived from microcalcifications was validated against near-infrared fluorescence molecular imaging and histology using an in vitro 3-dimensional hydrogel collagen platform, ex vivo human specimens, and a mouse model of atherosclerosis. RESULTS: Microcalcification size correlated inversely with collagen concentration. The F-18-fluoride ligand bound to microcalcifications formed by calcifying vascular smooth muscle cell derived extracellular vesicles in the in vitro 3-dimensional collagen system and exhibited an increasing signal with an increase in collagen concentration (0.25 mg/mL collagen -33.8x10(2)+/- 12.4x10(2) counts per minute; 0.5 mg/mL collagen -67.7x10(2)+/- 37.4x10(2) counts per minute; P=0.0014), suggesting amplification of the PET signal by smaller microcalcifications. We further incubated human atherosclerotic endarterectomy specimens with clinically relevant concentrations of F-18-fluoride. The F-18-fluoride ligand labeled microcalcifications in PET-positive, CT-negative regions of explanted human specimens as evidenced by F-18-fluoride PET/CT imaging, near-infrared fluorescence, and histological analysis. Additionally, the F-18-fluoride ligand identified micro and macrocalcifications in atherosclerotic aortas obtained from low-density lipoprotein receptor-deficient mice. CONCLUSIONS: Our results suggest that F-18-fluoride PET signal in PET-positive, CT-negative regions of human atherosclerotic plaques is the result of developing microcalcifications, and high surface area in regions of small microcalcifications may amplify PET signal.