Abstract 1207: In Vivo Molecular Imaging Links Macrophage Burden and Microcalcifications in Early Atherosclerosis

Abstract

Backround Microcalcifications may provoke plaque rupture leading to acute cardiovascular events. However, the mechanisms that initiate arterial calcification remain obscure. Methods and Results We tested the hypothesis that inflammation promotes osteogenesis in atherosclerotic plaques using in vivo molecular imaging in apoE −/− mice (n=40). A bisphosphonate-derivatized near-infrared fluorescent (NIRF) imaging agent (750 nm) visualized osteogenic activity that was otherwise undetectable by X-ray computed tomography. Flow cytometry validated target cells as osteoblasts. A spectrally distinct NIRF nanoparticle (680 nm) was coinjected to simultaneously image macrophages. Fluorescence reflectance mapping associated osteogenic activity with inflammation in aortas of apoE −/− mice. Intravital dual-channel fluorescent microscopy further monitored osteogenic changes in carotid plaques at 20 and 30 weeks of age and revealed that both macrophage burden and osteogenesis concomitantly increased during plaque progression and decreased after statin treatment (Figure ) . Fluorescence microscopy on cryosections colocalized NIRF osteogenic signals with alkaline phosphatase activity, bone-regulating proteins and hydroxyapatite nanocrystals detected by electron microscopy, while von Kossa staining showed no evidence of calcification. Real-time RT-PCR revealed that macrophage-conditioned media induced alkaline phosphatase mRNA expression in vascular smooth muscle cells. Conclusion This serial in vivo study detects osteogenic activity within macrophage-rich plaques, offering a cellular resolution tool to identify preclinical microcalcifications.