Cardiovascular calcification, a growing burden in Westernized countries, is not only a risk factor for cardiovascular events, but may itself contribute to cardiovascular risk. Research into treatment of cardiovascular calcification is lacking, as shown by clinical trials that have failed to demonstrate the reduction of calcific aortic stenosis. Hence the need to elucidate the pathways that contribute to cardiovascular calcification and to develop new therapeutic strategies to prevent or reverse calcification has driven our research investigations. We previously showed that early calcification/microcalcification associates with macrophage accumulation in vulnerable atherosclerotic plaques. Chronic renal disease (CRD) accelerates calcification and the subsequent release of matrix vesicles (MVs) — precursors of microcalcifications. We tested the hypothesis that macrophage-derived MVs contribute directly to microcalcifications. We showed that macrophages associated with regions of calcified vesicular structures in human carotid plaque samples (n=136 patients). In vitro, macrophages released MVs with high calcification potential. MVs expressed exosomal markers (CD9 and TSG101), and contained S100A9 and annexin V (Anx5). Silencing S100A9 in vitro and genetic deficiency in S100A9-/- mice reduced MV calcification, while stimulation with S100A9 increased calcification potential. Externalization of phosphatidylserine (PS) after Ca/P stimulation, and interaction of S100A9 and Anx5, indicated that a PS-Anx5-S100A9 membrane complex facilitates hydroxyapatite nucleation within the macrophage-derived MV membrane. These results supported the novel concept that macrophages release calcifying MVs, which contribute to accelerated formation of microcalcification, thus providing an alternative mechanism of calcification as opposed to osteogenic differentiation. This presentation will discuss studies that have used molecular imaging to advance knowledge of cardiovascular calcification, focusing in particular on the alternative mechanisms of vascular calcification via the release of calcifying matrix vesicles.