The collective global impact of the spectrum calcific cardiovascular diseases is serious but underappreciated health problem in the developed and rapidly developing regions of the world. Cardiovascular calcification is an independent risk factor for cardiovascular morbidity and mortality. Ectopic mineralization mainly affects the aorta, coronary arteries, peripheral arteries, and aortic valves, with fully-formed bone observed in atherosclerotic plaques and stenotic aortic valves. This disease of dysregulated metabolism is no longer viewed as a passive degenerative disorder, but instead as an active process triggered by pro-inflammatory cues. Hypercholesterolemia, metabolic syndrome, end-stage renal disease, diabetes mellitus and increased age accelerate cardiovascular calcification. Traditional imaging modalities such as computed tomography, although perfectly adept at identifying and quantifying advanced calcification, cannot detect the early stages of this disorder and offer limited insight into the mechanisms of mineral dysregulation. Here we present optical molecular imaging as a promising tool that simultaneously detects pathobiological processes associated with inflammation and early stages of calcification in vivo at the (sub)cellular levels. 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 investigations. We previously showed that early calcification/microcalcification associates with macrophage accumulation in vulnerable atherosclerotic plaques. Chronic renal disease (CRD) and mineral imbalance accelerates calcification and the subsequent release of matrix vesicles (MVs) — precursors of microcalcification. We tested the hypothesis that macrophage-derived MVs contribute directly to microcalcifications, which in turn may contribute to plaque rupture. 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 and aggregation 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, enriched in S100A9 and Anx5, which contribute to accelerated formation of microcalcification in CRD. This presentation will discuss studies that have used molecular imaging methods to advance knowledge of cardiovascular calcification, focusing in particular on the inflammation-dependent mechanisms of arterial and aortic valve calcification.