Abstract 648: Visualizing the Genesis of Microcalcifications via Matrix Vesicle Aggregation: Novel Implications for Plaque Vulnerability

Abstract

Background: Microcalcifications in the cap of vulnerable plaques contribute to plaque rupture. The mechanism by which microcalcifications form is poorly understood due to the inability to monitor initiating calcification events in vivo. We hypothesized that aggregation of cellular-derived matrix vesicles (MV) within atheroma lead to the formation of microcalcifications. Results: Transmission electron microscopy of ApoE-/- mouse plaques reveals MVs aggregating in regions of disrupted collagen (A). To image aggregation of calcifying MVs, we created a 3D collagen hydrogel system to simulate the atherosclerotic fibrous cap. Conditioned media from calcific human coronary artery smooth muscle cells was added to collagen hydrogels. Confocal microscopy reveals spherical microcalcifications (B, arrows) resembling those previously reported in human plaques. Super-resolution (< 200 nm) microscopy of the collagen hydrogels shows individual MVs aggregating (C, arrows) to form microcalcifications shown by a near-infrared calcium phosphate tracer (D). Scanning electron microscopy combined with x-ray spectroscopy confirms the presence of calcium phosphate mineral within MV aggregates (E, orange color). MV aggregation and calcification size is inversely related to the amount of collagen in our 3D model (p<0.05). Conclusion: To our knowledge, we are the first to directly visualize aggregation and calcification of MVs in real time. This approach presents a powerful tool to understand the genesis of microcalcifications from nucleating events. Aggregation of MVs within a thinning fibrous cap leads to the formation of microcalcifications, which further destabilize the cap.