Clinical evidence links arterial calcification and cardiovascular risk. Finite-element modelling of the stress distribution within atherosclerotic plaques has suggested that subcellular microcalcifications in the fibrous cap may promote material failure of the plaque, but that large calcifications can stabilize it. Yet the physicochemical mechanisms underlying such mineral formation and growth in atheromata remain unknown. Here, by using three-dimensional collagen hydrogels that mimic structural features of the atherosclerotic fibrous cap, and high-resolution microscopic and spectroscopic analyses of both the hydrogels and of calcified human plaques, we demonstrate that calcific mineral formation and maturation results from a series of events involving the aggregation of calcifying extracellular vesicles, and the formation of microcalcifications and ultimately large calcification zones. We also show that calcification morphology and the plaque’s collagen content – two determinants of atherosclerotic plaque stability - are interlinked.

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Background-The evolution of cell phenotypes and matrix architecture in cardiac valves during fetal maturation and postnatal adaptation through senescence remains unexplored. Methods and Results-We hypothesized that valvular interstitial (VIC) and endothelial cell (VEC) phenotypes, critical for maintaining valve function, change throughout life in response to environmental stimuli. We performed quantitative histological assessment of 91 human semilunar valves obtained from fetuses at 14 to 19 and 20 to 39 weeks' gestation; neonates minutes to 30 days old; children aged 2 to 16 years; and adults. A trilaminar architecture appeared by 36 weeks of gestation but remained rudimentary compared with that of adult valves. VECs expressed an activated phenotype throughout fetal development. VIC density, proliferation, and apoptosis were significantly higher in fetal than adult valves. Pulmonary and aortic fetal VICs showed an activated myofibroblast-like phenotype (␣-actin expression), abundant embryonic myosin, and matrix metalloproteinase-collagenases, which indicates an immature/activated phenotype engaged in matrix remodeling versus a quiescent fibroblast-like phenotype in adults. At birth, the abrupt change from fetal to neonatal circulation was associated with a greater number of ␣-actin-positive VICs in neonatal aortic versus pulmonary valves. Collagen content increased from early to late fetal stages but was subsequently unchanged, whereas elastin significantly increased postnatally. Collagen fiber color analysis revealed a progressive temporal decrease in thin fibers and a corresponding increase in thick fibers. Additionally, collagen fibers were more aligned in adult than fetal valves. Conclusions-Fetal