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.

Mesenchymal stem cell-like (MSC-like) cells reside in the vascular wall but their role in vascular regeneration and disease is poorly understood. Here, we show that Gli1+ cells located in the arterial adventitia are progenitors of vascular smooth muscle cells, and contribute to neointima formation and repair after acute injury to the femoral artery. Genetic fate tracing indicates that adventitial Gli1+ MSC-like cells migrate into the media and neointima during athero- and arteriosclerosis in ApoE−/− mice with chronic kidney disease. Our data indicate that Gli1+ cells are a major source of osteoblast-like cells during calcification in the media and intima. Genetic ablation of Gli1+ cells before induction of kidney injury dramatically reduced the severity of vascular calcification. These findings implicate Gli1+ cells as critical adventitial progenitors in vascular remodeling after acute and during chronic injury and suggest that they may be relevant therapeutic targets for mitigation of vascular calcification.

-No pharmacological therapy exists for calcific aortic valve disease (CAVD), which confers a dismal prognosis without invasive valve replacement. The search for therapeutics and early diagnostics is challenging since CAVD presents in multiple pathological stages. Moreover, it occurs in the context of a complex, multi-layered tissue architecture, a rich and abundant extracellular matrix phenotype, and a unique, highly plastic and multipotent resident cell population. -A total of 25 human stenotic aortic valves obtained from valve replacement surgeries were analyzed by multiple modalities, including transcriptomics and global unlabeled and label-based tandem-mass-tagged proteomics. Segmentation of valves into disease-stage-specific samples was guided by near infrared molecular imaging, and anatomical layer-specificity was facilitated by laser capture microdissection. Side-specific cell cultures were subjected to multiple calcifying stimuli, and their calcification potential and basal/stimulated proteomes were evaluated. Molecular (protein-protein) interaction networks were built and their central proteins and disease associations were identified. -Global transcriptional and protein expression signatures differed between the non-diseased, fibrotic, and calcific stages of CAVD. Anatomical aortic valve microlayers exhibited unique proteome profiles that were maintained throughout disease progression and identified glial fibrillary acidic protein (GFAP) as a specific marker of valvular interstitial cells (VICs) from the spongiosa layer. CAVD disease progression was marked by an emergence of smooth muscle cell activation, inflammation, and calcification-related pathways. Proteins overrepresented in the disease-prone fibrosa are functionally annotated to fibrosis and calcification pathways, and we found that , fibrosa-derived VICs demonstrated greater calcification potential than those from the ventricularis. These studies confirmed that the microlayer-specific proteome was preserved in cultured VICs, and that VICs exposed to ALPL-dependent and ALPL-independent calcifying stimuli had distinct proteome profiles, both of which overlapped with that of the whole tissue. Analysis of protein-protein interaction networks found a significant closeness to multiple inflammatory and fibrotic diseases. -A spatially- and temporally-resolved multi-omics, and network and systems biology strategy identifies the first molecular regulatory networks in CAVD, a cardiac condition without a pharmacological cure, and describes a novel means of systematic disease ontology that is broadly applicable to comprehensive omics studies of cardiovascular diseases.

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