-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.

Extracellular vesicles (EVs) including plasma membrane–derived microvesicles and endosomal-derived exosomes aggregate by unknown mechanisms, forming microcalcifications that promote cardiovascular disease, the leading cause of death worldwide. Here, we show a framework for assessing cell-independent EV mechanisms in disease by suggesting that annexin A1 (ANXA1)–dependent tethering induces EV aggregation and microcalcification. We present single-EV microarray, a method to distinguish microvesicles from exosomes and assess heterogeneity at a single-EV level. Single-EV microarray and proteomics revealed increased ANXA1 primarily on aggregating and calcifying microvesicles. ANXA1 vesicle aggregation was suppressed by calcium chelation, altering pH, or ANXA1 neutralizing antibody. ANXA1 knockdown attenuated EV aggregation and microcalcification formation in human cardiovascular cells and acellular three-dimensional collagen hydrogels. Our findings explain why microcalcifications are more prone to form in vulnerable regions of plaque, regulating critical cardiovascular pathology, and likely extend to other EV-associated diseases, including autoimmune and neurodegenerative diseases and cancer.

Calcific aortic valve disease (CAVD), once thought to be a degenerative disease, is now recognized to be an active pathobiological process, with chronic inflammation emerging as a predominant, and possibly driving, factor. However, many details of the pathobiological mechanisms of CAVD remain to be described, and new approaches to treat CAVD need to be identified. Animal models are emerging as vital tools to this end, facilitated by the advent of new models and improved understanding of the utility of existing models. In this paper, we summarize and critically appraise current small and large animal models of CAVD, discuss the utility of animal models for priority CAVD research areas, and provide recommendations for future animal model studies of CAVD.

Background: Microcalcifications in atherosclerotic plaques are destabilizing, predict adverse cardiovascular events, and are associated with increased morbidity and mortality. 18F-fluoride PET/CT imaging has demonstrated promise as a useful clinical diagnostic tool in identifying high risk plaques; however, there is confusion as to the underlying mechanism of signal amplification seen in PET-positive, CT-negative image regions. This study tested the hypothesis that 18F-fluoride PET/CT can identify early microcalcifications. Methods and Results: 18F-fluoride signal amplification derived from microcalcifications was validated against near infrared fluorescence (NIRF) molecular imaging and histology using an in vitro 3D hydrogel collagen platform, ex vivo human specimens, and a mouse model of atherosclerosis. Microcalcification size correlated inversely with collagen concentration. The 18F-fluoride ligand bound to microcalcifications formed by calcifying vascular smooth muscle cell-derived extracellular vesicles in the in vitro 3D collagen system and exhibited an increasing signal with an increase in collagen concentration (0.25mg/ml collagen - 33.8×102±12.4×102 CPM; 0.5 mg/ml collagen - 67.7×102±37.4×102 CPM, p=0.0014), suggesting amplification of the PET signal by smaller microcalcifications. We further incubated human atherosclerotic endarterectomy specimens with clinically-relevant concentrations of 18F-fluoride. The 18F-fluoride ligand labeled microcalcifications in PET-positive, CT-negative regions of explanted human specimens as evidenced by 18F-fluoride PET/CT imaging, NIRF and histological analysis. Additionally, the 18F-fluoride ligand identified micro- and macrocalcifications in atherosclerotic aortas obtained from LDLr-deficient mice. Conclusions: Our results suggest that 18F-fluoride PET signal in PET-positive, CT-negative regions of human atherosclerotic plaques is the result of developing microcalcifications, and high surface area in regions of small microcalcifications may amplify PET signal.