Strategies to reduce the risks of mRNA drug and vaccine toxicity.

mRNA formulated with lipid nanoparticles is a transformative technology that has enabled the rapid development and administration of billions of coronavirus disease 2019 (COVID-19) vaccine doses worldwide. However, avoiding unacceptable toxicity with mRNA drugs and vaccines presents challenges. Lipid nanoparticle structural components, production methods, route of administration and proteins produced from complexed mRNAs all present toxicity concerns. Here, we discuss these concerns, specifically how cell tropism and tissue distribution of mRNA and lipid nanoparticles can lead to toxicity, and their possible reactogenicity. We focus on adverse events from mRNA applications for protein replacement and gene editing therapies as well as vaccines, tracing common biochemical and cellular pathways. The potential and limitations of existing models and tools used to screen for on-target efficacy and de-risk off-target toxicity, including in vivo and next-generation in vitro models, are also discussed.

Major Facilitator Superfamily Domain Containing 5 Inhibition Reduces Lipoprotein(a) Uptake and Calcification in Valvular Heart Disease.

BACKGROUND: High circulating levels of Lp(a) (lipoprotein[a]) increase the risk of atherosclerosis and calcific aortic valve disease, affecting millions of patients worldwide. Although atherosclerosis is commonly treated with low-density lipoprotein-targeting therapies, these do not reduce Lp(a) or risk of calcific aortic valve disease, which has no available drug therapies. Targeting Lp(a) production and catabolism may provide therapeutic benefit, but little is known about Lp(a) cellular uptake. METHODS: Here, unbiased ligand-receptor capture mass spectrometry was used to identify MFSD5 (major facilitator superfamily domain containing 5) as a novel receptor/cofactor involved in Lp(a) uptake. RESULTS: Reducing MFSD5 expression by a computationally identified small molecule or small interfering RNA suppressed Lp(a) uptake and calcification in primary human valvular endothelial and interstitial cells. MFSD5 variants were associated with aortic stenosis (P=0.027 after multiple hypothesis testing) with evidence suggestive of an interaction with plasma Lp(a) levels. CONCLUSIONS: MFSD5 knockdown suppressing human valvular cell Lp(a) uptake and calcification, along with meta-analysis of MFSD5 variants associating with aortic stenosis, supports further preclinical assessment of MFSD5 in cardiovascular diseases, the leading cause of death worldwide.

Multiomics of Tissue Extracellular Vesicles Identifies Unique Modulators of Atherosclerosis and Calcific Aortic Valve Stenosis.

BACKGROUND: Fewer than 50% of patients who develop aortic valve calcification have concomitant atherosclerosis, implying differential pathogenesis. Although circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs are associated with early mineralization, but their cargoes, functions, and contributions to disease remain unknown. METHODS: Disease stage-specific proteomics was performed on human carotid endarterectomy specimens (n=16) and stenotic aortic valves (n=18). Tissue EVs were isolated from human carotid arteries (normal, n=6; diseased, n=4) and aortic valves (normal, n=6; diseased, n=4) by enzymatic digestion, (ultra)centrifugation, and a 15-fraction density gradient validated by proteomics, CD63-immunogold electron microscopy, and nanoparticle tracking analysis. Vesiculomics, comprising vesicular proteomics and small RNA-sequencing, was conducted on tissue EVs. TargetScan identified microRNA targets. Pathway network analyses prioritized genes for validation in primary human carotid artery smooth muscle cells and aortic valvular interstitial cells. RESULTS: Disease progression drove significant convergence (P<0.0001) of carotid artery plaque and calcified aortic valve proteomes (2318 proteins). Each tissue also retained a unique subset of differentially enriched proteins (381 in plaques; 226 in valves; q<0.05). Vesicular gene ontology terms increased 2.9-fold (P<0.0001) among proteins modulated by disease in both tissues. Proteomics identified 22 EV markers in tissue digest fractions. Networks of proteins and microRNA targets changed by disease progression in both artery and valve EVs revealed shared involvement in intracellular signaling and cell cycle regulation. Vesiculomics identified 773 proteins and 80 microRNAs differentially enriched by disease exclusively in artery or valve EVs (q<0.05); multiomics integration found tissue-specific EV cargoes associated with procalcific Notch and Wnt signaling in carotid arteries and aortic valves, respectively. Knockdown of tissue-specific EV-derived molecules FGFR2, PPP2CA, and ADAM17 in human carotid artery smooth muscle cells and WNT5A, APP, and APC in human aortic valvular interstitial cells significantly modulated calcification. CONCLUSIONS: The first comparative proteomics study of human carotid artery plaques and calcified aortic valves identifies unique drivers of atherosclerosis versus aortic valve stenosis and implicates EVs in advanced cardiovascular calcification. We delineate a vesiculomics strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Integration of vesicular proteomics and transcriptomics by network approaches revealed novel roles for tissue EVs in modulating cardiovascular disease.

Sortilin enhances fibrosis and calcification in aortic valve disease by inducing interstitial cell heterogeneity.

AIMS: Calcific aortic valve disease (CAVD) is the most common valve disease, which consists of a chronic interplay of inflammation, fibrosis, and calcification. In this study, sortilin (SORT1) was identified as a novel key player in the pathophysiology of CAVD, and its role in the transformation of valvular interstitial cells (VICs) into pathological phenotypes is explored. METHODS AND RESULTS: An aortic valve (AV) wire injury (AVWI) mouse model with sortilin deficiency was used to determine the effects of sortilin on AV stenosis, fibrosis, and calcification. In vitro experiments employed human primary VICs cultured in osteogenic conditions for 7, 14, and 21 days; and processed for imaging, proteomics, and transcriptomics including single-cell RNA-sequencing (scRNA-seq). The AVWI mouse model showed reduced AV fibrosis, calcification, and stenosis in sortilin-deficient mice vs. littermate controls. Protein studies identified the transition of human VICs into a myofibroblast-like phenotype mediated by sortilin. Sortilin loss-of-function decreased in vitro VIC calcification. ScRNA-seq identified 12 differentially expressed cell clusters in human VIC samples, where a novel combined inflammatory myofibroblastic-osteogenic VIC (IMO-VIC) phenotype was detected with increased expression of SORT1, COL1A1, WNT5A, IL-6, and serum amyloid A1. VICs sequenced with sortilin deficiency showed decreased IMO-VIC phenotype. CONCLUSION: Sortilin promotes CAVD by mediating valvular fibrosis and calcification, and a newly identified phenotype (IMO-VIC). This is the first study to examine the role of sortilin in valvular calcification and it may render it a therapeutic target to inhibit IMO-VIC emergence by simultaneously reducing inflammation, fibrosis, and calcification, the three key pathological processes underlying CAVD.

Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Promotes Macrophage Activation via LDL Receptor-Independent Mechanisms.

BACKGROUND: Activated macrophages contribute to the pathogenesis of vascular disease. Vein graft failure is a major clinical problem with limited therapeutic options. PCSK9 (proprotein convertase subtilisin/kexin 9) increases low-density lipoprotein (LDL)-cholesterol levels via LDL receptor (LDLR) degradation. The role of PCSK9 in macrophage activation and vein graft failure is largely unknown, especially through LDLR-independent mechanisms. This study aimed to explore a novel mechanism of macrophage activation and vein graft disease induced by circulating PCSK9 in an LDLR-independent fashion. METHODS: We used Ldlr-/- mice to examine the LDLR-independent roles of circulating PCSK9 in experimental vein grafts. Adeno-associated virus (AAV) vector encoding a gain-of-function mutant of PCSK9 (rAAV8/D377Y-mPCSK9) induced hepatic PCSK9 overproduction. To explore novel inflammatory targets of PCSK9, we used systems biology in Ldlr-/- mouse macrophages. RESULTS: In Ldlr-/- mice, AAV-PCSK9 increased circulating PCSK9, but did not change serum cholesterol and triglyceride levels. AAV-PCSK9 promoted vein graft lesion development when compared with control AAV. In vivo molecular imaging revealed that AAV-PCSK9 increased macrophage accumulation and matrix metalloproteinase activity associated with decreased fibrillar collagen, a molecular determinant of atherosclerotic plaque stability. AAV-PCSK9 induced mRNA expression of the pro-inflammatory mediators IL-1ß (interleukin-1 beta), TNFα (tumor necrosis factor alpha), and MCP-1 (monocyte chemoattractant protein-1) in peritoneal macrophages underpinned by an in vitro analysis of Ldlr-/- mouse macrophages stimulated with endotoxin-free recombinant PCSK9. A combination of unbiased global transcriptomics and new network-based hyperedge entanglement prediction analysis identified the NF-κB (nuclear factor-kappa B) signaling molecules, lectin-like oxidized LOX-1 (LDL receptor-1), and SDC4 (syndecan-4) as potential PCSK9 targets mediating pro-inflammatory responses in macrophages. CONCLUSIONS: Circulating PCSK9 induces macrophage activation and vein graft lesion development via LDLR-independent mechanisms. PCSK9 may be a potential target for pharmacologic treatment for this unmet medical need.

Acat1/Soat1 knockout extends the mutant Npc1 mouse lifespan and ameliorates functional deficiencies in multiple organelles of mutant cells.

Multiple membrane organelles require cholesterol for proper function within cells. The Niemann-Pick type C (NPC) proteins export cholesterol from endosomes to other membrane compartments, including the endoplasmic reticulum (ER), plasma membrane (PM), trans-Golgi network (TGN), and mitochondria, to meet their cholesterol requirements. Defects in NPC cause malfunctions in multiple membrane organelles and lead to an incurable neurological disorder. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), a resident enzyme in the ER, converts cholesterol to cholesteryl esters for storage. In mutant NPC cells, cholesterol storage still occurs in an NPC-independent manner. Here we report the interesting finding that in a mutant Npc1 mouse (Npc1nmf), Acat1 gene (Soat1) knockout delayed the onset of weight loss, motor impairment, and Purkinje neuron death. It also improved hepatosplenic pathology and prolonged lifespan by 34%. In mutant NPC1 fibroblasts, ACAT1 blockade (A1B) increased cholesterol content associated with TGN-rich membranes and mitochondria, while decreased cholesterol content associated with late endosomes. A1B also restored proper localization of syntaxin 6 and golgin 97 (key proteins in membrane trafficking at TGN) and improved the levels of cathepsin D (a key protease in lysosome and requires Golgi/endosome transport for maturation) and ABCA1 (a key protein controlling cholesterol release at PM). This work supports the hypothesis that diverting cholesterol from storage can benefit multiple diseases that involve cholesterol deficiencies in cell membranes.

Lipoprotein(a) Induces Vesicular Cardiovascular Calcification Revealed With Single-Extracellular Vesicle Analysis.

Lipoprotein(a) (Lp[a]) blood levels >50 mg/dL is a major cardiovascular disease risk factor in humans. Lp(a) associates with increased cardiovascular calcification, a critical pathology with no clinically available drug therapies. The mechanisms through which Lp(a) increases cardiovascular calcification risk remain undefined. We hypothesized that Lp(a) promotes the release of calcifying extracellular vesicles (EVs) that contribute to formation of microcalcification in cardiovascular tissues. Here, we show Lp(a) increased calcification in both primary human smooth muscle cells (SMCs) and valvular interstitial cells (VICs), potentially through inflammation-related mechanisms that were suppressed with E06 antibody that neutralizes pro-inflammatory oxidized phospholipids. Incubating human SMCs and VICs with Lp(a) altered the composition of EVs, increasing CD29+/tetraspanin- microvesicle release, demonstrated with a tailored single-EV microarray assay that can distinguish multivesicular body-derived exosomes and plasma membrane budded microvesicles at a single-vesicle level. Lp(a) stimulation led to release of SMC and VIC EVs that readily calcified in acellular 3D-collagen hydrogels mimicking formation of ectopic microcalcification occurring in extracellular matrix of human atherosclerotic arteries and stenotic aortic valves. Our study mechanistically demonstrates that Lp(a) partially mediates cardiovascular calcification formation via inducing the release of calcifying EVs. Additionally, we provide a customized method to assess calcifying EVs at a single-vesicle level that can be more broadly applied to assist in quantitatively differentiating exosome and microvesicle EV subpopulations.

Dynamin-related protein 1 inhibition reduces hepatic PCSK9 secretion.

AIMS: Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis. METHODS AND RESULTS: We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages. CONCLUSIONS: We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.

ApoC-III is a novel inducer of calcification in human aortic valves.

Calcific aortic valve disease (CAVD) occurs when subpopulations of valve cells undergo specific differentiation pathways, promoting tissue fibrosis and calcification. Lipoprotein particles carry oxidized lipids that promote valvular disease, but low-density lipoprotein-lowering therapies have failed in clinical trials, and there are currently no pharmacological interventions available for this disease. Apolipoproteins are known promoters of atherosclerosis, but whether they possess pathogenic properties in CAVD is less clear. To search for a possible link, we assessed 12 apolipoproteins in nonfibrotic/noncalcific and fibrotic/calcific aortic valve tissues by proteomics and immunohistochemistry to understand if they were enriched in calcified areas. Eight apolipoproteins (apoA-I, apoA-II, apoA-IV, apoB, apoC-III, apoD, apoL-I, and apoM) were enriched in the calcific versus nonfibrotic/noncalcific tissues. Apo(a), apoB, apoC-III, apoE, and apoJ localized within the disease-prone fibrosa and colocalized with calcific regions as detected by immunohistochemistry. Circulating apoC-III on lipoprotein(a) is a potential biomarker of aortic stenosis incidence and progression, but whether apoC-III also induces aortic valve calcification is unknown. We found that apoC-III was increased in fibrotic and calcific tissues and observed within the calcification-prone fibrosa layer as well as around calcification. In addition, we showed that apoC-III induced calcification in primary human valvular cell cultures via a mitochondrial dysfunction/inflammation-mediated pathway. This study provides a first assessment of a broad array of apolipoproteins in CAVD tissues, demonstrates that specific apolipoproteins associate with valvular calcification, and implicates apoC-III as an active and modifiable driver of CAVD beyond its potential role as a biomarker.

CROT (Carnitine O-Octanoyltransferase) Is a Novel Contributing Factor in Vascular Calcification via Promoting Fatty Acid Metabolism and Mitochondrial Dysfunction.

OBJECTIVE: Vascular calcification is a critical pathology associated with increased cardiovascular event risk, but there are no Food and Drug Administration-approved anticalcific therapies. We hypothesized and validated that an unbiased screening approach would identify novel mediators of human vascular calcification. Approach and Results: We performed an unbiased quantitative proteomics and pathway network analysis that identified increased CROT (carnitine O-octanoyltransferase) in calcifying primary human coronary artery smooth muscle cells (SMCs). Additionally, human carotid artery atherosclerotic plaques contained increased immunoreactive CROT near calcified regions. CROT siRNA reduced fibrocalcific response in calcifying SMCs. In agreement, histidine 327 to alanine point mutation inactivated human CROT fatty acid metabolism enzymatic activity and suppressed SMC calcification. CROT siRNA suppressed type 1 collagen secretion, and restored mitochondrial proteome alterations, and suppressed mitochondrial fragmentation in calcifying SMCs. Lipidomics analysis of SMCs incubated with CROT siRNA revealed increased eicosapentaenoic acid, a vascular calcification inhibitor. CRISPR/Cas9-mediated Crot deficiency in LDL (low-density lipoprotein) receptor-deficient mice reduced aortic and carotid artery calcification without altering bone density or liver and plasma cholesterol and triglyceride concentrations. CONCLUSIONS: CROT is a novel contributing factor in vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction, as such CROT inhibition has strong potential as an antifibrocalcific therapy.

Annexin A1-dependent tethering promotes extracellular vesicle aggregation revealed with single-extracellular vesicle analysis.

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.

Retinoids Repress Human Cardiovascular Cell Calcification With Evidence for Distinct Selective Retinoid Modulator Effects.

OBJECTIVE: Retinoic acid (RA) is a ligand for nuclear receptors that modulate gene transcription and cell differentiation. Whether RA controls ectopic calcification in humans is unknown. We tested the hypothesis that RA regulates osteogenic differentiation of human arterial smooth muscle cells and aortic valvular interstitial cells that participate in atherosclerosis and heart valve disease, respectively. Approach and Results: Human cardiovascular tissue contains immunoreactive RAR (RA receptor)-a retinoid-activated nuclear receptor directing multiple transcriptional programs. RA stimulation suppressed primary human cardiovascular cell calcification while treatment with the RAR inhibitor AGN 193109 or RARα siRNA increased calcification. RA attenuated calcification in a coordinated manner, increasing levels of the calcification inhibitor MGP (matrix Gla protein) while decreasing calcification-promoting TNAP (tissue nonspecific alkaline phosphatase) activity. Given that nuclear receptor action varies as a function of distinct ligand structures, we compared calcification responses to cyclic retinoids and the acyclic retinoid peretinoin. Peretinoin suppressed human cardiovascular cell calcification without inducing either secretion of APOC3 (apolipoprotein-CIII), which promotes atherogenesis, or reducing CYP7A1 (cytochrome P450 family 7 subfamily A member 1) expression, which occurred with cyclic retinoids all-trans RA, 9-cis RA, and 13-cis RA. Additionally, peretinoin did not suppress human femur osteoblast mineralization, whereas all-trans RA inhibited osteoblast mineralization. CONCLUSIONS: These results establish retinoid regulation of human cardiovascular calcification, provide new insight into mechanisms involved in these responses, and suggest selective retinoid modulators, like acyclic retinoids may allow for treating cardiovascular calcification without the adverse effects associated with cyclic retinoids.

Lipoprotein(a) and Oxidized Phospholipids Promote Valve Calcification in Patients With Aortic Stenosis.

BACKGROUND: Lipoprotein(a) [Lp(a)], a major carrier of oxidized phospholipids (OxPL), is associated with an increased incidence of aortic stenosis (AS). However, it remains unclear whether elevated Lp(a) and OxPL drive disease progression and are therefore targets for therapeutic intervention. OBJECTIVES: This study investigated whether Lp(a) and OxPL on apolipoprotein B-100 (OxPL-apoB) levels are associated with disease activity, disease progression, and clinical events in AS patients, along with the mechanisms underlying any associations. METHODS: This study combined 2 prospective cohorts and measured Lp(a) and OxPL-apoB levels in patients with AS (Vmax >2.0 m/s), who underwent baseline 18F-sodium fluoride (18F-NaF) positron emission tomography (PET), repeat computed tomography calcium scoring, and repeat echocardiography. In vitro studies investigated the effects of Lp(a) and OxPL on valvular interstitial cells. RESULTS: Overall, 145 patients were studied (68% men; age 70.3 ± 9.9 years). On baseline positron emission tomography, patients in the top Lp(a) tertile had increased valve calcification activity compared with those in lower tertiles (n = 79; 18F-NaF tissue-to-background ratio of the most diseased segment: 2.16 vs. 1.97; p = 0.043). During follow-up, patients in the top Lp(a) tertile had increased progression of valvular computed tomography calcium score (n = 51; 309 AU/year [interquartile range: 142 to 483 AU/year] vs. 93 AU/year [interquartile range: 56 to 296 AU/year; p = 0.015), faster hemodynamic progression on echocardiography (n = 129; 0.23 ± 0.20 m/s/year vs. 0.14 ± 0.20 m/s/year] p = 0.019), and increased risk for aortic valve replacement and death (n = 145; hazard ratio: 1.87; 95% CI: 1.13 to 3.08; p = 0.014), compared with lower tertiles. Similar results were noted with OxPL-apoB. In vitro, Lp(a) induced osteogenic differentiation of valvular interstitial cells, mediated by OxPL and inhibited with the E06 monoclonal antibody against OxPL. CONCLUSIONS: In patients with AS, Lp(a) and OxPL drive valve calcification and disease progression. These findings suggest lowering Lp(a) or inactivating OxPL may slow AS progression and provide a rationale for clinical trials to test this hypothesis.

Standardization of Human Calcific Aortic Valve Disease in vitro Modeling Reveals Passage-Dependent Calcification.

Aortic valvular interstitial cells (VICs) isolated from patients undergoing valve replacement are commonly used as in vitro models of calcific aortic valve disease (CAVD). Standardization of VIC calcification, however, has not been implemented, which impairs comparison of results from different studies. We hypothesized that different culture methods impact the calcification phenotype of human VICs. We sought to identify the key parameters impacting calcification in primary human VICs to standardize CAVD in vitro research. Here we report that in calcification media containing organic phosphate, termed osteogenic media (OM), primary human VICs exhibited a passage-dependent decrease in calcification potential, which was not observed in calcification media containing inorganic phosphate, termed pro-calcifying media (PM). We used Alizarin red staining to compare the calcification potential of VICs cultured in OM and PM between the first and fourth passages after cell isolation from human CAVD tissues. Human VICs showed consistent Alizarin red stain when cultured with PM in a passage-independent manner. VICs cultured in OM did not exhibit consistent calcification potential between donors in early passages and consistently lacked positive Alizarin red stain in late passages. We performed whole cell, cytoplasmic and nuclear fractionation proteomics to identify factors regulating VIC passage-dependent calcification in OM. Proteomics cluster analysis identified tissue non-specific alkaline phosphatase (TNAP) as a regulator of passage-dependent calcification in OM. We verified an association of TNAP activity with calcification potential in VICs cultured in OM, but not in PM in which VICs calcified independent of TNAP activity. This study demonstrates that media culture conditions and cell passage impact the calcification potential of primary human VICs and should be taken into consideration in cell culture models of CAVD. Our results help standardize CAVD modeling as part of a greater effort to identify disease driving mechanisms and therapeutics for this unmet medical need.

Cardiovascular calcification: artificial intelligence and big data accelerate mechanistic discovery.

Cardiovascular calcification is a health disorder with increasing prevalence and high morbidity and mortality. The only available therapeutic options for calcific vascular and valvular heart disease are invasive transcatheter procedures or surgeries that do not fully address the wide spectrum of these conditions; therefore, an urgent need exists for medical options. Cardiovascular calcification is an active process, which provides a potential opportunity for effective therapeutic targeting. Numerous biological processes are involved in calcific disease, including matrix remodelling, transcriptional regulation, mitochondrial dysfunction, oxidative stress, calcium and phosphate signalling, endoplasmic reticulum stress, lipid and mineral metabolism, autophagy, inflammation, apoptosis, loss of mineralization inhibition, impaired mineral resorption, cellular senescence and extracellular vesicles that act as precursors of microcalcification. Advances in molecular imaging and big data technology, including in multiomics and network medicine, and the integration of these approaches are helping to provide a more comprehensive map of human disease. In this Review, we discuss ectopic calcification processes in the cardiovascular system, with an emphasis on emerging mechanistic knowledge obtained through patient data and advances in imaging methods, experimental models and multiomics-generated big data. We also highlight the potential and challenges of artificial intelligence, machine learning and deep learning to integrate imaging and mechanistic data for drug discovery.