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Despite devastating clinical sequelae of calcific aortic valve disease that range from left ventricular remodeling to arrhythmias, heart failure, and early death, the molecular insights into disease initiation and progression are limited and pharmacotherapies remain unavailable. The pathobiology of calcific aortic valve disease is complex and comprehensive studies are challenging valvular calcification is heterogeneous and occurs preferentially on the aortic surface, along a fibrocalcific spectrum. Here, we review efforts to study (epi-)genomic, transcriptomic, proteomic, and metabolomic aspects of aortic valve calcification in combination with network medicine-/systems biology-based strategies to integrate multilayered omics datasets and prioritize druggable targets for experimental validation studies. Ultimately, such holistic approach efforts may open therapeutic avenues that go beyond invasive and costly valve replacement therapy.

The transition of healthy arteries and cardiac valves into dense, cell-rich, calcified, and fibrotic tissues is driven by a complex interplay of both cellular and molecular mechanisms. Specific cell types in these cardiovascular tissues become activated following the exposure to systemic stimuli including circulating lipoproteins or inflammatory mediators. This activation induces multiple cascades of events where changes in cell phenotypes and activation of certain receptors may trigger multiple pathways and specific alterations to the transcriptome. Modifications to the transcriptome and proteome can give rise to pathological cell phenotypes and trigger mechanisms that exacerbate inflammation, proliferation, calcification, and recruitment of resident or distant cells. Accumulating evidence suggests that each cell type involved in vascular and valvular diseases is heterogeneous. Single-cell RNA sequencing is a transforming medical research tool that enables the profiling of the unique fingerprints at single-cell levels. Its applications have allowed the construction of cell atlases including the mammalian heart and tissue vasculature and the discovery of new cell types implicated in cardiovascular disease. Recent advances in single-cell RNA sequencing have facilitated the identification of novel resident cell populations that become activated during disease and has allowed tracing the transition of healthy cells into pathological phenotypes. Furthermore, single-cell RNA sequencing has permitted the characterization of heterogeneous cell subpopulations with unique genetic profiles in healthy and pathological cardiovascular tissues. In this review, we highlight the latest groundbreaking research that has improved our understanding of the pathological mechanisms of atherosclerosis and future directions for calcific aortic valve disease.

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Cardiovascular disease is the most common cause of death worldwide, especially beyond the age of 65 years, with the vast majority of morbidity and mortality due to myocardial infarction and stroke. Vascular pathology stems from a combination of genetic risk, environmental factors, and the biologic changes associated with aging. The pathogenesis underlying the development of vascular aging, and vascular calcification with aging, in particular, is still not fully understood. Accumulating data suggests that genetic risk, likely compounded by epigenetic modifications, environmental factors, including diabetes and chronic kidney disease, and the plasticity of vascular smooth muscle cells to acquire an osteogenic phenotype are major determinants of age-associated vascular calcification. Understanding the molecular mechanisms underlying genetic and modifiable risk factors in regulating age-associated vascular pathology may inspire strategies to promote healthy vascular aging. This article summarizes current knowledge of concepts and mechanisms of age-associated vascular disease, with an emphasis on vascular calcification.

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.

Introduction: Inflammation is causally related to atherothrombosis. Interleukin-1β (IL-1β) and interleukin-18 (IL-18) require NLRP3 inflammasome for activation and have downstream effects on interleukin-6 (IL-6), a marker previously associated with high risk of coronary artery and cerebrovascular disease (CCVD). However, data pertaining to peripheral artery disease (PAD) are sparse and could offer druggable targets in this disease. Methods: We conducted a prospective cohort study of 4248 patients with type 2 diabetes or metabolic syndrome and prior coronary artery disease who participated in the NIH-funded Cardiovascular Inflammation Reduction Trial (CIRT). Participants were followed for up to 5 years for incident CCVD and symptomatic PAD events. Randomized treatment with low-dose methotrexate (vs. placebo) had no effect on event rates or plasma levels of inflammatory biomarkers. Baseline levels of IL-1β, IL-18, and IL-6 were tested for association with incident vascular events. Kaplan-Meier curves and Cox proportional hazards models (adjusted for traditional risk factors) were estimated. Results: In multivariable adjusted analyses, hazard ratios for the lowest (referent) to highest baseline quartiles of IL-6 were 1.0, 1.5, 1.8, and 2.0 (p-trend Conclusion: In this contemporary cohort of secondary prevention patients, elevated IL-6 was associated with both incident CCVD and PAD. These data support exploration of direct IL-6 inhibition for PAD prevention, a strategy currently being pursued to reduce risk of coronary artery and cerebrovascular disease.

Background: We hypothesize that macrophage heterogeneity is an unexploited source of therapeutic targets for vascular inflammation. Interferon-gamma (IFNγ) stimulated primary human macrophages M(IFNγ) is a widely used in vitro model for proinflammatory macrophages. However, typical activation-induced transcript profiling assumes a homogenous macrophage population. Our goal is to evaluate the extent of heterogeneity of activated macrophages to devise a strategy for precision medicine for inflammatory vascular disease. Methods: Using unbiased single-cell RNA sequencing (scRNA-seq), systems biology, and machine learning, we examined inter-subgroup differences of human primary M(IFNγ) (4 donors). Network analysis, kinetic proteomics, and in vitro assays (n=3-6) characterized the clusters, followed by validation in human carotid atherosclerotic plaques (n=13). scRNAseq data analysis in the L1000 CDS 2 drug-gene network computationally identified drugs that may potentiate or suppress each cluster. Results: The scRNA-seq demonstrated 3 distinct subpopulations: Clusters 1, 2, and 3 (C1, 2, and 3). C3 showed increased proinflammatory chemokine production, protein synthesis, and glycolysis. C1 was more efferocytotic/phagocytic, chemotactic, and less inflammatory. C2 is intermediate between C1 and C3. Histological analysis localized C1 and C3-like macrophages in different areas of the plaques ( Fig. 1A ). In addition, we used targeted scRNAseq (n=4) to analyze M(IFNγ) treated with an L1000-derived drug BI-2536 (Polo-like kinase inhibitor). As predicted, BI-2536 shifted the phenotypic heterogeneity of M(IFNγ) towards less inflammatory characteristics ( Fig. 1B ) which were further validated with bulk qPCR & ELISA (n=8). Conclusion: Our study presents a novel strategy for precision medicine that leverages single-cell data and gene interaction networks to identify modulators of macrophage heterogeneity as new anti-inflammatory therapies.

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.

Objective— Genome-wide association studies and preclinical studies demonstrated a role of sortilin in lipid metabolism, inflammation, and vascular calcification—all cardiovascular risk factors. We evaluated the association of serum sortilin levels with the risk of major adverse cerebrovascular and cardiovascular events (MACCE) and the severity of abdominal aortic calcification (AAC). Approach and Results— A cohort of community-dwelling men aged ≥50 years (n=830) was assessed. At baseline, sortilin levels were measured by ELISA, and AAC was assessed on lateral spine scans obtained by dual-energy X-ray absorptiometry. Men aged ≥60 years (n=745) were followed up prospectively for the incidence of MACCE. During the median follow-up of 7.9 years, 76 MACCE occurred. The unadjusted incidence of MACCE across increasing sortilin quartiles was 8.0, 7.4, 19.8, and 20.3 per 1000 person-years. In multivariate-adjusted analysis, sortilin associated with increased risk of MACCE (hazard ratio, 1.70 per SD; 95% confidence interval, 1.30–2.20; P P P 5) after adjustment for confounders (odds ratio, 1.43 per SD; 95% confidence interval, 1.10–1.85; P Conclusions— In older men, higher serum sortilin levels associated with higher MACCE risk and severe AAC independently of relevant confounders, including C-reactive protein and low-density lipoprotein cholesterol. This finding, however, needs to be validated in other cohorts.

Cardiovascular calcification is an insidious form of ectopic tissue mineralization that presents as a frequent comorbidity of atherosclerosis, aortic valve stenosis, diabetes, renal failure, and chronic inflammation. Calcification of the vasculature and heart valves contributes to mortality in these diseases. An inability to clinically image or detect early microcalcification coupled with an utter lack of pharmaceutical therapies capable of inhibiting or regressing entrenched and detectable macrocalcification has led to a prominent and deadly gap in care for a growing portion of our rapidly aging population. Recognition of this mounting concern has arisen over the past decade and led to a series of revolutionary works that has begun to pull back the curtain on the pathogenesis, mechanistic basis, and causative drivers of cardiovascular calcification. Central to this progress is the discovery that calcifying extracellular vesicles act as active precursors of cardiovascular microcalcification in diverse vascular beds. More recently, the omics revolution has resulted in the collection and quantification of vast amounts of molecular-level data. As the field has become poised to leverage these resources for drug discovery, new means of deriving relevant biological insights from these rich and complex datasets have come into focus through the careful application of systems biology and network medicine approaches. As we look onward toward the next decade, we envision a growing need to standardize approaches to study this complex and multifaceted clinical problem and expect that a push to translate mechanistic findings into therapeutics will begin to finally provide relief for those impacted by this disease.

Objective: Dysfunctional protein homeostasis (proteostasis) contributes to cardiovascular and metabolic disorders. We and others associated the mitochondrial fission protein, Dynamin-related protein 1 (DRP1) with cardiometabolic disease. Liver DRP1-deficiency reduces serum lipids and very-low density lipoprotein secretion in high-fat fed mice; whether DRP1 mediates these effects via proteostasis regulation is unknown. Approach and Results: Using mass spectrometry integrated with network analysis to map the human liver secretome, we found DRP1 associated with cardiovascular disease modules and lipid pathways. Electron microscopy revealed human liver DRP1 at mitochondria, cytosol, vesicles, endoplasmic reticulum (ER), and clustered at membrane tethered to ER exit sites. DRP1 small molecule inhibition (Mdivi-1) or CRISPR/Cas9-mediated DRP1 deletion in human liver cells, and Drp1 -liver deficiency in mice reduced autophagic flux without impairing the amino acid metabolome, or activating the autophagy inhibitor, mammalian target of rapamycin complex 1. DRP1 partially co-localized and co-immunoprecipitated with the ER trafficking and autophagy regulator, Syntaxin 17, in human liver tissue and cells. DRP1 inhibition reduced Proprotein convertase subtilisin/kexin type 9 (PCSK9) secretion in human liver cells and mice (-78.5%), and altered trafficking of the PCSK9-binding and ER maintenance chaperone, Glucose-regulated protein 94. Co-treating human liver cells with Mdivi-1 and proteasome inhibitor (MG132), non-transcriptionally increased intracellular PCSK9, while maintaining Mdivi-1-mediated reduced PCSK9 secretion. Conclusions: We propose a novel function of DRP1 in the regulation of proteostasis, wherein DRP1 may cluster, then tether and/or constrict nascent autophagy-associated membrane at the ER via its interaction with Syntaxin 17. DRP1 inhibition likely reduces lipoprotein and PCSK9 secretion in part by impairing autophagic flux leading to compensatory chaperone-mediated proteasomal degradation for ER maintenance. Proteostasis regulation and the cellular function of DRP1 is more complex than previously thought, potentially providing new avenues to therapeutically target cardiometabolic disease.

Objectives: Calcific aortic valve disease (CAVD) shares risk factors with atherosclerosis but has no pharmacotherapy. Bicuspid aortic valve (BAV) is the most common congenital cardiac defect (2% of humans), and individuals with BAVs acquire CAVD at a rate >15-25x of those with tricuspid aortic valves (TAVs). The causes and mechanisms of accelerated BAV-CAVD remain unclear. This study demonstrates the integrated transcriptome and proteome of BAV-CAVD. Methods: Quantification of gated chest CTs assessed aortic valve calcification. We performed transcriptomics using Illumina sequencing and global unlabeled proteomics by LC-MS/MS in a total of 55 human aortic valves (24 stenotic TAVs and 25 stenotic BAVs from valve replacement surgeries segmented into fibrotic and calcific portions, and 6 non-diseased aortic valves from heart transplants). Results: Calcific burden did not differ between BAVs and TAVs (Agatston score: p = 0.88), but BAV mineralization was significantly accelerated as BAVs underwent surgery 11.5±1.74 years earlier than TAVs (p < 0.01). Transcriptomics sequenced 11,223 genes and proteomics identified 1,798 proteins. Of these, 174 genes (fibrotic = 121, calcified = 53) and 98 proteins (fibrotic = 24, calcified = 74) were differentially expressed between BAV- and TAV-CAVD. Pathway analysis of proteins enriched in BAVs vs. TAVs found significant enhancement of iron metabolism and proteoglycan function in BAV-CAVD, while complement activation and collagen biosynthesis distinguished TAVs. When overrepresented proteins were mapped to the protein-protein interactome, network analysis identified disease stage- and valve type-specific subnetworks. While numerous protein-protein interactions existed between the fibrosis and calcification subnetworks of TAVs, a disconnection between these two pathological processes was identified in BAV subnetworks. Conclusions: Integrated multi-omics of CAVD stratified by disease stage and valve type identify unique contributors to BAV pathogenesis and shed light on molecular drivers of disease. Network analysis finds that while fibrocalcific responses are linked in TAVs, fibrosis and calcification may develop independently in BAVs, with vital implications for targeting of therapeutics.

Objectives: Vein graft (VG) failure rates after surgical bypass for PAD remain high, which can cause limb loss. Yet, the underlying mechanisms for VG lesion development remain obscure. We took a systems approach to explore key pathways in a mouse model. Approach and Results: We implanted the inferior vena cava into the left carotid artery of fat-fed Ldlr-/- mice and monitored lesion development up to 4 weeks post-surgery. Unbiased proteomic profiling followed by hierarchical clustering showed distinct clusters. One protein cluster is decreased in vein grafts with overt lesions (poor outcome) relative to the group of non-diseased veins devoid of lesions and vein grafts with minimal lesion (favorable outcome). Conversely, this can be viewed as increased in the “favorable” outcome group. Using the “favorable outcome” predominant protein cluster, network analysis and pathway enrichment identified PPARα pathway as a top-ranked key hub node with high betweeness centrality measure. This implies that PPARα may play a key regulatory role in limiting lesion severity. We examined whether network-based prediction of PPARα can also serve as a therapeutic target to attenuate the development of experimental VG lesions. A blinded in vivo loss-of-function study using PPARα siRNA encapsulated in macrophage-targeted lipid nanoparticles (LNP) demonstrated higher plaque volume as seen by 3D ultrasound and histologic morphometry in the test group (n=7) versus the control group (n=8). In contrast, a randomized drug study on another set of VG mice using the new PPARα-selective agonist pemafibrate demonstrated less plaque lesions and macrophage accumulation in the drug group (n=11) compared to the control group (n=11). In vitro validation studies (alongside loss-of-function) including high-throughput qPCR, single cell qPCR, CyTOF, ELISA, FACS, and metabolic assays all suggested that pemafibrate-induced PPARα activation promotes a phenotype shift of activated macrophages into a less inflammatory state through metabolic reprogramming. Conclusion: Systems-based target discovery on VG demonstrates that PPARα agonism reduces lesion development and inflammatory burden. Targeting this pathway might furnish a novel therapeutic option to prevent VG lesion development.