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Background: Vein graft failure remains a common clinical challenge. We applied a systems approach in mouse experiments to discover therapeutic targets for vein graft failure. Methods: Global proteomics and high-dimensional clustering on multiple vein graft tissues were used to identify potential pathogenic mechanisms. The PPARs (peroxisome proliferator-activated receptors) pathway served as an example to substantiate our discovery platform. In vivo mouse experiments with macrophage-targeted PPARα small interfering RNA, or the novel, selective activator pemafibrate demonstrate the role of PPARα in the development and inflammation of vein graft lesions. In vitro experiments further included metabolomic profiling, quantitative polymerase chain reaction, flow cytometry, metabolic assays, and single-cell RNA sequencing on primary human and mouse macrophages. Results: We identified changes in the vein graft proteome associated with immune responses, lipid metabolism regulated by the PPARs, fatty acid metabolism, matrix remodeling, and hematopoietic cell mobilization. PPARα agonism by pemafibrate retarded the development and inflammation of vein graft lesions in mice, whereas gene silencing worsened plaque formation. Pemafibrate also suppressed arteriovenous fistula lesion development. Metabolomics/lipidomics, functional metabolic assays, and single-cell analysis of cultured human macrophages revealed that PPARα modulates macrophage glycolysis, citrate metabolism, mitochondrial membrane sphingolipid metabolism, and heterogeneity. Conclusions: This study explored potential drivers of vein graft inflammation and identified PPARα as a novel potential pharmacological treatment for this unmet medical need.

Calcific aortic valve disease sits at the confluence of multiple world-wide epidemics of aging, obesity, diabetes, and renal dysfunction, and its prevalence is expected to nearly triple over the next 3 decades. This is of particularly dire clinical relevance, as calcific aortic valve disease can progress rapidly to aortic stenosis, heart failure, and eventually premature death. Unlike in atherosclerosis, and despite the heavy clinical toll, to date, no pharmacotherapy has proven effective to halt calcific aortic valve disease progression, with invasive and costly aortic valve replacement representing the only treatment option currently available. This substantial gap in care is largely because of our still-limited understanding of both normal aortic valve biology and the key regulatory mechanisms that drive disease initiation and progression. Drug discovery is further hampered by the inherent intricacy of the valvular microenvironment: a unique anatomic structure, a complex mixture of dynamic biomechanical forces, and diverse and multipotent cell populations collectively contributing to this currently intractable problem. One promising and rapidly evolving tactic is the application of multiomics approaches to fully define disease pathogenesis. Herein, we summarize the application of (epi)genomics, transcriptomics, proteomics, and metabolomics to the study of valvular heart disease. We also discuss recent forays toward the omics-based characterization of valvular (patho)biology at single-cell resolution; these efforts promise to shed new light on cellular heterogeneity in healthy and diseased valvular tissues and represent the potential to efficaciously target and treat key cell subpopulations. Last, we discuss systems biology- and network medicine-based strategies to extract meaning, mechanisms, and prioritized drug targets from multiomics datasets.

The morbidity and mortality rates of calcific aortic valve disease (CAVD) remain high while treatment options are limited. Here, we evaluated the role and therapeutic value of dual-specificity phosphatase 26 (DUSP26) in CAVD.Microarray profiling of human calcific aortic valves and normal controls demonstrated that DUSP26 was significantly up-regulated in calcific aortic valves. ApoE-/- mice fed a normal diet or a high cholesterol diet (HCD) were infected with adeno-associated virus serotype 2 carrying DUSP26 short-hairpin RNA to examine the effects of DUSP26 silencing on aortic valve calcification. DUSP26 silencing ameliorated aortic valve calcification in HCD-treated ApoE-/- mice, as evidenced by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased peak transvalvular jet velocity and mean transvalvular pressure gradient, as well as increased aortic valve area), and decreased levels of osteogenic markers (Runx2, osterix, and osteocalcin) in the aortic valves. These results were confirmed in osteogenic medium-induced human valvular interstitial cells. Immunoprecipitation, liquid chromatography-tandem mass spectrometry, and functional assays revealed that dipeptidyl peptidase-4 (DPP4) interacted with DUSP26 to mediate the procalcific effects of DUSP26. High N6-methyladenosine levels up-regulated DUSP26 in CAVD; in turn, DUSP26 activated DPP4 by antagonizing mouse double minute 2-mediated ubiquitination and degradation of DPP4, thereby promoting CAVD progression.DUSP26 promotes aortic valve calcification by inhibiting DPP4 degradation. Our findings identify a previously unrecognized mechanism of DPP4 up-regulation in CAVD, suggesting that DUSP26 silencing or inhibition is a viable therapeutic strategy to impede CAVD progression.

Despite the focus placed on cardiovascular research, the prevalence of vascular and valvular calcification is increasing and remains a leading contributor of cardiovascular morbidity and mortality. Accumulating studies provide evidence that cardiovascular calcification is an inflammatory disease in which innate immune signaling becomes sustained and/or excessive, shaping a deleterious adaptive response. The triggering immune factors and subsequent inflammatory events surrounding cardiovascular calcification remain poorly understood, despite sustained significant research interest and support in the field. Most studies on cardiovascular calcification focus on innate cells, particularly macrophages' ability to release pro-osteogenic cytokines and calcification-prone extracellular vesicles and apoptotic bodies. Even though substantial evidence demonstrates that macrophages are key components in triggering cardiovascular calcification, the crosstalk between innate and adaptive immune cell components has not been adequately addressed. The only therapeutic options currently used are invasive procedures by surgery or transcatheter intervention. However, no approved drug has shown prophylactic or therapeutic effectiveness. Conventional diagnostic imaging is currently the best method for detecting, measuring, and assisting in the treatment of calcification. However, these common imaging modalities are unable to detect early subclinical stages of disease at the level of microcalcifications; therefore, the vast majority of patients are diagnosed when macrocalcifications are already established. In this review, we unravel the current knowledge of how innate and adaptive immunity regulate cardiovascular calcification; and put forward differences and similarities between vascular and valvular disease. Additionally, we highlight potential immunomodulatory drugs with the potential to target calcification and propose avenues in need of further translational inquiry.

BACKGROUND AND AIMS: Calcific aortic valve disease is highly prevalent in patients with significant smoking history and is a marker of atherosclerosis. The aim of this study was to define the prognostic value of aortic valve calcification (AVC) derived from low dose, lung cancer screening computed tomography (LCSCT) for all-cause mortality in this higher risk population. METHODS: This is a single site, retrospective analysis of 1529 moderate-to-high atherosclerotic cardiovascular risk U.S. veterans (65 years [IQI: 61, 68] years; 96% male), who underwent clinically indicated LCSCT. CTs were scored for aortic valve calcification (AVC) and coronary artery calcification (CAC). The primary endpoint was all-cause mortality and secondary endpoints were nonfatal myocardial infarction (MI) and nonfatal cerebrovascular accident (CVA). RESULTS: Over 4-year follow-up, 227 patients (15%) died, 112 patients (7%) had nonfatal MI, and 52 patients (3%) had nonfatal CVA. AVC was predictive of all-cause mortality (HR per 100: 1.041 [1.030–1.052], p < 0.001), and this association remained significant after multivariate adjustment for traditional atherosclerotic risk factors, including CAC (1.021 [1.007–1.036], p = 0.003). After excluding patients with severe aortic stenosis (AS) or severe AVC (≥1274 AU in women and ≥2065 AU in men), in a subset of 765 patients who had echocardiograms, this association remained significant after multivariate analysis (HR per 100: 1.052 [1.010–1.095], p = 0.014). Despite controlling for CAC in the models, AVC was still associated with MI (HR per 100: 1.021 [1.004–1.039], p = 0.017) and with CVA (HR per 100: 1.027 [1.002–1.051], p = 0.032). CONCLUSIONS: Scoring AVC derived from LCSCT is predictive of mortality, nonfatal MI, and nonfatal CVA in patients at known risk for cardiovascular disease, independent of coronary calcification or severe aortic valve stenosis.

RATIONALE. Calcific Aortic Valve Disease (CAVD) affects more than 5.2 million people in the US. The only effective treatment is surgery and this comes with complications and no guarantee of long-term success. OBJECTIVE. Outcomes from pharmacological initiatives remain unsubstantiated and therefore the aim of this study is to determine if repurposing a selective XPO1 inhibitor drug (KPT-330) is beneficial in the treatment of CAVD. METHODS AND RESULTS. We show that KPT-330 prevents, attenuates and mitigates calcific nodule formation in heart valve interstitial cells (VICs) in vitro, and prevents CAVD in Klotho(−/−) mice. Using RNA-sequencing and Mass Spectrometry we show that KPT-330’s beneficial effect is mediated by inhibiting nuclear export of the transcription factor C/EBPβ in VICs, leading to repression of canonical Wnt signaling, in part through activation of the Wnt antagonist Axin1, and a subsequent decrease in pro-osteogenic markers and cell viability. CONCLUSIONS. Our findings have met a critical need to discover alterative, pharmacological-based therapies in the treatment of CAVD.

Journal of the American Heart Association 10(20), 1-29 (2021). doi:10.1161/JAHA.121.020834, Published by American Heart Association, New York, NY

Background: Heart valves regulate the unidirectional forward flow and prevent retrograde backflow of blood during the cardiac cycle. Cardiac valve disease (CVD) is observed in approximately 2.5% of the general population and the incidence increases to ~10% in elderly people. Patients with severe CVD require surgery and effective pharmacological treatments are currently not available. PROX1 is a transcription factor that regulates the development of lymphatic, venous, and lymphovenous valves (vascular valves). We identified that PROX1 is also expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 regulates cardiac valve development and disease is not known. Method and Results: We have discovered that mice lacking Prox1 in their VECs ( Prox1 ΔVEC ) develop enlarged aortic and mitral valves in which the expression of proteoglycans is increased (control, N=10; Prox1 ΔVEC , N=9, p Prox1 ΔVEC mice (control, N=5; Prox1 ΔVEC , N=9, p Prox1 ΔVEC mice. Specific knockdown of FOXC2 in VECs results in the thickening of aortic valves (control, N=10; shFoxc2 ΔVEC , N=8, p Foxc2 OE-VEC ) ameliorates the thickening of the aortic valves of Prox1 ΔVEC mice ( Prox1 ΔVEC , N=9; Foxc2 OE-VEC ; Prox1 ΔVEC , N=8, p Pdgfb ) is increased in the valve tissue of Prox1 ΔVEC mice and in PROX1 deficient sheep mitral valve VECs (MVECs) (siCtrl , N=4; siProx1 , N=4, p Prox1 ΔVEC mice (control , N=4; Pdgfb GOF , N=3, p Conclusion: Together these data suggest that PROX1 maintains the extracellular matrix composition of cardiac valves by regulating the expressions of FOXC2 and PDGF-B in VECs.

Visual Abstract, Highlights • This study is the first step towards the creation of a metabolomic map of calcified human aortic valves. • The study highlights an independent association of LysoPA with CAVS severity. • The study demonstrates that LysoPA levels are associated with faster CAVS progression rate., Summary This study outlines the first step toward creating the metabolite atlas of human calcified aortic valves by identifying the expression of metabolites and metabolic pathways involved at various stages of calcific aortic valve stenosis progression. Untargeted analysis identified 72 metabolites and lipids that were significantly altered (p

Background: Mediastinal ionizing radiotherapy is associated with an increased risk of valvular disease, which demonstrates pathological hallmarks similar to calcific aortic valve disease (CAVD). Despite advances in radiotherapy techniques, the prevalence of comorbidities such as radiation-associated valvular disease is still increasing due to improved survival of patients receiving radiotherapy. However, the mechanisms of radiation-associated valvular disease are largely unknown. CAVD is considered to be an actively regulated disease process, mainly controlled by valvular interstitial cells (VICs). We hypothesize that radiation exposure catalyzes the calcific response of VICs and, therefore, contributes to the development of radiation-associated valvular disease.Methods and Results: To delineate the relationship between radiation and VIC behavior (morphology, calcification, and matrix turnover), two different in vitro models were established: (1) VICs were cultured two-dimensional (2D) on coverslips in control medium (CM) or osteogenic medium (OM) and irradiated with 0, 2, 4, 8, or 16 Gray (Gy); and (2) three-dimensional (3D) hydrogel system was designed, loaded with VICs and exposed to 0, 4, or 16 Gy of radiation. In both models, a dose-dependent decrease in cell viability and proliferation was observed in CM and OM. Radiation exposure caused myofibroblast-like morphological changes and differentiation of VICs, as characterized by decreased αSMA expression. Calcification, as defined by increased alkaline phosphatase activity, was mostly present in the 2D irradiated VICs exposed to 4 Gy, while after exposure to higher doses VICs acquired a unique giant fibroblast-like cell morphology. Finally, matrix turnover was significantly affected by radiation exposure in the 3D irradiated VICs, as shown by decreased collagen staining and increased MMP-2 and MMP-9 activity.Conclusions: The presented work demonstrates that radiation exposure enhances the calcific response in VICs, a hallmark of CAVD. In addition, high radiation exposure induces differentiation of VICs into a terminally differentiated giant-cell fibroblast. Further studies are essential to elucidate the underlying mechanisms of these radiation-induced valvular changes.

Calcific aortic valve disease (CAVD) is a highly prevalent condition that comprises a disease continuum, ranging from microscopic changes to profound fibro-calcific leaflet remodelling, culminating in aortic stenosis, heart failure, and ultimately premature death. Traditional risk factors, such as hypercholesterolaemia and (systolic) hypertension, are shared among atherosclerotic cardiovascular disease and CAVD, yet the molecular and cellular mechanisms differ markedly. Statin-induced low-density lipoprotein cholesterol lowering, a remedy highly effective for secondary prevention of atherosclerotic cardiovascular disease, consistently failed to impact CAVD progression or to improve patient outcomes. However, recently completed phase II trials provide hope that pharmaceutical tactics directed at other targets implicated in CAVD pathogenesis offer an avenue to alter the course of the disease non-invasively. Herein, we delineate key players of CAVD pathobiology, outline mechanisms that entail compromised endothelial barrier function, and promote lipid homing, immune-cell infiltration, and deranged phospho-calcium metabolism that collectively perpetuate a pro-inflammatory/pro-osteogenic milieu in which valvular interstitial cells increasingly adopt myofibro-/osteoblast-like properties, thereby fostering fibro-calcific leaflet remodelling and eventually resulting in left ventricular outflow obstruction. We provide a glimpse into the most promising targets on the horizon, including lipoprotein(a), mineral-binding matrix Gla protein, soluble guanylate cyclase, dipeptidyl peptidase-4 as well as candidates involved in regulating phospho-calcium metabolism and valvular angiotensin II synthesis and ultimately discuss their potential for a future therapy of this insidious disease.

Aortic aneurysm/dissection (AAD) is a life-threatening disorder lacking effective pharmacotherapeutic remedies. Aldehyde dehydrogenase 2 (ALDH2) polymorphism is tied with various risk factors for AAD including hypertension, atherosclerosis, and hypercholesterolaemia although direct correlation between the two remains elusive.Two independent case-control studies were conducted involving 307 AAD patients and 399 healthy controls in two geographically distinct areas in China. Our data revealed that subjects carrying mutant ALDH2 gene possessed a ∼50% reduced risk of AAD compared with wild-type (WT) alleles. Using 3-aminopropionitrile fumarate (BAPN)- and angiotensin II (Ang II)-induced AAD animal models, inhibition of ALDH2 was found to retard development of AAD. Mechanistically, ALDH2 inhibition ablated pathological vascular smooth muscle cell (VSMC) phenotypical switch through interaction with myocardin, a determinant of VSMC contractile phenotype. Using microarray and bioinformatics analyses, ALDH2 deficiency was found to down-regulate miR-31-5p, which further altered myocardin mRNA level. Gain-of-function and loss-of-function studies verified that miR-31-5p significantly repressed myocardin level and aggravated pathological VSMC phenotypical switch and AAD, an effect that was blunted by ALDH2 inhibition. We next noted that ALDH2 deficiency increased Max expression and decreased miR-31-5p level. Moreover, ALDH2 mutation or inhibition down-regulated levels of miR-31-5p while promoting myocardin downstream contractile genes in the face of Ang II in primary human VSMCs.ALDH2 deficiency is associated with a lower risk of AAD in patients and mice, possibly via suppressing VSMC phenotypical switch in a miR-31-5p-myocardin-dependent manner. These findings favour a role for ALDH2 and miR-31-5p as novel targets for AAD therapy.

Objectives This study hypothesized that compensatory mitral leaflet area (MLA) adaptation occurs in patients with persistent atrial fibrillation (AF) without left ventricular (LV) dysfunction but has limitations that augment mitral regurgitation (MR). The study also explored whether asymmetrical annular dilation is matched by relative leaflet enlargement. Background Functional MR occurs in patients with AF and isolated annular dilation, but the relationship of MLA adaptation with annular area (AA) is unknown. Methods Three-dimensional echocardiographic images were acquired from 86 patients with quantified MR: 53 with nonvalvular persistent AF (23 MR+ with moderate or greater MR, 30 MR−) without LV dysfunction or dilation and 33 normal controls. Comprehensive 3-dimensional analysis included total diastolic MLA, adaptation ratios of MLA to annular area and MLA to leaflet closure area, and annular and tenting geometry. Results Total MLA was 22% larger in patients with AF than in controls, thus paralleling the increased AA. However, as AA increased, adaptive indices (MLA/AA ratio and ratio of MLA to closure area) plateaued, becoming lowest in MR+ patients (ratio of MLA to closure area = 1.63 ± 0.17 controls, 1.60 ± 0.11 MR−, 1.32 ± 0.10 MR+; p Conclusions MLA adaptively increases in AF with isolated annular dilation and normal LV function. This compensatory enlargement becomes insufficient with greater annular dilation, and the leaflets fail to match asymmetrical annular remodeling, thereby increasing MR. These findings can potentially help optimize therapeutic options and motivate basic studies of adaptive growth processes.

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.

Calcific aortic valve disease (CAVD), and its clinical manifestation that is calcific aortic valve stenosis, is the leading cause for valve disease within the developed world, with no current pharmacological treatment available to delay or halt its progression. Characterized by progressive fibrotic remodelling and subsequent pathogenic mineralization of the valve leaflets, valve disease affects 2.5% of the western population, thus highlighting the need for urgent intervention. Whilst the pathobiology of valve disease is complex, involving genetic factors, lipid infiltration, and oxidative damage, the immune system is now being accepted to play a crucial role in pathogenesis and disease continuation. No longer considered a passive degenerative disease, CAVD is understood to be an active inflammatory process, involving a multitude of pro-inflammatory mechanisms, with both the adaptive and the innate immune system underpinning these complex mechanisms. Within the valve, 15% of cells evolve from haemopoietic origin, and this number greatly expands following inflammation, as macrophages, T lymphocytes, B lymphocytes, and innate immune cells infiltrate the valve, promoting further inflammation. Whether chronic immune infiltration or pathogenic clonal expansion of immune cells within the valve or a combination of the two is responsible for disease progression, it is clear that greater understanding of the immune systems role in valve disease is required to inform future treatment strategies for control of CAVD development.

Mitral stenosis (MS) is a consequence of rheumatic heart disease that leads to heart failure requiring mechanical intervention. Percutaneous mitral commissurotomy (PMC) is the treatment of choice for the intervention, and currently there are no soluble markers associated with hemodynamic improvement after PMC. This study aims to determine the changes in cytokine/chemokine plasma levels, as well as T cell activation after PMC, and to investigate their association with immediate hemodynamic improvement and clinical outcomes. Plasma samples from eighteen patients with well-defined MS who underwent PMC and 12 healthy controls were analyzed using BioPlex immunoassay. We observed that 16 out of the 27 (60%) molecules assessed were altered in patients' plasma pre-PMC as compared to control group. Of those, IL-1β, IL-12, IL-6, IL-4, PDGF, and CCL11 showed significant decrease after PMC. Stratifying the patients according to adverse outcome after a 28-month median follow up, we detected a significant reduction of IL-1β, IL-12, IL-6, IL-4, IFN-γ, CXCL-10, VEGF, FGF and PDGF post-PMC in patients without events, but not in those who presented adverse events during the follow-up. Patients with adverse outcomes had lower IL-10 pre-PMC, as compared to the ones without adverse events. In addition, the frequency of CD8+ activated memory cells was increased after PMC, while the frequency of CD4+ activated memory cells did not change. Our results show an association between the decrease of specific cytokines and changes in T cell activation with hemodynamic improvement post-PMC, as well as with long-term outcomes, suggesting their possible use as soluble markers for hemodynamic recovery after MS intervention.

Rheumatic heart valve disease (RHVD) is a post-infectious sequel of acute rheumatic fever resulting from an abnormal immune response to a streptococcal pharyngitis that triggers valvular damage. RHVD is the leading cause of cardiovascular death in children and young adults, mainly in women from low and middle-income countries. It is known that long-term inflammation and high degree of fibrosis leads to valve dysfunction due to anatomic disruption of the valve apparatus. However, since public and private investments in RHVD studies are practically inexistent the number of publications is scarce. This disease shows different natural history and clinical presentations as compared to other degenerative heart valve diseases. Although more than five decades passed after the pioneering studies on the pathogenesis of RHVD, it is still unclear how self-tolerance mechanisms fail in this disease, and how humoral and cellular inflammatory responses are interconnected. Despite that pathological mechanisms have been already proposed for RHVD, none of them are able to explain the preferential involvement of the mitral valve. This review focuses on pathophysiology and underlying mechanisms of RHVD.

Extracellular vesicles (EVs) are composed of a lipid bilayer containing transmembrane and soluble proteins. Subtypes of EVs include ectosomes (microparticles/microvesicles), exosomes, and apoptotic bodies that can be released by various tissues into biological fluids. EV cargo can modulate physiological and pathological processes in recipient cells through near- and long-distance intercellular communication. Recent studies have shown that origin, amount, and internal cargos (nucleic acids, proteins, and lipids) of EVs are variable under different pathological conditions, including cardiovascular diseases (CVD). The early detection and management of CVD reduce premature morbidity and mortality. Circulating EVs have attracted great interest as a potential biomarker for diagnostics and follow-up of CVD. This review highlights the role of circulating EVs as biomarkers for diagnosis, prognosis, and therapeutic follow-up of CVD, and also for drug delivery. Despite the great potential of EVs as a tool to study the pathophysiology of CVD, further studies are needed to increase the spectrum of EV-associated applications.

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.

Rheumatic heart disease (RHD) is a major burden in low- to mid-income countries, where each year it accounts for over a million premature deaths associated with severe valve disease. Life-saving valve replacement procedures are not available to the majority of affected RHD patients, contributing to an increased risk of death in young adults and creating a devastating impact. In December 2017, a group of representatives of major cardiothoracic societies and industry, discussed the plight of the millions of patients who suffer from RHD. A comprehensive solution based on this global partnership was outlined in "The Cape Town Declaration on Access to Cardiac Surgery in the Developing World". The key challenge in controlling RHD is related to identification and removal of barriers to the translation of existing knowledge into policy, programs, and practice to provide high-quality care for patients with RHD. This review provides an overview on RHD by emphasizing the disease medical and economic burdens worldwide, risk factors, recent advance for early disease detection, and overall preventive strategies.

Introduction: Vascular calcification is the top cause of mortality worldwide. Vascular smooth muscle cells (SMCs) are major contributors to vascular calcification. There is an urgent need for a large scale and inexhaustible source of functional SMCs to study mechanisms of cardiovascular calcification. Here, we describe a new platform utilizing iPSCs differentiated towards SMCs that can calcify, to model ectopic calcification and identify mechanisms of SMCs’ contribution in this pathological process. Methods: We differentiated human iPSCs towards an induced-SMC (iSMC) phenotype in a 10-day protocol. We verified successful iSMC differentiation through immunofluorescent staining for SMC marker proteins αSMA and MYH11, FACS using CD140b, and proteomics analysis on whole cell lysate at days 0, 3, 5, 7, and 10. A set of 19 canonical SMC proteins were used to generate a Pearson’s correlation over the differentiation time course. After differentiation, we cultured the iSMCs in osteogenic media (OM) for 14 days. Alizarin Red S staining performed on day 14 confirmed the presence of calcification. Three independent experiments were performed for all data analyses. Results: Typical SMC morphology with positive staining for αSMA and MYH11 was detected at day 10 by immunofluorescence (Fig 1A). FACS analysis on markers for iPSCs (TRA-1-60), ECs (CD31), and SMCs (CD140b) demonstrated that 93.9% of cells were positive for CD140b after 10 days of differentiation (Fig 1B, C). Time course proteomics showed a strong Pearson’s correlation with of iSMCs with SMC marker proteins (R = 0.89) by day 10 (Figure 1D). Calcification in osteogenic media was observed by day 14 in culture using Alizarin Red S staining (Fig 1E, F), with 4.8-fold higher calcification in OM as compared to normal media (NM). Conclusions: Investigated iSMCs have promising future applications in cardiovascular research and may serve as an inexhaustible resource for studying vascular SMC calcification.

Introduction: Acute rheumatic fever (ARF) results from autoimmune responses to group A streptococcus. ARF can cause chronic rheumatic heart valve disease (RHVD), a major public health burden in low- and middle-income countries. Although ARF affects both sexes equally, females have a higher prevalence of RHVD. We identified a target protein, ProTα, that may contribute to pathophysiology of and gender predisposition in RHVD. Estradiol regulates ProTα, which influences transcriptional activity of the estrogen receptor (ER). Methods: Human heart valves (n=120) obtained from heart transplantation and valve replacement surgeries, including non-diseased mitral and aortic valves (NDMV and NDAV, n=17/group), rheumatic mitral and aortic valves (RMVD, n=45; RAVD, n=8) and calcifying valves from patients with calcific aortic valve disease (CAVD, n=33) underwent proteomic and network analyses, and immunohistochemical assessment of CD68, CD4, CD8 and ProTα. ProTα levels were measured in plasma and peripheral mononuclear cells (PBMCs) from RHVD patients and healthy donors (n=14/group) by ELISA and flow cytometry. Results: A comparison of proteomes from NDMV and NDAV established the baseline differences between valve types. 12 proteins were enriched (q Conclusion: We propose that ProTα is a novel contributor to the immunopathogenesis of RHVD and may regulate sex predisposition in this disease.

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.

Calcium is an integral part of human physiology. The presence of calcium mineral in soft tissue, however, reflects disease. The occurrence of mineral in cardiovascular system is the best predictor of morbidity and mortality, and in addition to serving as a reliable biomarker, the presence of stiff calcific mineral in otherwise soft tissues actively contributes to the leading causes of death in the world. Once thought to derive from passive tissue degeneration, active mechanisms that resemble bone formation have been shown to mediate cardiovascular calcification. The recognition that active cellular and molecular mechanisms mediate mineralization provide hope that interventions can prevent or reverse cardiovascular calcification, but the prevalence of mineral in arterial walls across human history suggests that soft tissues inherently predisposed to calcify. Mineral within arterial walls can be observed in 4000-year-old mummified human remains and across populations with varying lifestyles and diets. As our understanding of cardiovascular calcification continues to evolve, we may find ways to avoid the fate of our ancestors and confront this major contributor to cardiovascular disease. In this chapter, we provide an overview of the history of cardiovascular calcification discovery and its impact on human health.

Calcification is a highly prevalent pathology occurring in arteries and heart valves that contributes to cardiovascular disease, the leading cause of death worldwide. Arterial calcification impairs vascular resistance, thereby contributing to heart failure, and increases atherosclerotic plaque rupture risk contributing to heart attack and stroke. Calcification of the aortic valve leads to narrowing of the valve opening, reduced blood flow, and heart failure. There are presently no approved drug therapies that prevent or regress cardiovascular calcification. Both arterial and valvular calcification occur via active processes with multiple mechanisms attributed to initiating the disease pathologies. The extent to which various mechanisms drive calcific disease and just how distinct vascular and valvular calcification are is unknown. Vascular and valvular calcification do not always occur simultaneously, as such they may be regulated by distinct disease-driving mechanisms. Outlined here are vascular and valvular calcification mechanisms including mechanistic differences between these two critical cardiovascular pathologies.

Objective: Vascular calcification is a cardiovascular risk factor and accelerated in diabetes mellitus. Previous work has established a role for calcification-prone extracellular vesicles in promoting vascular calcification. However, the mechanisms by which diabetes mellitus provokes cardiovascular events remain incompletely understood. Our goal was to identify that increased S100A9 promotes the release of calcification-prone extracellular vesicles from human macrophages in diabetes mellitus. Approach and Results: Human primary macrophages exposed to high glucose (25 mmol/L) increased S100A9 secretion and the expression of receptor for advanced glycation end products (RAGE) protein. Recombinant S100A9 induced the expression of proinflammatory and osteogenic factors, as well as the number of extracellular vesicles with high calcific potential (alkaline phosphatase activity, P −/− S100a9 −/− mice and mice treated with S100a9 siRNA encapsulated in macrophage-targeted lipid nanoparticles showed decreased inflammation and microcalcification in atherosclerotic plaques, as gauged by molecular imaging and comprehensive histological analysis. In human carotid plaques, comparative proteomics in patients with diabetes mellitus and histological analysis showed that the S100A9-RAGE axis associates with osteogenic activity and the formation of microcalcification. Conclusions: Under hyperglycemic conditions, macrophages release calcific extracellular vesicles through mechanisms involving the S100A9-RAGE axis, thus contributing to the formation of microcalcification within atherosclerotic plaques.

Background: 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 because 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. Methods: 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 anatomic 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. Results: Global transcriptional and protein expression signatures differed between the nondiseased, fibrotic, and calcific stages of CAVD. Anatomic aortic valve microlayers exhibited unique proteome profiles that were maintained throughout disease progression and identified glial fibrillary acidic protein as a specific marker of valvular interstitial cells 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 in vitro, fibrosa-derived valvular interstitial cells demonstrated greater calcification potential than those from the ventricularis. These studies confirmed that the microlayer-specific proteome was preserved in cultured valvular interstitial cells, and that valvular interstitial cells exposed to alkaline phosphatase–dependent and alkaline phosphatase–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. Conclusions: 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.

The sorting receptor Sortilin functions in the regulation of glucose and lipid metabolism. Dysfunctional lipid uptake, storage, and metabolism contribute to several major human diseases including atherosclerosis and obesity. Sortilin associates with cardiovascular disease; however, the role of Sortilin in adipose tissue and lipid metabolism remains unclear. Here we show that in the low-density lipoprotein receptor-deficient (Ldlr−/−) atherosclerosis model, Sortilin deficiency (Sort1−/−) in female mice suppresses Niemann-Pick type C1-Like 1 (Npc1l1) mRNA levels, reduces body and white adipose tissue weight, and improves brown adipose tissue function partially via transcriptional downregulation of Krüppel-like factor 4 and Liver X receptor. Female Ldlr−/−Sort1−/− mice on a high-fat/cholesterol diet had elevated plasma Fibroblast growth factor 21 and Adiponectin, an adipokine that when reduced is associated with obesity and cardiovascular disease-related factors. Additionally, Sort1 deficiency suppressed cholesterol absorption in both female mice ex vivo intestinal tissue and human colon Caco-2 cells in a similar manner to treatment with the NPC1L1 inhibitor ezetimibe. Together our findings support a novel role of Sortilin in energy regulation and lipid homeostasis in female mice, which may be a potential therapeutic target for obesity and cardiovascular disease.

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.

Aortic stenosis is a growing worldwide health burden with no approved therapies beyond surgical or transcatheter valve replacement procedures. Calcification of aortic valve leaflets is a major contributor to aortic stenosis, subsequent heart failure and ultimately death. A small number of genetic contributors to valvular calcification have been identified, with elevated lipoprotein(a) (Lp(a)) being one of the strongest risk factors for calcific aortic valve disease (CAVD).1 Despite >1 billion people globally having elevated Lp(a) (>30 to 50 mg/dL or >75 to 125 nmol/L),2 the normal physiological function of Lp(a) and how Lp(a) mechanistically promotes CAVD progression have not been established. As such, identifying the roles that Lp(a) plays in human disease is a major unmet medical need with far-reaching therapeutic potential. In their Heart paper, Capoulade et al 3 assessed Lp(a) complexes with apolipoproteins and oxidised phospholipids (OxPLs) in patients from the Aortic Stenosis Progression Observation: Measuring Effects of Rousvastatin trial. This new clinical report demonstrates the novel finding that complexes of Lp(a) with apolipoprotein CIII (apoCIII) are associated with accelerated CAVD progression, in addition to validating4 the association of apolipoprotein(a) (apo(a))-OxPL complexes with accelerated CAVD progression. Lp(a) is a low-density lipoprotein-like particle with apo(a) covalently …

OBJECTIVE: Understanding message delivery among vascular cells is essential for deciphering the intercellular communications in cardiovascular diseases. MicroRNA (miR)-92a is enriched in endothelial cells (ECs) and circulation under atheroprone conditions. Macrophages are the primary immune cells in atherosclerotic lesions that modulate lesion development. Therefore, we hypothesize that, in response to atheroprone stimuli, ECs export miR-92a to macrophages to regulate their functions and enhance atherosclerotic progression. APPROACH AND RESULTS: We investigated the macrophage functions that are regulated by EC miR-92a under atheroprone microenvironments. We first determined the distributions of functional extracellular miR-92a by fractionating the intra- and extra-vesicular compartments from endothelial conditioned media and mice serum. The results indicate that extracellular vesicles are the primary vehicles for EC miR-92a transportation. Overexpression of miR-92a in ECs enhanced the pro-inflammatory responses and low-density-lipoprotein uptake, while impaired the migration, of co-cultured macrophage. Opposite effects were found in macrophages co-cultured with ECs with miR-92a knockdown. Further analyses demonstrated that intra-vesicular miR-92a suppressed the expression of target gene Krüppel-like factor 4 (KLF4) in macrophages, suggesting a mechanism by which intra-vesicular miR-92a regulates recipient cell functions. Indeed, the overexpression of KLF4 rescued the EC miR-92a-induced macrophage atheroprone phenotypes. Furthermore, an inverse correlation of intra-vesicular miR-92a in blood serum and KLF4 expression in lesions was observed in atherosclerotic animals, indicating the potential function of extracellular miR-92a in regulating vascular diseases. CONCLUSIONS: EC miR-92a can be transported to macrophages through extracellular vesicles to regulate KLF4 levels, thus leading to the atheroprone phenotypes of macrophage and hence atherosclerotic lesion formation.

Background Mitral leaflet enlargement has been identified as an adaptive mechanism to prevent mitral regurgitation in dilated left ventricles (LVs) caused by chronic aortic regurgitation (AR). This enlargement is deficient in patients with functional mitral regurgitation, which remains frequent in the population with ischemic cardiomyopathy. Maladaptive fibrotic changes have been identified in post-myocardial infarction (MI) mitral valves. It is unknown if these changes can interfere with valve growth and whether they are present in other valves. Objectives This study sought to test the hypothesis that MI impairs leaflet growth, seen in AR, and induces fibrotic changes in mitral and tricuspid valves. Methods Sheep models of AR, AR + MI, and controls were followed for 90 days. Cardiac magnetic resonance, echocardiography, and computed tomography were performed at baseline and 90 days to assess LV volume, LV function, mitral regurgitation and mitral leaflet size. Histopathology and molecular analyses were performed in excised valves. Results Both experimental groups developed similar LV dilatation and dysfunction. At 90 days, mitral valve leaflet size was smaller in the AR + MI group (12.8 ± 1.3 cm2 vs. 15.1 ± 1.6 cm2, p = 0.03). Mitral regurgitant fraction was 4% ± 7% in the AR group versus 19% ± 10% in the AR + MI group (p = 0.02). AR + MI leaflets were thicker compared with AR and control valves. Increased expression of extracellular matrix remodeling genes was found in both the mitral and tricuspid leaflets in the AR + MI group. Conclusions In these animal models of AR, the presence of MI was associated with impaired adaptive valve growth and more functional mitral regurgitation, despite similar LV size and function. More pronounced extracellular remodeling was observed in mitral and tricuspid leaflets, suggesting systemic valvular remodeling after MI.

Background: Chronic kidney disease (CKD) increases cardiovascular risk. Underlying mechanisms, however, remain obscure. The uremic toxin indoxyl sulfate is an independent cardiovascular risk factor in CKD. We explored the potential impact of indoxyl sulfate on proinflammatory activation of macrophages and its underlying mechanisms. Methods: We examined in vitro the effects of clinically relevant concentrations of indoxyl sulfate on proinflammatory responses of macrophages and the roles of organic anion transporters and organic anion transporting polypeptides (OATPs). A systems approach, involving unbiased global proteomics, bioinformatics, and network analysis, then explored potential key pathways. To address the role of Delta-like 4 (Dll4) in indoxyl sulfate–induced macrophage activation and atherogenesis in CKD in vivo, we used 5/6 nephrectomy and Dll4 antibody in low-density lipoprotein receptor–deficient (Ldlr-/-) mice. To further determine the relative contribution of OATP2B1 or Dll4 to proinflammatory activation of macrophages and atherogenesis in vivo, we used siRNA delivered by macrophage-targeted lipid nanoparticles in mice. Results: We found that indoxyl sulfate–induced proinflammatory macrophage activation is mediated by its uptake through transporters, including OATP2B1, encoded by the SLCO2B1 gene. The global proteomics identified potential mechanisms, including Notch signaling and the ubiquitin-proteasome pathway, that mediate indoxyl sulfate-triggered proinflammatory macrophage activation. We chose the Notch pathway as an example of key candidates for validation of our target discovery platform and for further mechanistic studies. As predicted computationally, indoxyl sulfate triggered Notch signaling, which was preceded by the rapid induction of Dll4 protein. Dll4 induction may result from inhibition of the ubiquitin-proteasome pathway, via the deubiquitinating enzyme USP5. In mice, macrophage-targeted OATP2B1/Slco2b1 silencing and Dll4 antibody inhibited proinflammatory activation of peritoneal macrophages induced by indoxyl sulfate. In low-density lipoprotein receptor–deficient mice, Dll4 antibody abolished atherosclerotic lesion development accelerated in Ldlr-/- mice. Moreover, coadministration of indoxyl sulfate and OATP2B1/Slco2b1 or Dll4 siRNA encapsulated in macrophage-targeted lipid nanoparticles in Ldlr-/- mice suppressed lesion development. Conclusions: These results suggest that novel crosstalk between OATP2B1 and Dll4-Notch signaling in macrophages mediates indoxyl sulfate–induced vascular inflammation in CKD.

Background: Microcalcifications in atherosclerotic plaques are destabilizing, predict adverse cardiovascular events, and are associated with increased morbidity and mortality. 18 F-fluoride positron emission tomography (PET)/computed tomography (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 18 F-fluoride PET/CT can identify early microcalcifications. Methods: 18 F-fluoride signal amplification derived from microcalcifications was validated against near-infrared fluorescence molecular imaging and histology using an in vitro 3-dimensional hydrogel collagen platform, ex vivo human specimens, and a mouse model of atherosclerosis. Results: Microcalcification size correlated inversely with collagen concentration. The 18 F-fluoride ligand bound to microcalcifications formed by calcifying vascular smooth muscle cell derived extracellular vesicles in the in vitro 3-dimensional collagen system and exhibited an increasing signal with an increase in collagen concentration (0.25 mg/mL collagen −33.8×10 2 ±12.4×10 2 counts per minute; 0.5 mg/mL collagen −67.7×10 2 ±37.4×10 2 counts per minute; P =0.0014), suggesting amplification of the PET signal by smaller microcalcifications. We further incubated human atherosclerotic endarterectomy specimens with clinically relevant concentrations of 18 F-fluoride. The 18 F-fluoride ligand labeled microcalcifications in PET-positive, CT-negative regions of explanted human specimens as evidenced by 18 F-fluoride PET/CT imaging, near-infrared fluorescence, and histological analysis. Additionally, the 18 F-fluoride ligand identified micro and macrocalcifications in atherosclerotic aortas obtained from low-density lipoprotein receptor-deficient mice. Conclusions: Our results suggest that 18 F-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.

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.

Vascular calcification is a major contributor to the progression of cardiovascular disease, one of the leading causes of death in industrialized countries. New evidence on the mechanisms of mineralization identified calcification-competent extracellular vesicles (EVs) derived from smooth muscle cells, valvular interstitial cells and macrophages as the mediators of calcification in diseased heart valves and atherosclerotic plaques. However, the regulation of EV release and the mechanisms of interaction between EVs and the extracellular matrix leading to the formation of destabilizing microcalcifications remain unclear. This review focuses on current limits in our understanding of EVs in cardiovascular disease and opens up new perspectives on calcific EV biogenesis, release and functions within and beyond vascular calcification. We propose that, unlike bone-derived matrix vesicles, a large population of EVs implicated in cardiovascular calcification are of exosomal origin. Moreover, the milieu-dependent loading of EVs with microRNA and calcification inhibitors fetuin-A and matrix Gla protein suggests a novel role for EVs in intercellular communication, adding a new mechanism to the pathogenesis of vascular mineralization. Similarly, the cell type-dependent enrichment of annexins 2, 5 or 6 in calcifying EVs posits one of several emerging factors implicated in the regulation of EV release and calcifying potential. This review aims to emphasize the role of EVs as essential mediators of calcification, a major determinant of cardiovascular mortality. Based on recent findings, we pinpoint potential targets for novel therapies to slow down the progression and promote the stability of atherosclerotic plaques.

Epidemiological evidence conclusively demonstrates that calcium burden is a significant predictor of cardiovascular morbidity and mortality; however, the underlying mechanisms remain largely unknown. These observations have challenged the previously held notion that calcification serves to stabilize the atherosclerotic plaque. Recent studies have shown that microcalcifications that form within the fibrous cap of the plaques lead to the accrual of plaque-destabilizing mechanical stress. Given the association between calcification morphology and cardiovascular outcomes, it is important to understand the mechanisms leading to calcific mineral deposition and growth from the earliest stages. We highlight the open questions in the field of cardiovascular calcification and include a review of the proposed mechanisms involved in extracellular vesicle-mediated mineral deposition.

Calcific aortic valve stenosis (CAVS) is common in the ageing population and set to become an increasing economic and health burden. Once present, it inevitably progresses and has a poor prognosis in symptomatic patients. No medical therapies are proven to be effective in holding or reducing disease progression. Therefore, aortic valve replacement remains the only available treatment option. Improved knowledge of the mechanisms underlying disease progression has provided us with insights that CAVS is not a passive disease. Rather, CAVS is regulated by numerous mechanisms with a key role for calcification. Aortic valve calcification (AVC) is actively regulated involving cellular and humoral factors that may offer targets for diagnosis and intervention. The discovery that the vitamin K-dependent proteins are involved in the inhibition of AVC has boosted our mechanistic understanding of this process and has opened up novel avenues in disease exploration. This review discusses processes involved in CAVS progression, with an emphasis on recent insights into calcification, methods for imaging calcification activity, and potential therapeutic options.

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.

Background: No pharmacological therapy exists for calcific aortic valve disease (CAVD), which confers a dismal prognosis without valve replacement. The search for therapeutics and early diagnostics is challenging since CAVD presents in multiple pathological stages. Methods: A total of 25 human stenotic aortic valves obtained from valve replacement surgery were analyzed by multiple modalities, including transcriptomics and global unlabeled and tandem-mass-tagged proteomics by liquid chromatography-mass spectrometry. Results: Global transcriptional and protein expression signatures differed between the non-diseased, fibrotic, and calcific stages of CAVD, with consistent trends in gene and protein expression across disease stages. Anatomical aortic valve microlayers exhibited unique proteome profiles that were maintained throughout disease progression, and revealed 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, in vitro , 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 TNAP-dependent and TNAP-independent calcifying stimuli had distinct proteome profiles, both of which overlapped with that of the whole tissue. Network analysis of protein-protein interaction networks found a significant closeness to multiple inflammatory and fibrotic diseases. Conclusions: A spatially- and temporally-resolved multi-omics and systems biology strategy identifies the first molecular regulatory networks in CAVD, a cardiac condition without a pharmacological cure, and describes a strategy for endophenotype characterization that is broadly applicable to comprehensive omics studies of cardiovascular diseases.