Superficial plaque erosion causes many acute coronary syndromes. However, mechanisms of plaque erosion remain poorly understood, and we lack directed therapeutics for thrombotic complication. Human eroded plaques can harbor neutrophil extracellular traps (NETs) that propagate endothelial damage at experimental arterial lesions that recapitulate superficial erosion. Clonal Hematopoiesis of Indeterminate Potential (CHIP) denotes age-related clonal expansion of bone marrow-derived cells harboring somatic mutations in the absence of overt hematological disease. CHIP heightens the risk of cardiovascular disease, with the greatest increase seen in individuals with JAK2

Background: Clonal hematopoiesis of indeterminate potential (CHIP) refers to somatic mutations in hematopoietic cells with a minimum allele fraction ≥ 2% without hematological disease. CHIP depends on age and increases the risk of coronary artery disease (CAD). In their 70s women’s prevalence of cardiovascular disease catches up to that of men. In Canakinumab Antiinflammatory Thrombosis Outcome Study (CANTOS) neutralizing interleukin-1β (IL-1β) in chronic CAD, subjects with TET2 CHIP showed greater reduction of cardiovascular events. However, this substudy did not investigate sex. Hypothesis: We hypothesized that sex modifies the association between CHIP mutations and risk of CAD, and the effects of IL-1β inhibition in CHIP-related atherosclerosis in mice. Methods: Sex differences on the association of CAD and CHIP mutations were investigated using 2 cohorts: the UK and Mass General Brigham Biobanks. CHIP genes showing sex differences in human data were deleted in hematopoietic cells of female and male atherosclerosis-susceptible Ldlr -/- mice. Mice consumed a 0.2% cholesterol high-fat diet with or without an anti-mouse IL-1β monoclonal antibody for 9 weeks. We assessed atherosclerosis by histology and performed mechanistic studies using single-cell RNA sequencing (scRNAseq) of atherosclerotic aortic arch and in vitro bone-marrow derived macrophages. Results: Women with TET2 CHIP had larger hazard ratio for CAD than men in both biobanks. Overall CHIP and other mutations did not demonstrate sex differences in the association with CAD. In Ldlr -/- mice deficient for Tet2 in hematopoietic cells, IL-1β inhibition reduced atherosclerosis in female but not male mice. ScRNAseq revealed more inflammatory cells and more expression of inflammatory genes in hematopoietic Tet2 -/- females than in males. IL-1β inhibition limited these sex differences. In vitro experiments demonstrated that Tet2 deficiency prevents the interaction between estrogen-receptor alpha and histone H3 controlling epigenetic regulation in mouse macrophages. Conclusions: Females with TET2 CHIP have accentuated CAD risk and IL-1β blockade limits Tet2 -augmented atherosclerosis in female but not male mice. These results highlight the importance of study of sex as a biological variable, uncover a new mechanism of Tet2 CHIP on acceleration of atherosclerosis, and inform the design of trials refining allocation of anti-inflammatory therapies in TET2 CHIP.

BackgroundSuperficial plaque erosion causes many acute coronary syndromes. However, mechanisms of plaque erosion remain poorly understood, and we lack directed therapeutics for thrombotic complication. Human eroded plaques can harbor neutrophil extracellular traps (NETs) that propagate endothelial damage at experimental arterial lesions that recapitulate superficial erosion. Clonal Hematopoiesis of Indeterminate Potential (CHIP) denotes age-related clonal expansion of bone marrow-derived cells harboring somatic mutations in the absence of overt hematological disease. CHIP heightens the risk of cardiovascular disease, with the greatest increase seen in individuals with JAK2V617F. Neutrophils from mice and humans with JAK2V617F undergo NETosis more readily than Jak2WT (wild-type) cells. We hypothesized that JAK2V617F, by increasing propensity to NETosis, exacerbates aspects of superficial erosion.Methods and resultsWe generated Jak2V617F and Jak2WT mice with heterozygous Jak2V617F in myeloid cells. We induced areas of denuded endothelium that recapitulate features of superficial erosion and assessed endothelial integrity, cellular composition of the erosion, thrombosis rates, and response to ruxolitinib, a clinically available JAK1/2 inhibitor, in relation to genotype. Following experimental erosion, Jak2V617F mice have greater impairment of endothelial barrier function and increased rates of arterial thrombosis. Neointimas in Jak2V617F mice exhibit increased apoptosis, NETosis, and platelet recruitment. Jak2V617F mice treated with ruxolitinib show increased endothelial continuity and reduced apoptosis in the neointima comparable to levels in Jak2WT.ConclusionsThese observations provide new mechanistic insight into the pathophysiology of superficial erosion, the heightened risk for myocardial infarction in JAK2V617F CHIP, and point the way to personalized therapeutics based on CHIP status.

Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the presence of a cancer-associated somatic mutation in white blood cells in the absence of overt hematological malignancy. It arises most commonly from loss-of-function mutations in the epigenetic regulators DNMT3A and TET2 . CHIP predisposes to both hematological malignancies and atherosclerotic cardiovascular disease in humans. Here we demonstrate that loss of Dnmt3a in myeloid cells increased murine atherosclerosis to a similar degree as previously seen with loss of Tet2 . Loss of Dnmt3a enhanced inflammation in macrophages in vitro and generated a distinct adventitial macrophage population in vivo which merges a resident macrophage profile with an inflammatory cytokine signature. These changes surprisingly phenocopy the effect of loss of Tet2 . Our results identify a common pathway promoting heightened innate immune cell activation with loss of either gene, providing a biological basis for the excess atherosclerotic disease burden in carriers of these two most prevalent CHIP mutations.

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.

Development of abdominal aortic aneurysms (AAA) enhances lesion group-2 innate lymphoid cell (ILC2) accumulation and blood IL5. ILC2 deficiency in Rorafl/fl Il7rCre/+ mice or induced ILC2 depletion in Icosfl-DTR-fl/+ Cd4Cre/+ mice expedites AAA growth, increases lesion inflammation, but leads to systemic IL5 and eosinophil (EOS) deficiency. Mechanistic studies show that ILC2 protect mice from AAA formation via IL5 and EOS. IL5 or ILC2 from wild-type (WT) mice, but not ILC2 from Il5-/- mice induces EOS differentiation in bone-marrow cells from Rorafl/fl Il7rCre/+ mice. IL5, IL13, and EOS or ILC2 from WT mice, but not ILC2 from Il5-/- and Il13-/- mice block SMC apoptosis and promote SMC proliferation. EOS but not ILC2 from WT or Il5-/- mice block endothelial cell (EC) adhesion molecule expression, angiogenesis, dendritic cell differentiation, and Ly6Chi monocyte polarization. Reconstitution of WT EOS and ILC2 but not Il5-/- ILC2 slows AAA growth in Rorafl/fl Il7rCre/+ mice by increasing systemic EOS. Besides regulating SMC pathobiology, ILC2 play an indirect role in AAA protection via the IL5 and EOS mechanism.

AIMS:Blood eosinophil (EOS) counts and EOS cationic protein (ECP) levels associate positively with major cardiovascular disease (CVD) risk factors and prevalence. This study investigates the role of EOS in cardiac hypertrophy. METHODS AND RESULTS:A retrospective cross-section study of 644 consecutive inpatients with hypertension examined the association between blood EOS counts and cardiac hypertrophy. Pressure overload- and β-adrenoreceptor agonist isoproterenol-induced cardiac hypertrophy was produced in EOS-deficient ΔdblGATA mice. This study revealed positive correlations between blood EOS counts and left ventricular (LV) mass and mass index in humans. ΔdblGATA mice showed exacerbated cardiac hypertrophy and dysfunction, with increased LV wall thickness, reduced LV internal diameter, and increased myocardial cell size, death, and fibrosis. Repopulation of EOS from wild-type (WT) mice, but not those from IL4-deficient mice ameliorated cardiac hypertrophy and cardiac dysfunctions. In ΔdblGATA and WT mice, administration of ECP mEar1 improved cardiac hypertrophy and function. Mechanistic studies demonstrated that EOS expression of IL4, IL13, and mEar1 was essential to control mouse cardiomyocyte hypertrophy and death and cardiac fibroblast TGF-β signalling and fibrotic protein synthesis. The use of human cardiac cells yielded the same results. Human ECP, EOS-derived neurotoxin, human EOS, or murine recombinant mEar1 reduced human cardiomyocyte death and hypertrophy and human cardiac fibroblast TGF-β signalling. CONCLUSION:Although blood EOS counts correlated positively with LV mass or LV mass index in humans, this study established a cardioprotective role for EOS IL4 and cationic proteins in cardiac hypertrophy and tested a therapeutic possibility of ECPs in this human CVD.

Background: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low- and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown. Methods: Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTα (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcus pyogenes proteomes were used to identify mimic epitopes. Results: A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTα plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTα colocalized with CD8 + T cells in RHVD. Expression of ProTα and estrogen receptor alpha correlated strongly in circulating CD8 + T cells from patients with RHVD. Recombinant ProTα induced expression of the lytic proteins perforin and granzyme B by CD8 + T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTα increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8 + T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTα. In silico analysis of RHVD and S pyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8 + T-cell activation in vitro. Conclusions: ProTα-dependent CD8 + T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. ProTα facilitated recognition of type 1 collagen mimic epitopes by CD8 + T cells, suggesting mechanisms provoking autoimmunity.

Introduction: Fewer than 50% of patients develop vascular and valvular calcification, implying differential pathogenesis. Tissue-entrapped extracellular vesicles (EVs) are implicated in mineralization but their contents and functions are unstudied. We investigated entrapped EV cargoes in human cardiovascular disease. Methods: Human carotid endarterectomy specimens and stenotic aortic valves were obtained from 53 patients. Disease stage-specific proteomics was performed on whole tissue (non-diseased/fibrotic/calcified areas). Tissue EVs were enriched by gradient fractionation then underwent proteomics and miRNA-seq. miR targets were predicted by TargetScan, pathway analyses utilized BioCarta/KEGG/Reactome, and protein-protein interaction networks employed STRING. Results: Disease progression drove significant convergence (p<0.0001) of atherosclerotic plaque and valve proteomes (2,318 proteins). 548 and 158 proteins were exclusively altered (q<0.05) by disease in plaques or valves, respectively. Vesicular GO terms increased 2.2x (p<0.01) amongst proteins altered by disease in both tissues (202). Proteomics found 24 EV markers in the low-density fractions of plaques and valves, confirmed by electron microscopy and nanoparticle tracking. EV omics quantified 1,104 proteins and 123 miR cargoes. Networks of proteins and miR targets shared by plaque and valve EVs revealed common regulation of Rho GTPase and MAPK signaling. 179 proteins and 5 miRs were altered between plaque and valve EVs (q<0.05); multi-omics integration found that EVs modulated cellular contraction and p53-mediated transcriptional regulation in plaques and valves, respectively. Conclusions: This first comparative proteomics study of human valves and arteries finds shared EV functionality in both diseases. Using novel means to examine tissue EV molecular cargoes, we also reveal critical divergent tissue-specific roles for EVs in mediating cardiovascular disease.

Objective: Accelerated atherosclerosis in diabetes constitutes an ongoing challenge despite optimal medical therapies. This study aimed to identify evolutionarily conserved lesion-based regulatory signaling networks in diabetic versus nondiabetic conditions during the development of atherosclerosis in an initial translational effort to provide insights for targets. Approach and Results: Serial 3-mm coronary artery segments of hypercholesterolemic Yorkshire swine and diabetic-hypercholesterolemic swine were characterized as mild, moderate, or severe phenotypic manifestations of coronary atherosclerosis based on histopathologic examination. Lesional RNA sequencing was performed (n=3–8 lesions per group) corresponding to increasing phenotypic severity. Differentially expressed genes, transcription factors, upstream regulators, and hubs were validated using the NanoString technology and a human atherosclerotic specimen cohort. Despite similar stage histopathologic characterization of lesions, genome-wide transcriptomics revealed gene sets and nodal signaling pathways uniquely expressed in diabetic lesions including signaling pathways for Th17, IL (interleukin)-17F, TWEAK (TNF [tumor necrosis factor]-related weak inducer of apoptosis), CD27, and PI3K/Akt. In contrast, pathways of nondiabetic lesions involved TREM-1 and Th1 and Th2 responses during the initiation stage, whereas networks for mitochondrial dysfunction, oxidative phosphorylation, and lipid metabolism emerged with progression. RNA sequencing data were validated in a human atherosclerosis specimen cohort using machine learning algorithms. F8 , MAPKAPK3 , and ITGB1 emerged as powerful genes for clustering diabetic versus nondiabetic lesions and for separating different degrees of atherosclerosis progression. Conclusions: This study identifies evolutionarily conserved gene signatures and signaling pathways in a stage-specific manner that successfully distinguishes diabetes- and non–diabetes-associated atherosclerosis. These findings establish new molecular insights and therapeutic opportunities to address accelerated atherosclerotic lesion formation in diabetes.

Aims Recent evidence suggests that 'vulnerable plaques', which have received intense attention as underlying mechanism of acute coronary syndromes over the decades, actually rarely rupture and cause clinical events. Superficial plaque erosion has emerged as a growing cause of residual thrombotic complications of atherosclerosis in an era of increased preventive measures including lipid lowering, antihypertensive therapy, and smoking cessation. The mechanisms of plaque erosion remain poorly understood, and we currently lack validated effective diagnostics or therapeutics for superficial erosion. Eroded plaques have a rich extracellular matrix, an intact fibrous cap, sparse lipid, and few mononuclear cells, but do harbour neutrophil extracellular traps (NETs). We recently reported that NETs amplify and propagate the endothelial damage at the site of arterial lesions that recapitulate superficial erosion in mice. We showed that genetic loss of protein arginine deiminase (PAD)-4 function inhibited NETosis and preserved endothelial integrity. The current study used systemic administration of targeted nanoparticles to deliver an agent that limits NETs formation to probe mechanisms of and demonstrate a novel therapeutic approach to plaque erosion that limits endothelial damage. Methods and results We developed Collagen IV-targeted nanoparticles (Col IV NP) to deliver PAD4 inhibitors selectively to regions of endothelial cell sloughing and collagen IV-rich basement membrane exposure. We assessed the binding capability of the targeting ligand in vitro and evaluated Col IV NP targeting to areas of denuded endothelium in vivo in a mouse preparation that recapitulates features of superficial erosion. Delivery of the PAD4 inhibitor GSK484 reduced NET accumulation at sites of intimal injury and preserved endothelial continuity. Conclusions NPs directed to Col IV show selective uptake and delivery of their payload to experimentally eroded regions, illustrating their translational potential. Our results further support the role of PAD4 and NETs in superficial erosion.

Rationale: Blood eosinophil count and ECP (eosinophil cationic protein) associate with human cardiovascular diseases. Yet, whether eosinophils play a role in cardiovascular disease remains untested. The current study detected eosinophil accumulation in human and murine abdominal aortic aneurysm (AAA) lesions, suggesting eosinophil participation in this aortic disease. Objective: To test whether and how eosinophils affect AAA growth. Methods and Results: Population-based randomized clinically controlled screening trials revealed higher blood eosinophil count in 579 male patients with AAA than in 5063 non-AAA control (0.236±0.182 versus 0.211±0.154, 10 9 /L, P <0.001). Univariate (odds ratio, 1.381, P <0.001) and multivariate (odds ratio, 1.237, P =0.031) logistic regression analyses indicated that increased blood eosinophil count in patients with AAA served as an independent risk factor of human AAA. Immunostaining and immunoblot analyses detected eosinophil accumulation and eosinophil cationic protein expression in human and murine AAA lesions. Results showed that eosinophil deficiency exacerbated AAA growth with increased lesion inflammatory cell contents, matrix-degrading protease activity, angiogenesis, cell proliferation and apoptosis, and smooth muscle cell loss using angiotensin-II perfusion–induced AAA in Apoe −/− and eosinophil-deficient Apoe −/− ΔdblGATA mice. Eosinophil deficiency increased lesion chemokine expression, muted lesion expression of IL (interleukin) 4 and eosinophil-associated-ribonuclease-1 (mEar1 [mouse EOS-associated-ribonuclease-1], human ECP homolog), and slanted M1 macrophage polarization. In cultured macrophages and monocytes, eosinophil-derived IL4 and mEar1 polarized M2 macrophages, suppressed CD11b + Ly6C hi monocytes, and increased CD11b + Ly6C lo monocytes. mEar1 treatment or adoptive transfer of eosinophil from wild-type and Il13 −/− mice, but not eosinophil from Il4 −/− mice, blocked AAA growth in Apoe −/− ΔdblGATA mice. Immunofluorescent staining and immunoblot analyses demonstrated a role for eosinophil IL4 and mEar1 in blocking NF-κB (nuclear factor-κB) activation in macrophages, smooth muscle cells, and endothelial cells. Conclusions: Eosinophils play a protective role in AAA by releasing IL4 and cationic proteins such as mEar1 to regulate macrophage and monocyte polarization and to block NF-κB activation in aortic inflammatory and vascular cells.

Objective: Vascular smooth muscle cell (VSMC) plasticity plays a critical role in the development of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging as important regulators in the vessel wall and impact cellular function through diverse interactors. However, the role of lncRNAs in regulating VSMCs plasticity and atherosclerosis remains unclear. Approach and Results: We identified a VSMC-enriched lncRNA cardiac mesoderm enhancer-associated noncoding RNA (CARMN) that is dynamically regulated with progression of atherosclerosis. In both mouse and human atherosclerotic plaques, CARMN colocalized with VSMCs and was expressed in the nucleus. Knockdown of CARMN using antisense oligonucleotides in Ldlr −/− mice significantly reduced atherosclerotic lesion formation by 38% and suppressed VSMCs proliferation by 45% without affecting apoptosis. In vitro CARMN gain- and loss-of-function studies verified effects on VSMC proliferation, migration, and differentiation. TGF-β1 (transforming growth factor-beta) induced CARMN expression in a Smad2/3-dependent manner. CARMN regulated VSMC plasticity independent of the miR143/145 cluster, which is located in close proximity to the CARMN locus. Mechanistically, lncRNA pulldown in combination with mass spectrometry analysis showed that the nuclear-localized CARMN interacted with SRF (serum response factor) through a specific 600–1197 nucleotide domain. CARMN enhanced SRF occupancy on the promoter regions of its downstream VSMC targets. Finally, knockdown of SRF abolished the regulatory role of CARMN in VSMC plasticity. Conclusions: The lncRNA CARMN is a critical regulator of VSMC plasticity and atherosclerosis. These findings highlight the role of a lncRNA in SRF-dependent signaling and provide implications for a range of chronic vascular occlusive disease states.

Background Fewer than 50% of patients develop calcification of both atherosclerotic plaques and aortic valves, implying differential pathogenesis. While circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs associate with early mineralization, but their contents, function, and contributions to disease remain unknown. Results Global proteomics of human carotid artery endarterectomies and calcified aortic valves from a total of 27 donors/patients revealed significant over-representation of proteins with vesicle-associated pathways/ontologies common to both diseases. We exploited enzymatic digestion, serial (ultra)centrifugation and OptiPrep density-gradient separation to isolate EV populations from diseased arteries and valves. Mass spectrometry found 22 EV marker proteins to be highly enriched in the four least-dense OptiPrep fractions while extracellular matrix proteins predominated in denser fractions, as confirmed by CD63 immunogold electron microscopy and nanoparticle tracking analysis. Proteomics and miRNA-sequencing of OptiPrep-enriched tissue EVs quantified 1,104 proteins and 123 miR cargoes linked to 5,182 target genes. Pathway networks of proteins and miR targets common to artery and valve tissue EVs revealed a shared regulation of Rho GTPase and MAPK intracellular signaling cascades. 179 proteins and 5 miRs were significantly altered between artery and valve EVs; multi-omics integration determined that EVs differentially modulated cellular contraction and p53-mediated transcriptional regulation in diseased vascular vs. valvular tissue. Conclusions Our findings delineate a strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Multi-omics and network approaches implicated tissue-resident EVs in human cardiovascular disease.

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 <0.001) in macrophages. Treatment with a RAGE antagonist or silencing with S100A9 siRNA in macrophages abolished these responses, suggesting that stimulation of the S100A9-RAGE axis by hyperglycemia favors a procalcific environment. We further showed that an imbalance between Nrf-2 (nuclear factor 2 erythroid related factor 2) and NF-κB (nuclear factor-κB) pathways contributes to macrophage activation and promotes a procalcific environment. In addition, streptozotocin-induced diabetic Apoe −/− 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.

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<0.05) in NDMV that may indicate the mitral valve predisposition to RVHD. Proteome analysis of non-diseased and diseased valves (NDMV vs RMVD, NDAV vs RAVD) identified a total of 213 proteins enriched in RHVD (q<0.05). The expression of the 12 NDMV-enriched proteins were evaluated across the 213 proteins enriched in diseased valves, resulting in the identification of ProTα common to both the RMVD and RAVD proteomes, but absent in CAVD. Additional KEGG pathway enrichment analyses linked 6 among 213 proteins with the estrogen signaling pathway (p<0.05). Immunoreactive ProTα colocalized with T CD8 cells in RHVD. ProTα and ERα also correlated strongly in circulating T CD8 cells in RHVD (p<0.05). ProTα plasma levels were higher in RHVD patients than healthy individuals (2 fold, p<0.05). Conclusion: We propose that ProTα is a novel contributor to the immunopathogenesis of RHVD and may regulate sex predisposition in this disease.

Long noncoding RNAs (lncRNAs) play important roles in regulating diverse cellular processes in the vessel wall, including atherosclerosis. RNA-Seq profiling of intimal lesions revealed a lncRNA, VINAS (Vascular INflammation and Atherosclerosis lncRNA Sequence), that is enriched in the aortic intima and regulates vascular inflammation. Aortic intimal expression of VINAS fell with atherosclerotic progression and rose with regression. VINAS knockdown reduced atherosclerotic lesion formation by 55% in LDL receptor–deficient (LDLR–/–) mice, independent of effects on circulating lipids, by decreasing inflammation in the vessel wall. Loss- and gain-of-function studies in vitro demonstrated that VINAS serves as a critical regulator of inflammation by modulating NF-κB and MAPK signaling pathways. VINAS knockdown decreased the expression of key inflammatory markers, such as MCP-1, TNF-α, IL-1β, and COX-2, in endothelial cells (ECs), vascular smooth muscle cells, and bone marrow–derived macrophages. Moreover, VINAS silencing decreased expression of leukocyte adhesion molecules VCAM-1, E-selectin, and ICAM-1 and reduced monocyte adhesion to ECs. DEP domain containing 4 (DEPDC4), an evolutionary conserved human ortholog of VINAS with approximately 74% homology, showed similar regulation in human and pig atherosclerotic specimens. DEPDC4 knockdown replicated antiinflammatory effects of VINAS in human ECs. These findings reveal a potentially novel lncRNA that regulates vascular inflammation, with broad implications for vascular diseases.

Introduction: In the current era of intense LDL lowering, superficial erosion may be on the rise as a cause of ACS (25-30%). Experimental data on human atheromata support a role for neutrophils and the formation of neutrophil extracellular traps (NETs) in the pathogenesis of superficial erosion and subsequent thrombosis. The common mutation of Janus kinase 2 ( Jak2 V617F ) borne by clones of leukocytes derived from bone marrow stem cells sensitizes neutrophils to undergo NETosis and linked NET formation to increased thrombosis. Jak2 V617F associates with accelerated atherosclerosis and venous thrombosis in patients with myeloproliferative neoplasms (MPN) and in individuals harboring this mutation but without a demonstrable MPN. Hypothesis We hypothesized that i) the Jak2 V617F associated with clonal hematopoiesis and increased atherosclerotic and thrombotic risk in humans, predisposes to NETosis and subsequent endothelial injury and thrombosis at sites of flow disturbance in arteries with erosion-like intimas; and ii) the clinically approved Jak-1,2 inhibitor, ruxolitinib (Rux), can preserve endothelial integrity and reduce thrombosis in mice with myeloid Jak2 V617F . Methods: We generated cohorts of mice harboring Jak2 V617F or wild-type Jak2 . We used a surgical preparation recapitulating features of superficial erosion and assessed endothelial integrity, thrombosis, and NET formation in relation to genotype and in vivo Rux treatment. Results and Conclusions: Evans blue extravasation following introduction of flow disturbance at sites of erosion-prone intimas showed significant impairment of endothelial barrier in the group of mice bearing the Jak2 V617F mutation compared to control (WT) mice ( ** p<0.005). We further observed more thrombosis in mice bearing the Jak2 V617F mutation. Immunohistochemical evaluation of the regions of experimental superficial erosion revealed reduced endothelial continuity and increased NET accumulation in Jak2 V617F vs. WT mice. Rux treatment mitigated the adverse effects of Jak2 V617F on both NET accumulation and endothelial integrity, supporting the translational potential of these observations in individuals with clonal hematopoiesis due to Jak2 V617F and acute coronary syndromes.

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) describes somatic mutations of bone marrow derived blood cells in the absence of overt hematological disease. CHIP correlates with aging and atherosclerotic cardiovascular diseases in humans. Tet2 mutations, common in CHIP, augment atherosclerosis. Tet2 -deficient macrophages over-express Interleukin-(IL)1β mRNA in response to LDL. IL-1β neutralization reduces recurrent events in patients post myocardial infarction with residual inflammatory risk.We previously showed that IL-1β neutralization reduces evolution of established mouse atherosclerosis. Hypothesis: This study tested the hypothesis that IL-1β neutralization will reduce early accelerated atherosclerosis caused by Tet2 deficiency in hematopoietic cells. Methods: We transplanted lethally irradiated LDL-R -/- atherosclerotic prone mice with either Tet2 +/+ or Tet2 - /- bone marrow cells. Chimeric mice consumed a western diet and received a selective anti-mouse IL-1β monoclonal antibody or isotype-matched control. After nine weeks of treatment, we analyzed atherosclerotic lesions morphologically and by single cell mRNA analysis. Results: IL-1β neutralization reduced accelerated atherosclerosis associated with Tet2 deficiency in female but not in male mice (Table). Single cell RNA-sequencing analysis of the dissociated aortic arch revealed differences in macrophage expression of genes involved in innate immune response interleukin signaling pathways between male and female mice. Conclusions: This unexpected sex-dependence of accelerated atherogenesis in Tet2 - /- bone-marrow mice on IL-1β signaling lays the ground for further mechanistic exploration.

IgE-mediated activation of Nhe1 (Na + -H + exchanger-1) induces aortic cell extracellular acidification and promotes cell apoptosis. A pH-sensitive probe pHrodo identified acidic regions at positions of macrophage accumulation, IgE expression, and cell apoptosis in human and mouse abdominal aortic aneurysm (AAA) lesions. Ang II (angiotensin II)–induced AAA in Nhe1-insufficient Apoe −/− Nhe1 +/− mice and Apoe −/− Nhe1 +/+ littermates tested Nhe1 activity in experimental AAA, because Nhe1 −/− mice develop ataxia and epileptic-like seizures and die early. Nhe1 insufficiency reduced AAA incidence and size, lesion macrophage and T-cell accumulation, collagen deposition, elastin fragmentation, cell apoptosis, smooth muscle cell loss, and MMP (matrix metalloproteinase) activity. Nhe1 insufficiency also reduced blood pressure and the plasma apoptosis marker TCTP (translationally controlled tumor protein) but did not affect plasma IgE. While pHrodo localized the acidic regions to macrophage clusters, IgE expression, and cell apoptosis in AAA lesions from Apoe −/− Nhe1 +/+ mice, such acidic areas were much smaller in lesions from Apoe −/− Nhe1 +/− mice. Nhe1-FcεR1 colocalization in macrophages from AAA lesions support a role of IgE-mediated Nhe1 activation. Gelatin zymography, immunoblot, and real-time polymerase chain reaction analyses demonstrated that Nhe1 insufficiency reduced the MMP activity, cysteinyl cathepsin expression, IgE-induced apoptosis, and NF-κB activation in macrophages and blocked IgE-induced adhesion molecule expression in endothelial cells. A near-infrared fluorescent probe (LS662) together with fluorescence reflectance imaging of intact aortas showed reduced acidity in AAA lesions from Nhe-1-insufficient mice. This study revealed extracellular acidity at regions rich in macrophages, IgE expression, and cell apoptosis in human and mouse AAA lesions and established a direct role of Nhe1 in AAA pathogenesis.