Atherosclerosis

BACKGROUND: Coronary artery disease (CAD) and Impaired Cognitive (IC) disease share sociodemographic, genetic, and clinical factors, but the association of IC with statin use in CAD remains unclear. OBJECTIVES: To determine the association between IC and statin use in CAD based on APO (e) genotype, sex, and lipid levels. DESIGN, SETTING, AND PARTICIPANTS: We performed a retrospective study of AllofUS (AoU) participants with CAD (Age [&ge;]60 yrs) enrolled from 2017 to 2023. We defined CAD as having a history of angina/myocardial infarction/chronic ischemic heart disease or having percutaneous coronary intervention/CABG, and IC defined as mild cognitive impairment or all cause dementia, using ICD/SNOMED codes. MEASURES: We assessed the association between IC and statin use using logistic regression analysis, while adjusting for clinical factors, sociodemographics, and APO (e) genotypes before and after propensity score matching. We further performed stratified analysis by sex, and APO (e) genotypes. We finally assessed the association between IC and statin users, based magnitude on the change in lipid levels before CAD and after IC (TC: Total cholesterol, LDL: low density lipoprotein, HDL: High Density Lipoprotein). Significance was defined at p < 0.05. RESULTS: The cohort included 22,089 participants with CAD and 1343 with IC. Thirty-nine percent of participants were females, 77% were European, 13% were African American, and 9% were of Admixed American ancestry. The proportion of IC was higher (6.8% vs 3.5%, p<0.001) in statin users (n=17,191) vs non-statin users (n=4,898). IC was significantly associated with statin use (OR:1.70;1.40-2.10, p = 4.9e-7) after adjustment for clinical factors, sociodemographics, and APO (e) genotypes. After propensity-score matching between IC and CAD, we observed an association between IC and statin use (OR:1.55;1.24-1.94, p =1e-4). In stratified analysis, the association between IC and statin use was strongest in the APO e3/e3 group (OR:2.04;1.53-2.75, p = 1e-6), and in females (OR:2.20;1.60-3.06, p = 2.e-6) compared to males (OR:1.43;1.10-1.90, p = 0.01). We finally observed an increased magnitude of association between IC and statin users having higher HDL increase (> 10 mg/dl: OR:1.95;1.44-2.66, p=1e-5) as compared to statin users with lesser HDL increase (<=; 10mg/dl: OR:1.61;1.22-2.15, p=8e-4). CONCLUSION: In the AllofUS cohort, IC was significantly associated with statin use in CAD participants. We observed the strongest association in the APO e3/e3 group, among females, and with a greater increase in HDL levels in statin users.

BACKGROUND: MicroRNAs (miRNAs) regulate macrophage plasticity in atherosclerosis (AS). We tested the hypothesis that miR487a-3p and miR6855-3p accelerate AS by promoting macrophage inflammatory responses and metabolic dysregulation. METHODS: miRNA-seq and mRNA-seq were performed on peripheral monocytes from CAD patients and healthy controls. Macrophages in mouse aortas and human coronary arteries were characterized by flow cytometry and immunostaining. AS was evaluated in PCSK9-overexpressing mice with myeloid-specific deficiency of carboxypeptidase E (CPE) or ribonucleotide reductase subunit M2 (RRM2) fed a high-fat diet. RESULTS: miR487a-3p and miR6855-3p were the top differentially expressed miRNAs in peripheral monocytes from CAD patients versus controls. Both miRNAs were lipid-inducible, with transcription driven by ox-LDL via KLF5 and IRF1, respectively, and were secreted extracellularly. Plasma levels of both miRNAs were elevated in CAD patients, correlated positively with blood lipids and Gensini score, and exhibited diagnostic accuracy (AUC 0.83 each). Both miRNAs were predominantly expressed in coronary plaque macrophages, and their abundance correlated with lesion area. Overexpression of either miRNA promoted macrophage pro-inflammatory polarization, lipid metabolic dysregulation, foam cell formation, and endothelial cell apoptosis, whereas miRNA inhibition attenuated these ox-LDL-induced phenotypes. CPE and RRM2 were identified as direct targets of miR487a-3p and miR6855-3p, respectively, by integrating mRNA-seq and TargetScan predictions, with binding confirmed by dual-luciferase assays and miRNA pulldown. CPE or RRM2 overexpression partially reversed miRNA-induced macrophage dysfunction. Conversely, myeloid-specific deletion of Cpe or Rrm2 exacerbated AS in hypercholesterolemic mice. CONCLUSIONS: miR-487a-3p and miR-6855-3p are promising biomarkers for CAD diagnosis and prognosis. Mechanistically, they drive macrophage inflammation and lipid metabolic disruption, identifying them as potential therapeutic targets. Key Words: microRNA; atherosclerosis; macrophage; inflammation; lipid disorders

BACKGROUNDSmooth muscle cells (SMCs) comprise the majority of cells in human atherosclerotic lesions and are thought to be a major source of cholesterol-overloaded foam cells in human and mouse atheromas. However, the transcriptomic profile, specific markers, and biologic itinerary of SMC foam cells relative to macrophage foam cells remain poorly defined. METHODSSingle-cell RNA sequencing (scRNA-seq) was performed on fresh coronary artery segments from heart transplant recipients with early- to intermediate-stage atherosclerosis. Gene expression in a putative SMC foam cell cluster was compared with cultured SMCs loaded with aggregated low-density lipoprotein (agLDL) or cholesterol-methyl-{beta}-cyclodextrin (Chol-M{beta}CD). Candidate markers distinguishing SMC from macrophage foam cells were validated using additional publicly-available scRNA-seq datasets, Xenium spatial transcriptomics, and immunofluorescence microscopy of human coronary atheromas. Pathway analysis was performed using Gene Set Enrichment Analysis Hallmark gene sets. RESULTSA distinct SMC foam cell cluster derived from fibromyocytes ("lipomyocytes") was identified using markers induced by in vitro cholesterol loading. agLDL loading reproduced the lipomyocyte transcriptional profile, whereas Chol-M{beta}CD induced an inflammatory phenotype colocalizing with macrophages rather than lipomyocytes. Lipomyocytes highly expressed SERPINE1, encoding plasminogen activator inhibitor-1 (PAI-1), and CFH, encoding complement factor H, which were validated in human coronary lesions by spatial transcriptomics and immunofluorescence microscopy. Compared with macrophage foam cells, lipomyocytes demonstrated distinct pathway activation, including enrichment of extracellular matrix, coagulation and angiogenesis pathways. CONCLUSIONSSMC foam cells, or lipomyocytes, represent a distinct foam cell phenotype with unique markers and biologic programs that differ from macrophage foam cells during atherosclerotic plaque development. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LISmooth muscle cell (SMC) foam cells, or lipomyocytes, arise from fibromyocytes and exhibit a transcriptomic profile that is markedly distinct from that of macrophage foam cells. C_LIO_LIIn vitro loading of SMCs with aggregated LDL recapitulates the gene expression profile of SMC foam cells in human coronary atheromas, whereas loading with cyclodextrin-bound cholesterol does not. C_LIO_LIPlasminogen activator inhibitor 1 (PAI-1, encoded by SERPINE1) and Complement Factor H are specific markers of SMC foam cells and are not expressed by macrophage foam cells. C_LI What Are the Clinical Implications?O_LISMCs contribute a substantial proportion, and potentially the majority, of foam cells in atherosclerotic lesions. C_LIO_LIDefining the biological trajectory of SMC foam cells within plaques is critical for understanding their roles in plaque progression, rupture and thrombosis, and for establishing their relevance as a distinct therapeutic target to reduce major cardiovascular events. C_LI

INTRODUCTIONMost randomized controlled trials (RCTs) found that omega-3 fatty acids have little to no effect on cardiovascular disease risk. However, a few suggested that a specific omega-3 fatty acid, eicosapentaenoic acid (EPA), reduces cardiovascular disease risk in patients with high triglycerides (TG). It is unclear whether EPA is beneficial in the general population or how it affects triglyceride-rich lipoproteins (TRL) and related traits. Using two-sample Mendelian randomization (MR), this study aimed to evaluate whether EPA has a protective effect on ischemic heart disease (IHD), TRL, and related traits in a general population. METHODSAssociations of genetic variants with plasma EPA (EPIC-Norfolk, INTERVAL; n=14,267), and the outcomes IHD (Aragam et al., cases/n=181,522/1,165,690; FinnGen, N cases/n=31,640/218792), TRL, and related traits (Karjalainen et al.; n=68,559) were based on summaries from previous genome wide association studies (GWAS) of participants of European descent. Using eight proposed instruments associated with plasma EPA (P<5*10-5), inverse-variance weighted (IVW), MR-Egger, and weighted median (WM) estimators were used to determine the effect of a period shift in the natural log of plasma EPA one standard deviation, or EPA, on these outcomes. RESULTSUsing IVW, EPA was associated with higher odds of IHD (OR=1.05; 95% CI=1.00, 1.10), but the CI included the null value. The WM estimate was similar, and the MR-Egger estimate was closer to the null (OR=1.01; 95% CI: 0.90, 1.11). EPA was associated with lower serum TG and lower large to small very low-density lipoprotein (VLDL) particle concentrations, but with increases in very small VLDL, intermediate density lipoproteins, and low-density lipoproteins. Although the distribution changed from larger to smaller TRL, there was no change in apolipoprotein B. EPA was also associated with increases in very large to medium high-density lipoprotein (HDL) particles and no change in small HDL, consistent with an increase in apolipoprotein A-I. EPA was also associated with increases in both remnant cholesterol and total serum cholesterol. DISCUSSIONThis study suggests that EPA may not have a beneficial effect on IHD in the general population of European ancestry. Rather, EPA appears to remodel TRL, possibly through lipolysis of large particles without full clearance of the resulting smaller particles, and this may have mixed implications for cardiovascular disease risk. A cardiovascular outcome trial of EPA monotherapy in a general population that collects lipid/lipoprotein subfractions would be needed to confirm these findings.

Background: Vascular smooth muscle cells (VSMCs) phenotypic plasticity can modulate atherosclerosis progression. Although several gene regulatory steps towards pro-inflammatory phenotypes have been well-studied, epitranscriptomic changes during this transition and their regulatory roles remain unexplored. Methods and Results: Primary human VSMCs were stimulated with TGF-{beta}1 to induce an atheroprotective, contractile, and matrix-producing state and with IL1-{beta} plus PDGF-BB to induce a highly energetic, pro-inflammatory state, confirmed by Illumina bulk RNA sequencing and proteomics. Untargeted screening of mRNA base modifications using Oxford Nanopore Technologies direct RNA sequencing and xPore analysis revealed enhanced uridine modification within a GUUUU motif in pro-inflammatory VSMCs. Modified uridines were enriched in 3'-UTR and accessible RNA structures, with implications on Poly(A) tail dynamics and miRNA binding. Conclusions: Atheroprotective and pro-atherogenic treatments induce distinct epitranscriptomic landscapes composed of different modification types, often co-localized in the same transcript. Modified uridines in mRNAs are abundant in a high-energy, pro-inflammatory VSMC state and associated with post-transcriptional regulation. In summary, epitranscriptomics adds a novel regulatory layer to VSMC phenotypic transitions critical for atherosclerosis development and progression.

Background and aimsAtherosclerotic plaque rupture is a major cause of myocardial infarction and stroke. However, the precise drivers of plaque destabilisation remain elusive. We hypothesised that antigen-driven, autoimmune-like T cell responses are central to the destabilisation and rupture of atherosclerotic plaques. MethodsTo dissect T cell responses specifically in unstable compared to stable plaques, we leveraged near-infrared autofluorescence (NIRAF) imaging-guided dissection of human carotid plaques. We also used our tandem stenosis model reflecting plaque instability as seen in patients to differentiate between unstable and stable plaques in mice. To explore T cell involvement, we studied T cell differentiation states and T cell receptor (TCR) repertoires by single-cell multi-omics. Then, testing if antigen-driven CD8+ T cell responses drive plaque instability in mice, we applied a combination of AAV8-PCSK9-induced atherosclerosis, tandem stenosis and TCR transgenic mice. Finally, we leveraged data from the AtheroExpress Biobank Study to link T cell immunity to histology-defined instability and cardiovascular outcomes. ResultsT cell responses in unstable versus stable atherosclerosis were distinct. Unstable human plaques contained highly expanded, autoimmune-like CD8 T cells with markedly increased cytotoxic signatures, reduced exhaustion and distinct clonal repertoires compared to stable regions. Most plaque CD8 T cells exhibited a pronounced tissue-resident transcriptional program. Moreover, the transcriptional signature of these plaque resident T cells was distinct from multiple other human tissues. Autoimmune-like cytotoxic and tissue-resident CD8+ T cell responses were also evident in murine atherosclerosis, where restricting the activation of antigen-driven CD8+ T cells prevented plaque destabilisation. Importantly, analysis of carotid endarterectomy samples from >1000 patients identified that intraplaque cytotoxic CD8 T cell gene signatures strongly correlated with histological instability and predicted future strokes. ConclusionsIntegrated human, murine and clinical analyses demonstrate that autoimmune-like, cytotoxic CD8 T cell responses are central drivers of plaque instability and major cardiovascular events. Targeting pathogenic CD8 T cell responses may thus offer a compelling immunomodulatory strategy to stabilise plaques and reduce the risks of stroke and myocardial infarction. Graphical AbstractO_ST_ABSKey QuestionC_ST_ABSRupture of unstable atherosclerotic plaques is a typical cause of myocardial infarction and stroke. To understand the underlying cause and to prevent plaque rupture, we addressed the central hypothesis that autoimmune-like T cell responses drive plaque destabilisation and rupture. Key FindingsCD8+ T cells are clonally expanded with increased cytotoxic signatures in unstable versus stable plaques (mice and humans) and require antigen recognition to drive plaque instability. Cytotoxic CD8+ T cell signatures in excised plaques correlate with increased future cardiovascular events. Take Home MessageAutoimmune-like adaptive immune reactions, dominated by CD8+ T cells, are a major driver of plaque instability/rupture. Therefore, targeting pathogenic CD8 T cell responses offers a compelling immunomodulatory strategy to stabilise plaques and reduce the risk of myocardial infarction and stroke. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/720043v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@bd83dcorg.highwire.dtl.DTLVardef@1bee3borg.highwire.dtl.DTLVardef@1b56bc0org.highwire.dtl.DTLVardef@1b5176f_HPS_FORMAT_FIGEXP M_FIG C_FIG

IntroductionAccurate stratification of hard atherosclerotic cardiovascular disease (ASCVD) risk remains challenging despite advances in prevention. Liver function biomarkers (LFBs), particularly gamma-glutamyl transferase (GGT), have been linked to cardiovascular outcomes, yet their contribution to hard ASCVD risk prediction is not well defined. MethodsThis study analyzed data from the National Health and Nutrition Examination Survey (NHANES, 2005-2018) to assess cross-sectional associations between LFBs and 10-year hard ASCVD risk estimated by the ACC/AHA Pooled Cohort Equations. Multivariable regression, restricted cubic splines, and mediation analyses were applied to examine independent and dose-response relationships. External validation was performed in the China Health and Retirement Longitudinal Study (CHARLS) and NHANES using machine learning models (CoxBoost, Naive Bayes and Random Forest). ResultsAmong 5,731 NHANES participants, GGT showed an independent linear association with hard ASCVD risk (P-trend = 0.003), partly mediated by systolic blood pressure (44.8%), HbA1c (19.0%), and high density lipoprotein cholesterol (13.4%). Machine learning (ML) models incorporating GGT, alkaline phosphatase (ALP), and globulin alongside traditional risk factors improved predictive accuracy, with Naive Bayes achieving an AUC of 0.751 in NHANES validation. ConclusionsGGT is an independent and biologically plausible biomarker of hard ASCVD risk, acting through cardiometabolic pathways. Incorporating LFBs into risk prediction models, particularly with machine learning, enhances risk stratification and may facilitate early identification of high-risk individuals.

BackgroundVascular smooth muscle cells (VSMCs) play a central role in atherosclerotic coronary artery disease (CAD). Oxidised low-density lipoprotein cholesterol (ox-LDL) induces VSMCs dysfunction but the underlying molecular mechanisms are unclear. CAD genome-wide association studies (GWAS) have identified hundreds of disease-associated loci but their biological roles remain poorly defined. We hypothesised that ox-LDL drives pro-atherogenic changes in VSMCs by altering gene regulatory programs involving causal CAD variants. MethodsEx-vivo human coronary VSMCs were exposed to ox-LDL and profiled using RNA-seq, ATAC-seq, and H3K27ac ChIPmentation. Enhancer-gene links were inferred by integrating these data with Hi-C using the Activity-by-Contact (ABC) model. Variant effect predictions were done using AlphaGenome and key target genes functionally tested by CRISPR/Cas9 knockout. ResultsOx-LDL induced widespread transcriptional reprogramming in coronary VSMCs, with 1,487 upregulated and 1,864 downregulated genes (FDR < 0.05). Single-cell RNA-seq meta-analysis demonstrated that ox-LDL-associated programmes enriched in pro-inflammatory and synthetic-inflammatory VSMC clusters in vivo. ATAC-seq identified [~]22k differentially accessible regions following ox-LDL exposure (FDR < 0.05). Integration of ATAC-seq, H3K27ac, and Hi-C using the ABC framework showed that ox-LDL-driven chromatin remodelling was concentrated at distal enhancers, which linked to 2,008 differentially expressed genes via 4,243 peak-gene connections. ABC enhancers were significantly enriched for CAD variants compared with non-vascular disease controls, with stronger enrichment in dynamically accessible enhancers. AlphaGenome predicted larger regulatory effects of prioritised CAD variants in smooth muscle cells than in a non-vascular comparator, and motif analyses indicated allele-dependent transcription factor binding at prioritised enhancer variants. Locus-level prioritisation nominated candidate enhancer-mediated mechanisms at the SPECC1L and MAP1S loci, and CRISPR knockout of the target genes GUCD1 and BACH1 rescued ox-LDL-induced growth arrest/senescence phenotypes in human coronary artery VSMCs. ConclusionsOur unbiased multi-omics framework shows that ox-LDL rewires VSMC regulatory programmes that influence CAD genetic risk. Enhancer-gene mapping refines effector-gene assignment at CAD loci and prioritises regulatory targets in coronary VSMCs.

Atherosclerosis is linked to an increased risk of cognitive decline, with chronic inflammation being a common feature of both pathologies. IL-12 activates STAT4 to regulate myeloid cell functions, and blockade of this pathway alleviates cognitive impairment in Alzheimers models. However, the mechanisms connecting vascular pathology to neuroinflammation remain unclear. Here, we examine whether STAT4 functions as a common mediator of neuroinflammation in atherosclerosis. We demonstrate that LysMCre-specific STAT4 deficiency ameliorates deficits in long-term memory in low-density lipoprotein-deficient (Ldlr-/-) mice fed a high-fat diet (HFD-C). STAT4 deficiency moderately reduces Ser199-phosphorylated Tau burden. Atherosclerosis alters brain immune composition, characterized by increased numbers of CD45+ leukocytes, activated microglia, and activated T and B cells, whereas STAT4 deficiency attenuates these effects. Nanostring gene-expression pathway analysis further highlights the importance of STAT4 in regulating multiple neuroinflammatory pathways and the Rhodopsin-like receptor signaling, which is associated with synaptic plasticity. LysMCre-specific STAT4 deficiency supports microglial efferocytosis in atherosclerotic Ldlr-/- mice and increases the number of efferocytotic macrophages. Accordingly, STAT4 deficiency also reduced neuronal death. Overall, our data reveal an important role for myeloid-driven STAT4 expression in the pathogenesis of cognitive decline associated with atherosclerosis, mediated through impaired efferocytosis and enhanced leukocyte activation, leading to increased brain neuroinflammation.

BACKGROUNDInactivation of ANGPTL3 (angiopoietin-like protein 3, A3) is a proven therapeutic strategy for lowering plasma lipid levels independently of the LDL receptor (LDLR), yet the optimal approach to inactivate A3 remains unclear. A3 is proteolytically cleaved and circulates as full-length (A3-FL), N-terminal (A3-Nter) and C-terminal (A3-Cter) fragments. The specific contribution of each form of A3, and of its paralog, ANGPTL8 (A8), in modulating circulating levels of ApoB-Containing Lipoproteins (ABCLs) remain poorly defined. Clarifying these relationships will inform next-generation A3-directed therapies. METHODSWe performed liver perfusion studies to directly compare the number and composition of VLDL particles secreted from mice with and without A3. To amplify effects on cholesterol metabolism, we generated Ldlr-/- mice expressing wildtype A3 (A3-WT), A3-FL or A3-Nter, with or without co-expression of A8, and analyzed plasma lipids, circulating A3 and A8 complexes, and intravascular lipase activities. Complementary in vitro assays and structural modeling were used to assess relative endothelial lipase (EL) inhibition by A3 alone or in complex with A8. RESULTSLiver perfusion studies revealed that A3 inactivation does not alter the rates of hepatic secretion of VLDL in wildtype or Ldlr-/- mice. Inactivation of A8 alone lowered plasma LDL-cholesterol (C) levels by [~]20%, an effect dependent upon the expression of both EL and A3. Maximal inhibition of lipoprotein lipase (LPL) required co-expression of A8 plus both A3-FL and A3-Nter, indicating that A3 cleavage, in addition to A8 expression, is essential for maximal LPL inhibition. In contrast, A8 expression, but not A3 cleavage, was required for optimal EL inhibition. CONCLUSIONSA8 acts in concert with A3 to differentially modulate LPL- and EL-mediated lipolysis, which antagonizes hepatic clearance of newly-secreted atherogenic ABCLs. This mechanistic framework refines our understanding of A3-targeted lipid lowering and highlights the therapeutic potential of dual A3- plus A8-directed strategies to treat dyslipidemia and prevent atherosclerotic cardiovascular disease. Clinical perspectiveO_ST_ABSWhat is new?C_ST_ABSO_LIInactivation of A3 lowers circulating ABCL levels without altering hepatic secretion rates of VLDL-ApoB or -TG. C_LIO_LIProteolytic cleavage of A3 is required for maximal inhibition of LPL. C_LIO_LIInactivation of A8 lowers LDL-C levels through an A3- and EL-dependent, but LDLR-independent, mechanism. C_LI What are the clinical implications?O_LICombining A8 inhibition with A3-inactivating therapies offers a strategy to achieve greater reduction in LDL-C levels and atherosclerotic cardiovascular risk. C_LI

STRUCTERED ABSTRACTO_ST_ABSBACKGROUNDC_ST_ABSCoronary artery bypass graft (CABG) is a widely performed procedure for coronary artery disease (CAD), yet its association with Impaired Cognition (IC), i.e., mild-cognitive impairment or all-cause dementia, while accounting for APO ({varepsilon}) genotype, remains unclear. METHODSWe analyzed AllofUS participants with CAD (Age[&ge;]60 yrs) from 2017-2023. We defined CAD as a history of angina/myocardial infarction/chronic ischemic heart disease or having percutaneous coronary intervention/CABG, and IC as mild cognitive impairment or all-cause dementia using ICD/SNOMED codes. We performed logistic regression analyses to assess the association between CABG and IC, adjusting for clinical factors (age, sex, hypertension, diabetes, hyperlipidemia, depression, stroke, smoking, alcohol use, statin/antihypertensive/antidiabetic use), social determinants (self-reported race/ethnicity, income, employment), and APO ({varepsilon}) genotypes. We further performed stratified analyses across APO ({varepsilon}) genotypes ({varepsilon}2/{varepsilon}2, {varepsilon}2/{varepsilon}3 {varepsilon}3/{varepsilon}3, {varepsilon}2/{varepsilon}4, {varepsilon}3/{varepsilon}4, {varepsilon}4/{varepsilon}4). We defined significance at p [&le;] 0.05. RESULTSWe included 22,349 with CAD and identified 908 with IC after CAD till 2023. 40% were females, 70% were White, 12% were Black, and 9% were Hispanic. The proportion of IC was higher (5.1% vs 3.5%, p=1e-08) in CABG (n=8,135) vs non-CABG (n=14,214). After adjusting for clinical factors, social determinants, and APO ({varepsilon}) genotypes, CABG (1.23;1.06-1.41, p = 0.005) was associated with IC. In APO ({varepsilon}) stratified analysis, the association of CABG with IC was strongest in the APO {varepsilon}2/{varepsilon}3 group (1.91;1.21-3.02, p = 0.005). CONCLUSIONIn the AllofUS cohort, we observed an association between CABG and IC in CAD participants, with the strongest association in the APO {varepsilon}2/{varepsilon}3 group. Key MessageO_ST_ABSWhat is already known on this topicC_ST_ABSCoronary artery disease (CAD) and Impaired Cognitive (IC) disease, i.e., mild cognitive impairment and all-cause dementia, share genetic, sociodemographic, and clinical factors, including cardiovascular conditions like coronary artery bypass grafting (CABG) procedure. What this study addsWe observed an association between CABG and IC in CAD participants after adjusting for sociodemographic, clinical factors, and APO ({varepsilon}) effects. Further, when CAD participants were stratified across APO ({varepsilon}) groups, CABG was significantly associated with IC in the APO {varepsilon}2/{varepsilon}3 group. How this study might affect research, practice or policyOur observations highlight the role of APO ({varepsilon}) genotype evaluation in CAD patients for IC risk assessment.

Background: Familial hypercholesterolemia (FH) is most frequently caused by pathogenic variants in LDLR, but phenotypic variability suggests the influence of genetic modifiers. Methods: We investigated a large multigenerational family with FH, combining clinical data, lipid profiles, and genetic analysis with functional studies. LDLR and PCSK9 variants were characterized according to ACMG/ClinGen guidelines. Functional assays in CHO-ldlA7 cells assessed LDLR activity, while plasma PCSK9 levels were quantified by ELISA. Results: LDLR c.2479G>A variant was associated with FH in the family. The presence of loss of function c.137G>T and c.2023del variants at PCSK9 appears to mitigate the effect of the LDLR variant. Conclusions: This study provides evidence that PCSK9 variants can counteract the deletereous effect of a LDLR variant associated with FH. These findings highlight the importance of gene/gene interactions in the clinical variability of FH and their potential implications for precision medicine.

BackgroundSex-related differences in cardiovascular disease suggest the presence of intrinsic vasoprotective mechanisms, with estrogen recognized as an important modulator of endothelial function. Building on existing evidence, the present study provides mechanistic insights into how estrogen and nitric oxide (NO) signaling regulate selective pathways of oxLDL uptake, mitochondrial dynamics, and inflammatory responses during early atherogenesis. MethodsWe combined an in vitro endothelial cell-macrophage co-culture model with in vivo studies in low-density lipoprotein receptor-knockout (LDLr-/-) mice to investigate the role of estrogen in early atherosclerotic processes. Human aortic endothelial cells (HAECs) were exposed to oxidized low-density lipoprotein (oxLDL) in the presence or absence of 17{beta}-estradiol (E2) and the nitric oxide (NO*) donor S-nitroso-N-acetylcysteine (SNAC). Key outcomes included oxLDL uptake, mitochondrial oxidative stress, mitochondrial dynamics, and inflammatory signaling. In vivo, male and female LDLr-/- mice were exposed to a short-term high-fat diet with or without SNAC treatment. Plasma lipid levels, blood pressure, aortic lesion formation, and cardiac remodeling were evaluated. ResultsE2 reduced oxLDL uptake and oxidative stress, effects recapitulated by SNAC; however, these responses involved distinct entry pathways, with E2 preferentially modulating lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) dependent uptake and SNAC targeting caveolae-associated mechanisms. In parallel, both E2 and SNAC reduced Scavenger Receptor Class B Type 1 (SR-B1) expression, suggesting an additional modulation on oxLDL transcytosis via this mechanism. Endothelial cells exposed to oxLDL exhibited altered mitochondrial regulatory proteins, including superoxide dismutase 2 (SOD-2), dynamin-related protein 1 (Drp-1), and optic atrophy protein 1 (OPA-1). Despite reducing oxidative stress, E2 increased the expression of adhesion molecules and enhanced monocyte adhesion in response to oxLDL exposure, particularly when combined with SNAC. Strikingly, E2 also modulated macrophage responses, increasing interleukin receptor antagonist (IL-1ra) expression and reducing GDF15, macrophage inhibitory factor (MIF), macrophage inflammatory protein 3 alfa (MIP-3), and matrix metalloproteinase 9 (MMP-9) levels, consistent with a less pro-inflammatory macrophage profile. In vivo, HFD increased plasma lipid levels and atherosclerotic lesion area in LDLr-/- mice, whereas SNAC partially attenuated these effects without affecting plasma lipid levels. In vivo, female LDLr-/- mice developed approximately 50% smaller aortic lesions than males, despite comparable or higher plasma lipid levels. A dyslipidemia led to increased blood pressure and a hypertensive phenotype in both males and females. SNAC treatment reduced lesion burden in both sexes and prevented diet-induced hypertension in females. ConclusionEstrogen limits early atherogenic injury by reducing endothelial uptake of oxLDL, preserving mitochondrial homeostasis, and modulating inflammatory signaling. Together, the E2 and NO pathways regulate early atherosclerosis through distinct yet complementary mechanisms, offering a potential framework for vascular-protective strategies.

Background: Unruptured intracranial aneurysms (UIAs) pose a significant risk of subarachnoid hemorrhage. Both hypertension and hyperhomocysteinemia are recognized as independent risk factors for vascular disease; however, their combined impact (H-type hypertension) on aneurysm instability and rupture remains unclear. Methods: We analyzed a prospective cohort of 358 adults with UIAs (475 aneurysms) using high-resolution vessel-wall MRI (HRVWI) for cross-sectional and longitudinal assessment. H-type hypertension was defined as hypertension with plasma homocysteine ?10 ?mol/L. Multivariable logistic regression assessed associations with AWE and aneurysm growth (longitudinal sub-cohort: n = 82, 89 aneurysms). Mendelian randomization (MR) analyses evaluated the causal role of homocysteine in hypertension and aSAH. Proteomic profiling identified potential molecular mechanisms. Results: AWE occurred in 33.7% of aneurysms, which were larger, irregular, and had higher PHASES scores. Elevated homocysteine (10.3 vs 9.5 ?mol/L, p = 0.004) and H-type hypertension (43.8% vs 28.3%, p < 0.001) were associated with AWE. After adjustment, H-type hypertension independently predicted AWE (OR = 3.18) and aneurysm growth (OR = 3.63). MR analyses showed homocysteine increased aSAH (OR = 1.39) and hypertension risk (OR = 1.10), while hypertension increased aSAH risk (OR = 1.58). Mediation analysis did not support hypertension as a mediator (p = 0.20). Proteomic analyses identified key pathways related to inflammation?immune dysregulation, extracellular matrix remodeling, and signaling activation as potential mediators. Conclusions: H-type hypertension amplifies aneurysmal-wall instability and growth. Combined control of blood pressure and homocysteine merits prospective evaluation for UIA prevention.

Intraplaque haemorrhage (IPH) is a hallmark of advanced atherosclerosis and a major risk factor for ischemic stroke and myocardial infarction. Current IPH classification focusses on extravascular erythrocyte presence as a proxy of acute bleeding, where iron detection generally indicates older haemorrhages. While often used interchangeably, a comprehensive analysis of transcriptional and metabolic context and impact of iron and erythrocyte deposition on the plaque is still lacking. Here, we investigate iron as a late-stage IPH hallmark in human atherosclerotic plaques. We analysed erythrocyte-rich, iron-rich, and non-IPH regions in human carotid endarterectomy plaques by re-analysing a published transcriptomic dataset of 43 patient samples. In addition, we performed histological and immune phenotyping to define plaque traits associated with iron versus erythrocyte accumulation. Finally, we performed spatial metabolic profiling to functionally define iron-rich regions. Although iron and erythrocyte deposits frequently co-localised, both co-related with different histological traits. While iron- and erythrocyte-rich regions shared transcriptomic features of advanced plaques compared with non-IPH regions, direct comparison showed differences in gene expression profiles. Iron deposition was associated with increased myeloid cell accumulation and a unique spatial metabolic signature distinct from erythrocyte-rich and non-IPH regions. While sharing many characteristics with IPH plaques, the molecular, cellular and metabolic landscape of iron-rich regions is marked by features of plaque remodelling and repair. This makes iron deposition a unique hallmark of late-stage IPH, extending the current erythrocyte-based definition of IPH.

DOT1L-catalyzed H3K79 methylation is a hallmark of actively transcribed genes and has been extensively studied in developmental and disease contexts. While DOT1L inhibition has emerged as a promising therapeutic strategy in cancer, its role in pro-atherogenic endothelial inflammation remains unclear. To investigate this, we utilized an in vivo partial carotid artery ligation model and observed increased DOT1L expression and H3K79me3 level. Consistently, in vitro studies employing a 3D-printed human coronary artery model and TNF- stimulation corroborated these results, showing elevated DOT1L expression and H3K79me3 deposition, while levels of H3K79me and me2 remained unchanged. Further analyses identified key DOT1L-containing complex (DotCom) components, AF10 and AF9 (upregulated) and AF17 (downregulated), as contributors to the enhanced H3K79me3 landscape. CUT&RUN sequencing showed prominent H3K79me3 enrichment at the RELA (NF-{kappa}B p65) promoter, corresponding with increased NF-{kappa}B p65 expression and activation. Notably, inhibition/knockdown of the methyltransferase DOT1L or overexpression of the demethylase FBXL10 significantly reduced H3K79me3 levels, thereby suppressing NF-{kappa}B p65 expression and attenuating endothelial inflammation, independent of canonical NF-{kappa}B p65 activation. These findings establish DOT1L-mediated H3K79me3 as a crucial epigenetic regulator of endothelial inflammation, highlighting a potential therapeutic avenue for mitigating NF-{kappa}B p65-driven pro-atherogenic endothelial dysfunction.

Introduction: Menopause may coincide with rising Lp(a) levels, a causal risk factor for atherosclerotic cardiovascular disease (ASCVD). Characterizing changes in Lp(a) across menopause may inform risk stratification and testing recommendations. Methods: We examined changes in serum Lp(a) levels by menopausal status among women with Lp(a) measured at visits 1 and 2 in the UK Biobank. Lp(a) analyses were examined by menopausal status: those who underwent menopause (N=415), those who remained premenopausal (N=532), and those who remained postmenopausal (N=3,615) between visits. We examined the change in Lp(a) between visits stratified by visit 1 Lp(a) levels. The primary outcome was incident Lp(a) 125 nmol/L at visit 2, estimated using Poisson regression with adjustment for baseline age. Results: Data were available for 4,562 women (mean age at visit 1 = 57{+/-}7 years; median Lp(a) at visit 1 = 22 (IQR: 47) nmol/L; median time between visits = 4 (IQR: 1) years). At visit 1, median Lp(a) was slightly higher in postmenopausal women (23 nmol/L) than premenopausal women (19 nmol/L). Overall, median changes in Lp(a) between visits 1 and 2 were modest. Among women with intermediate visit 1 Lp(a) levels (75-125 nmol/L), those who transitioned through menopause experienced a median increase of 34.9 (-6.7, 53.0) nmol/L between visits, an approximately fourfold greater increase than for women who remained pre- (7.9 nmol/L) or postmenopausal (8.0 nmol/L). Further, 56% of women with intermediate visit 1 Lp(a) levels who transitioned through menopause between visits had incident Lp(a) 125 nmol/L at visit 2, compared with 29% and 28% of women who remained pre- or postmenopausal, representing an age-adjusted risk ratio of 2.26 (95% CI: 1.31, 3.90). Conclusion: Relying on a single lifetime Lp(a) measurement may miss clinically relevant increases during menopause. Repeat testing in women as they age may improve identification of those at high risk for ASCVD.

Bakground and Purpose Antiplatelet resistance is a recognized risk factor for recurrent ischemic stroke, yet evidence supporting platelet function test?guided antiplatelet therapy modification in stroke prevention remains limited. We investigated whether VerifyNow-guided antiplatelet therapy modification reduces recurrent ischemic stroke in patients with atherothrombotic or lacunar infarction. Methods This retrospective observational study enrolled consecutive patients with atherothrombotic or lacunar infarction at a single center (April 2023-March 2025). Of 302 patients, 243 were analyzed: 122 in the modified group, whose antiplatelet agent was selected based on VerifyNow Aspirin Reaction Units and P2Y12 Reaction Units, and 121 in the unmodified group, whose agent was empirically selected. The mean follow-up period was 1.62 {+/-} 0.61 years. In the modified group, when both aspirin and clopidogrel showed inadequate inhibition, prasugrel or cilostazol was selected. The primary endpoint was recurrent ischemic stroke; the secondary endpoint was intracranial hemorrhage. Cox proportional hazards models with inverse probability weighting were used to adjust for confounders. Results Recurrent ischemic stroke occurred in 1 patient (0.8%) in the modified group versus 8 (6.6%) in the unmodified group (log-rank P=0.018). After adjustment, the modified group had a significantly lower risk of recurrent stroke (HR, 0.10; 95% CI, 0.012-0.84; P=0.033). Intracranial hemorrhage occurred in 0 (0%) and 1 (0.8%) patients, respectively. Conclusions In Japanese patients with atherothrombotic or lacunar infarction, VerifyNow-guided antiplatelet therapy modification was associated with a significantly lower incidence of recurrent ischemic stroke without increased hemorrhagic risk. Given the single-center retrospective design and small sample size, validation in a multicenter randomized controlled trial is warranted.

BackgroundElevated lipoprotein(a) [Lp(a)] is a known risk factor for several cardiovascular-related diseases established from multiple genetic and observational studies. However, the underlying mechanisms mediating the effects of Lp(a) levels on cardiovascular disease risk and major adverse cardiovascular events (MACE) are unclear. The aim of this study was to identify proteins downstream of Lp(a) using mendelian randomization (MR) - a genetic causal inference approach. MethodsA two-sample MR was performed by initially identifying Lp(a) genetic instruments based on data from genome wide association studies (GWAS) of Lp(a) blood concentrations. These instruments were then tested for association with proteins from proteomic pQTL data (Olink from UK Biobank, 2940 proteins and SomaScan from deCODE, 4907 proteins). ResultsA total of 521 proteins associated with Lp(a) were identified. Using pathway enrichment analysis, the following MACE-relevant pathways were identified comprising a total of 91 Lp(a) downstream proteins: oxidized phospholipid-related, chemotaxis of immune cells and endothelial cell activation, pro-inflammatory monocyte activation, neutrophil activity, coagulation, and lipid metabolism. ConclusionThe results suggest that the influence of Lp(a) treatments is primarily through modifying inflammation rather than lipid-lowering, thus providing insight into the mechanistic framework which mediates the effects of elevated Lp(a) on atherosclerotic cardiovascular disease.

BackgroundType 2 diabetes mellitus (T2DM) has long been considered a risk factor for cerebral small vessel disease (cSVD), yet the exact relationship between glycemic markers and cSVD remains unclear. This study explores the genetic overlap and causal associations between T2DM, glycemic indices, and cSVD phenotypes using genome-wide association studies (GWAS). MethodsUsing large consortium-based GWAS data, we examined relationships between T2DM, glycemic indicators (glycated hemoglobin, fasting glucose, 2-hour glucose after oral challenge, and fasting insulin), and cSVD phenotypes (white matter hyperintensity volume, lacunar stroke, cerebral microbleeds, and enlarged perivascular spaces). Our multi-level genomic strategy included: 1) identifying pleiotropic single nucleotide polymorphisms (SNPs) through PLEIO and eQTL analysis, 2) assessing genome-wide genetic correlations using LDSC and GNOVA, and 3) determining causal relationships with two-sample and multivariable Mendelian randomization analyses. ResultsWe identified 14 pleiotropic SNPs with significant shared associations among T2DM, glycemic indicators, and cSVD phenotypes. Notably, MICB gene expression was elevated in brain, vascular, and pancreatic tissues, while three HLA genes (HLA-DQA1, HLA-DRB1 and HLA-DRB5) showed reduced expression. Genetic correlation analysis revealed positive correlations between T2DM, fasting glucose, and postprandial glucose with multiple cSVD phenotypes including WMH, lacunar stroke, and perivascular spaces. Mendelian randomization demonstrated that T2DM, 2-hour glucose, and HbA1c level causally increased lacunar stroke risk (OR 1.16 [1.09-1.23], OR 1.46 [1.20-1.77], OR 1.52 [1.04-2.23], respectively). Multivariable Mendelian randomization analysis confirmed that T2DM and postprandial glucose maintained a robust direct effect on lacunar stroke independent of other cSVD phenotypes, while HbA1c did not retain significance after conditioning on cSVD imaging markers. ConclusionsOur multi-level genomic analysis reveals links between T2DM, glycemic traits, and cSVD through specific genetic variants, genome-wide correlations, and causal relationships. The involvement of immune-related genes suggests potential biological mechanisms. The causal effect of postprandial glucose on lacunar stroke suggests that impaired glucose tolerance may be a relevant therapeutic target for lacunar stroke prevention.