Atherosclerosis

BackgroundOverweight and obese individuals often exhibit lower mortality rates or better prognoses than lean or normal-weight individuals with stroke and other diseases, a phenomenon called the "obesity paradox". Carotid atherosclerosis is the primary cause of ischemic stroke, and body weight serves as a reliable surrogate for adiposity in mice. MethodsPhenotypic and genetic connections of carotid atherosclerosis with body weight were evaluated in 299 F2 mice derived from BALB/cJ and LP/J Apoe knockout (Apoe-/-) mice. F2 mice were fed a Western diet for 12 weeks. Atherosclerotic lesion sizes in left carotid arteries, body weight, coat color, plasma lipids, glucose, small dense LDL ApoB, and malondialdehyde were measured, and 11,000 single nucleotide polymorphism (SNP) markers were genotyped. ResultsCarotid lesion sizes inversely correlated with body weight in both sexes. Genome-wide scans identified two significant quantitative trait loci (QTLs) for carotid atherosclerosis on chromosomes (Chr) 6 and 15 in an additive sex model, and five QTLs on Chr 6, 7, 12, 13, and 15 in an interactive sex model. Adjusting for body weight variation downgraded Chr 15 QTL (Cath5) in both models, whereas other QTLs upgraded in the additive sex model and downgraded in the interactive sex model. Human syntenic region of Cath5 associated with carotid intima-medial thickness (cIMT) and waist-to-hip ratio (WHR). ConclusionsThese findings indicate that the obesity paradox in carotid atherosclerosis is partially driven by shared genetic components that exert opposing effects on adiposity and plaque development and act through sex-dependent mechanisms.

Background: Phenotyping of atherosclerotic plaque vulnerability has largely relied on histopathology that captures structural features, but does not fully account for clinical presentation. Proteomic profiling could uncover molecular readouts of vulnerability that refine plaque phenotyping and provide mechanistic insights. Yet, the proteomic signatures associated with plaque rupture and symptomatic presentation are poorly characterized. Methods: We profiled paired carotid plaque tissue and preoperative plasma from 88 patients undergoing carotid endarterectomy (51 symptomatic, 37 asymptomatic) using the Olink Explore 3072 platform. We related plaque protein abundance to symptomatic presentation and quantitative histopathological features, and compared the performance of histopathology- vs. proteomics-based models for discriminating symptomatic disease. Next, we developed proteomic signatures of cellular abundance and explored their associations with plaque phenotypes by using plaque single-cell RNA-sequencing (scRNA-seq) data. Finally, we assessed plaque-plasma concordance across 2,837 shared proteins. Results: Across 2,837 plaque proteins, 19 were differentially expressed in symptomatic plaques related to distinct clinical events, highlighting pathways related to neutrophil degranulation and innate immune system. FGFBP1 showed the strongest association with symptomatic presentation (log2 fold change = 1.14; P = 1.82 x 10^-6). Proteins associated with a composite vulnerability index based on histopathology were enriched for inflammatory pathways, including TNF signaling through NF{kappa}B, complement activation, and IL6-JAK-STAT3 signaling. Individual proteins also mapped to specific histopathological features, including CXCL8 associated with macrophage burden and lipid core size, and EPHB4 and PKN3 with neovascularization. A proteomics-based model discriminated symptomatic from asymptomatic plaques substantially better than a histopathology-based model (AUC 0.83 vs. 0.66; P = 0.026). Integration with scRNA-seq data enabled the development of cell-class signatures that correlated with histopathology readouts, including macrophage burden, smooth muscle cell content, and neovascularization. Plaque and plasma protein levels showed limited overall correspondence (median {rho}=0.11), although selected proteins, including FGFBP1, demonstrated concordant associations in plasma. Conclusions: Deep proteomic profiling of human carotid plaques identifies molecular signatures of symptomatic atherosclerosis that extend beyond conventional histopathology. These signatures implicate neutrophil activation and inflammatory signaling pathways as key determinants of plaque vulnerability. Although plaque and plasma proteomes are largely distinct, selected proteins may represent promising circulating biomarkers for future risk stratification.

Background: Preoperative risk assessment in vascular surgery relies on clinical scores and lipids that do not capture atherosclerotic disease activity. Circulating fatty acid synthase (cFAS) is a liver-derived enzyme whose concentration correlates with arterial plaque FAS content independent of LDL. The 5-item modified frailty index (mFI-5) is a validated predictor of postoperative mortality. Whether cFAS predicts outcomes after vascular surgery, and whether combining it with the mFI-5 improves risk discrimination, have not been examined. Methods: We studied 657 patients undergoing elective vascular surgery at a single center (2014 to 2023). cFAS was classified as non-detectable (n = 306) or, among detectable values, by tertiles (n = 117 each). Multivariable Cox models assessed associations with major adverse events (MAE), major adverse cardiovascular events (MACE), major adverse limb events (MALE), reintervention, and mortality, and Harrell's C-statistic quantified the incremental discrimination gained by adding cFAS and the mFI-5 to standard clinical covariates. Results: High serum cFAS was independently associated with 5-year MAE (adjusted hazard ratio [aHR] 1.94; 95% CI 1.31- 2.85), mortality (aHR 1.77; 1.05 to 3.00), MALE (aHR 4.53; 2.04 to 10.05), and reintervention (aHR 2.50; 1.37 to 4.57), but not MACE. Severe frailty (mFI-5 of 3 or higher) was associated with MACE (aHR 2.69; 1.29 to 5.58) and MAE (aHR 2.46; 1.30 to 4.65) but not limb endpoints at 1 year. Adding cFAS raised the 1-year MALE C-statistic from 0.649 to 0.764; the combined model yielded the highest discrimination. Conclusions: cFAS and mFI-5 were independently and additively associated with adverse outcomes after elective vascular surgery. cFAS was associated with limb events and mortality, the mFI-5 with cardiovascular events. Combining them improved discrimination over standard covariates.

Background: Spontaneous coronary artery dissection (SCAD) causes acute coronary syndrome that predominantly affects women. It is not known why SCAD occurs in specific coronary artery segments. We aimed to identify anatomical and hemodynamic factors that lead to SCAD. Methods: We studied 36 women with angiographically-confirmed SCAD from more than 20 hospital sites and 75 sex- and ethnicity-matched control participants with normal coronary anatomy. Coronary arteries were reconstructed from computed tomography coronary angiography (CTCA) to quantify vessel geometry (curvature, diameter, torsion) and flow-derived metrics (time-averaged endothelial shear stress [TAESS], topological shear variation index [TSVI], oscillatory shear index [OSI], and relative residence time [RRT]) at the tree (left/right), territory (LAD, LCx, RCA), and lesion levels. Results: Compared with controls, SCAD-affected coronary arteries had greater curvature and higher TAESS and TSVI at the whole-tree level (all p?0.007). At the vessel (territory) level, SCAD-affected arteries were smaller in average diameter and showed higher curvature, TAESS, and TSVI than matched control vessels (all p?0.047). Within the same patient, SCAD lesion segments were characterized by smaller diameter, lower torsion, and higher TAESS and TSVI than non-affected segments from the same coronary tree (all p?0.001; curvature borderline). A model combining curvature, TAESS, and TSVI discriminated SCAD from controls with AUC 0.95 (left tree) and 0.97 (right tree); adding diameter yielded AUCs >0.91 at the territory level. Conclusions: SCAD was associated with a reproducible multi-scale signature of smaller vessel caliber and higher, more variable endothelial shear stress supporting a hemodynamic contribution to SCAD clustering in specific coronary arteries and segments.

People living with HIV-1 (PWH) receiving antiretroviral therapy (ART) exhibit an increased cardiovascular disease (CVD) risk. Coronary atherosclerotic plaque formation is linked to chronic immune activation, a process fueled in PWH by additional mechanisms likely including residual HIV-1 production and microbial translocation from the gut during ART. Our previous studies demonstrated that aldehyde dehydrogenase (ALDH), an enzyme converting vitamin A into retinoic acid (RA), is upregulated in myeloid cells upon exposure to viral/bacterial/fungal products and that RA promotes HIV-1 production. To explore the link between ALDH/RA pathway and CVD risk, we used PBMC/plasma from ART-treated PWH (PWH+ART; n=51) and people without HIV-1 (Pw/oH; n=63) from the Canadian HIV/Aging Cohort Study, with/without subclinical coronary atherosclerosis, measured by coronary computed tomography angiography. ALDH expression was analyzed by flow cytometry on monocyte subsets, dendritic cells, and CD4+ T-cells. Unsupervised t-SNE-guided FlowSOM analysis associated top ALDH activity with a monocyte phenotype. The frequency of ALDH+ monocytes and plasma levels of RA and retinol binding protein 4 were increased in PWH+ART versus Pw/oH, with the highest RA levels coinciding with detectable plasma levels of soluble HIV-1 gp120. Multivariate regression models linked ALDH activity in monocytes to subclinical coronary atherosclerosis (i.e., total plaque volume, low attenuated plaque volume, coronary artery calcification score) presence/burden in PWH+ART, independently of Framingham risk score. Finally, ALDH+ monocyte frequency and RA levels positively correlated with pericoronary fat attenuation index, an emerging CVD predictor. Thus, ALDH/RA pathway may represent a new marker of metabolic/immune dysfunction contributing to CVD risk in ART-treated PWH.

Background: Spontaneous cervical artery dissection (sCeAD) is a rare vasculopathy whose pathophysiology remains incompletely understood. Impaired vascular extracellular matrix integrity, including elastic fibers, may contribute to its development. We investigated whether serum fibrillin-1 and soluble elastin fragments (sELF) differ between patients with sCeAD and controls during the acute and chronic stages. Methods: Patients with acute sCeAD were prospectively enrolled at four German stroke centers. Blood samples were collected at baseline and after 6{+/-}1 months. Patients with a first acute ischemic stroke unrelated to sCeAD and healthy individuals served as controls. Serum fibrillin-1 and sELF concentrations were measured using enzyme-linked immunosorbent assays. Results: 61 patients with sCeAD, 53 patients with first non-CeAD ischemic stroke, and 79 healthy controls were included. After sex-matching, serum fibrillin-1 concentrations were significantly lower in patients with acute sCeAD than in healthy controls (97 [60; 192] vs. 176 [113; 269] ng/mL; p=0.009). Fibrillin-1 concentrations were also lower in both male and female patients with sCeAD than in respective healthy controls. In patients with sCeAD, fibrillin-1 concentrations increased significantly after 6 months compared with baseline (171 [130; 270] vs. 104 [67; 205] ng/mL; p=0.021). Serum fibrillin-1 concentrations were higher in men than in women across all study groups. No significant differences in sELF concentrations were observed between groups or time points. Discussion: Serum fibrillin-1 concentrations were lower during acute sCeAD and increased significantly during follow-up, whereas sELF concentrations remained unchanged. These findings support an association between circulating fibrillin-1 and acute sCeAD and warrant further investigation of its role in sCeAD pathophysiology. Pronounced sex-related differences in fibrillin-1 concentrations highlight the importance of sex-specific analyses in future.

Background: Growing evidence suggests persistent thrombotic endothelial damage (together with elevated (fibrinaloid) microclot complexes (FMCs)) and immune dysfunction in the pathophysiology of Long COVID. Recently we proposed that there are different FMC phenotypes. Here we seek to determine the nature of these FMCs and aggregates in platelet-poor plasma (PPP) by using different markers, as well as thromboelastography (TEG) to assess for hypercoagulability of samples. Material and Methods: Whole-blood and PPP from control (n=19) and Long COVID (n=20) participants were assessed by thromboelastography. FMCs were quantified by imaging flow cytometry of Thioflavin-T (ThT)-stained PPP, 10X diluted PPP, and resuspended PPP pellets. The resuspended pellets were separately stained with a CD62P-PE antibody or Hoechst 33342 to label aggregates and FMCs containing amyloid, platelet, and nuclear material. ThT and CellMask Red were co-stained for confocal microscopy. ThT and myeloperoxidase (MPO), and ThT, Congo Red, and Hoechst were co-stained for fluorescence and polarized microscopy. Whole-blood smears were imaged by scanning electron microscopy (SEM). Results: Long COVID samples showed pronounced hypercoagulability in both whole blood and PPP, with shortened R, K and TMRTG and elevated alpha-angle and MRTG, but unchanged MA and TTG, indicating altered clotting kinetics. Persistence of this phenotype in PPP implicates soluble plasma constituents. ThT-positive FMCs were significantly increased in Long COVID across undiluted, diluted, and resuspended pellet samples; counts were processing-sensitive and a substantial ThT-positive population remained in the supernatant after centrifugation, indicating heterogeneity in density. Across probes, leukocyte material was the most abundant, then platelet material, and ThT-positive FMCs were the least abundant, with the three populations exhibiting unique morphology and occupying distinct size domains. Platelet-derived material was significantly elevated in Long COVID, whereas nuclear material was not. Co-stained samples subject to confocal, fluorescence, and polarized microscopy imaging showed that FMCs are heterogeneous, including events positive for ThT, CellMask, Hoechst, MPO, and Congo Red, and also a distinct subset of membrane-free, ThT-only events. Conclusion: In this Long COVID cohort, plasma is characterised by hypercoagulability and an increased burden of ThT-positive FMCs that are numerically minor relative to, and morphologically distinct from, aggregates and amyloidogenic FMCs marked with platelet- and leukocyte-derived material. The increased burden of platelet debris in PPP is likely indicative of persistent platelet activity. The existence of membrane-free, ThT-only FMCs, in addition to FMCs associated with cellular material, confirms an amyloid-dominated FMC population. Furthermore, positive Congo Red signal further confirms the amyloid nature of FMCs in PPP.

Preeclampsia (PE) is a hypertensive pregnancy-specific disorder and a leading cause of maternal and fetal mortality. A common feature of PE placentas and maternal plasma is dyslipidemia, or abnormal lipid levels, which can increase oxidative stress and endothelial dysfunction. However, the precise transcriptional, post-transcriptional, and epigenetic mechanisms underlying these abnormalities remain poorly characterized. Identifying such changes may clarify disease mechanisms and identify lipid-related PE biomarkers. We conducted a large-scale meta-analysis integrating public placental datasets from NCBI GEO, comprising four DNA methylation (DNAm) datasets (n = 172), three RNA-sequencing datasets (n = 92), and an independent RNA microarray validation cohort (n =146). We evaluated differential DNAm (limma), gene expression (DESeq2), transcript-level shifts (Swish), and alternative splicing (rMATS) in PE versus control placentas, with all analyses stratified by fetal sex via an interaction term model. We also performed placental cell-type deconvolution to quantify PE-associated cell-type proportion changes. Our results demonstrated that lipid-related regulation changes in PE placentas occur primarily at the gene and transcript level, with DNAm showing no changes. We also identified significant isoform switching in PE that were undetected by differential gene expression analysis, and primarily driven by alternative transcription initiation and termination sites rather than alternative splicing. A subset of these isoform switches mapped to pathways dysregulated in PE and were predicted to cause functional protein changes. An interaction term model identified several sex-specific differentially expressed genes (DEGs) in PE, including a subset of male-specific downregulated genes involved in oxidative metabolism. However, many of the remaining sex-specific DEGs across both sexes were previously uncharacterized in the literature. These findings suggest that transcriptional and isoform-level regulation play a role in PE-associated dyslipidemia, with certain regulatory pathways displaying fetal sex-specific patterns. Highlights- Preeclampsia-associated dyslipidemia manifests at the gene and transcript level - Reciprocal isoform switches were missed by standard gene-level analyses - Alternative transcript initiation and termination drove isoform switching - Sex-interaction modeling identified sex-specific transcriptional shifts in PE

Background: DNA methylation (DNAm) may capture cumulative genetic, environmental, and lifestyle influences on cardiovascular health. Composite DNAm score based on the American Heart Association Life's Essential 8 (LE8) framework have been linked to clinical events, but their association with early vascular changes and intergenerational effects is unclear. Methods: We studied up to 1432 participants from the multigenerational Young Finns Study (YFS-3G), including parents (G0) and adult offspring (G1). DNAm was measured using Illumina EPIC arrays in 2011 and/or 2018, and carotid intima--media thickness (cIMT) was assessed in 2018. The LE8 DNAm score was calculated as a weighted sum of methylation levels. Associations with cIMT were evaluated in intergenerational, prospective, and cross-sectional settings, adjusting for demographic, technical, and biological covariates and conventional cardiovascular risk factors. Results: Higher parental LE8 DNAm score was associated with lower offspring cIMT ({beta} = -0.022 mm/SD; p-value = 0.02), although the association was attenuated after adjustment for parental cardiovascular risk factors. In G1, a higher baseline DNAm score was associated with lower cIMT measured seven years later ({beta} = -0.030 mm/SD; p-value = 1.1 x 10-5). This association remained significant after adjustment for follow-up cardiovascular risk factors (p-value=0.009) but not after additional adjustment for prior cIMT. Cross-sectionally, higher DNAm score was associated with lower cIMT in both generations, with attenuation after risk factor adjustment in G1 but not G0. Associations with carotid plaque were not significant. Genes associated with the DNAm score were enriched for immune and inflammatory pathways. Conclusions: An LE8-derived DNAm score was associated with lower cIMT across the life course and, to a lesser extent, across generations. These findings suggest that blood DNAm reflects cumulative cardiovascular health and vascular burden and may complement conventional cardiovascular risk assessment.

Objective: One of the strongest genetic associations with coronary artery disease (CAD) risk maps to the metalloproteinase 'a disintegrin and metalloproteinase with thrombospondin motifs 7' (ADAMTS-7) locus. The protein was shown to promote plaque formation and instability. We aimed to generate and evaluate an antibody-based strategy targeting ADAMTS-7 therapeutically to reduce atherosclerotic plaque formation. Approach and Results: A truncated form of human ADAMTS-7 was produced in Nicotiana benthamiana and used as antigen for antibody generation by hybridoma technology. Eight monoclonal antibodies (mAbs) were screened, among which ADAMTS-7-mAb32 (mAb32) demonstrated the highest affinity, as confirmed by surface plasmon resonance analyses and immunoblotting against full-length ADAMTS-7. In vitro, mAb32 inhibited interactions of ADAMTS-7 with its substrates TIMP-1 and SVEP1 in a dose- and time-dependent manner, as assessed by time-resolved Forster resonance energy transfer assays. To assess therapeutic efficacy in vivo, Apoe-/- mice were fed a Western diet for ten weeks and treated with weekly injections of mAb32 or control IgG over the last six weeks. En face aortic Oil Red O staining revealed significantly reduced plaque area in the treatment group, without changes in plasma cholesterol levels or body weight. No evidence of liver or kidney toxicity was observed. Conclusion: Monoclonal antibody-based inhibition of ADAMTS-7 reduced atherosclerotic burden in vivo without affecting lipid metabolism, supporting ADAMTS-7 as a viable therapeutic target in CAD. Further development of mAb32 may provide a cholesterol-independent treatment strategy for atherosclerosis.

Background Asymptomatic carotid artery stenosis (ACAS) increases the risk of stroke, and is associated with cognitive decline. This association might be driven by underlying atherosclerotic disease instead of the stenosis itself, which could explain why studies on the cognitive benefits of carotid revascularisation are inconclusive. Methods Between 2007-2012, dementia-free participants of the population-based Rotterdam Study underwent carotid ultrasound, and additional carotid MRI if intimal media thickness was >2.5mm. All participants underwent repeated cognitive assessments and were followed for dementia until January 2022. We determined the effect of ACAS and plaque without stenosis on all-cause dementia using multivariable Cox models, and on change in cognition (g-factor) using multivariable linear mixed-effects models. Results Of 4267 participants (mean age 67.5 years, 55.5% women), 483 (11.3%) had plaque without stenosis, 989 (23.2%) had 1-49% stenosis, 107 (2.5%) had 50-99% stenosis, and 15 (0.4%) had occlusion. During a mean follow-up of 9.7 years, 391 participants developed dementia. Compared to individuals without carotid atherosclerosis, risk of dementia was increased in the presence of plaque without stenosis (HR: 1.39 [95%CI: 1.05-1.83]) and occlusion (HR: 4.61 [1.82-11.66]), but not with stenosis (HR 1-49% stenosis: 1.08 [0.84-1.39]; 50-99% stenosis: 1.18 [0.70-1.99]). Neither carotid plaques nor stenosis affected cognitive decline. Results did not differ consistently by plaque characteristics. Conclusion Risk of dementia was increased with asymptomatic carotid artery plaque and occlusion, but not significantly with 50-99% stenosis. These results are in line with detrimental effects of generalised atherosclerotic disease and severe haemodynamic impairment on cognitive decline and dementia risk.

NOTCH3 is a transmembrane receptor highly expressed in vascular mural cells where it contributes to blood vessel formation and homeostasis. NOTCH3 expression declines in the vasculature with aging, and dysregulated NOTCH3 signalling is implicated in pulmonary arterial hypertension, cancer progression and CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy). RNA-based approaches targeting NOTCH3 are emerging as potential therapeutic strategies, however, the consequences of NOTCH3 suppression in mature vascular smooth muscle cells (VSMCs) remain incompletely understood. Here, we investigated the molecular and functional effects of siRNA-mediated NOTCH3 knockdown in human aortic smooth muscle cells. Transfection with NOTCH3-targeting siRNA efficiently suppressed NOTCH3 transcript and protein levels. Quantitative proteomics revealed remodelling of extracellular matrix (ECM), cytoskeletal and metabolic pathways, with enrichment of collagen biosynthesis and inhibition of glycolytic signalling. Specifically, NOTCH3 knockdown increased ECM components, including COL3A1, elevated F-actin, and upregulated the actin regulator, CTTN. In parallel, glycolytic capacity was reduced, accompanied by decreased expression of the glycolytic enzyme ENO2. Despite reduced VEGFA and alteration in angiogenic signalling proteins, endothelial network formation in co-cultures, as well as VSMC proliferation and migration remained unaffected. Finally, NOTCH3 interactome analysis revealed key collagen and actin-regulating proteins. These findings identify NOTCH3 as an important regulator of ECM homeostasis, cytoskeletal organisation, and glycolytic metabolism. The preservation of primary cellular functions despite molecular remodelling highlights the adaptive capacity of VSMCs. These findings demonstrate that therapeutic modulation of NOTCH3 may alter vascular cell biology which warrants consideration during development of RNA-based therapeutics for CADASIL and other NOTCH3-associated diseases.

Background Statins and ezetimibe are the preferred lipid-lowering therapies (LLTs) for children with heterozygous familial hypercholesterolaemia (HeFH). Proprotein convertase subtilisin/kexin type 9 inhibitors (PCSK9i) are newer add-on therapies for individuals not achieving low-density lipoprotein-cholesterol (LDL-C) targets. We evaluated the efficacy and safety of PCSK9i in children aged <18 years with HeFH. Methods Systematic review and pairwise meta-analyses of randomised-controlled trials (RCTs) of evolocumab, alirocumab and inclisiran. Comprehensive bibliographic searches were conducted in February 2026. Risk of bias was assessed with Cochrane RoB 2. Results Of 2798 unique records screened, three RCTs were included (n=451, mean age 13 years, follow-up 24 to 47 weeks). Each trial evaluated either evolocumab, alirocumab or inclisiran against placebo as add-on to baseline LLT. Participants had elevated LDL-C (>3.4 mmol/L [130 mg/dL]) despite stable LLT. Overall risk of bias was low. PCSK9i reduced LDL-C by an average of 35.44% (95% CI -41.74 to -29.14, I2=50.8%) and by 1.63 mmol/L [62.93 mg/dL] (95% CI -1.86 to -1.39, I2=18.6%) compared with placebo. There was no evidence of differences between PCSK9i and placebo in tolerability, growth and maturation, and overall incidence of adverse events. Conclusions PCSK9i add-on therapy leads to substantial reductions in LDL-C in paediatric patients with HeFH failing to achieve LDL-C targets with standard LLT. While the findings of this review support the use of PCSK9i in a subset of children and young people with HeFH, limited trial numbers and short follow-up periods underscore the need for future high-quality studies evaluating long-term safety, effectiveness and cost-effectiveness.

Background and aimsAtherosclerotic plaques form preferentially at vascular sites exposed to disturbed blood flow, yet the protein changes underlying this site-specific plaque development remain unclear. Mouse models are widely used to study atherosclerosis but yield only limited amounts of tissue, previously restricting proteomic studies. However, recent advances in mass spectrometry now enable proteomic profiling of very small tissue samples. We aimed to utilise this to uncover site-specific protein changes in aortic regions prone or resistant to plaque formation. MethodsAortic arches from apolipoprotein E-deficient (ApoE-/-) mice fed a Western diet (WD) for 16 weeks were dissected into plaques from the major branches and inner curvature and visibly healthy regions. Proteins were extracted, enzymatically digested, and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). ResultsMore than 4000 proteins were identified per sample despite their small size (< 1 mg tissue). Principal component analysis showed clustering by both disease status and anatomical location within the aortic arch, indicating distinct proteomes. Proteins known to drive atherosclerosis - including vascular cell adhesion molecule 1 (Vcam1), apolipoprotein B (Apob), lipoprotein lipase (Lpl), and galectin 3 (Lgals3) - were most abundant in advanced plaques and decreased progressively across anatomical regions, reaching their lowest levels in healthy regions furthest from the plaques. Enrichment analysis highlighted pathways related to the extracellular matrix, immune system, hemostasis, and lipoprotein transport as central to disease progression. ConclusionsThis study demonstrates the feasibility of region-resolved proteomics in individual murine aortas and provide new molecular insights into the site-specific nature of atherosclerotic plaque development.

Trained immunity and clonal hematopoiesis are two newly identified immunological phenomena that contribute to the pathophysiology of atherosclerotic cardiovascular disease. These two phenomena share some convergent molecular mechanisms, such as IL-1{beta} being a central regulator and involvement of epigenetic enzymes. Therefore, we hypothesize that presence of clonal hematopoiesis driver mutations (CHDMs) can predispose to an increased capacity to build trained immunity. We previously characterized how the presence of CHDMs relates to immune cell function and vasculometabolic complications in a cohort of older individuals with overweight and obesity. From this cohort we now selected 17 individuals with CH due to DNMT3A mutations and 15 without any known CHDMs. We performed in depth immune characterization via flow cytometry, functional assays with monocytes and neutrophils, and we measured the capacity to build trained immunity using {beta}-glucan and oxLDL as stimuli. We corroborated our previous findings of lower ex vivo cytokine production capacity of PBMCs from individuals with DNMT3A mutations. Importantly, presence of DNMT3A CHDMs associated with higher trained immunity response. Moreover, we demonstrated that individuals with DNMT3A mutations were characterized with higher CD10+ mature neutrophils and a lower neutrophil MPO release upon TLR2 stimulation. In conclusion, presence of DNMT3A CHDMs is associated with increased susceptibility to build a hyperresponsive trained monocyte phenotype. The exact molecular mechanisms behind this phenomena requires further investigation.

IntroductionPreeclampsia is characterized by a pro-inflammatory, anti-migratory and anti-angiogenic placental phenotype. Impaired spiral artery remodeling stemming from trophoblast dysfunction is a key pathogenic mechanism. Little is known about the processes that govern trophoblast function normally and in preeclampsia. In preeclampsia, placental Let-7b-5p is reduced. The objectives of this study were to determine the normal function of Let-7b-5p in human trophoblast cells, to examine whether the ssRNA sensors, Toll-like receptor (TLR) 7 and/or TLR8 are mediators of trophoblast Let-7b-5p function, and whether disruption of this pathway promotes a preeclampsia-like phenotype in the trophoblast. MethodsThe human first trimester trophoblast cell line, Sw.71, was transfected with a Let-7b- 5p mimic, a Let-7b-5p inhibitor, or scramble control. Cells were treated with or without the TLR7 inhibitor IRS661 or the TLR8 inhibitor CUCPT9a. Trophoblast migration was measured using a two-chamber assay and interactions with human endometrial endothelial cells (HEECs) was measured using a 3D matrigel model. Trophoblast anti-angiogenic sFlt-1 release was measured by ELISA and sFLT1 mRNA measured by RT-qPCR. ResultsTransfection of trophoblast cells with a Let-7b-5p mimic elevated migration through activation of TLR7 and TLR8, while in a TLR7-dependent manner, the Let-7b-5p mimic negatively regulated sFlt-1 production. Furthermore, inhibition of trophoblast Let-7b-5p reduced migration, elevated FLT1 mRNA expression and sFlt-1 release, and reduced trophoblast-endometrial endothelial cell interactions. ConclusionsThis study highlights a role for TLR7/TLR8-activating Let-7b-5p in promoting normal trophoblast function and endothelial interactions and that disruption in this miR-driven signaling pathway may be relevant to processes driving a pre-eclamptic placental phenotype. HighlightsTrophoblast migration is positively driven by Let-7b-5p activating TLR7 and TLR8 Let-7b-5p, via TLR7, negatively regulates trophoblast anti-angiogenic sFlt-1 production. Inhibition of trophoblast Let-7b-5p reduces trophoblast migration and normal interactions with endometrial endothelial cells, while sFlt-1 production is elevated. TLR7/TLR8-activating Let-7b-5p promotes normal trophoblast function and endothelial interactions and disruption in this miR-driven signaling pathway may promote a preeclamptic placental phenotype.

Background and Aims Atrial fibrillation (AF) is associated with dementia and cognitive decline; it remains unclear whether the association is independent of cardiovascular comorbidities, particularly stroke. We aimed to establish stroke-dependent and stroke-independent effects of AF on multiple layers of brain health. Methods We investigated stroke-dependent and stroke-independent associations of AF with brain MRI imaging measures, cognitive performance, incident dementia, and biomarkers of neuronal and glial injury among 502 099 participants in the UK Biobank. Genetic analyses were performed to support causal inference. Results AF was associated with lower global and regional grey matter volume. After full adjustment, AF remained associated with lower grey matter volume (-0.13 SD, 95%CI: -0.15 to -0.10), greater white matter hyperintensity burden (0.06 SD, 0.03 to 0.10), and higher mean diffusivity (0.06 SD, 0.03 to 0.10). Genetic analyses were consistent with stroke-independent associations between AF and grey matter loss. AF was associated with poorer cognitive performance and higher concentrations of neurofilament light chain, a marker of neuronal injury. Associations with vascular and all-cause dementia were attenuated by adjustment for cardiovascular risk-factors and stroke. Genetic analyses supported ischaemic stroke as a major driver of dementia risk in AF. Conclusion Associations with grey matter and cognitive impairment persisted after accounting for stroke, whereas the association with dementia was largely explained by ischaemic stroke. This highlights the potential for AF to impact brain health through both stroke-dependent and stroke-independent mechanisms; stroke prevention is necessary but may not be sufficient to fully address the broader burden of AF-associated brain injury.

Background: Adults aged [&ge;]75 years represent a rapidly growing population at risk of acute myocardial infarction (AMI), yet they remain markedly underrepresented in statin trials for primary prevention. The limited evidence base, together with multimorbidity, functional heterogeneity, and competing mortality risks, has contributed to uncertainty regarding the potential role of statins in very old adults. This study evaluated the association between baseline statin exposure and incident AMI among community-dwelling adults aged [&ge;]75 years without prior cardiovascular disease. Methods: We conducted a retrospective population-based cohort study using linked primary-care, hospital, laboratory, and pharmacy dispensing data from the Community of Madrid. Statin exposure was ascertained during a 24-month exposure-assessment period from 1 January 2018 to 31 December 2019 and classified at a landmark date of 1 January 2020, when outcome follow-up began. Participants were classified as exposed if they had received at least two statin dispensations during the exposure-assessment period and had no record of prior lipid-lowering therapy before 2018. Individuals with prior cardiovascular disease, type 1 diabetes, cancer, dementia, or advanced chronic kidney disease were excluded. Missing data were addressed using multiple imputation. The association between baseline statin exposure and incident AMI was estimated using multivariable Cox proportional hazards regression. Propensity-score matching and Fine-Gray competing-risk regression, with all-cause mortality as the competing event, were performed as sensitivity analyses. Results: Among 174,014 individuals included in the final cohort, 32,698 (18.8%) met the criteria for baseline statin exposure. The mean age was 82.5 years. During a median follow-up of 5 years, AMI occurred in 533 (1.63%) statin-exposed individuals and 2722 (1.93%) non-exposed individuals (p=0.0003). The observed absolute risk difference was 0.30 percentage points (95% CI, 0.14-0.45), corresponding to an estimated observational number needed to treat of 338 over 5 years (95% CI, 222-708). In the fully adjusted Cox model, baseline statin exposure was associated with a lower risk of incident AMI (HR, 0.805; 95% CI, 0.731-0.887). In the full-cohort Fine-Gray model accounting for competing mortality, baseline statin exposure remained associated with a lower cumulative incidence of AMI (sHR, 0.823; 95% CI, 0.748-0.905). After propensity-score matching, the association remained in the competing-risk analysis (subdistribution HR, 0.852; 95% CI, 0.738-0.983). Conclusions: In this large population-based cohort of adults aged [&ge;]75 years without prior cardiovascular disease, baseline statin exposure was associated with a lower incidence of AMI across several analytical approaches. The observed absolute risk difference was modest, and the findings should be interpreted in light of the observational design, residual confounding, and the potential for selection related to survival to the landmark date. Further randomized evidence is needed to determine whether this association reflects a causal effect of statin therapy in very old adults. Keywords: Statins; primary prevention; acute myocardial infarction; aged [&ge;]75 years; landmark analysis; competing risks; propensity-score matching; real-world data.

Introduction: Severe hypercholesterolemia has four primary causes: monogenic familial hypercholesterolemia (FH), polygenic hypercholesterolemia (PRS), severely elevated Lp(a) concentration, and hypercholesterolemia due to environmental/lifestyle/behavioral factors (i.e., no known genetic etiology). Here, we explore patient and clinician perspectives about the identification and management of each of these causes. Methods: Patients with severe hypercholesterolemia with a primary language of English or Spanish and clinicians (primary care, genetic counseling, cardiology) across two health systems (Geisinger, Mount Sinai) participated in semi-structured interviews. Analysis was completed using an a priori codebook informed by Proctor?s implementation outcomes to identify themes influencing the identification and management of the underlying causes of severe hypercholesterolemia. Results: A total of 28 patients and 25 clinicians participated. Patients emphasized the importance of receiving results directly from their clinician, requested take-home resources that mirrored the information from their clinician, were motivated to seek multidisciplinary care, and anticipated all results would be actionable, but that high-risk PRS and elevated Lp(a) may require more support (e.g., specialists, education) to act on. Clinicians stressed the importance of integrating workflows (e.g., test ordering) with the electronic health record, highlighted LDL-C levels and multidisciplinary care coordination as key to management, explained how they would tailor care to individual patients, and expressed a more limited understanding of Lp(a) and PRS result types based on their clinical experiences and, therefore, hesitation about the recommended clinical actions. Conclusions: Patients and clinicians identified complementary determinants influencing the identification and management of the underlying cause of severe hypercholesterolemia. Participants welcomed risk information and requested a higher level of informational support and specialty expertise to appropriately manage high Lp(a) and PRS results. Integrating genomic information into risk assessments will require a partnership between general practitioners and specialists to provide a multidisciplinary approach to the identification and management of the underlying causes of severe hypercholesterolemia.

BackgroundAtherosclerosis is driven by macrophage foam cell formation resulting from excessive oxidized low-density lipoprotein (oxLDL) accumulation and chronic vascular inflammation. This study evaluated the therapeutic potential of Aegeline, Atorvastatin, and their combined in mitigating oxLDL-induced inflammatory responses, cholesterol accumulation, and oxLDL uptake in human THP-1 macrophages. MethodsTHP-1 monocytes were differentiated into macrophages using a 72-hour differentiation protocol followed by a 48-hour resting period, confirmed via CD14 surface marker characterization. Macrophages were exposed to DiI-oxLDL and treated with Aegeline, Atorvastatin, or their combination. Key inflammatory cytokines and chemokines (CRP, TNF-, IL-6, and IL-8) were measured using ELISA. Cholesterol efflux capacity and cellular oxLDL uptake were quantitatively assessed using fluorescence retention assays and immunofluorescence imaging. ResultsDifferentiation of THP-1 monocytes to macrophages resulted in marked down-regulation of CD14 expression. DiI-oxLDL exposure triggered significant pro-inflammatory mediator secretion (p<0.001) and excessive intracellular cholesterol accumulation. Single-agent treatment with Aegeline or Atorvastatin significantly attenuated oxLDL-induced elevations of CRP, TNF-, IL-6, and IL-8. Atorvastatin alone strongly suppressed CRP expression back to physiological baseline levels (p=ns vs. control). Notably, the combination of Aegeline and Atorvastatin demonstrated enhanced, broad-spectrum anti-inflammatory efficacy, achieving superior suppression of TNF- (p=ns vs. control), IL-6, and IL-8 compared to monotherapies. Furthermore, both agents promoted cholesterol efflux and suppressed oxLDL uptake, with the combination treatment producing the lowest residual intracellular cholesterol levels (p<0.001). ConclusionAegeline and Atorvastatin effectively suppress oxLDL-induced macrophage inflammatory cascades and intracellular lipid overload. While Atorvastatin monotherapy exerts robust control over CRP and oxLDL loading, combining Aegeline with Atorvastatin provides synergistic efficacy, enhancing cholesterol efflux and restoring pro-inflammatory cytokine expression toward physiological levels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/744794v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1d90d88org.highwire.dtl.DTLVardef@1079202org.highwire.dtl.DTLVardef@2d659org.highwire.dtl.DTLVardef@4685af_HPS_FORMAT_FIGEXP M_FIG C_FIG