Atherosclerosis

Cardiovascular diseases, including atherosclerosis, are major causes of morbidity and mortality worldwide. Here, we investigate the role of the kynurenine pathway (KP) in macrophages in the context of atheroma plaque microenvironment and its impact on atherogenesis. Using an in vitro model of primary human macrophages, we observed that exposure to plaque homogenates induces a marked increase in the early steps of the KP which impacts on kynurenine production. This was confirmed by immunostaining on human plaque of carotid arteries. Further investigation into the underlying molecular mechanisms revealed that LXR signaling contributes to this plaque microenvironment-induced KP activation. We showed that kynurenine released from macrophages affected endothelial cells, leading to increased expression of ICAM-1 and VCAM-1 in an AhR-dependent manner. Consistently with the proatherogenic effects, in a cohort of atherosclerotic patients, we observed higher levels of plasma kynurenine in patients with lower extremity arterial disease. In line with the results of in vitro investigations, the plasma kynurenine levels were associated plaque oxysterol content. Using a multiple logistic regression model, we showed that plasma kynurenine was independently associated with lower extremity arterial disease in atherosclerotic patients. Altogether, our data indicate that the activation of KP in macrophages in the context of atheroma plaque is partially mediated by LXR axis and leads to the release of kynurenine. This, in turn, contributes to the exacerbation of both local and peripheral atherosclerosis particularly through the activation of endothelial cells.

Immunization of ApoE-/- mice expressing human HLA-A 02:01 with p210, an apoB100-derived peptide, reduces atherosclerotic plaque development by inducing a p210-specific CD8+ T cell population. Studying Class-I MHC/CD8+ T cell signaling offers a promising approach to understanding the mechanism behind the athero-protective effects of p210 immunization. We aimed to identify a p210-specific CD8+ T cell population in human carotid atherosclerotic plaques from blood-positive HLA-A 02:01 patients undergoing surgical carotid endarterectomy (CEA). The study included 22 consecutive patients who were HLA-A 02:01 (+) out of 49 enrolled (reflecting an estimated prevalence of about 30% HLA-A02:01(+) in the Caucasian population). Immunohistochemistry staining used a PE-marked A 02:01-KTTKQSFDL Pentamer on fixed endarterectomy plaques. Both HLA-A 02:01 (+) and (-) patient plaques were used, with the latter serving as an internal negative control. Presence of pentamer (+) CD8 T cells indicated a p210-specific CD8+ T cell population. Patients positive for HLA-A 02:01 showed an average of 3.40 {+/-} 2.17 x 10^3 HPF p210-specific CD8+ T cells (61.80%, 3% of total CD3+) in the shoulders of atherosclerotic plaques post-CEA, significantly higher than in controls (p < 0.0001). The proportion of p210-specific CD8+ T cells was lower in plaques displaying morphological features of instability. This study, for the first time, identifies a p210-specific CD8+ T cell population in human carotid atherosclerotic plaques from HLA-A02:01(+) patients, suggesting a role for autoimmunity in atherosclerosis development and supporting the potential efficacy of p210 immunization in HLA-A02:01 (+) individuals to reduce atherosclerosis. The variation in this specific T cell population within human plaques correlates with plaque vulnerability, highlighting p210-specific CD8+ T cells as a potential target for future therapies.

RationaleThe CC-chemokine ligand-2 (CCL2)/ CC-chemokine receptor-2 (CCR2) axis governs monocyte recruitment to atherosclerotic lesions. Coherent evidence from experimental studies employing genetic deletion of CCL2 or CCR2 and human epidemiological studies support a causal involvement of the CCL2/CCR2 axis in atherosclerosis. Still, preclinical studies testing pharmacological inhibition of CCL2 or CCR2 in atheroprone mice apply widely different approaches and report inconsistent results, thus halting clinical translation. ObjectiveTo systematically review and meta-analyze preclinical studies pharmacologically targeting the CCL2/CCR2 axis in atherosclerosis in an effort to inform the design of future trials. Methods and ResultsWe identified 14 studies testing CCL2/CCR2 inhibition using 11 different pharmacological agents in mouse models of atherosclerosis. In meta-analyses, blockade of CCL2 or CCR2 attenuated atherosclerotic lesion size in the aortic root or arch (g=-0.75 [-1.17 to -0.32], p=6x10-4; N=171/171 mice in experimental/control group), the carotid (g=-2.39 [-4.23 to -0.55], p=0.01; N=24/25) and the femoral artery (g=-2.38 [-3.50 to -1.26], p=3x10-5; N=10/10). Furthermore, CCL2/CCR2 inhibition reduced intralesional macrophage accumulation and increased smooth muscle cell content and collagen deposition, consistent with a plaque-stabilizing effect. While there was heterogeneity across studies, the effects of CCL2/CCR2 inhibition on lesion size correlated with reductions in plaque macrophage accumulation, in accord with a prominent role of CCL2/CCR2 signaling in monocyte recruitment. Subgroup analyses revealed similar efficacy of both CCL2- and CCR2-inhibiting approaches across different atherosclerosis models in reducing lesion size and intralesional macrophage accumulation, but stronger atheroprotective effects in carotid and femoral arteries, as compared to the aorta. ConclusionsPharmacological targeting of CCL2 or CCR2 lowers atherosclerotic lesion burden and confers plaque stability in mice across different vascular territories, drug candidates, and models of atherosclerosis. Our findings in conjunction with recent human data highlight the translational potential of targeting the CCL2/CCR2 axis in atherosclerosis and can inform future clinical trials. Subject codesatherosclerosis, inflammation, vascular biology, translational studies

Atherosclerosis is a chronic disease of the vascular wall driven by lipid accumulation and inflammation in the intimal layer of arteries [1], [2], and its main complications, myocardial infarction and stroke, are the leading cause of mortality worldwide [3]. Recent studies have identified Triggering receptor expressed on myeloid cells 2 (TREM2), a lipid-sensing receptor regulating several key myeloid cell functions [4], as a highly expressed marker of macrophage foam cells in experimental and human atherosclerosis [5]. However, the function of TREM2 in the development of atherosclerosis is unknown. Here, we show that hematopoietic or global TREM2 deficiency increases necrotic core formation in early experimental atherosclerosis. We further demonstrate that TREM2 is essential for the efferocytosis capacities of macrophages, and to the survival of lipid-laden macrophages, altogether indicating a crucial role of TREM2 in maintaining the balance between foam cell death and their clearance in atherosclerotic lesions, thereby controlling plaque necrosis.

ObjectiveDeamidation of the NGR (Asn-Gly-Arg) motif to the isoDGR (isoAsp-Gly-Arg) motif in fibronectin (IsoDGR-fibronectin) enhances in vitro monocyte and endothelial cell activation. Blocking isoDGR reduces macrophage influx in murine tissues. Although macrophage influx is an important feature of human plaque destabilization, the role for plasma and plaque isoDGR-fibronectin in macrophage influx in the atherosclerotic plaque and thereby increasing plaque vulnerability has not been investigated in large human cohorts. DesignIsoDGR-fibronectin levels in plasma and plaques were measured in carotid endarterectomy (CEA) patients from the Athero Express biobank cohort and associated with macrophage and other vulnerable plaque characteristics in the carotid plaque of the same patient. MethodsLevels of isoDGR-fibronectin were measured using an ELISA. Carotid plaque characteristics were visualized with immunohistochemistry staining and scored semi-quantitatively. Baseline characteristics were analysed with Pearsons Chi-squared test and Mann-Whitney U-test when applicable. Univariate and multivariate logistics regression analyses were used to identify associations with adverse plaque characteristics. ResultsPlasma isoDGR-fibronectin was measured in 730 CEA patients. Patients with moderate/heavy plaque macrophage staining had higher levels of isoDGR-fibronectin than patients with no/minor macrophage staining (multivariate OR 1.40 (95%CI 1.04 - 1.90, p=0.028)). Of the 730 CEA patients, 348 had plaque samples available for isoDGR-fibronectin measurements. In the multivariate analysis, higher plaque levels of isoDGR-fibronectin were associated with moderate/high plaque macrophage staining (OR 1.22 (95%CI 1.00 - 1.56, p=0.049)), >40% fat in plaque (OR 1.1.44 (95% CI 1.14 - 1.86, p=0.004)) and intraplaque haemorrhage (OR 1.38 (95% 1.12 - 1.72, p=0.003)). ConclusionIn this large human cohort study high plasma and plaque levels of isoDGR-fibronectin were associated with more plaque macrophages and other adverse plaque characteristics. This suggests the involvement of isoDGR-fibronectin in human plaque destabilization that may lead to new potential treatment modalities.

Interindividual differences for outcomes of lipid-lowering therapy are well known. Alterations in cellular pathways may contribute to the phenomenon. To address this question, we employed an automated multiplexed analysis pipeline to systematically characterize alterations in cellular lipid trafficking in leukocytes from 400 subjects of the FINRISK 2012 Study. Of individuals receiving high-intensity statin therapy those with lower cellular lipid trafficking scores displayed higher circulating concentrations of several pro-atherogenic lipoproteins and had higher odds for myocardial infarction and stroke when compared to the rest of the subjects with equivalent treatment. Most subjects with a poor lipid trafficking score did not reach low density lipoprotein cholesterol (LDL-C) target levels on statin monotherapy. Lipid trafficking scores showed synergy with a polygenic risk score for LDL-C, improving the association with pro-atherogenic lipoprotein profile when combined. Our results suggest that quantification of cellular lipid trafficking can aid in treatment selection and risk assessment in dyslipidemia. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/23287344v3_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@d0ff02org.highwire.dtl.DTLVardef@115f036org.highwire.dtl.DTLVardef@14d9c0corg.highwire.dtl.DTLVardef@3ff32a_HPS_FORMAT_FIGEXP M_FIG C_FIG

AimsNucleotide-binding oligomerization domain-containing protein (NOD)1 is an intracellular pattern recognition receptor that initiates immune responses upon ligation of molecules such as bacterial peptidoglycan containing a D-glutamyl-meso-diaminopimelic acid (iE-DAP) moiety. NOD1 ligation has been shown to promote vascular inflammation and atherosclerosis. In this study, we investigate the functional role of NOD1 in atherosclerotic plaques and characterize the vascular cells responsible for NOD1 expression and function. Methods and resultsNOD1 was mainly expressed in a subtype of vascular smooth muscle cells (SMC) in human atherosclerotic lesions. In ex vivo cultures, human endarterectomy specimens reacted to NOD1 ligand by activation of mitogen-activated protein kinase (MAPK) pathways, leading to cytokine expression. Levels of NOD1 mRNA were higher in carotid endarterectomy specimens obtained from symptomatic patients compared to asymptomatic ones. NOD1high SMC were also found in arteries of atherosclerosis-prone Ldlr-/- mice. Challenging these mice with a NOD1 agonist resulted in transmural vascular inflammation, severe arterial damage, accelerated atherogenesis throughout the aorta, and evidence of occlusive coronary artery disease. In rats, mechanic injury to carotid arteries promoted NOD1high SMC expansion and neointima formation. In vitro, neointima derived NOD1high SMCs responded to NOD1 ligand exposure by enhanced migration, increased iNOS+ cells and amplified CCL5 production. ConclusionOur findings show that NOD1 promotes vascular inflammation, vascular injury responses and atherosclerosis by acting on a NOD1high subtype of SMC.

BackgroundPreviously we reported a time dependent change in atherosclerotic carotid plaque characteristics, with decreasing destabilising characteristics between 2002 -2011. This observation was considered confirmative with a parallel improved adherence to medication and decrease in overall cardiovascular mortality in Western Europe. In the present study, we investigated if these time dependent changes in plaque characteristics still occurred over the last decade. MethodsUsing the Athero Express biobank, atherosclerotic plaques of 1,277 consecutive carotid endarterectomy patients included between 2010 and 2021 were analysed to examine time dependent changes in histological plaque characteristics in intervals of 2 years. These results were compared with our previous time dependent plaque composition data between 2002-2011. ResultsIn contrast to the period 2002-2011, the period 2010-2021 showed a significant increase in vulnerable plaque characteristics: large lipid cores, intraplaque hemorrhage, macrophages, and calcification. When adjusted for confounders related to these plaque characteristics, such as time to operation and pre-operative type of symptoms, the adjusted odds ratios per 2 years increase in time were 1,177 (95% confidence interval, 1,070-1,293; p<0,001) for calcification, 1,352 (95% confidence interval, 1,229-1,487; p<0,000) for intraplaque hemorrhage, 1,277 (95% confidence interval, 1,159-1,407; p<0,001) for plaques consisting of >40% of fat and 1,388 (95% confidence interval, 1,262-1,528; p<0,001) for macrophages. Use of statins increased in both the 2002-2011 and 2010-2021 period. ConclusionOur study did not support a further increase in plaque stabilizing features in carotid atherosclerotic plaques between 2010-2021. In contrast, plaques even showed features of destabilisation in the present cohort.

BackgroundThe causal relationship between atherosclerosis and the development of aneurysms remains controversial. This study aims to analyze the genetic predictive association between atherosclerosis and intracranial aneurysms, as well as to explore intermediate biomarkers that may mediate the causal relationship between these two conditions. MethodsThis study utilized head imaging data for a cross-sectional analysis, investigating the prevalence of cerebral atherosclerosis and intracranial aneurysms in 13,739 patients. We performed Mendelian randomization analysis on two samples to explore the genetic predictive association between atherosclerosis and intracranial aneurysms. Furthermore, we examined the role of the matrix metalloproteinase family as an intermediary biomarker in the causal relationship between these two conditions and validated biomarker in serum and in situ samples from clinical patients. ResultsCross-sectional study revealed that patients with intracranial aneurysms exhibited a higher prevalence of cerebral atherosclerosis compared to controls (14.155% vs. 7.069%, P<0.001). Two-sample Mendelian randomization analysis demonstrated that genetically predicted peripheral atherosclerosis significantly increased the risk of intracranial aneurysms, encompassing both unruptured (ORIVW = 1.711, PIVW = 0.002) and ruptured intracranial aneurysms (ORIVW = 1.533, PIVW = 9.41x10^-4). Mendelian randomization further indicated that peripheral atherosclerosis leads to elevated circulating MMP12 levels ({beta}IVW = 0.083, PIVW = 0.008), which were subsequently associated with increased risks of unruptured (ORIVW = 1.136, PIVW = 0.010) and ruptured intracranial aneurysms (ORsimple-mode = 1.217, Psimple-mode = 0.038). ELISA assays confirmed elevated MMP12 levels in the plasma of patients with carotid artery stenosis and intracranial aneurysms. In situ analyses revealed upregulated MMP12 expression in both plaques and intracranial aneurysm tissues. ConclusionOur research clarifies the causal relationship between atherosclerosis and intracranial aneurysms, identifies and validates MMP12 as a key biomarker, and provides new methods and evidence to explain the association between these two diseases. What is already known on this topicPrevious studies have suggested a significant association between atherosclerosis and the development of aneurysms. What this study addsThis study provides robust evidence and causal inferences regarding this relationship, identifying intermediate biomarkers of the diseases. How this study might affect research, practice or policyThese findings suggest that preventing atherosclerosis and controlling MMP12 levels may help reduce the risk of intracranial aneurysms.

Background and aimsOncostatin M (OSM) signaling is implicated in atherosclerosis, however the mechanism remains unclear. We investigated the impact of common genetic variants in OSM and its receptors, OSMR and LIFR, on overall plaque vulnerability, plaque phenotype, intraplaque OSMR and LIFR expression, coronary artery calcification burden and cardiovascular disease susceptibility. Methods and resultsWe queried Genotype-Tissue Expression data and found that rs13168867 (C allele) was associated with decreased OSMR expression and that rs10491509 (A allele) was associated with increased LIFR expression in arterial tissues. No variant was significantly associated with OSM expression. We associated these two variants with plaque characteristics from 1,443 genotyped carotid endarterectomy patients in the Athero-Express Biobank Study. After correction for multiple testing, rs13168867 was significantly associated with an increased overall plaque vulnerability ({beta}=0.118 {+/-} s.e.=0.040, p=3.00x10-3, C allele). Looking at individual plaque characteristics, rs13168867 showed strongest associations with intraplaque fat ({beta}=0.248 {+/-} s.e.=0.088, p=4.66 x 10-3, C allele) and collagen content ({beta}=-0.259 {+/-} s.e.=0.095, p=6.22 x 10-3, C allele), but these associations were not significant after correction for multiple testing. rs13168867 was not associated with intraplaque OSMR expression. Neither was intraplaque OSMR expression associated with plaque vulnerability and no known OSMR eQTLs were associated with coronary artery calcification burden, or cardiovascular disease susceptibility. No associations were found for rs10491509 in the LIFR locus. ConclusionsOur study suggests that rs1316887 in the OSMR locus is associated with increased plaque vulnerability, but not with coronary calcification or cardiovascular disease risk. It remains unclear through which precise biological mechanisms OSM signaling exerts its effects on plaque morphology. However, the OSM-OSMR/LIFR pathway is unlikely to be causally involved in lifetime cardiovascular disease susceptibility.

Background and AimsMetabolism dictates macrophage function and plays a central role in atherosclerotic plaque progression. The kynurenine pathway, which metabolizes the majority of the essential amino acid tryptophan, plays a pivotal role in regulating immune responses and supporting NAD+ synthesis, essential for cellular energy metabolism. Higher circulating kynurenine levels are associated with cardiovascular disease, yet their role in atherosclerotic plaques is unclear. This study aims to investigate the underlying mechanisms driving increased kynurenine concentrations in plaques and to determine whether kynurenine serves as a mere biomarker of low-grade inflammation or reflects specific macrophage-driven metabolic alterations that could position it as a potential therapeutic target. MethodsWe used histological and transcriptomic data from two biobanks: the Athero Express Biobank (AE; n=91) and Maastricht human plaque study (MaasHPS, n= 26). Macrophages were identified through CD68 staining in AE, and M1/M2-like macrophage subtypes were distinguished by iNOS/CD68 and arginase/CD68 expression in MAASHPS. Primary human monocyte-derived cultured macrophages were polarized into M1- and M2-like phenotypes for using IFN-{gamma} and IL-4, respectively. Tryptophan, kynurenine and/or NAD+ concentrations in plaques were quantified usingliquid chromatography and metabolomics analyses. ResultsKynurenine concentrations were significantly higher in plaques with greater macrophage density (p = 0.023). Transcriptomic analysis in AE revealed upregulation of IDO2, AFMID, and KYNU in plaques with increased macrophage infiltration (p < 0.05), but not IDO1 (p = 0.16). In the MAASHPS biobank, higher IDO1, KYNU, and KMO expression correlated negatively with M2 marker positive macrophages (p < 0.001), while HAAO correlated positively (p < 0.01). In vitro, M1-like macrophages showed increased IDO1 and reduced QPRT expression compared to M2-like macrophages. We found that this disruption in kynurenine pathway gene expression led to decreased NAD+ concentrations in M1-like macrophages compared to M2-like macrophages in vitro. ConclusionHigher kynurenine levels in atherosclerotic plaques are increased by the increased presence of M1 macrophages, likely driven by both an increased IDO1 activity and reduced QPRT gene expression. This leads to decreased concentrations of NAD+, potentially determining the phenotype of the macrophages. Future studies should address whether modulation of the kynurenine pathway restores NAD+ metabolism and leads to a decrease in inflammation and an increased stable plaque phenotype.

BackgroundInflammatory processes are a key cause of atherosclerosis and cardiovascular diseases. Complement system has been implicated but evidence is less clear. We tested whether atherosclerosis severity, plaque types, and vascular region are associated with mRNA expression of genes encoding proteins of the complement system and investigated the cell-specific expression of the most differentially expressed transcripts in plaque macrophages, fibroblasts, and endothelial, smooth muscle, Schwann, mast, plasma, T-, and B-cells. MethodsTotal mRNA was isolated, and gene expression analyzed from 29 carotid, 15 abdominal aortic, and 24 femoral plaque samples, and 28 atherosclerosis-free control left internal thoracic artery samples of 95 patients, as well as from 97 whole blood and 97 peripheral mononuclear cell samples of 97 patients. Genome-wide transcriptomic analyses were done using RNA bead microarray platforms. Differential expression was compared between plaques with normal arteries, unstable with stable plaques, as well as CAD patients with CAD-free patients. ResultsA total of 33 out of 90 (37%) transcripts of the complement system were differentially expressed in atherosclerotic plaques as compared to histologically normal arteries. In aortic, carotid, and femoral plaques 38, 36 and 29 transcripts, respectively, were differentially expressed, of which 25 were shared by the plaques of all arterial beds. Among the most interesting gene-level findings, we observed that transcripts of the integrin gene ITGB2 are highly upregulated mostly in macrophages and T cells while having top centrality in the network and top enriched genesets related to cell junctions. ConclusionsThis is the first study exploring the association of local complement expression across multiple arterial beds and histologically diverse plaque samples. The local effect of complement-mediated inflammation is indicated in inflammatory plaque tissue but not in smooth muscle cell-dominated plaques. Taken together, gene expression of the complement system components in artery cells is associated with advanced atherosclerosis.

Background and aimsThe accumulation of erythrocyte membranes within an atherosclerotic plaque may contribute to the deposition of free cholesterol and thereby the enlargement of the necrotic core. Erythrocyte membranes can be visualized and quantified in the plaque by immunostaining for the erythrocyte marker glycophorin C. Consequently, we hypothesized, that the accumulation of erythrocytes quantified by glycophorin C is a marker for plaque vulnerability and may therefore reflect intraplaque hemorrhage (IPH), vulnerability of plaques and predict pre-procedural neurological symptoms. MethodsWe employed the CellProfiler-integrated slideToolKit workflow to visualize and quantify glycophorin C, defined as the total plaque area that is positive for glycophorin C, in single slides of culprit lesions obtained from the Athero-Express Biobank of 1,819 consecutive asymptomatic and symptomatic patients who underwent carotid endarterectomy. Our assessment included the evaluation of various parameters such as lipid core, calcifications, collagen content, SMC content, and macrophage burden. These parameters were evaluated using a semi-quantitative scoring method, and the resulting data was dichotomized as predefined criteria into categories of no/minor or moderate/heavy staining. In addition, the presence or absence of IPH was also scored. ResultsThe prevalence of IPH and pre-procedural neurological symptoms were 62.4% and 87.1%, respectively. The amount of glycophorin staining was significantly higher in samples from men compared to samples of women (median 7.15 (IQR:3.37, 13.41) versus median 4.06 (IQR:1.98, 8.32), p<0.001). Glycophorin C was associated with IPH adjusted for clinical confounders (OR 1.90; 95% CI 1.63, 2.21; p=<0.001). Glycophorin C was significantly associated with ipsilateral pre-procedural neurological symptoms (OR:1.27, 95%CI:1.06-1.41, p=0.005). Sex-stratified analysis, showed that this was also the case for men (OR 1.37; 95%CI 1.12, 1.69; p=0.003), but not for women (OR 1.15; 95%CI 0.77, 1.73; p=0.27). Glycophorin C was associated with classical features of a vulnerable plaque, such as a larger lipid core, a higher macrophage burden, less calcifications, a lower collagen and SMC content. There were marked sex differences, in men, glycophorin C was associated with calcifications and collagen while these associations were not found in women. ConclusionsThe accumulation of erythrocytes in atherosclerotic plaque quantified and visualized by glycophorin C was independently associated with the presence of IPH, preprocedural symptoms in men, and with a more vulnerable plaque composition in both men and women. These results strengthen the notion that the accumulation of erythrocytes quantified by glycophorin C can be used as a marker for plaque vulnerability.

BackgroundSex and plaque histology are intertwined, with fibrous atherosclerotic plaques being more prevalent in women and pointing to general smooth muscle cell plasticity and estrogen signaling. Plaque erosion, a significant contributor to acute coronary syndromes (ACSs), is linked to fibrous plaques and is more prevalent in women as compared to men. We hypothesize that the molecular drivers of histologically determined fibrous plaques differ between men and women. MethodsHuman end-stage atherosclerotic plaques were isolated from consecutive patients who underwent carotid endarterectomy and were included in the Athero-Express biobank. Fibrous plaques from both female and male patients were histologically assessed and further processed to obtain protein, bulk RNA, single-cell RNA and DNA methylation data. We leveraged sex-differential expression and deconvolution analyses to uncover sex-biased molecular mechanisms and cellular dynamics which were experimentally validated using an EndMT in vitro model. ResultsOut of 1,889 atherosclerotic plaques (1,309 male and 580 female), fibrous lesions were observed in 50% of female (n=290) and 31% of male patients (n=416). Compared to patients with atheromatous plaques (n=494), women with fibrous plaques exhibited a higher prevalence of smoking (41% vs. 33%), while men with fibrous plaques presented more often with diabetes (29% vs. 20%). Transcriptional and proteomic phenotyping highlighted more immune-dependent and inflammatory processes in male fibrous plaques. Genes and proteins with higher abundance in female fibrous plaques pointed to endothelial-to-mesenchymal transition (EndMT) and extracellular matrix remodelling. Using single-cell RNA sequencing, we identified a dominant role of endothelial and smooth muscle cells in female plaques, and more macrophages in males. Finally, at the cellular level, we show that sex - specific, smoking-mediated promoter methylation changes may explain these differences. ConclusionsPatients with end-stage fibrous atherosclerotic plaques have a distinct clinical profile, with men more often having diabetes and women more often smoking. This clinical profile associates with sex differences that point to different cellular and molecular compositions of fibrous plaques. These mechanisms might be candidate pathways to understand plaque erosion from a molecular point of view and may provide promising targets for atherosclerosis therapies, as they account for the sex-specific differences in plaque phenotype. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/24314739v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@f3f474org.highwire.dtl.DTLVardef@3606d4org.highwire.dtl.DTLVardef@fa8801org.highwire.dtl.DTLVardef@1f719f4_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background and AimsNeutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. MethodsWe generated myeloid-specific Stat4{Delta}LysMLdlr-/-, neutrophil-specific Stat4{Delta}S100A8 Ldlr-/-, and control Stat4fl/flLdlr-/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat Pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4{Delta}LysMLdlr-/- and Stat4fl/flLdlr-/- bone marrow cells into aged atherosclerotic Apoe-/- mice and detected by flow cytometry. ResultsSTAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in Stat4{Delta}LysMLdlr-/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. ConclusionsOur work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.

Background and aimsCurrent FH VCEP specifications of ACMG/AMP guidelines for familial hypercholesterolemia (FH) variant interpretation assign a higher evidence weight to functional data obtained with flow cytometry than microscopy assays, due to lack of existing evidence. This restricts the use of microscopy-derived functional data for variant classification. We aimed to systematically compare functional data of LDLR variants obtained by high-content microscopy and flow cytometry to determine their concordance and assess whether microscopy-based assays could support a higher evidence level. MethodsFifty LDLR variants with available flow cytometry and high-content microscopy data were compared for LDL uptake activity, including 21 newly characterized variants by microscopy in this study. Variants were grouped by FH VCEP functional thresholds (<70% activity, abnormal function; >90% activity, normal function) and results were integrated with UK Biobank data to assess associations with lipid traits. ResultsFirst, we validated our scalable microscopy assay with FH VCEP-classified control variants. Then we compared functional activity measured by microscopy and flow cytometry assays for 50 variants, which showed significant correlation (r = 0.66, p<0.0001) and a close average agreement (Bland-Altman bias = -0.05). Applying FH VCEP functional classification thresholds yielded broadly consistent classification in both methods, with minor shifts among categories. Integration with UK Biobank data showed that carriers of variants with reduced LDLR activity (<70% and <50%) had higher LDL-C, total cholesterol and ApoB levels compared to those with normal activity (>90%) for both microscopy and flow cytometry assays, with more pronounced differences observed at the <50% LDLR activity threshold. ConclusionHigh-content microscopy provides reliable and scalable measurements of LDLR function, showing high concordance with flow cytometry and consistent associations with lipid phenotypes. These findings support reconsideration of the evidence weight assigned to validated microscopy assays within FH VCEP variant classification frameworks, namely to Strong (Level 1).

Background and aimsLipids play a critical role in atherosclerosis. Low-density lipoprotein (LDL)-cholesterol and certain lipid classes like sphingomyelins are associated with inflammation and poor cardiovascular outcomes. Phosphatidylserine (PS), on the other hand, is a negatively charged anti-inflammatory phospholipid class involved in efferocytosis. In this study, we sought to investigate its anti-atherosclerotic properties through a combination of complementary human lipidomics analyses, in vitro assays and in vivo experiments in Apoe-/- mice. MethodsHuman lipidomics studies were performed on the 300OB cohort comprising 300 obese and overweight individuals at risk of cardiovascular disease. In vitro assays were carried out using human monocytes and macrophages, and in vivo experiments included histopathological, immunophenotyping and single-cell transcriptomic analyses. ResultsIn humans, we identified PS as an anti-inflammatory and atheroprotective biomarker. Hence, we developed a high-density lipoprotein (HDL)-like formulation enriched in PS to exploit its properties in a targeted fashion in mice. In vitro, this formulation potently inhibited inflammatory cytokine production on human myeloid cells. Our in-depth in vivo experiments provided evidence of the formulations potent plaque-stabilizing and anti-inflammatory actions. These effects were mediated by a shift in the monocyte/macrophage compartment toward homeostatic/repairing phenotypes. ConclusionsCollectively, our results demonstrate that HDL-associated PS potently suppresses inflammation and atheroprogression, and holds promise as a viable approach to improve immunomodulatory therapies.

BackgroundAtherosclerosis is characterized by the buildup of fatty plaques that thicken and stiffen arterial walls. Macrophages (M{varphi}s) significantly contribute to this process through their scavenger receptor CD36. PIM1 is a serine/threonine kinase known to modulate immune responses and cell metabolism. However, its role in M{varphi} lipid handling and atherogenesis is not well defined. This study examines the role of PIM1 in regulating CD36 expression and function in M{varphi}s during foam cell formation and atherosclerosis progression. MethodsWe performed in vitro studies by treating murine peritoneal M{varphi}s from Pim1-/- and wild-type (WT) mice with oxidized low-density lipoprotein (oxLDL). We measured CD36, PIM1, and plaque-associated proteins and mRNA levels, oxLDL binding and uptake rates, as well as foam cell formation. For in vivo studies, we fed M{varphi}-specific PIM1-deficient (Apoe-/- Lyz2Cre/+Pim1fl/fl) and their littermate control (Apoe-/-Pim1fl/fl) mice a high-fat diet for 12 weeks. We then evaluated the plaque formation in their aortic sinuses and arches. ResultsDeletion of Pim1 in M{varphi}s reduced CD36 protein expression by up to 96.7% compared to WT controls. This led to a 49.6% decrease in foam cell formation and a 25.5% reduction in cellular cholesterol after oxLDL treatment. Pharmacological inhibition of PIM kinase activity in WT M{varphi}s also impaired oxLDL handling, with a 64.5% reduction in binding and a 57.9% in uptake. Bulk RNA-seq revealed that Pim1 deficiency downregulated PPAR{gamma} signaling. Treatment with a PPAR{gamma} agonist restored CD36 levels in the PIM1 knockdown M{varphi}s, suggesting that PIM1 regulates CD36 through PPAR{gamma}. Moreover, PIM1 M{varphi}-specific deficiency caused a 69.4% reduction in atherosclerotic plaque formation. ConclusionPIM1 acts as a key upstream regulator of CD36 by enhancing PPAR{gamma} activity in M{varphi}s. The PIM1-CD36 axis promotes oxLDL binding, uptake, and foam cell formation. Targeting the PIM1/PPAR{gamma}/CD36 pathway could offer new ways to modulate M{varphi} lipid metabolism and reduce atherosclerotic plaque progression. Non-standard Abbreviations and AcronymsELISA: enzyme-linked immunosorbent assay; HFD: high-fat diet; M{varphi}s: macrophages; MCP-1: monocyte chemoattractant protein-1; ORO: oil red O; oxLDL: oxidized low-density lipoprotein; PBS: phosphate-buffered saline; PPAR{gamma}: peroxisome proliferator-activated receptor gamma; WT: wild type.

BackgroundHDL particles can carry microRNAs (miRNAs), capable of regulating gene expression connected to HDL functions. Despite links to some cardiovascular risk factors, miRNA association with incident acute myocardial infarction (AMI) remains unclear. ObjectivesOur aim was to elucidate the association between HDL-bound miRNAs (HDL-miRNAs) and incident AMI using a non-targeted approach in a population-based study. MethodsWe conducted a case-cohort study including 247 participants from the REGICOR cohort in northeastern Spain (51 AMI cases and a random sample of 196 participants, including seven overlapping AMI cases). HDL-miRNAs were isolated from apolipoprotein B-depleted serum and quantified by whole-genome miRNA sequencing. Associations between HDL-miRNAs and incident AMI were assessed using multivariable Cox proportional hazards model. For AMI-associated HDL-miRNAs (p-value <0.10), we retrieved their experimentally validated targets and assessed pathway enrichment of these targets via over-representation analysis. ResultsTwo HDL-miRNAs were associated with incident AMI after FDR correction: miR-628-3p (HR 1.69, 95% CI 1.30 to 2.19) and miR-28-3p (HR 1.58, 95% CI 1.21 to 2.06). Nine additional HDL-miRNAs were nominally associated with AMI incidence (p-value <0.05), eight with a direct association (miR-93-5p, miR-26b-5p, miR-106a-5p, miR-126-3p, miR-15b-5p, let-7a-5p, let-7e-5p, and let-7f-5p) and one with an inverse association (miR-361-5p). These miRNAs regulate the expression of genes in pathways involved in cholesterol regulation, particularly cholesterol efflux and homeostasis. The AMI group exhibited higher variance and a greater number of significant and strong correlations. ConclusionsTwo HDL-miRNAs (miR-628-3p and miR-28-3p) were significantly associated with AMI incidence. A tighter coregulatory network in cases was observed, underscoring their potential clinical utility in risk prediction and cardiovascular prevention. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIIn a population-based case-cohort study we profiled the HDL-bound miRNome and found two miRNAs (miR-628-3p and miR-28-3p) associated with incident AMI. C_LIO_LIThe use of HDL-enriched serum fractions provided a focused analysis on HDL functionality. These miRNAs regulate the expression of genes in pathways involved in cholesterol efflux and homeostasis (ABCA1, ARL4C, SIRT1, NFKBIA, ANXA2, LRP6) and show a tighter coregulatory network among significant miRNAs among cases, supporting biological coherence. C_LI What Are the Clinical Implications?O_LIHDL-miRNA signatures may complement traditional risk factors to refine AMI risk stratification and provide a rationale for HDL-guided, miRNA-targeted preventive interventions using HDL-like delivery platforms. C_LI

BACKGROUNDThe atherogenic properties of lipoproteins are well-characterized, in contrast to their putative atheroprotective effects which remain underinvestigated. METHODSWe conducted this Mendelian randomization (MR) study to analyze the causal relationship between free cholesterol (FC) levels/total lipids/FC to total lipids ratio in lipoprotein subfractions and emergency coronary revascularization (for acute coronary syndromes [ACS]) (no controls excluded). RESULTSPositive causal association between the FC to total lipid ratio/FC levels in small HDL and emergency coronary revascularization (for ACS) suggest that spontaneous FC efflux from small HDL mediates pro-atherosclerotic effects independently. Replacing FC levels with the FC-to-total-lipids ratio converts LDL from pro-atherogenic to anti-atherosclerotic particles. Or reduces pro-atherogenic effects in VLDL. In contrast, applying the same replacement in HDL particles reduces their anti-atherogenic properties. The most remarkable finding is the negative causal relationship between the FC to total lipids ratio in (large LDL [P=1.88E-02, OR:0.78, 95%CI:0.64-0.96], medium LDL [P=1.76E-02, OR:0.78, 95%CI:0.64-0.96]) and emergency coronary revascularization (for ACS). This indicates that combined spontaneous FC diffusion and reverse cholesterol transport (RCT) can shift from pro-atherogenic to anti-atherogenic effects. The loss of bidirectional FC diffusion reduces the anti-atherosclerotic effect. CONCLUSIONSHDL can exhibit pro-atherosclerotic effects through spontaneously diffused FC. However, the bidirectional exchange of FC between HDL and other lipoproteins/cells--coupled with RCT and its alternative pathways--may attenuate or even reverse atherosclerosis, implying that the lack of this specific mechanism could drive proatherogenic processes. Notably, excessive cholesteryl ester (CE) production can counteract these protective mechanisms, potentially restoring a pro-atherosclerotic state.