Arteriosclerosis, Thrombosis, and Vascular Biology

BackgroundPeripheral arterial disease (PAD) is the 3rd leading type of atherosclerotic disease (ASD) morbidity. Arterial stiffness is intimately connected to the onset and progression of peripheral artery disease (PAD). The role of arterial stiffening on flow-mediated atherosclerotic plaque formation is not well understood. The objective of this study is to discover endothelial cell (EC) pathways under PAD conditions and test the modifiability of these pathways on ASD. MethodsPAD conditions in mice were conferred by partial carotid ligation to induce disturbed Flow (D-flow) in pre-stiffened Fibulin-5 knockout (KO) mice that lack normal elastin function. EC pathways, including Connective tissue growth factor (CTGF/CCN) were quantified by gene analysis and histology. Atherogenic mice had PCSK9 infection + high fat diet. CTGF was inhibited in an EC-specific knockout (ECKOCTGF) and with a CTGF antibody (FG-3149). Human vascular tissue was used to validate PAD biomechanics and CTGF upregulation. ResultsBiomechanical testing demonstrated that d-flow KO arteries mimic biomechanics of PAD arteries. RNA microarray, qPCR, and immunohistochemistry identified EC plasticity in these arteries compared to WT and KO under stable flow. Under atherogenic conditions, KO arteries demonstrated vulnerable plaques not seen in WT animals. CTGF expression was increased by d-flow, in KO arteries, in aged (18 months) WT arteries, and vascular tissue under d-flow. CTGF inhibition by ECKOCTGF favorably improved plaque characteristics in male but not female animals. FG-3149 treatment of ECWTCTGF male animals delivered similar benefits to plaque characteristics and arterial compliance. ConclusionECs in PAD arteries exist under a complex hemodynamic environment that integrates stiffness and d-flow into an atherogenic and inflammatory environment. Stiffness + d-flow stimulates precocious onset of EC plasticity and a vulnerable plaque phenotype. CTGF is a matricellular protein that can tune fibro-inflammatory pathways. CTGF is a prominent mediator of D-flow-mediated arterial remodeling and focal atherosclerotic plaque remodeling. Inhibition of CTGF improves plaque phenotype and arterial compliance. CTGF-associated pathways hold promise as therapeutic targets for PAD patients.

BACKGROUNDVascular calcification significantly influences the onset and outcome of cardiovascular events, yet no effective treatment currently exists. Dysfunction of osteoclastic macrophages contributes to the formation of calcification. Our previous studies have shown that sonodynamic therapy (SDT) can rapidly reverse atherosclerotic plaques by targeting macrophages. This study aimed to investigate the effect of SDT on reducing early or mild vascular calcification by modulating the function of osteoclastic macrophages. METHODSThirty-two patients with symptomatic femoropopliteal peripheral artery disease (PAD) were recruited to evaluate changes in vessel CT values and the target-to-background ratio (TBR) using positron emission tomography/computed tomography (PET/CT) 30 days post-SDT. An early calcification model was established in ApoE-/- mice, followed by SDT intervention. Frozen plaque sections from the mice were collected for mass spectrometry imaging (MSI)-based spatial metabolic analysis in situ. The NHGRI-EBI GWAS Catalog database and the human single-cell eQTL database (scQTLbase) were employed to analyze the causal relationship between key enzyme genes involved in phosphatidic acid (PA) synthesis in macrophages and vascular calcification using two-sample Mendelian randomization. To investigate cell ossification, calcification, and underlying mechanisms, RAW264.7 mouse macrophages were treated with a medium containing receptor activator of nuclear factor kappa-B ligand (RANKL), while mouse aortic vascular smooth muscle cells (MOVAS cells) were exposed to a calcification medium. RESULTSSDT significantly reduced the number of mildly calcified sites and the target-to-background ratio (TBR) of these sites in patients with femoropopliteal peripheral artery disease (PAD). In ApoE-/- mice, SDT alleviated early calcification of atherosclerotic plaques. MSI revealed that SDT altered the composition and distribution of lipid metabolites in atherosclerotic plaques, notably increasing the content of PA in the early calcified regions. Analysis of single-cell sequencing databases showed that key enzyme genes involved in PA synthesis--PLD1, PLD3, AGPAT4, and diacylglycerol kinase E (DGKE)--were enriched in macrophages of human coronary artery plaques. Mendelian randomization analysis indicated that DGKE negatively regulated coronary artery calcification. In vitro studies demonstrated that PA mediates SDT to promote M1 macrophage fusion and enhance carbonic anhydrase II (CA2) expression, thereby improving osteoclastic function and alleviating early calcification of MOVAS cells via the reactive oxygen species (ROS)-DGKE-PA pathway. In vivo, the CA2 inhibitor acetazolamide impaired the effects of SDT and exacerbated early calcification of atherosclerotic plaques in ApoE-/- mice. CONCLUSIONThis study demonstrates that PA-mediated SDT promotes M1 macrophage fusion and CA2 expression, improving osteoclastic function and alleviating early calcification through the ROS-DGKE-PA pathway. REGISTRATIONURL: https://www.clinicaltrials.gov; Unique identifier: NCT03457662. What Is New?1. SDT reduces early calcification in patients with symptomatic femoropopliteal PAD and in ApoE-/- mouse models of early calcification. 2. SDT upregulates the expression of PA in the early calcified regions of ApoE-/- mouse models. 3. PA-mediated SDT facilitates M1 macrophage fusion and enhances CA2 expression, thereby improving osteoclastic function and alleviating early calcification through the ROS-DGKE-PA pathway. What Are the Clinical Implications?1. Targeting M1 macrophage DGKE-PA may serve as a potential intervention for treating early vascular calcification. 2. The combination of MSI and Mendelian randomization analysis proves to be an effective method for exploring key signaling lipids in disease. 3. PA-mediated SDT represents a promising approach for the effective reduction of early vascular calcification.

Monocyte chemoattractant protein-1 (MCP-1) recruits monocytes to the atherosclerotic plaque. While experimental,1-6 genetic,7 and observational8,9 data support a key role of MCP-1 in atherosclerosis, the translational potential of targeting MCP-1 signaling for lowering vascular risk is limited by the lack of data on plaque MCP-1 activity in human atherosclerosis. Here, we measured MCP-1 levels in human plaque samples from 1,199 patients undergoing carotid endarterectomy and explored associations with histopathological, molecular, and clinical features of plaque vulnerability. MCP-1 plaque levels were associated with histopathological hallmarks of plaque vulnerability (large lipid core, low collagen, high macrophage burden, low smooth muscle cell burden, intraplaque hemorrhage) as well as molecular markers of plaque inflammation and matrix turnover, clinical plaque instability, and periprocedural stroke during plaque removal. Collectively, our findings highlight a role of MCP-1 in human plaque vulnerability and suggest that interfering with MCP-1 signaling in patients with established atherosclerosis could lower vascular risk.

Histopathological studies have revealed key processes of atherosclerotic plaque thrombosis. However, the diversity and complexity of lesion types highlight the need for improved sub- phenotyping. We hypothesized that unbiased clustering of plaques based on gene expression results in an alternative categorization of late-stage atherosclerotic lesions. We analyzed the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These novel plaque phenotypes associated with clinical presentation (p<0.001) and showed differences in cellular compositions. Validation in coronary segments showed that the molecular signature of these plaques was linked to coronary ischemia. One of the plaque types with most severe clinical symptoms pointed to both inflammatory and fibrotic cell lineages. This highlighted plaque phenotype showed high expression of genes involved in active inflammatory processes, neutrophil degranulation, matrix turnover, and metabolism. For clinical translation, we did a first promising attempt to identify circulating biomarkers that mark these newly identified plaque phenotypes. In conclusion, the definition of the plaque at risk for a thrombotic event can be fine-tuned by in- depth transcriptomic based phenotyping. These differential plaque phenotypes prove clinically relevant for both carotid and coronary artery plaques and point to differential underlying biology of symptomatic lesions.

Background and AimsProliferation of arterial smooth muscle cells (SMCs) and their modulation to alternative mesenchymal phenotypes is a central mechanism in the growth of atherosclerotic lesions. The underlying processes have been studied extensively in mouse models, but a detailed analysis of when and where modulated SMCs accumulate in human atherosclerosis is lacking. The present study mapped modulated SMC subtypes during the progression of human coronary atherosclerosis and explored their associations with disease processes in human carotid plaques. MethodsMultiplex immunostaining protocols based on single-cell RNA sequencing-validated markers were established to detect SMCs, modulated SMCs, and macrophages in sections of left anterior descending arteries from forensic autopsies. The material comprised 44 arterial segments from 38 individuals, encompassing samples with normal intima, eccentric intimal thickening, pathological intimal thickening, and fibroatheroma. A similar analysis of carotid endarterectomy samples allowed examination of the involvement of modulated SMCs in fibrosis, calcification, and apoptosis. Coronary and carotid sections were analyzed by machine learning-assisted cell classification, enabling phenotyping of entire plaques at high microscopic resolution. ResultsCells co-expressing contractile and modulated SMC markers were present in normal human coronary arteries, but fully modulated SMCs, with complete loss of detectable contractile protein expression, did not accumulate substantially until the fibroatheroma stage, where they were located preferentially around the necrotic core. SMC subtypes showed no preferential co-localization with areas of fibrosis or calcification; however, osteoprotegerin secreted by modulated SMCs was found bound to calcium deposits. Modulated SMCs accounted for 35-53% of all apoptotic remnants for which a cell origin could be determined. ConclusionsFully modulated SMCs expand at the fibroatheroma stage, localize around the necrotic core region, and account for many apoptotic remnants in plaques.

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

Micro- and nanoplastics (MNPs) are increasingly detected in human tissues, yet their causal contribution to cardiovascular disease remains poorly understood. Here we show that oral exposure to polyethylene (PE) and polyvinyl chloride (PVC) -- the most abundant polymers found in human atheromas -- accelerates atherosclerosis in ApoE-/-mice through distinct, polymer-specific molecular mechanisms. While both polymers increased plaque burden and reduced contractile smooth muscle cell (SMC) markers, single-cell transcriptomic profiling revealed divergent phenotypic trajectories. PE exposure drives SMCs toward a chondromyocyte-like cell (CMC) state, characterized by upregulated osteogenic signaling and markedly increased vascular calcification. Conversely, PVC exposure promotes a fibromyocyte-like program associated with altered collagen metabolism and accelerated cell migration without enhancing calcification. These distinct SMC programs are reflected in the transcriptional signatures of symptomatic human carotid plaques, suggesting clinical relevance for polymer-specific vascular remodeling. Our findings establish a causal link between common environmental plastics and accelerated atherosclerosis, demonstrating that MNP-induced vascular risk is mediated by divergent SMC fate decisions. These results provide a mechanistic framework for assessing the cardiovascular impact of global plastic pollution and identifying potential therapeutic targets to mitigate MNP-associated vascular toxicity.

Enzymatically oxygenated phospholipids (eoxPL) formed by lipoxygenases (LOX) and cyclooxygenase (COX) in platelets and leukocytes are pro-coagulant in multiple model systems. However, their generation in arterial thrombotic disease, and how their levels are modulated by common therapies is unknown. Here, eoxPL were first characterized in isolated platelets and leukocytes from an arterial vascular disease cohort, a healthy cohort administered low dose aspirin, and from retrieved human arterial thrombi. In both cohorts, aspirin reduced platelet COX-1-derived eoxPL, while elevating diacyl 12-LOX-derived eoxPL in males, through enhanced Lands cycle esterification. Conversely, P2Y12 inhibition reduced 12-LOX-derived eoxPL in leukocytes. Complex aspirin-dependent gender and seasonal effects on platelet eoxPL were seen in healthy subjects. Limb or coronary (STEMI) thrombi showed a platelet eoxPL signature while carotid thrombi had a white cell profile. Mice genetically lacking leukocyte 12/15-LOX, which are deficient in eoxPL, generated smaller carotid thrombi in vivo. In summary, pro-coagulant eoxPL generation is altered in human arterial vascular disease by commonly used cardiovascular therapies. These changes to the phospholipid composition of blood cells in humans at risk of thrombotic events may be clinically significant where the pro-coagulant membrane plays a central but poorly understood role in driving elevated thrombotic risk. Key PointsO_LIeoxPL generation is altered in health and arterial vascular disease by aspirin or P2Y12 inhibitors, and shows gender and seasonal variation. C_LIO_LIAspirin regulates eoxPL by inhibiting cyclooxygenase and modulating Lands cycle. C_LIO_LIThe eoxPL profile of human arterial thrombi identifies platelet and leukocyte involvement. C_LIO_LIMice deficient in LOX-derived eoxPL form smaller arterial thrombi in vivo. C_LI

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.

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.

BackgroundAtherosclerosis (ATH) is a chronic systemic inflammatory disease affecting the vessel wall, wherein regulating non-coding RNAs play a crucial role. We previously demonstrated that miR-125b is upregulated in ATH and is a main regulator of cholesterol metabolism in macrophages. Herein we hypothesized that inhibiting miR-125b may attenuate ATH. Methods and resultsIn the ApoE-/- mice model fed with a high fat diet for 14 weeks, we inhibited miR-125b using an antagomiR over a 4-week period. We observed a significant reduction in plaque size, accompanied by diminished infiltration of F4/80 macrophages and attenuation of NF-{kappa}B+ activation within plaques. We explored the mechanism using a Vas-on-Chip adhesion assay using Human Aortic Endothelial Cells (HAoEC) stimulated with TNF. We observed an impairment in the trafficking of miR-125b transfected THP-1 monocytes, accompanied by the downregulation of the CD11b/CD18 integrin and the CCR7 receptor. Furthermore, we demonstrated a direct regulation of the CCR7 receptor by miR-125b using a reporter plasmid construct (p_CCR7.WT) containing the 3UTR region of CCR7 gene fused with a luciferase coding sequence. In addition, miR-125b transfected monocytes inhibited CCR7 cell migration induced by the CCL21 ligand but did not affect migration induced by others ligands such as MCP1. Finally, we confirmed the downregulation of CCR7 in coronary plaques in both ApoE-/- mice and patients with coronary artery disease. ConclusionsInhibiting miR-125b offers a novel therapeutic approach for ameliorating ATH that results in a reduction of macrophage content and plaque lesion size. This improvement occurs through the enhancement of monocyte trafficking via CCR7 that facilitates the exit of foam cells from the plaque. CLINICAL PERSPECTIVEO_ST_ABSWhat is New?C_ST_ABS- We found evidences of a new therapeutic approach for atherosclerosis, in which miR-125b inhibition reduces macrophage content and plaque size. - We described the molecular mechanism underlying miR-125b, which involves regulating of monocyte trafficking to plaques and the downregulation of the chemokine receptor CCR7. CCR7 plays a crucial role in facilitating the egress of macrophages and foam cells from plaques, and its downregulation contribute to progression of ATH. - The results have been validated in a cohort of patients with coronary artery disease, where CCR7 expression was reduced in plaques. What are the clinical implications?- We highlight the pivotal role of monocyte trafficking in the inflammatory mechanism of atherosclerosis. Managing miR-125b/CCR7 signaling may improve the resolution of ATH promoting the exit of foam cells from plaque. - Inhibition of miR-125b in plaque macrophages represents a novel and promising therapeutic approach for cardiovascular disease.

Atherosclerotic plaque rupture remains a leading cause of adverse cardiovascular events, yet the molecular drivers of plaque vulnerability are incompletely understood. To address this challenge, we developed an integrated approach that combines histomorphology-guided spatial proteomics with machine learning to map protein signatures across spatially distinct plaque subregions. Our analysis revealed that vulnerability signatures concentrate in the necrotic core and fibrous cap subregions, and are significantly enriched for ossification, inflammation, cholesterol metabolism, and extracellular matrix degradation pathways. When comparing the vulnerability status across subregions, we found that the necrotic core has the most distinctive vulnerability-associated proteome, with 454 proteins significantly altered between stable and vulnerable states. We identified a mechanistic link between inflammation and oxidative stress, PCSK9 upregulation, and vascular smooth muscle cell dysfunction in vulnerable plaques. This finding suggests arterial PCSK9 as a therapeutic target beyond its established role in hepatic lipid metabolism. By employing machine learning, we developed and independently validated a seven-protein tissue panel (receiver operating characteristic- area under the curve = 0.86) and found a 12-protein serum panel to predict plaque vulnerability status. Thus, plaque vulnerability signatures are spatially concentrated in specific subregions and highlight actionable biomarkers and therapeutic targets for advanced carotid artery disease.

Omics technologies enable deep profiling of human atherosclerosis, but have mostly been applied in small studies lacking integration with other data modalities. AtherOMICS is a biobanking project linking multi-omic atherosclerotic plaque phenotyping with blood biobanking, in vivo imaging, and clinical data. Since August 2022, 246 patients scheduled to undergo carotid or femoral endarterectomy at LMU Klinikum (Munich, Germany), have been enrolled (median age 73 years, 32% female). Plaques undergo systematic histological characterization of lipid core, calcification, intraplaque hemorrhage, fibrous cap, macrophages, and smooth muscle cells. Single-nuclei RNAseq (n=17) has highlighted macrophages as the dominant intraplaque cell type, whereas paired plaque and plasma affinity-based proteomics (n=88) quantified 2,841 shared proteins showing low plaque-plasma correlation (median {rho}=0.10). Cross-modality image integration (histology, ex vivo MRI, in vivo MRI, CTA) proved feasible. Taken together, AtherOMICS enables multimodal characterization of human atherosclerosis aiming for the discovery of athero-specific drug targets, molecular signatures of atheroprogression, and non-invasive biomarkers of plaque vulnerability.

BackgroundThe pathways linking lipoprotein(a) (Lp[a]) to atherosclerotic cardiovascular disease (ASCVD) are unclear. This study aimed to discover Lp(a)-associated plasma proteins and estimate their associations with incident ASCVD. MethodsWe analyzed 48,859 UK Biobank participants with measured Lp(a) and proteomic profiles, with replication in 9,416 individuals in the Atherosclerosis Risk in Communities (ARIC) study cohort utilizing a separate proteomic platform. Linear models assessed associations between Lp(a) and protein concentrations adjusted for age, sex, cigarette smoking, diabetes diagnosis, body mass index, systolic blood pressure, hypertension, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, triglycerides, estimated glomerular filtration rate, statin prescription, and the first 10 components of genetic ancestry. Multiple testing correction was performed using the Benjamini-Hochberg FDR method (P < 0.05). We examined how the protein effect sizes from the primary analysis using the outcome of Lp(a) aligned with those for the outcomes of an LPA genetic risk score (GRS) and LDL-C. Cox proportional hazards models quantified hazard ratios (HRs) for protein associations with incident ASCVD. ResultsParticipants were a mean age of 57 years (SD 8.22), 93.9% European, and 53.8% male, with median follow-up of 8.9 years (IQR 8.3-9.7). Of 1,459 circulating proteins, 164 were significantly associated with Lp(a) after FDR correction, with enrichment for lipid degradation, metabolism, and insulin secretion. In the ARIC study, 10 proteins were replicated with consistent effect estimates. Of these replicated proteins, there were no significant associations observed with an LPA GRS. Only REG4 and VWC2 showed concordant associations with LDL-C (P < 0.001), consistent with their association with Lp(a). Five proteins exhibited concordant associations with Lp(a) and incident ASCVD (ITIH3, DLL1, REG4, VWC2, CBLN4). ITIH3 was positively associated with coronary artery disease (HR 1.13, 95% CI 1.04-1.23), peripheral artery disease (HR 1.42, 95% CI 1.19-1.69), major adverse limb events (HR 1.65, 95% CI 1.14-2.40), carotid stenosis (HR 1.45, 95% CI 1.13-1.85), and ischemic stroke (HR 1.33, 95% CI 1.13-1.55). CBLN4 uniquely showed inverse associations with Lp(a) and disease: higher levels were linked to lower risk of CAD (HR 0.88, 95% CI 0.80-0.96), PAD (HR 0.78, 95% CI 0.64-0.96), and ischemic stroke (HR 0.72, 95% CI 0.60-0.85). ConclusionUsing high-throughput proteomics, we discovered and replicated 10 proteins associated with circulating Lp(a), several of which were independent of genetically-predicted Lp(a). While Lp(a) is highly heritable, these atherogenic proteins represent a non-heritable Lp(a) axis. Clinical PerspectiveO_ST_ABSWhats New?C_ST_ABSO_LITen proteins associated with circulating lipoprotein(a) levels were identified and independently replicated in an external cohort, with associations independent of a genetic risk score. C_LIO_LIFive proteins (ITIH3, CBLN4, FLL1, REG4, VWC2) were concordantly associated with lipoprotein(a) and incident atherosclerotic cardiovascular disease. C_LI Clinical ImplicationsO_LIThese proteins may represent pathways through which lipoprotein(a) drives atherosclerosis beyond traditional lipid mechanisms. C_LIO_LIFuture mechanistic studies should investigate the roles of these proteins to guide the development of targeted strategies for preventing atherosclerotic cardiovascular disease. C_LI

BackgroundElevated lipoprotein (a) [Lp(a)] is associated with a higher risk of atherosclerotic cardiovascular disease (ASCVD) events. Although Lp(a) is a genetically determined risk factor, the metabolomic and proteomic features that may mediate or be associated with this risk are unknown. MethodsIn young, healthy Coronary Artery Risk Development in Young Adults (CARDIA) participants, we defined the relationships between year 7 (Y7) Lp(a) and metabolomic (n=563)/proteomic (n=184) features, derived quantitative Lp(a)-omic scores from these features, and related these quantitative scores to ASCVD phenotypes. ResultsCARDIA participants had a mean age of 32 years at Y7 in this study and a median follow-up of 27.1 years. In the overall cohort (n=3920), Y7 Lp(a) levels were associated prospectively with ASCVD phenotypes at year 25 (Y25), including hs-CRP, coronary artery calcification (CAC), and incident CHD. In the subcohort (n=2290) that had measurements of Lp(a), proteomics, and metabolomics, Y7 Lp(a) levels were associated with distinct proteomic and metabolomic features indicative of immune responses, lipoprotein metabolism, atherogenesis, and arginine/steroid biosynthesis. Using machine learning approaches, Lp(a) metabolomic, proteomic, and transomic quantitative scores were derived. The Y7 Lp(a) transomic score was more strongly associated with Y25 incident CAC (standardized {beta} = 0.29, p=0.04), hs-CRP (standardized {beta} = 0.18, p =0.0008), and incident any CHD (standardized {beta} = 0.51, p = 0.05), than the Y7 Lp(a) concentration itself (no significant associations). ConclusionsTo our knowledge, this is the first study to identify relationships between Lp(a) and associated metabolomic/proteomic features in young, healthy adults and joint associations with ASCVD phenotypes. The multi-omics approaches employed here provide insight into the pathobiology of Lp(a)-driven ASCVD and enable more nuanced mechanistic risk assessment compared with Lp(a) concentrations alone.

BackgroundUnderstanding the pathophysiology of unstable atherosclerosis is imperative to prevent myocardial infarction and stroke. We used multi-omics integration to identify key molecular targets with diagnostic and therapeutic potential. MethodsBiobank of Karolinska Endarterectomies encompassing patients with symptomatic (S) and asymptomatic (AS) carotid atherosclerosis, was the main resource. Plaques, peripheral blood monocytes and plasma sampled locally from around plaque or periphery of n>700 individuals, were profiled by transcriptomics, proteomics and metabolomics. A supervised feature-selection method DIABLO was used for per patient data integration. Multi-omics layers were integrated separately across local and peripheral disease sites, and their intersection, with stratification for symptomatology. Identified analytes were investigated using scRNAseq, clinical and outcome data. ResultsIn peripheral circulation, FABP4, IL6, Bilirubin and Sphingomyelin were the most prominent analytes. F11, ANGPTL3, ICOSLG, ITGB1 and Sphingomyelin were enriched in the local disease site, while FABP4, C1R, IL6, Bilirubin and Sphingomyelin appeared at the intersection. Coagulation, necroptosis, inflammation and cholesterol metabolism were confirmed as key pathways determining symptomatology. Clinical analyses showed an impact of lipid-lowering therapy on ICOSLG expression, anti-hypertensives on plasma FABP4 and BLVRB levels, anti-diabetics on plasma Sphingomyelins, while no medications affected ANGPTL3. Association with future adverse events was shown for plasma Bilirubin, Sphingomyelin, ANGPTL3 and ICOSLG plaque levels. Open-source target genetic analyses suggested causal involvement of F11, C1S, EGFR, IL6, ANGPTL3 in the disease. ConclusionsUsing an innovative, deep-data framework, this study provides confirmatory and novel information on mechanisms behind atherosclerotic instability. The findings raise possibilities for translational prioritizations to aid personalized medicine. Structured Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/25327328v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@1372bdborg.highwire.dtl.DTLVardef@1203b97org.highwire.dtl.DTLVardef@1283922org.highwire.dtl.DTLVardef@1beead9_HPS_FORMAT_FIGEXP M_FIG C_FIG Key QuestionThis study performed first-of-a-kind, orthogonal, per-patient multi-omics integration from a large carotid stenosis biobank, with an aim to identify key molecular signatures and pathways of human atherosclerotic instability. Key FindingThe complex multi-omics design coupled with deep-data analyses, enabled the discovery of numerous confirmatory and novel molecular signatures implicated in patient symptomatology. Extended validation analyses elucidated their cellular sources, associations with plaque morphology, clinical biochemistry, medication and long-term patient outcomes. Take-home MessageThe findings are interesting for further investigation with respect to druggable targeting or plasma biomarkers, altogether leading to improved patient phenotyping and precision medicine potential in cardiovascular disease.

BackgroundAcute coronary syndrome (ACS) is caused by arterial thrombosis and is associated with sustained activation of coagulation. Clotting requires interactions of coagulation factors with aminophospholipids (aPL): phosphatidylserine (PS) and phosphatidylethanolamine (PE) on membrane surfaces. The aPL composition of circulating membranes in coronary disease has not been characterized. Furthermore, the contribution of external-facing aPL to elevated thrombotic risk in ACS is unknown. Methods and resultsThrombin generation was measured on platelet, leukocyte and extracellular vesicles (EV) from patients with ACS (n = 24), stable coronary artery disease (CAD, n = 18), risk factor positive (RF, n = 23) and healthy controls (HC, n = 24). The aPL composition on the surface of EV, platelets and leukocytes was determined using lipidomics. Leukocytes, platelets and EV externalized PE- and PS-containing fatty acids ranging from C16:0-20:4. These included both diacyl and plasmalogen forms, with significant increases stimulated by agonist activation. Thrombin generation on the surface of EV and leukocytes was higher in ACS than HC. Also, thrombin generation was higher for EV from CAD and RF, than HC. EV counts were higher in CAD and ACS compared with HC. Thrombin generation correlated positively with plasma EV counts and membrane surface area. ConclusionThe aPL membrane of EV and leukocytes may contribute to the activation of coagulation in CAD and ACS. Targeting EV formation/clearance and the aPL surface of EV and leukocyte membranes represents a novel anti-thrombotic target in CAD and ACS. Condensed abstractAcute coronary syndrome (ACS) is associated with sustained activation of coagulation, requiring procoagulant aminophospholipids (aPL). However, the aPL composition of circulating membranes and their contribution to thrombotic risk in ACS is undetermined. Lipidomics demonstrated that leukocytes, platelets and extracellular vesicles (EV) externalized aPL-containing fatty acids ranging from C16:0-20:4. Thrombin generation on the surface of EV and leukocytes was higher in ACS patients than healthy controls (HC). EV counts were higher in ACS compared with HC and correlated positively with thrombin generation. In summary, aPL in the outer membranes of EV and leukocytes may contribute to elevated thrombotic risk in ACS. Highlights What is new?O_LIThe aPL profile of platelets, leukocytes and EV in patients with ACS, CAD, RF and HC is defined for the first-time using lipidomics. C_LIO_LIThrombin generation on the surface of unstimulated leukocytes, is elevated in patients with ACS compared with HC. C_LIO_LIThrombin generation on the surface of EV is elevated in patients with ACS, CAD and RF compared with HC. C_LIO_LIEV counts in patients with ACS/CAD/RF were elevated compared with HC and correlate positively with thrombin generation. C_LI Clinical Perspective What are the clinical implications?O_LIThe membranes of EV and leukocytes may contribute to the activation of coagulation in ACS. C_LIO_LIThe aPL in EV and leukocyte membranes represent a novel target for reducing thrombotic risk in ACS. C_LIO_LITargeting EV formation/clearance could reduce thrombotic risk in CAD and ACS. C_LI

Background and rationaleAs an adjunct to coronary intervention, the Liquid Biopsy System (LBS, PlaqueTec, UK) enables accurate intracoronary blood sampling at discrete sites simultaneously. We investigated variation between local coronary and remote (peripheral) blood levels of a panel of atherosclerosis-associated proteins and examined how this might relate to cardiovascular risk assessment. Methods and ResultsIn a previous proof-of-concept trial, coronary blood samples were collected using the LBS in 28 patients. For 12 of these patients, sampling was conducted across the uninstrumented lesion, prior to percutaneous coronary intervention (PCI). Peripheral blood samples were also collected, at baseline and after PCI. Protein levels in coronary and peripheral plasma samples were analysed by proximity extension assay (PEA, Olink). Before PCI, in 10 out of 12 patients, coronary levels of hepatocyte growth factor (HGF), pappalysin-1 (PAPPA) and spondin-1 (SPON1) were elevated compared with peripheral levels, in some cases >10-fold. Following PCI, involving iatrogenic plaque rupture prior to stenting, peripheral levels of these proteins were elevated to a similar degree as coronary levels. In 2 patients, peripheral elevations of HGF, PAPPA and SPON1 (all >90th centile) were observed at baseline, prior to PCI. The protein pattern that was identified, consisting of high levels of a combination of HGF, PAPPA and SPON1 was absent in healthy control peripheral blood, but when investigated in baseline peripheral blood samples from reference cardiovascular and COVID-19 patient cohorts, was associated with the occurrence of major adverse cardiovascular events (MACE) and mortality. ConclusionsFrom investigation of coronary and peripheral blood samples, we identified a novel inflammatory protein signature, which when present in peripheral blood appears to portend worse outcomes. Measurement of these proteins could therefore aid identification of individuals at high risk of cardiovascular events or death. Translational PerspectiveThrough sampling of local coronary blood, we discovered a novel protein biosignature consisting of a combination of elevated levels of HGF, PAPPA and SPON1. When this biosignature was assessed in peripheral samples from reference cardiovascular and COVID-19 cohorts, it associated with the occurrence of MACE and mortality. The biosignature protein levels correlated with markers of mast cell and neutrophil activity but not with CRP, possibly indicating a specific inflammatory status. Early detection of this protein signal has potential clinical utility to identify specific patients at increased risk of poor outcomes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=129 HEIGHT=200 SRC="FIGDIR/small/23288168v2_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1f282dborg.highwire.dtl.DTLVardef@6d1bbdorg.highwire.dtl.DTLVardef@138302eorg.highwire.dtl.DTLVardef@1e350f3_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundMonocytes play a pivotal role in pathology of abdominal aortic aneurysm (AAA) and can display an immunophenotypic heterogeneity with functionally distinct subpopulations. Alterations in monocyte subsets have been described in CVD, and some are associated with cardiovascular risk, but their profile in AAA is poorly understood. AimWe aimed to comprehensively define associations of circulating monocyte phenotypes with AAA risk and AAA morphology. MethodsMonocyte subsets (CD14++CD16-; CD14++/CD16+; CD14+/CD16++) were analyzed in a prospective, observational study in patients with AAA (n=34) and varicose veins (n=34) by using flow cytometry. ResultsClassical monocytes were 1.6-fold lower (P=0.001) in AAA, while intermediate and non-classical monocytes were 1.8 (P=0.019) and 1.9-fold (P=0.025) higher in AAA, respectively. The differences remained significant after adjusting for age, sex and peripheral artery disease. A lower proportion of classical monocytes (HR: 0.73, P=0.002) and increases in intermediate (HR: 1.41, P=0.006) and non-classical monocytes (HR: 1.54, P=0.030) were associated with a higher risk of AAA. Non-classical monocytes showed an inverse correlation with AAA diameter (Pearson correlation =-0.64, P=0.001) and AAA volume (Pearson correlation =-0.50, P=0.003). ConclusionThe present study revealed age- and sex-independent shifts in monocytes, all of which were associated with risk of AAA disease. Non-classical monocytes were inversely correlated with AAA diameter and volume and thus may be surrogate markers for AAA morphology. Whats new?O_LIClassical monocytes are lower in patients with late-stage AAA. C_LIO_LINon-classical monocytes showed the strongest increase in AAA disease. C_LIO_LIA reduction in classical monocytes is associated with increased risk of AAA. C_LIO_LIAn increase in non-classical and intermediate monocytes is associated with an increased risk of AAA. C_LIO_LILowering in non-classical monocytes may be a surrogate marker for AAA morphology, particlarly AAA volume. C_LIO_LIIntermediate monocytes showed a positive correlation with the thickness of the intraluminal thrombus. C_LI What are the clinical implications?O_LIThe increase in non-classical monocytes could be a novel surrogate marker for AAA volume, which could be useful when AAA diameter is insufficient or to monitor a saccular AAA, as there is a weaker correlation between diameter and risk of rupture in this type of AAA. C_LIO_LIThe decrease in classical monocytes could be a useful surrogate marker for AAA volume and provide additional information on AAA diameter. C_LIO_LIMonocyte shifts and their association with AAA disease may be relevant for the diagnosis of AAA and should be verified in larger cohorts. C_LIO_LIThe mechanisms behind the decrease in classical monocytes and the increase in intermediate and non-classical subsets should be investigated in further in vitro studies as they offer therapeutic potential. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=142 HEIGHT=200 SRC="FIGDIR/small/24317518v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1be3fa4org.highwire.dtl.DTLVardef@1445a1eorg.highwire.dtl.DTLVardef@78706dorg.highwire.dtl.DTLVardef@11d13bb_HPS_FORMAT_FIGEXP M_FIG C_FIG

RationaleCardiovascular disease (CVD) is the leading cause of mortality for women in the USA. Current clinical biomarkers are inadequate to determine CVD risk in women, especially Black women, who disproportionately suffer from CVD. MethodsClinical data and LC-MS lipidomics from two independent study cohorts were used to identify novel circulating markers of CVD risk in White and Black women. Machine learning assessed predictive efficacy of identified lipids, and targeted oxylipid analysis provided insight into dysregulated inflammatory pathways. ResultsSelect phospholipids and triglycerides containing acyl chains in the arachidonic acid (ARA) pathway were predictive of systolic blood pressure (BP) after adjusting for biological factors including age, obesity, and glycemic status in White and Black women. Oxylipid levels indicated increased conversion of ARA through the COX and LOX enzymes to pro-inflammatory cytokines in Black women. ConclusionARA-containing phospholipid are independent predictors of CVD risk in White and Black women. Predisposition to CVD risk in Black women may further be explained by increased production of pro-inflammatory oxylipids relative to White women, regardless of blood pressure status. Future studies investigating the clinical utility of phospholipid ARA abundance as a marker of CVD risk in White and Black women are warranted.