Identification of metabolic components of carotid plaque in high-risk patients utilizing liquid chromatography-tandem mass spectrometry.

OBJECTIVE: Carotid atherosclerosis is a chronic progressive vascular disease that can be complicated by stroke in severe cases. Prompt diagnosis and treatment of high-risk patients are quite difficult due to the lack of reliable clinical biomarkers. This study aimed to explore potential plaque metabolic markers of stroke-prone risk and relevant targets for pharmacological intervention. METHOD: Carotid intima and plaque sample tissues were obtained from 20 patients with cerebrovascular symptoms of carotid origin. An untargeted metabolomics approach based on liquid chromatography-tandem mass spectrometry was utilized to characterize the metabolic profiles of the tissues. Multivariate and univariate analysis tools were used. RESULTS: A total of 154 metabolites were significantly altered in carotid plaque when compared with thickened intima. Of these, 62 metabolites were upregulated, whereas 92 metabolites were downregulated. Support vector machines identified the 15 most important metabolites, such as N-(cyclopropylmethyl)-N'-phenylurea, 9(S)-HOTrE, ACar 12:2, quinoxaline-2,3-dithiol, and l-thyroxine, as biomarkers for high-risk plaques. Metabolic pathway analysis showed that abnormal purine and nucleotide metabolism, amino acid metabolism, glutathione metabolism, and vitamin metabolism may contribute to the occurrence and progression of carotid atherosclerotic plaque. CONCLUSIONS: Our study identifies the biomarkers and related metabolic mechanisms of carotid plaque, which is stroke-prone, and provides insights and ideas for the precise prevention and targeted intervention of the disease.

Microplastics and Nanoplastics in Atheromas and Cardiovascular Events.

BACKGROUND: Microplastics and nanoplastics (MNPs) are emerging as a potential risk factor for cardiovascular disease in preclinical studies. Direct evidence that this risk extends to humans is lacking. METHODS: We conducted a prospective, multicenter, observational study involving patients who were undergoing carotid endarterectomy for asymptomatic carotid artery disease. The excised carotid plaque specimens were analyzed for the presence of MNPs with the use of pyrolysis-gas chromatography-mass spectrometry, stable isotope analysis, and electron microscopy. Inflammatory biomarkers were assessed with enzyme-linked immunosorbent assay and immunohistochemical assay. The primary end point was a composite of myocardial infarction, stroke, or death from any cause among patients who had evidence of MNPs in plaque as compared with patients with plaque that showed no evidence of MNPs. RESULTS: A total of 304 patients were enrolled in the study, and 257 completed a mean (±SD) follow-up of 33.7±6.9 months. Polyethylene was detected in carotid artery plaque of 150 patients (58.4%), with a mean level of 21.7±24.5 µg per milligram of plaque; 31 patients (12.1%) also had measurable amounts of polyvinyl chloride, with a mean level of 5.2±2.4 µg per milligram of plaque. Electron microscopy revealed visible, jagged-edged foreign particles among plaque macrophages and scattered in the external debris. Radiographic examination showed that some of these particles included chlorine. Patients in whom MNPs were detected within the atheroma were at higher risk for a primary end-point event than those in whom these substances were not detected (hazard ratio, 4.53; 95% confidence interval, 2.00 to 10.27; P<0.001). CONCLUSIONS: In this study, patients with carotid artery plaque in which MNPs were detected had a higher risk of a composite of myocardial infarction, stroke, or death from any cause at 34 months of follow-up than those in whom MNPs were not detected. (Funded by Programmi di Ricerca Scientifica di Rilevante Interesse Nazionale and others; ClinicalTrials.gov number, NCT05900947.).

Identifying lipid traces of atherogenic mechanisms in human carotid plaque.

BACKGROUND AND AIMS: Lipids play an important role in atherosclerotic plaque development and are interesting candidate predictive biomarkers. However, the link between circulating lipids, accumulating lipids in the vessel wall, and plaque destabilization processes in humans remains largely unknown. This study aims to provide new insights into the role of lipids in atherosclerosis using lipidomics and mass spectrometry imaging to investigate lipid signatures in advanced human carotid plaque and plasma samples. METHODS: We used lipidomics and desorption electrospray ionization mass spectrometry imaging (DESI-MSI) to investigate lipid signatures of advanced human carotid plaque and plasma obtained from patients who underwent carotid endarterectomy (n = 14 out of 17 whose plaque samples were analyzed by DESI-MSI). Multivariate data analysis and unsupervised clustering were applied to identify lipids that were the most discriminative species between different patterns in plaque and plasma. These patterns were interpreted by quantitative comparison with conventional histology. RESULTS: Lipidomics detected more than 300 lipid species in plasma and plaque, with markedly different relative abundances. DESI-MSI visualized the spatial distribution of 611 lipid-related m/z features in plaques, of which 330 m/z features could be assigned based on exact mass, comparison to the lipidomic data, and high mass resolution MSI. Matching spatial lipid patterns to histological areas of interest revealed several molecular species that were colocalized with pertinent disease processes in plaque including specific sphingomyelin and ceramide species with calcification, phospholipids and free fatty acids with inflammation, and triacylglycerols and phosphatidylinositols with fibrin-rich areas. CONCLUSIONS: By comparing lipid species in plaque and plasma, we identified those circulating species that were also prominently present in plaque. Quantitative comparison of lipid spectral patterns with histology revealed the presence of specific lipid species in destabilized plaque areas, corroborating previous in vitro and animal studies.

Spatially Resolved Metabolites in Stable and Unstable Human Atherosclerotic Plaques Identified by Mass Spectrometry Imaging.

BACKGROUND: Impairments in carbohydrate, lipid, and amino acid metabolism drive features of plaque instability. However, where these impairments occur within the atheroma remains largely unknown. Therefore, we sought to characterize the spatial distribution of metabolites within stable and unstable atherosclerosis in both the fibrous cap and necrotic core. METHODS: Atherosclerotic tissue specimens from 9 unmatched individuals were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and Human Metabolome Database, we confidently annotated 170 of these metabolites and found over 60 of these were different between stable and unstable atheromas. We then integrated these results with an RNA-sequencing data set comparing stable and unstable human atherosclerosis. RESULTS: Upon integrating our mass spectrometry imaging results with the RNA-sequencing data set, we discovered that pathways related to lipid metabolism and long-chain fatty acids were enriched in stable plaques, whereas reactive oxygen species, aromatic amino acid, and tryptophan metabolism were increased in unstable plaques. In addition, acylcarnitines and acylglycines were increased in stable plaques whereas tryptophan metabolites were enriched in unstable plaques. Evaluating spatial differences in stable plaques revealed lactic acid in the necrotic core, whereas pyruvic acid was elevated in the fibrous cap. In unstable plaques, 5-hydroxyindoleacetic acid was enriched in the fibrous cap. CONCLUSIONS: Our work here represents the first step to defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this will be a valuable resource and open new avenues of research in cardiovascular disease.

Carotid contrast-enhanced ultrasonography combined with sirtuin-3 in the diagnosis of plaques in carotid atherosclerosis.

BACKGROUND: Carotid atherosclerosis (CAS) is one of the main causes of ischemic stroke. Currently, the clinical evidence for contrast-enhanced ultrasonography (CEUS) as a method for diagnosing CAS is still inadequate. Sirtuin-3 (SIRT3) is associated with the inflammation response; however, few studies have evaluated SIRT3 in CAS. OBJECTIVES: To investigate the role of SIRT3 in CAS patients and its diagnostic value for unstable plaques when combined with CEUS. MATERIAL AND METHODS: This is a prospective observational study including 517 CAS patients who were admitted to our hospital from January 2015 to December 2020. All patients received a normal Doppler ultrasound, CEUS and magnetic resonance imaging (MRI). The latter was used as the gold standard in evaluating plaque conditions. Serum SIRT3 levels were measured using an enzyme-linked immunosorbent assay (ELISA). Serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-ch), low-density lipoprotein cholesterol (LDL-ch), C-reactive protein (CRP), and interleukin (IL)-6 levels were measured and recorded. RESULTS: Patients with severe CAS showed significantly higher levels of CRP, IL-6, TC, and LDL-ch, a higher frequency of unstable plaques, as well as a lower level of HDL-ch. In patients with severe CAS and CAS patients with stable plaques, the levels of SIRT3 were markedly lower. Patients with a high expression of SIRT3 showed significantly lower levels of CRP, IL-6, TC and LDL-ch, and higher levels of HDL-ch, as well as a lower frequency of unstable plaques. Receiver operating characteristic (ROC) curves showed that the combination of CEUS and SIRT3 could achieve high sensitivity and specificity in the diagnosis of unstable plaques. High levels of C-reactive protein, IL-6, TC, TG and LDL-ch, as well as low levels of SIRT3 and HDL-ch, and current smoking were risk factors of unstable plaques in CAS patients. CONCLUSIONS: A low expression of SIRT3 predicted a higher risk for unstable plaques in CAS patients. The combination of CEUS and SIRT3 is a potential strategy for diagnosing unstable plaques.

The Initial Human Atherosclerotic Lesion and Lipoprotein Modification-A Deep Connection.

Atherosclerosis research typically focuses on the evolution of intermediate or advanced atherosclerotic lesions rather than on prelesional stages of atherogenesis. Yet these early events may provide decisive leads on the triggers of the pathologic process, before lesions become clinically overt. Thereby, it is mandatory to consider extracellular lipoprotein deposition at this stage as the prerequisite of foam cell formation leading to a remarkable accumulation of LDL (Low Density Lipoproteins). As progression of atherosclerosis displays the characteristic features of a chronic inflammatory process on the one hand and native LDL lacks inflammatory properties on the other hand, the lipoprotein must undergo biochemical modification to become atherogenic. During the last 25 years, evidence was accumulated in support of a different concept on atherogenesis proposing that modification of native LDL occurs through the action of ubiquitous hydrolytic enzymes (enzymatically modified LDL or eLDL) rather than oxidation and contending that the physiological events leading to macrophage uptake and reverse transport of eLDL first occur without inflammation (initiation with reversion). Preventing or reversing initial atherosclerotic lesions would avoid the later stages and therefore prevent clinical manifestations. This concept is in accordance with the response to retention hypothesis directly supporting the strategy of lowering plasma levels of atherogenic lipoproteins as the most successful therapy for atherosclerosis and its sequelae. Apart from but unquestionable closely related to this concept, there are several other hypotheses on atherosclerotic lesion initiation favoring an initiating role of the immune system ('vascular-associated lymphoid tissue' (VALT)), defining foam cell formation as a variant of lysosomal storage disease, relating to the concept of the inflammasome with crystalline cholesterol and/or mitochondrial DAMPs (damage-associated molecular patterns) being mandatory in driving arterial inflammation and, last but not least, pointing to miRNAs (micro RNAs) as pivotal players. However, direct anti-inflammatory therapies may prove successful as adjuvant components but will likely never be used in the absence of strategies to lower plasma levels of atherogenic lipoproteins, the key point of the perception that atherosclerosis is not simply an inevitable result of senescence. In particular, given the importance of chemical modifications for lipoprotein atherogenicity, regulation of the enzymes involved might be a tempting target for pharmacological research.

The potential role of T2*-weighted multi-echo data image combination as an imaging marker for intraplaque hemorrhage in carotid plaque imaging.

BACKGROUND: Carotid atherosclerotic plaques with intraplaque hemorrhage (IPH) are associated with elevated stroke risk. IPH is predominantly imaged based on paramagnetic properties of the upstream hemoglobin degradation product methemoglobin. This is an explorative observational study to test the feasibility of a spoiled gradient echo based T2* weighted MRI sequence (3D MEDIC) for carotid plaque imaging, and to compare signs suggestive of the downstream degradation product hemosiderin on 3D MEDIC with signs of methemoglobin on a T1wBB sequence. METHODS: Patients with recent TIA or stroke were selected based on the presence on non-calcified plaque components on CTA to promote an enriched prevalence of IPH in the material. Patients (n = 42) underwent 3T MRI with 3D MEDIC and 2D turbo spin echo T1w black blood (T1wBB). Images were independently evaluated by two neuroradiologists and Cohens Kappa was used for inter-reader agreement for each sequence. RESULTS: The technical feasibility for 3D MEDIC, was 34/42 patients (81%). Non-calcified plaque components with susceptibility effect without simultaneous T1-shortening-a combination suggestive of hemosiderin, was seen in 13/34 of the plaques. An equally large group display elevated T1w signal in combination with signal loss on 3D MEDIC, a combination suggestive of both hemosiderin and methemoglobin. Cohen's kappa for inter-reader agreement was 0.64 (CI 0.345-0.925) for 3D MEDIC and 0.94 (CI 0.81-1.00) for T1wBB. CONCLUSIONS: 3D MEDIC shows signal loss, without elevated T1w signal on T1wBB, in non-calcified tissue in many plaques in this group of patients. If further studies, including histological verification, confirm that the 3D MEDIC susceptibility effect is indeed caused by hemosiderin, 3D MEDIC could aid in the detection of IPH, beyond elevation of T1w signal.

Identification of vulnerable plaques and patients by intracoronary near-infrared spectroscopy and ultrasound (PROSPECT II): a prospective natural history study.

BACKGROUND: Near-infrared spectroscopy (NIRS) and intravascular ultrasound are promising imaging modalities to identify non-obstructive plaques likely to cause coronary-related events. We aimed to assess whether combined NIRS and intravascular ultrasound can identify high-risk plaques and patients that are at risk for future major adverse cardiac events (MACEs). METHODS: PROSPECT II is an investigator-sponsored, multicentre, prospective natural history study done at 14 university hospitals and two community hospitals in Denmark, Norway, and Sweden. We recruited patients of any age with recent (within past 4 weeks) myocardial infarction. After treatment of all flow-limiting coronary lesions, three-vessel imaging was done with a combined NIRS and intravascular ultrasound catheter. Untreated lesions (also known as non-culprit lesions) were identified by intravascular ultrasound and their lipid content was assessed by NIRS. The primary outcome was the covariate-adjusted rate of MACEs (the composite of cardiac death, myocardial infarction, unstable angina, or progressive angina) arising from untreated non-culprit lesions during follow-up. The relations between plaques with high lipid content, large plaque burden, and small lumen areas and patient-level and lesion-level events were determined. This trial is registered with ClinicalTrials.gov, NCT02171065. FINDINGS: Between June 10, 2014, and Dec 20, 2017, 3629 non-culprit lesions were characterised in 898 patients (153 [17%] women, 745 [83%] men; median age 63 [IQR 55-70] years). Median follow-up was 3·7 (IQR 3·0-4·4) years. Adverse events within 4 years occurred in 112 (13·2%, 95% CI 11·0-15·6) of 898 patients, with 66 (8·0%, 95% CI 6·2-10·0) arising from 78 untreated non-culprit lesions (mean baseline angiographic diameter stenosis 46·9% [SD 15·9]). Highly lipidic lesions (851 [24%] of 3500 lesions, present in 520 [59%] of 884 patients) were an independent predictor of patient-level non-culprit lesion-related MACEs (adjusted odds ratio 2·27, 95% CI 1·25-4·13) and non-culprit lesion-specific MACEs (7·83, 4·12-14·89). Large plaque burden (787 [22%] of 3629 lesions, present in 530 [59%] of 898 patients) was also an independent predictor of non-culprit lesion-related MACEs. Lesions with both large plaque burden by intravascular ultrasound and large lipid-rich cores by NIRS had a 4-year non-culprit lesion-related MACE rate of 7·0% (95% CI 4·0-10·0). Patients in whom one or more such lesions were identified had a 4-year non-culprit lesion-related MACE rate of 13·2% (95% CI 9·4-17·6). INTERPRETATION: Combined NIRS and intravascular ultrasound detects angiographically non-obstructive lesions with a high lipid content and large plaque burden that are at increased risk for future adverse cardiac outcomes. FUNDING: Abbott Vascular, Infraredx, and The Medicines Company.

Atheroma-Specific Lipids in ldlr-/- and apoe-/- Mice Using 2D and 3D Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

Atherosclerosis is the major contributor to cardiovascular diseases. It is a spatially and temporally complex inflammatory disease, in which intravascular accumulation of a plethora of lipids is considered to play a crucial role. To date, both the composition and local distribution of the involved lipids have not been thoroughly mapped yet. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) enables analyzing and visualizing hundreds of lipid molecules within the plaque while preserving each lipid's specific location. In this study, we aim to identify and verify aortic plaque-specific lipids with high-spatial-resolution 2D and 3D MALDI-MSI common to high-fat-diet-fed low-density lipoprotein receptor deficient (ldlr-/-) mice and chow-fed apolipoprotein E deficient (apoe-/-) mice, the two most widely used animal models for atherosclerosis. A total of 11 lipids were found to be significantly and specifically colocalized to the plaques in both mouse models. These were identified and belong to one sphingomyelin (SM), three lysophosphatidic acids (LPA), four lysophosphatidylcholines (LPC), two lysophosphatidylethanolamines (LPE), and one lysophosphatidylinositol (LPI). While these lysolipids and SM 34:0;2 were characteristic of the atherosclerotic aorta plaque itself, LPI 18:0 was mainly localized in the necrotic core of the plaque.

Viewing atherosclerosis through a crystal lens: How the evolving structure of cholesterol crystals in atherosclerotic plaque alters its stability.

Reducing the residual risk of cardiovascular (CV) events in patients with atherosclerosis continues to be a challenge. Thus, understanding how cholesterol spontaneously self assembles into metastable structures that evolve into flat plate cholesterol crystals (CCs) in atherosclerotic plaque, and why they fundamentally change the nature of the disease provides a paradigm for the development of additional therapies. Specifically, flat plate CCs that form within lysosomes of macrophages may become large enough to disrupt lysosomal membranes leading to the release of cathepsin B and CCs fragments directly into the cytosol. In the cytosol, the surface of flat plate CCs can be recognized by complosome that together with cathepsin B may trigger pyrin domain-containing inflammasome. In addition, flat plate CCs in the cytosol may trigger caspase 8 initiating apoptosis. In the interstitial space, the surface of flat plate CCs can be recognized by complement and receptors on proinflammatory cells, and larger fragments can induce "frustrated phagocytosis" that together perpetuate inflammatory injury. In addition, rapid transition of metastable CCs into large flat plate CCs within lipid rich plaques can lead to traumatic injury by expansion of the plaque's necrotic core causing plaque disruption or rupture that may precipitate further inflammation. Other crystalloids in plaque including monosodium urate and calcium phosphate crystals can augment these processes. Thus, therapies that further limit the deposition of cholesterol in the vascular bed, slow the formation of flat plate CCs and inhibit crystal-induced inflammation may lead to further reduce CV risk in patients with established CV disease.

Expression of ectopic trypsin in atherosclerotic plaques and the effects of aprotinin on plaque stability.

BACKGROUND: Our previous research revealed that trypsin is abundantly expressed in atherosclerotic plaques and its distribution overlaps with that of matrix metalloproteinase-9 (MMP-9). This study was performed to explore the possible roles of trypsin in vulnerable atherosclerotic plaque formation. METHODS AND RESULTS: Twenty-four rabbits were randomly assigned to a normal (control) group, an atherosclerosis (experimental) group and a trypsin inhibitor (aprotinin) group. In the 13th feeding week, the aprotinin group was treated with 5 mg/kg/day aprotinin via ear vein for 4 weeks. At the end of the 16th week, coronary arterial and aortic expression of trypsin, proteinase-activated receptor-2 (PAR-2), activated MMP-9, and pro-inflammatory cytokines were significantly greater in the experimental group than in the control group. Aprotinin decreased trypsin expression and activation in plaques, blocked PAR-2 and MMP-9 activation, and decreased cytokine expression; it also increased fibrous cap thickness, decreased the intima-media thickness and intimal/medial ratio, thus significantly ameliorating plaque vulnerability. Upregulated trypsin, MMP-9 and PAR-2 were also found in coronary intimal atherosclerotic plaques of patients undergoing coronary artery bypass grafting. CONCLUSIONS: Ectopic trypsin was significantly upregulated in atherosclerotic plaques, which increased pro-inflammatory cytokine levels by activating PAR-2 and promoted plaque instability by activating proMMP-9, thereby promoting atherosclerosis and plaque vulnerability. In addition, the high trypsin expression in human coronary intimal atherosclerotic plaques suggests that targeting trypsin may be a new strategy for acute coronary syndrome prevention.

sLRP1 (Soluble Low-Density Lipoprotein Receptor-Related Protein 1): A Novel Biomarker for P2Y12 (P2Y Purinoceptor 12) Receptor Expression in Atherosclerotic Plaques.

OBJECTIVE: Recent studies suggest that the P2Y12 (P2Y purinoceptor 12) receptor of vascular smooth muscle cells in atherosclerotic plaques aggravates atherosclerosis, and P2Y12 receptor inhibitors such as CDL (clopidogrel) may effectively treat atherosclerosis. It is imperative to identify an effective biomarker for reflecting the P2Y12 receptor expression on vascular smooth muscle cells in plaques. Approach and Results: We found that there was a positive correlation between the level of circulating sLRP1 (soluble low-density lipoprotein receptor-related protein 1) and the number of LRP1+ α-SMA+ (α-smooth muscle actin), P2Y12+, or P2Y12+ LRP1+ cells in plaques from apoE-/- mice fed a high-fat diet. Furthermore, activation of the P2Y12 receptor increased the expression and shedding of LRP1 in vascular smooth muscle cells by inhibiting cAMP (3'-5'-cyclic adenosine monophosphate)/PKA (protein kinase A)/SREBP-2 (sterol regulatory element binding transcription factor 2). Conversely, genetic knockdown or pharmacological inhibition of the P2Y12 receptor had the opposite effects. Additionally, CDL decreased the number of lesional LRP1+ α-SMA+ cells and the levels of circulating sLRP1 by activating cAMP/PKA/SREBP-2 in apoE-/- mice fed a high-fat diet. CONCLUSIONS: Our study suggests that sLRP1 may be a biomarker that reflects the P2Y12 receptor level in plaques and has the potential to be an indicator for administering P2Y12 receptor inhibitors for patients with atherosclerosis.

Attenuation Coefficient Parameter Computations for Tissue Composition Assessment of Carotid Atherosclerotic Plaque in Vivo.

Quantitative ultrasound has been used to assess carotid plaque tissue composition. Here, we compute the attenuation coefficient (AC) in vivo with the optimum power spectral shift estimator (OPSSE) and reference phantom method (RPM), extract AC parameters and form parametric maps. Differences between OPSSE and RPM AC parameters are computed. Relationships between AC parameters, surgical scores and histopathology assessments are examined. Kendall's τ correlations between OPSSE AC and surgical scores are significant, including those between cholesterol and Standard Deviation (adjusted p = 0.038); thrombus and Minimum (adjusted p = 0.002), Maximum (adjusted p = 0.021) and Standard Deviation (adjusted p = 0.001); ulceration and Average (adjusted p = 0.033), Median (unadjusted p = 0.013), Maximum (unadjusted p = 0.039) and Mode (adjusted p = 0.009). The strongest correlations with histopathology are percentage cholesterol and Median OPSSE (unadjusted p = 0.007); percentage hemorrhage and Minimum OPSSE (adjusted p < 0.001); hemosiderin score and Median OPSSE (adjusted p = 0.010); and percentage calcium and Percentage Non-physical RPM Pixels (unadjusted p = 0.014). Kruskal-Wallis H and Dunn's post hoc tests have the ability to distinguish between groups (p < 0.05). Results suggest AC parameters may assist in vivo evaluation of carotid plaque vulnerability.

Legumain is upregulated in acute cardiovascular events and associated with improved outcome - potentially related to anti-inflammatory effects on macrophages.

BACKGROUND AND AIMS: We have previously found increased levels of the cysteine protease legumain in plasma and plaques from patients with carotid atherosclerosis. This study further investigated legumain during acute cardiovascular events. METHODS: Circulating levels of legumain from patients and legumain released from platelets were assessed by enzyme-linked-immunosorbent assay. Quantitative PCR and immunoblotting were used to study expression, while localization was visualized by immunohistochemistry. RESULTS: In the SUMMIT Malmö cohort (n = 339 with or without type 2 diabetes and/or cardiovascular disease [CVD], and 64 healthy controls), the levels of circulating legumain were associated with the presence of CVD in non-diabetics, with no relation to outcome. In symptomatic carotid plaques and in samples from both coronary and intracerebral thrombi obtained during acute cardiovascular events, legumain was co-localized with macrophages in the same regions as platelets. In vitro, legumain was shown to be present in and released from platelets upon activation. In addition, THP-1 macrophages exposed to releasate from activated platelets showed increased legumain expression. Interestingly, primary peripheral blood mononuclear cells stimulated with recombinant legumain promoted anti-inflammatory responses. Finally, in a STEMI population (POSTEMI; n = 272), patients had significantly higher circulating legumain before and immediately after percutaneous coronary intervention compared with healthy controls (n = 67), and high levels were associated with improved outcome. CONCLUSIONS: Our data demonstrate for the first time that legumain is upregulated during acute cardiovascular events and is associated with improved outcome.