Stable Atherosclerotic Plaques Research Articles

Identification of Critical Genes Differentiating Stable and Unstable Atherosclerotic Plaques: A Bioinformatic and Computational Analysis.

Identification of biomarkers to distinguish between stable and unstable plaque formation would be very useful to predict plaque vulnerability. We downloaded microarray profiles of gene set enrichment (GSE) accession numbers including GSE71226 and GSE20680 (group A: containing healthy vs stable plaque samples) and GSE62646 and GSE34822 (group B: containing stable vs unstable plaque samples) from Gene expression omnibus (GEO) database. Differentially expressed genes were compared in both data sets of each group. Ten and 12 key genes were screened in groups A and B, respectively. Gene Ontology (GO) enrichment was applied by the plugin "BiNGO" (Biological networks gene ontology tool) of the Cytoscape. The key genes were mostly enriched in the biological process of positive regulation of the cellular process. The protein-protein interaction and co-expression network were analyzed by the STRING (search tool for the retrieval of interacting genes/proteins) and GeneMANIA (gene multiple association network integration algorithm) plugin of Cytoscape, respectively, which showed that Epidermal growth factor (EGF), Heparin-binding EGF like growth factor (HBEGF), and Matrix metalloproteinase 9 (MMP9) were at the core of the network. Further validation of key genes using two datasets showed that Phosphodiesterase 5A (PDE5A) and Protein S (PROS1) were decreased in unstable plaques, while Suppressor of cytokine signaling (SOCS3), HBEGF, and Leukocyte immunoglobulin-like receptor B4 (LILRB4) were increased. The present study used several datasets to identify key genes associated with stable and unstable atherosclerotic plaque.

Characterization of the proteome of stable and unstable carotid atherosclerotic plaques using data-independent acquisition mass spectrometry

BackgroundCurrently, noninvasive imaging techniques and circulating biomarkers are still insufficient to accurately assess carotid plaque stability, and an in-depth understanding of the molecular mechanisms that contribute to plaque instability is still lacking.MethodsWe established a clinical study cohort containing 182 patients with carotid artery stenosis. After screening, 39 stable and 49 unstable plaques were included in the discovery group, and quantitative proteomics analysis based on data independent acquisition was performed for these plaque samples. Additionally, 35 plaques were included in the validation group to validate the proteomics results by immunohistochemistry analysis.ResultsA total of 397 differentially expressed proteins were identified in stable and unstable plaques. These proteins are primarily involved in ferroptosis and lipid metabolism-related functions and pathways. Plaque validation results showed that ferroptosis- and lipid metabolism-related proteins had different expression trends in stable plaques versus unstable fibrous cap regions and lipid core regions. Ferroptosis- and lipid metabolism-related mechanisms in plaque stability were discussed.ConclusionsOur results may provide a valuable strategy for revealing the mechanisms affecting plaque stability and will facilitate the discovery of specific biomarkers to broaden the therapeutic scope.

EXPRESSION OF GENES OF BIOMOLECULES ASSOCIATED WITH THE ETIOPATHOGENESIS OF ATHEROSCLEROSIS IN ATHEROSCLEROTIC PLAQUES OF CORONARY ARTERIES

HighlightsThe study showed differences in the expression of a number of genes in atherosclerotic plaques of different types in patients with coronary atherosclerosis. The obtained data can become the basis for the development of test systems in order to determine the dynamics of the atherosclerotic process and detect signs of destabilization of the atherosclerotic plaque as early as possible. Aim. To study the differential expression of genes encoding molecules associated with the etiopathogenesis of atherosclerosis by the method of genome-wide RNA sequencing in stable atherosclerotic plaque of fibrous type and unstable atherosclerotic plaque of dystopic-necrotic type.Methods. The study was performed on samples of atherosclerotic plaques of patients with coronary atherosclerosis without acute coronary syndrome with stable angina pectoris of functional class II–IV (FC) at the age of 45-65 years. Tissue sampling of atherosclerotic plaques was performed intraoperatively in the presence of indications. Genome-wide RNA sequencing was performed using Illumina’s TruSeq RNA Sample Preparation Kit (Illumina, USA).Results. An increase in the level of gene expression in stable atherosclerotic plaques was noted for A2M, ADAMTS13, CSF3, CX3CL1, CXCL1, FGF2, GDF15, ICAM1, IL1A, IL1B, IL6, IL10, PDGFA, PTX3. There was an eightfold statistically significant increase in the level of CFD, CXCL16, FABP4, FLT3, IFNG, IL7, IL15, SELL, TGFA, THBD, TNNT1, VCAM1 and VEGFA gene expression (p<0,001) in unstable atherosclerotic plaques of dystrophic-necrotic type.Conclusion. The study showed differences in the expression of a number of genes in atherosclerotic plaques of different types in patients with coronary atherosclerosis. The obtained data can become the basis for the development of test systems in order to determine the dynamics of the atherosclerotic process and detect signs of destabilization of the atherosclerotic plaque as early as possible.

The Effect of PCSK9 Inhibition on the Stabilization of Atherosclerotic Plaque Determined by Biochemical and Diagnostic Imaging Methods.

Atherosclerosis is a multifactorial, progressive, chronic inflammatory disease. Ultrasound and magnetic resonance imaging are the most accurate predictors of atherosclerotic plaque instability (MRI). Cytokines such as osteopontin, osteoprotegerin, and metalloproteinase 9 could be used as the most recent markers to identify and track the efficacy of anti-atherosclerotic therapy. Patients with USG and MRI-verified unstable atherosclerotic plaque were included in the study. Biomarker concentrations were measured and compared before and after PCSK9 inhibitor therapy. Additionally, concentrations prior to treatment were correlated with MRI images of the carotid artery. After treatment with alirocumab, the concentrations of MMP-9 (p < 0.01) and OPN, OPG (p < 0.05) decreased significantly. Furthermore, the results of OPN, OPG, and MMP 9 varied significantly depending on the type of atherosclerotic plaque in the MRI assay. In stable atherosclerotic plaques, the concentrations of OPN and OPG were greater (p < 0.01), whereas the concentration of MMP9 correlated with the instability of the plaque (p < 0.05). We demonstrated, probably for the first time, that alirocumab therapy significantly decreased the serum concentration of atherosclerotic plaque markers. In addition, we demonstrated the relationship between the type of atherosclerotic plaque as determined by carotid MRI and the concentration of these markers.

Combining idebenone and rosuvastatin prevents atherosclerosis by suppressing oxidative stress and NLRP3 inflammasome activation

Atherosclerosis is a progressive inflammatory disease activated by excessive oxidized low-density lipoprotein (ox-LDL). Statins are the first-line choice to reduce the risk of cardiovascular disease. However, statin-associated side effects prompt dose reduction or discontinuation. Idebenone could protect against atherosclerosis by scavenging reactive oxygen species (ROS). Although both idebenone and statins have certain efficacy, neither of them can achieve a completely satisfactory effect. Here, we aim to investigate the anti-atherosclerotic effect of the combination of idebenone and statins. Apolipoprotein E knockout (ApoE−/−) mice were given idebenone (400 mg/kg/d), rosuvastatin (10 mg/kg/d) or a combination of idebenone and rosuvastatin. Histological and immunohistochemical staining were used to analyze the size and composition of the plaque. In vivo and in vitro experiments were conducted to explore the possible mechanism. Idebenone and rosuvastatin both reduced plaque burden and increased the stability of atherosclerotic plaques in the ApoE−/− mice. Mice receiving the combination therapy had even reduced and more stable atherosclerotic plaques than mice treated with idebenone or rosuvastatin alone. NLRP3 and IL-1β were further downregulated in mice receiving combination therapy compared with mice treated with monotherapy. The combination treatment also markedly reduced oxidative stress and cell apoptosis in vivo and in vitro. In conclusion, our data demonstrate that the combination of idebenone and rosuvastatin works synergistically to inhibit atherosclerosis, and that the use of both substances together is more effective than using either substance alone. From a therapeutic point, combining idebenone and rosuvastatin appears to be a promising strategy to further prevent atherosclerosis.

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

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. 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. 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. 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.

Abstract 147: Spatially Resolved Metabolites In Stable And Unstable Human Atherosclerotic Plaques Identified By Mass Spectrometry Imaging

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. Atherosclerotic tissue specimens were scored based on the Stary classification scale and subdivided into stable and unstable atheromas. After performing mass spectrometry imaging (MSI) on these samples, we identified over 850 metabolite-related peaks. Using MetaboScape, METASPACE, and HMDB, 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 dataset comparing stable and unstable human atherosclerosis. Upon integrating our MSI results with the RNA-seq dataset, 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. 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-hydroxyindole-acetic acid was enriched in the fibrous cap. Our work herein 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.

Potential circulating biomarkers of atherosclerotic plaque vulnerability in patients in the early recovery period of atherothrombotic stroke

Despite the informative value of ultrasound examination of atherosclerotic vascular lesions, predicting­ the plaque vulnerabili­ty remains difficult. Circulating blood biomarkers could provide additional criteria that would allow better determination of the risk of recurrent stroke. The aim of our study was to estimate the level of C-reactive protein (CRP), low-density lipoproteins (LDL) and lipoprotein-associated phospholipase A2 (Lp-PLA2) in the blood of patients in the early recovery period of atherothrombotic stroke depending on the density of atherosclerotic plaque according to duplex scanning of cerebral vessels. Clinical and laboratory analysis of 69 men and 61 women aged (60.42 ± 7.40) years in the early recovery period of atherothrombotic stroke was conducted. Depending on the structure of the atherosclerotic plaque the examinees were divided into two groups with stable (n = 80) and unstable (n = 50) atherosclerotic layers. The blood lipid spectrum was examined on a biochemical analyzer Screen master, the level of CRP was determined with a diagnostic kit and that of Lp-PLA2 by ELISA. Significantly higher levels of LDL, CRP and Lp-PLA2 were observed in patients with unstable atherosclerotic plaque compared to patients with stable atherosclerotic plaque in the early recovery period of atherothrombotic stroke. The increased level of LDL, CRP, and Lp-PLA2 in patients with cerebral atherosclerosis can be considered as an indicator of the vulnerability of atherosclerotic plaques, prone to rupture, and as a prognostic marker of repeated acute ischemic events. Keywords: atherosclerotic plaque, C-reactive protein, lipoprotein-associated phospholipase A2, low density lipoproteins, recovery period of ischemic stroke

RNA Sequencing Reveals the Differentially Expressed circRNAs between Stable and Unstable Carotid Atherosclerotic Plaques.

This study aimed to identify circular RNA profiles (circRNAs) via high-throughput RNA sequencing and distinguish the differentially expressed (DE) circRNAs between stable and unstable plaques. RNA sequencing was performed on unstable and stable carotid plaque samples obtained from patients with carotid artery stenosis. DE circRNAs were screened, and six DE circRNAs were verified using quantitative real-time PCR (qRT-PCR). Functional evaluation of the DE circRNAs was conducted via Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. We screened 344 DE circRNAs in unstable plaques, consisting of 342 upregulated and 2 downregulated circRNAs. GO analysis showed that the host genes of the upregulated circRNAs were related to ER to Golgi transport vesicle membrane, endocytic vesicle membrane, and Ran GTPase binding. KEGG analysis revealed that the host genes of the upregulated circRNAs were primarily associated with protein processing in endoplasmic reticulum, lysine degradation, homologous recombination, epithelial cell signaling in Helicobacter pylori infection, and yersinia infection. The results of qRT-PCR verified three upregulated DE circRNAs and two downregulated DE circRNAs in unstable plaques. Hsa-circ-0001523, hsa-circ-0008950, hsa-circ-0000571, hsa-circ-0001946, and hsa-circ-0000745 may be involved in regulating the stability of atherosclerotic plaques and serves as a therapeutic target for unstable plaques.

Single-cell atlas reveals different immune environments between stable and vulnerable atherosclerotic plaques.

Regardless of the degree of stenosis, vulnerable plaque is an important cause of ischemic stroke and thrombotic complications. The changes of the immune microenvironment within plaques seem to be an important factor affecting the characteristics of the plaque. However, the differences of immune microenvironment between stable and vulnerable plaques were remained unknown. In this study, RNA-sequencing was performed on superficial temporal arteries from 5 traumatic patients and plaques from 3 atherosclerotic patients to preliminary identify the key immune response processes in plaques. Mass cytometry (CyTOF) technology was used to explore differences in immune composition between 9 vulnerable plaques and 12 stable plaques. Finally, immunofluorescence technique was used to validate our findings in the previous analysis. Our results showed that more CD86+CD68+ M1 pro-inflammatory macrophages were found in vulnerable plaques, while CD4+T memory cells were mainly found in stable plaques. In addition, a CD11c+ subset of CD4+T cells with higher IFN-r secretion was found within the vulnerable plaque. In two subsets of B cells, CD19+CD20-B cells in vulnerable plaques secreted more TNF-a and IL-6, while CD19-CD20+B cells expressed more PD-1 molecules. In conclusion, our study suggested that M1-like macrophages are the major cell subset affecting plaque stability, while functional B cells may also contribute to plaque stability.

Adipokines and adipocytokines in men with coronary atherosclerosis and overweight

Aim To study concentrations of adipokines and their associations with proinflammatory cytokines in overweight men with coronary atherosclerosis.Material and methods This study included 79 men aged 45-60 years with atherosclerosis who had undergone coronary endarterectomy during a coronary bypass surgery, and were overweight (body weight index (BWI), 25.0-29.9 kg /m2). Based on a histological analysis of plaques, the patients were divided into two subgroups: 43 men with stable atherosclerotic plaques and 36 men with unstable plaques in coronary arteries. The control group consisted of 40 age- and BWI-matched men without clinical manifestations of IHD. Blood concentrations of adipokines, including adiponectin, adipsin, lipocalin-2, resistin, and plasminogen 1 activator inhibitor were measured by a multiplex analysis with a MILLIPLEX MAP Human Adipokine Panel 1. Concentrations of proinflammatory cytokines, including tumor necrosis factor α (TNF- α), interleukin (IL)-1β, IL-6, and C-reactive protein (CRP) were measured by enzyme immunoassay.Results The blood concentration of lipocalin -2 was higher in patients with coronary atherosclerosis and stable or unstable atherosclerotic plaques than in the control group (p&lt;0.01). Both subgroups of men with coronary atherosclerosis were characterized by significant differences from the control group in concentrations of TNF-α (p&lt;0.05), CRP, and IL-6 (p&lt;0.01). The most significant direct correlations were found between adipokines and TNF-α, IL-6, and CRP (p&lt;0.01). Results of a logistic regression analysis showed that relative odds for the presence of significant coronary stenoses increased with increasing blood concentrations of lipocalin-2 (OR=1.005, 95 % CI: 1.002-1.008, р=0.011) and IL-6 (OR=1.582 , 95 % CI: 1.241-2.017, р=0.001).Conclusion The changes in blood concentrations of adipokines associated with higher levels of proinflammatory cytokines may represent a factor that increases the probability of clinically significant coronary stenosis in overweight men with coronary atherosclerosis.

The Search for Associations of Serum Proteins with the Presence of Unstable Atherosclerotic Plaque in Coronary Atherosclerosis.

To study the associations of blood proteins with the presence of unstable atherosclerotic plaques in the arteries of patients with coronary atherosclerosis using quantitative proteomics. The studies involved two groups of men with coronary atherosclerosis (group 1 (St) had only stable atherosclerotic plaques; group 2 (Ns) had only unstable atherosclerotic plaques, according to histological analysis of tissue samples); the average age of patients was 57.95 ± 7.22. Protein concentrations in serum samples were determined using the PeptiQuant Plus Proteomics Kit. The identification of protein fractions was carried out by monitoring multiple reactions on a Q-TRAP 6500 mass spectrometer combined with a liquid chromatograph. Mass spectrometric identification revealed in serum samples from patients with unstable atherosclerotic plaques a reduced concentration of proteins in the blood: α-1-acid glycoprotein, α-1-antichymotrypsin, α-1-antitrypsin, ceruloplasmin, hemopexin, haptoglobin, apolipoprotein B-100, apolipoprotein L1, afamin and complement component (C3, C7, C9). Moreover, at the same time a high concentration complements factor H and attractin. The differences were considered significant at p &lt; 0.05. It was found that the instability of atherosclerotic plaques is associated with the concentration of proteins: afamin, attractin, components of the complement system, hemopexin and haptoglobin. The data of our study showed the association of some blood proteins with the instability of atherosclerotic plaques in coronary atherosclerosis. Their potential role in the development of this disease and the possibility of using the studied proteins as biomarkers requires further research.

Identification of immune cell infiltration and diagnostic biomarkers in unstable atherosclerotic plaques by integrated bioinformatics analysis and machine learning.

ObjectiveThe decreased stability of atherosclerotic plaques increases the risk of ischemic stroke. However, the specific characteristics of dysregulated immune cells and effective diagnostic biomarkers associated with stability in atherosclerotic plaques are poorly characterized. This research aims to investigate the role of immune cells and explore diagnostic biomarkers in the formation of unstable plaques for the sake of gaining new insights into the underlying molecular mechanisms and providing new perspectives for disease detection and therapy.MethodUsing the CIBERSORT method, 22 types of immune cells between stable and unstable carotid atherosclerotic plaques from RNA-sequencing and microarray data in the public GEO database were quantitated. Differentially expressed genes (DEGs) were further calculated and were analyzed for enrichment of GO Biological Process and KEGG pathways. Important cell types and hub genes were screened using machine learning methods including least absolute shrinkage and selection operator (LASSO) regression and random forest. Single-cell RNA sequencing and clinical samples were further used to validate critical cell types and hub genes. Finally, the DGIdb database of gene–drug interaction data was utilized to find possible therapeutic medicines and show how pharmaceuticals, genes, and immune cells interacted.ResultsA significant difference in immune cell infiltration was observed between unstable and stable plaques. The proportions of M0, M1, and M2 macrophages were significantly higher and that of CD8+ T cells and NK cells were significantly lower in unstable plaques than that in stable plaques. With respect to DEGs, antigen presentation genes (CD74, B2M, and HLA-DRA), inflammation-related genes (MMP9, CTSL, and IFI30), and fatty acid-binding proteins (CD36 and APOE) were elevated in unstable plaques, while the expression of smooth muscle contraction genes (TAGLN, ACAT2, MYH10, and MYH11) was decreased in unstable plaques. M1 macrophages had the highest instability score and contributed to atherosclerotic plaque instability. CD68, PAM, and IGFBP6 genes were identified as the effective diagnostic markers of unstable plaques, which were validated by validation datasets and clinical samples. In addition, insulin, nivolumab, indomethacin, and α-mangostin were predicted to be potential therapeutic agents for unstable plaques.ConclusionM1 macrophages is an important cause of unstable plaque formation, and CD68, PAM, and IGFBP6 could be used as diagnostic markers to identify unstable plaques effectively.

Differential expression of extracellular matrix-related genes in tissue of stable and unstable atherosclerotic plaques

Differential expression of extracellular matrix-related genes in tissue of stable and unstable atherosclerotic plaques

The Profile of Circulating Blood microRNAs in Outpatients with Vulnerable and Stable Atherosclerotic Plaques: Associations with Cardiovascular Risks.

Non-coding RNAs reflect many biological processes in the human body, including athero-sclerosis. In a cardiology outpatient department cohort (N = 83), we aimed to compare the levels of circulating microRNAs in groups with vulnerable plaques (N = 22), stable plaques (N = 23) and plaque-free (N = 17) depending on coronary computed tomography angiography and to evaluate associations of microRNA levels with calculated cardiovascular risks (CVR), based on the SCORE2 (+OP), ACC/AHA, ATP-III and MESA scales. Coronary computed tomography was performed on a 640-slice computed tomography scanner. Relative plasma levels of microRNA were assessed via a real-time polymerase chain reaction. We found significant differences in miR-143-3p levels (p = 0.0046 in plaque-free vs. vulnerable plaque groups) and miR-181b-5p (p = 0.0179 in stable vs. vulnerable plaques groups). Analysis of microRNA associations with CVR did not show significant differences for SCORE2 (+OP) and ATPIII scales. MiR-126-5p and miR-150-5p levels were significantly higher (p < 0.05) in patients with ACC/AHA risk >10% and miR-145-5p had linear relationships with ACC/AHA score (adjusted p = 0.0164). The relative plasma level of miR-195 was higher (p < 0.05) in patients with MESA risk > 7.5% and higher (p < 0.05) in patients with zero coronary calcium index (p = 0.036). A linear relationship with coronary calcium was observed for miR-126-3p (adjusted p = 0.0484). A positive correlation with high coronary calcium levels (> 100 Agatson units) was found for miR-181-5p (p = 0.036). Analyzing the biological pathways of these microRNAs, we suggest that miR-143-3p and miR-181-5p can be potential markers of the atherosclerosis process. Other miRNAs (miR-126-3p, 126-5p, 145-5p, 150-5p, 195-5p) can be considered as potential cardiovascular risk modifiers, but it is necessary to validate our results in a large prospective trial.

Abstract 441: Spatially Resolved Metabolites In Stable And Vulnerable Human Atherosclerotic Plaques Identified By Mass Spectrometry Imaging

There is a growing interest in elucidating the mechanisms that govern atherosclerotic plaque instability, which has so far highlighted the role of dysregulations in lipid, carbohydrate, and amino acid metabolism. However, where these impairments occur in a plaque has yet to be revealed. This is critically important because lipid deposition, cell composition, and matrix stiffness are heterogeneous throughout human atheromas, particularly in regions of the fibrous cap and near the necrotic core. Furthermore, lesional cells are constantly bombarded with various intrinsic and extrinsic metabolic insults that influence cell phenotype. Mass spectrometry imaging (MSI) is a powerful technology for the visualization of the spatial localization and relative abundance of hundreds to thousands of analytes in thin tissue sections without the need for a priori knowledge of analytes present in the tissue. Imaging is performed using matrix-assisted laser desorption/ionization (MALDI) to interrogate small areas from a tissue surface after the addition of a chemical matrix to the section which serves to extract, co-crystallize, and ionize molecules from the sample. In the present study, we performed MSI of late-stage, stable and vulnerable human atherosclerotic plaques using MALDI-MSI to visualize the distribution of metabolites in the fibrous cap and necrotic core. This platform was able to identify 856 metabolites assigned with chemical formulas. We were then able to confidently annotate 189 metabolites using a data filtering system whereby we queried detected and quantified metabolites from the Human Metabolome Database (HMDB). Coupled with RNAseq examining human stable and unstable atheromas, we identified differences in pathways related to nitric oxide metabolism, collagen catabolism, arginine metabolism, tryptophan metabolism, lipid metabolism, and reactive oxygen species. This work represents the first study to begin defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this work will be a valuable resource for the scientific community and will ultimately open new avenues of research in cardiovascular disease.

Osteomodulin Gene Expression Is Associated With Plaque Calcification, Stability, and Fewer Cardiovascular Events in the CPIP Cohort.

Stable atherosclerotic plaques are characterized by thick fibrous caps of smooth muscle cells, collagen, and macrocalcifications. Identifying factors of plaque stability is necessary to design drugs to prevent plaque rupture and symptoms. Osteomodulin, originally identified in bones, is expressed by bone synthesizing osteoblasts and involved in mineralization. In the present study, we analyzed osteomodulin expression in human carotid plaques, its link with plaque phenotype, calcification, and future cardiovascular events. Osteomodulin gene expression (OMD; n=82) was determined by RNA sequencing and osteomodulin protein levels by immunohistochemistry (n=45) in carotid plaques obtained by endarterectomy from patients with or without cerebrovascular symptoms from the CPIP (Carotid Plaque Imaging Project) cohort, Skåne University Hospital, Sweden. Plaque components were assessed by immunohistochemistry, RNA sequencing, and multiplex analysis. Patients were followed for cardiovascular events or cardiovascular death during a median of 57 or 70 months, respectively, using national registers. OMD levels were increased in plaques from asymptomatic patients compared to symptomatics. High OMD levels were associated with fewer cardiovascular events during follow-up. OMD correlated positively with smooth muscle α-actin (ACTA2; r=0.73, P=10-13) and collagen (COL1A2; r=0.4, P=0.0002), but inversely with CD68 gene expression (r=-0.67, P=10-11), lipids (r=-0.37, P=0.001), intraplaque hemorrhage (r=-0.32, P=0.010), inflammatory cytokine, and matrix metalloproteinase plaque contents. OMD was positively associated with MSX2 (Msh Homeobox 2) (r=0.32, P=0.003), a marker of preosteoblast differentiation, BMP4 (bone morphogenetic protein) (r=0.50, P=0.000002) and BMP6 (r=0.47, P=0.000007), plaque calcification (r=0.35, P=0.016), and was strongly upregulated in osteogenically stimulated smooth muscle cells, which was further increased upon BMP stimulation. Osteomodulin protein was present in calcified regions. Osteomodulin protein levels were associated with plaque calcification (r=0.41, P=0.006) and increased in macrocalcified plaques. These data show that osteomodulin mRNA and protein levels are associated with plaque calcification in human atherosclerosis. Furthermore, osteomodulin mRNA, but not protein levels, is associated with plaque stability.

Pharmacological Inhibition of Factor XIIa Attenuates Abdominal Aortic Aneurysm, Reduces Atherosclerosis, and Stabilizes Atherosclerotic Plaques.

Background 3F7 is a monoclonal antibody targeting the enzymatic pocket of activated factor XII (FXIIa), thereby inhibiting its catalytic activity. Given the emerging role of FXIIa in promoting thromboinflammation, along with its apparent redundancy for hemostasis, the selective inhibition of FXIIa represents a novel and highly attractive approach targeting pathogenic processes that cause thromboinflammation-driven cardiovascular diseases. Methods The effects of FXIIa inhibition were investigated using three distinct mouse models of cardiovascular disease—angiotensin II-induced abdominal aortic aneurysm (AAA), an ApoE −/− model of atherosclerosis, and a tandem stenosis model of atherosclerotic plaque instability. 3F7 or its isotype control, BM4, was administered to mice (10 mg/kg) on alternate days for 4 to 8 weeks, depending on the experimental model. Mice were examined for the development and size of AAAs, or the burden and instability of atherosclerosis and associated markers of inflammation. Results Inhibition of FXIIa resulted in a reduced incidence of larger AAAs, with less acute aortic ruptures and an associated fibro-protective phenotype. FXIIa inhibition also decreased stable atherosclerotic plaque burden and achieved plaque stabilization associated with increased deposition of fibrous structures, a >2-fold thicker fibrous cap, increased cap-to-core ratio, and reduction in localized and systemic inflammatory markers. Conclusion Inhibition of FXIIa attenuates disease severity across three mouse models of thromboinflammation-driven cardiovascular diseases. Specifically, the FXIIa-inhibiting monoclonal antibody 3F7 reduces AAA severity, inhibits the development of atherosclerosis, and stabilizes vulnerable plaques. Ultimately, clinical trials in patients with cardiovascular diseases such as AAA and atherosclerosis are warranted to demonstrate the therapeutic potential of FXIIa inhibition.

Analysis of differential expression of lipid metabolism genes in atherosclerotic plaques in patients with coronary atherosclerosis

Aim. The goal of the study was to analyze the differential expression of lipid metabolism-related genes in the atherosclerotic plaques of different types in patients with coronary atherosclerosis.Material and Methods. The study was performed on the specimens of atherosclerotic plaques in 45–65-year-old patients with coronary atherosclerosis with stable exertional angina functional class II-IV without acute coronary syndrome. Coronary atherosclerosis was verified by coronary angiography. Atherosclerotic plaque tissue was sampled intraoperatively when indicated. Whole-genome sequencing of ribonucleic acid (RNA) was performed using the TruSeq RNA Sample Preparation Kit (Illumina, USA).Results. We analyzed the differences in the expression of 12 genes including LDLR, APOB, PCSK9, LDLRAP1, LIPA, STAP1, ABCA1, APOA1, APOE, LPL, SCARB1, and SREBF2 depending on the type of atherosclerotic plaques. The expression level of APOE gene was eight times higher in unstable atherosclerotic plaques of dystrophic-necrotic type (p &lt; 0.0001). The expression levels of LDLR and APOB genes were eight times higher in stable atherosclerotic plaques (p &lt; 0.0001). We did not find differences in the expression levels of the ABCG5, ABCG8, APOC3, CETP, CLPS, CYP7A1, and PNPLA5 genes.Conclusion. The study showed the differences in the activity of individual metabolism-related genes in the atherosclerotic plaques of different types in patients with coronary atherosclerosis. Obtained data may become the basis for the development of test systems aimed at predicting the development of atherosclerotic process and its complications.

The Role of Hematological Indices in Patients with Acute Coronary Syndrome

Background: Acute coronary syndrome (ACS) including ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI), and unstable angina - is an umbrella term for life-threatening situations that occur when the blood supply to the heart is altered due to destabilization of a previously stable atherosclerotic plaque. This alteration hinders the heart muscle from functioning properly and even could lead to death. This study aimed to identify the role of hematological indices in patients with acute coronary syndrome.&#x0D; Methods: This prospective cohort study was carried out on 75 patients; they were divided into 3 groups each of them included 25 patients. Group I represent patients with myocardial infarction (MI), group II represents patients with unstable angina (UA) and control group included 25 subjects. All patients were subjected to complete history taking, clinical examination and routine laboratory investigation. Patients also were subjected to electrocardiography, transthoracic echo Doppler and coronary angiography.&#x0D; Results: Regarding hematological indices for prediction of MI, the ROC revealed: NLR ≥ 4.45 with sensitivity 96%, specificity 92%, Area under the curve 0. 99 and accuracy 94%. RDW ≥ 14.15 with sensitivity 96% specificity 72%, Area under the curve 0.973 and accuracy 84%. PLR ≥ 88.8 with sensitivity 96%, specificity 96%, Area under the curve 0.994 and accuracy 96%. MPV ≥ 9.65 with sensitivity 68%, specificity 52%, Area under the curve 0.646 and accuracy 60%. PDW ≥ 15.15 with sensitivity 96%, specificity 96%, Area under the curve 0.998 and accuracy 96%.&#x0D; Conclusions: There is a high demand for a reliable, accessible, noninvasive, and hematological prognostic marker in ACS, which would identify patients of high cardiovascular risk in secondary prevention and tailor the therapy to their needs. The inflammatory processes play a key role in the development of atherosclerosis, destabilization of atherosclerotic plaques and formation of clots on the plaque surface. There is a significance role of NLR, PLR, PDW, MPV, and RDW in the prognosis of ACS.