Human Atherosclerotic Plaques Research Articles

Evidence of an anti-inflammatory effect of PCSK9 inhibitors within the human atherosclerotic plaque

Background and aimsPreclinical evidence suggests that proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors hold anti-inflammatory properties independently of their ability to lower LDL-cholesterol (C). However, whether PCSK9 inhibitors exert anti-inflammatory effects within the atherosclerotic plaque in humans is unknown. We explored the impact of PCSK9 inhibitors, used as monotherapy, compared with other lipid-lowering drugs (oLLD) on the expression of inflammatory markers within the plaque, assessing also the subsequent incidence of cardiovascular events. MethodsIn an observational study, we recruited 645 patients on stable therapy for at least six months and undergoing carotid endarterectomy, categorizing patients according to the use of PCSK9 inhibitors only (n = 159) or oLLD (n = 486). We evaluated the expression of NLRP3, caspase-1, IL-1β, TNFα, NF-kB, PCSK9, SIRT3, CD68, MMP-9, and collagen within the plaques in the two groups through immunohistochemistry, ELISA, or immunoblot. A composite outcome including non-fatal myocardial infarction, non-fatal stroke, and all-cause mortality was assessed during a 678 ± 120 days follow-up after the procedure. ResultsPatients treated with PCSK9 inhibitors had a lower expression of pro-inflammatory proteins and a higher abundance of SIRT3 and collagen within the plaque, a result obtained despite comparable levels of circulating hs-CRP and observed also in LDL-C-matched subgroups with LDL-C levels <100 mg/dL. Patients treated with PCSK9 inhibitors showed a decreased risk of developing the outcome compared with patients on oLLD, also after adjustment for multiple variables including LDL-C (adjusted hazard ratio 0.262; 95% CI 0.131–0.524; p < 0.001). The expression of PCSK9 correlated positively with that of pro-inflammatory proteins, which burden was associated with a higher risk of developing the outcome, independently of the therapeutic regimen. ConclusionsThe use of PCSK9 inhibitors is accompanied by a beneficial remodelling of the inflammatory burden within the human atheroma, an effect possibly or partly independent of their LDL-C lowering ability. This phenomenon might provide an additional cardiovascular benefit.

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.

The inflammatory oxidant peroxynitrous acid modulates the structure and function of the recombinant human V3 isoform of the extracellular matrix proteoglycan versican

Continued oxidant production during chronic inflammation generates host tissue damage, with this being associated with pathologies including atherosclerosis. Atherosclerotic plaques contain modified proteins that may contribute to disease development, including plaque rupture, the major cause of heart attacks and strokes. Versican, a large extracellular matrix (ECM) chondroitin-sulfate proteoglycan, accumulates during atherogenesis, where it interacts with other ECM proteins, receptors and hyaluronan, and promotes inflammation. As activated leukocytes produce oxidants including peroxynitrite/peroxynitrous acid (ONOO−/ONOOH) at sites of inflammation, we hypothesized that versican is an oxidant target, with this resulting in structural and functional changes that may exacerbate plaque development. The recombinant human V3 isoform of versican becomes aggregated on exposure to ONOO−/ONOOH. Both reagent ONOO−/ONOOH and SIN-1 (a thermal source of ONOO−/ONOOH) modified Tyr, Trp and Met residues. ONOO−/ONOOH mainly favors nitration of Tyr, whereas SIN-1 mostly induced hydroxylation of Tyr, and oxidation of Trp and Met. Peptide mass mapping indicated 26 sites with modifications (15 Tyr, 5 Trp, 6 Met), with the extent of modification quantified at 16. Multiple modifications, including the most extensively nitrated residue (Tyr161), are within the hyaluronan-binding region, and associated with decreased hyaluronan binding. ONOO−/ONOOH modification also resulted in decreased cell adhesion and increased proliferation of human coronary artery smooth muscle cells. Evidence is also presented for colocalization of versican and 3-nitrotyrosine epitopes in advanced (type II-III) human atherosclerotic plaques. In conclusion, versican is readily modified by ONOO−/ONOOH, resulting in chemical and structural modifications that affect protein function, including hyaluronan binding and cell interactions.

MPO activity determined by molecular MRI is associated with ruptured and destabilised human atherosclerotic plaques

Abstract Funding Acknowledgements Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): National Health &amp; Medical Research Council - Postgraduate Scholarship National Heart Foundation - PhD Scholarship University of New South Wales - RTP Scholarship Introduction Intraplaque MPO activity is associated with unstable atherosclerosis and elevated following acute cardiovascular events. Imaging probes for the detection of MPO exist, including MPO-Gd, a gadolinium-based probe activated and retained in the presence of MPO-specific chlorinating species. To date, the utility of MPO activity in plaque rupture has not been examined. We sought to assess the association between intraplaque MPO activity and ruptured human plaques. Method Carotid endarterectomy specimens (CEA) were collected from 30 patients undergoing surgery. Following pre-surgical in vivo MRI for plaque characterisation, ex vivo plaques were activated using MPO-Gd. MPO-Gd retention was then correlated to AHA plaque grading both histologically and by in vivo MRI as well as liquid chromatography mass spectrometry (LC-MS/MS) through the addition of hydroethidine tissue sections and it’s conversion to the MPO-specific adduct 2-chloroethidium (2-Cl-E+). Results MPO-Gd retention in CEA was greater in both histologically and MRI-graded ruptured and destabilised AHA type VI plaques compared to types III-V (T1 percentage recovery from baseline: VI: 52% ± 5.7 vs. V: 77% ± 7.8, IV: 83% ± 8.2 and III: 84% ± 6.8; p &amp;lt; 0.0001). This association was confirmed by comparing histological AHA grade to MPO activity determined by LC-MS/MS (2-Cl-E+ corrected to internal standard per mgp: VI: 0.2 ± 0.2 vs. V: 0.00 ± 0.01, IV: 0.01 ± 0.01, and III: 0.03 ± 0.03; p = 0.005). Discussion We demonstrate that MPO activity determined by MPO-Gd and compared to histology is increased in ruptured human plaques. These results highlight the unique and specific role of intraplaque MPO activity and its potential application as a molecular probe in the detection of vulnerable atherosclerosis.

Transcriptome-wide association study reveals novel susceptibility genes for coronary atherosclerosis.

Genetic risk factors substantially contributed to the development of coronary atherosclerosis. Genome-wide association study (GWAS) has identified many risk loci for coronary atherosclerosis, but the translation of these loci into therapeutic targets is limited for their location in non-coding regions. Here, we aimed to screen the potential coronary atherosclerosis pathogenic genes expressed though TWAS (transcriptome wide association study) and explore the underlying mechanism association. Four TWAS approaches (PrediXcan, JTI, UTMOST, and FUSION) were used to screen genes associated with coronary atherosclerosis. Enrichment analysis of TWAS-identified genes was applied through the Metascape website. The summary-data-based Mendelian randomization (SMR) analysis was conducted to provide the evidence of causal relationship between the candidate genes and coronary atherosclerosis. At last, the cell type-specific expression of the intersection genes was examined by using human coronary artery single-cell RNA-seq, interrogating the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity. We identified 19 genes by at least three approaches and 1 gene (NBEAL1) by four approaches. Enrichment analysis enriching the genes identified at least by two TWAS approaches, suggesting that these genes were markedly enriched in asthma and leukocyte mediated immunity reaction. Further, the summary-data-based Mendelian randomization (SMR) analysis provided the evidence of causal relationship between NBEAL1 gene and coronary atherosclerosis, confirming the protecting effects of NBEAL1 gene and coronary atherosclerosis. At last, the single cell cluster analysis demonstrated that NBEAL1 gene has differential expressions in macrophages, plasma cells and endothelial cells. Our study identified the novel genes associated with coronary atherosclerosis and suggested the potential biological function for these genes, providing insightful guidance for further biological investigation and therapeutic approaches development in atherosclerosis-related diseases.

TM6SF2 reduces lipid accumulation in vascular smooth muscle cells by inhibiting LOX-1 and CD36 expression

TM6SF2, predominantly expressed in the liver and intestine, is closely associated with lipid metabolism. We have demonstrated the presence of TM6SF2 in VSMCs within human atherosclerotic plaques. Subsequent functional studies were conducted to investigate its role in lipid uptake and accumulation in human vascular smooth muscle cells (HAVSMCs) using siRNA knockdown and overexpression techniques. Our results showed that TM6SF2 reduced lipid accumulation in oxLDL-stimulated VSMCs, likely through the regulation of lectin-like oxLDL receptor 1 (LOX-1) and scavenger receptor cluster of differentiation 36 (CD36) expression. We concluded that TM6SF2 plays a role in HAVSMC lipid metabolism with opposing effects on cellular lipid droplet content by downregulation of LOX-1 and CD36 expression.

Spatial Transcriptional Mapping Reveals Site-Specific Pathways Underlying Human Atherosclerotic Plaque Rupture

BackgroundAtherosclerotic plaque ruptures, triggered by blood flow–associated biomechanical forces, cause most myocardial infarctions and strokes. ObjectivesThis study aims to investigate the exact location and underlying mechanisms of atherosclerotic plaque ruptures, identifying therapeutic targets against cardiovascular events. MethodsHistology, electron microscopy, bulk and spatial RNA sequencing on human carotid plaques were studied in proximal, most stenotic, and distal regions along the longitudinal blood flow direction. Genome-wide association studies were used to examine heritability enrichment and causal relationships of atherosclerosis and stroke. Associations between top differentially expressed genes (DEGs) and preoperative and postoperative cardiovascular events were examined in a validation cohort. ResultsIn human carotid atherosclerotic plaques, ruptures predominantly occurred in the proximal and most stenotic regions but not in the distal region. Histologic and electron microscopic examination showed that proximal and most stenotic regions exhibited features of plaque vulnerability and thrombosis. RNA sequencing identified DEGs distinguishing the proximal and most stenotic regions from the distal region which were deemed as most relevant to atherosclerosis-associated diseases as shown by heritability enrichment analyses. The identified pathways associated with the proximal rupture-prone regions were validated by spatial transcriptomics, firstly in human atherosclerosis. Of the 3 top DEGs, matrix metallopeptidase 9 emerged particularly because Mendelian randomization suggested that its high circulating levels were causally associated with atherosclerosis risk. ConclusionsOur findings show plaque site–specific transcriptional signatures associated with proximal rupture-prone regions of carotid atherosclerotic plaques. This led to the geographical mapping of novel therapeutic targets, such as matrix metallopeptidase 9, against plaque rupture.

Abstract 105: Lipid Regulation of Senescent Effector CD8 T Cells in Type 2 Diabetes

Introduction: Atherosclerotic plaques from patients with Type 2 Diabetes (T2D) present a more severe pathology than non-T2D (ND) plaques and response to cholesterol-lowering is compromised. An increased level of circulating senescent CD8 T cells in T2D patients is associated with increased risk for myocardial infarction. However, whether senescent T cells infiltrate human atherosclerotic plaques and how they are regulated by lipid lowering is unknown. Hypothesis: Senescent CD8+ T cells are increased in atherosclerotic plaques and circulation of T2D, and are normalized by aggressive lipid lowering in T2D. Methods: We performed unbiased scRNA-seq analysis (75,049 total cells) of immune cells in plaques from T2D (n=12) and ND patients (n=10) to identify senescent T cells in human atherosclerotic plaques. Using CyTOF we tested the effect of aggressive lipid lowering on circulating CD8 T cells in T2D (n=16) and ND (n=6) patients. Results: We identified senescent T cells within atherosclerotic plaques and found that senescent CD8 T cells largely accumulate in plaques from T2D patients (p&lt;0.001). Plaque CD8 T cells showed a terminally differentiated effector memory phenotype with high expression of CX3CR1 and low expression of SELL and CCR7 genes. CD8 T cell clusters express senescence markers ( B3GAT1,KLRG1,KLRD1,KLRF1 ) and presented senescence associated secretory phenotype with high expression of cytotoxic ( GZMB,PRF1,GNLY ) and proatherogenic ( IFNG,TNFA,CCL5 ) genes. CyTOF analysis showed higher levels of circulating senescent CD57+CD28-CD8 T cells in patients with T2D vs ND controls (p&lt;0.001). Potent lipid lowering with PCSK9 inhibitor + statin/ezetimibe, which reduced LDL-C levels by &gt;70% in all patients at 4 weeks, normalized the frequency of circulating senescent CD8 T cells (p&lt;0.001) in T2D patients to the levels found in ND. Conclusion: Our results show that senescent CD8 T cells accumulated in blood and plaques of T2D patients and that potent lipid lowering diminished circulating aged CD8 T cells, suggesting that CD8 T cell senescence can be improved by aggressive lipid control in high-risk T2D subjects. Further analyses are needed to understand the role of lipid metabolism in the T cell senescence and the impact on atherosclerosis progression.

Abstract 101: Crosstalk Of Pluripotency And Innate Immunity In Smooth Muscle Cell Phenotypic Transitions

Objectives: Smooth muscle cell ( SMC ) phenotypic transitions play a critical role in the pathogenesis of cardiovascular diseases. However, the molecular mechanisms responsible for these transitions are not well understood. We previously found that the embryonic stem cell/pluripotency factor OCT4 is reactivated in SMC in human and mouse atherosclerotic plaques and plays an atheroprotective role by regulating SMC investment into the protective fibrous cap. Loss of OCT4 in SMC is associated with SMC de-differentiation toward a macrophage-like phenotypic state resulting in increased lipid accumulation and phagocytosis. However, the endogenous molecular mechanism responsible for atheroprotective SMC-OCT4-dependent effects remains unknown. Methods and Results: Herein, we performed combinatory bioinformatics analyses on the in vivo and in vitro wild-type and SMC- Oct4 knockout mouse samples and found that loss of Oct4 in SMC resulted in up-regulation of the innate immune receptor TLR4 in numerous experimental conditions. Notably, the TLR4 promoter does not have any putative OCT4 binding sites. First - Using publicly available OCT4 ChIPseq datasets and long non-coding ( lnc )RNA databases, we identified several lncRNAs, including IGF2AS, MALAT1, PLATR10, BACE1, and SRA based on the OCT4 occupancy at promoter and enhancer regions of lncRNA, which in turn, directly target TLR4. To confirm the OCT4-dependent activation of the identified lncRNAs in SMC, qRT-PCR was performed in mouse aortic SMC transfected with Oct4 -blocking siRNA. We found that the Oct4 knockdown in SMC led to significant decreases in all five selected lncRNAs and increases in TLR4 and its downstream gene, TRIF. Second - Intriguingly, we also demonstrated that TLR4 signaling, in turn, promotes hydroxymethylation of the OCT4 promoter and reactivates OCT4 in SMC. Third - Using a Tlr4 -reporter and SMC-lineage tracing mouse model, we also demonstrated that medial differentiated SMC from healthy arteries, but not phenotypically modulated lesion SMC, express TLR4. Conclusions: Together, these results indicate a role of TLR4-signaling in SMC both upstream and downstream of OCT4, suggesting that pluripotency/innate immunity crosstalk plays a critical role in SMC phenotypic transitions.

Abstract 695: Serine Hydroxymethyltransferase 2 Regulates Mitochondrial Function And Ceramide Metabolism In Macrophages During Atherosclerosis

Increasing evidence suggests a critical role for amino acid metabolism in cardiovascular disease, highlighting potential therapeutic targets and our need for further understanding. Plasma serine has been found to be a biomarker for cardiovascular disease, while glycine is negatively correlated with coronary artery disease. Furthermore, attenuated plasma glycine:serine is associated with unstable atherosclerotic plaques. Mitochondrial Serine hydroxymethyl transferase (SHMT) catalyzes the bulk formation of glycine from serine in most cell types and drives one-carbon metabolism in the cytosol (SHMT1) or mitochondria (SHMT2). SHMT2 is necessary for mitochondrial protein translation and critical in development since global knockout mice are nonviable. We found LPS-treated macrophages have reduced SHMT2 expression concomitant with attenuated intracellular glycine:serine. Deletion of macrophage SHMT2 in vitro enhanced intracellular serine, which contributes to the rate-limiting step of ceramide de novo biosynthesis. C16:0 ceramide is highly abundant in atherosclerotic plaques and is positively associated with plaque severity, and SHMT2-deleted macrophages treated with C16:0 fatty acid enhances intracellular ceramide. Furthermore, deletion of SHMT2 reduced mitochondrial respiration and enhanced glycolysis similar to LPS-treated macrophages. Moreover, we found SHMT2 is highly expressed in plaque-associated macrophages in both human and mouse atherosclerotic plaques. We therefore hypothesized that SHMT2 regulates macrophage inflammatory phenotype in atherosclerosis through alterations in mitochondrial function and ceramide biosynthesis. To determine whether macrophage-expressed SHMT2 contributes to atherosclerosis, we developed a novel Shmt2 -floxed mouse crossed with LysM-Cre ( Shmt2 MKO ) and Apoe -/- mice. After 12 weeks on Western diet, we observed no differences in plasma glycine:serine or circulating lipids. Nevertheless, Shmt2 MKO / Apoe -/- mice had significantly enhanced plaque area compared to littermate controls. Our findings, representing the first investigation of conditional SHMT2 knockout mice, suggest a novel immunometabolic role for SHMT2 in macrophages in atherosclerosis.

Loss Of Nicotinamide Nucleotide Transhydrogenase Promotes Redox Dependent Endothelial Activation In Atherosclerosis

Heart disease is the leading cause of death in the United States and atherosclerosis accounts for nearly 75% of all deaths from heart disease. Atherosclerosis is a chronic inflammatory disease and mitochondrial reactive oxygen species (mtROS) are critical contributors to disease development. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that maintains mitochondrial NADPH pools and supports the enzymatic degradation of mtROS. Preliminary data indicates that NNT expression is decreased in the endothelium of human atherosclerotic plaques and we sought to test the hypothesis that the loss of NNT increases mtROS and promotes atherosclerosis by enhancing endothelial and vascular dysfunction. Previous we have demonstrated that the loss of NNT was associated with increased vascular ROS production, plaque formation, and plaque size in response to treatment with PCSK9 and high fat diet (HFD). We now show that the loss of NNT in endothelial specific NNT knockout mice promotes enhanced endothelial VCAM-1 expression in response to HFD and disturbed flow. Similarly, in vitro, the loss of NNT promoted both VCAM-1 and ICAM-1 expression in response to oxLDL. Increased adhesion molecule expression associated with the loss of NNT also augmented monocyte adhesion in response to oxLDL consistent with vascular inflammation observed in early atherosclerosis. Additionally, macrophages isolated from NNT knockout mice display enhanced M1 polarization, decreased lipid uptake, and impaired lipid utilization. Co- treatment with the mitochondria targeted antioxidant MitoEbselen is able to normalize both endothelial and macrophage function, underscoring a critical role for mtROS in promoting an inflammatory phenotype in these cells. In both global and endothelial cell specific NNT knockout mice the loss of NNT was associated with increased necrotic core formation in response to PCSK9 treatment and 16 weeks of high fat diet. Taken together, these data indicate that decreased NNT expression promotes endothelial activation, increased inflammatory cell trafficking, macrophage metabolic dysfunction and M1 polarization that could contribute to an acceleration of necrotic core development and severe atherosclerotic disease. NIH P20GM121307. NIH 1 R01 HL145171-01A1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Evaluating acoustic and thermal properties of a plaque phantom.

The aim of this study is to evaluate the acoustic and thermal properties of a plaque phantom. This is very important for the effective implementation of ultrasound not only in diagnosis but especially in treatment for the future. An evaluation of acoustic and thermal properties of plaque phantoms to test their suitability mainly for ultrasound imaging and therapy was presented. The evaluation included measurements of the acoustic propagation speed using pulse-echo technique, ultrasonic attenuation coefficient using through transmission immersion technique, and absorption coefficient. Moreover, thermal properties (thermal conductivity, volumetric specific heat capacity and thermal diffusivity) were measured with the transient method using a needle probe. It was shown that acoustic and thermal properties of atherosclerotic plaque phantoms fall well within the range of reported values for atherosclerotic plaque and slightly different for thermal diffusivity and volumetric specific heat capacity for soft tissues. The mean value of acoustic and thermal properties and their standard deviation of plaque phantoms were 1523 ± 23m/s for acoustic speed, 0.50 ± 0.02W/mK for thermal conductivity, 0.30 ± 0.21db/cm-MHz for ultrasonic absorption coefficient and 1.63 ± 0.46db/cm-MHz for ultrasonic attenuation coefficient. This study demonstrated that acoustic and thermal properties of atherosclerotic plaque phantoms were within the range of reported values. Future studies should be focused on the optimum recipe of the atherosclerotic plaque phantoms that mimics the human atherosclerotic plaque (agar 4% w/v, gypsum 10% w/v and butter 10% w/v) and can be used for HIFU therapy.

Tongmai Zhuke decoction restrains the inflammatory reaction of macrophages for carotid artery atherosclerosis by up-regulating lincRNA-Cox2

ABSTRACT To explore the mechanism of Tongmai Zhuke decoction for promoting blood circulation by taking carotid artery atherosclerosis (CAA) as an example, two sets of in-depth transcriptomic data as well as two sets of single-cell RNA sequencing data related to the macrophages in CAA were included. STAR and DCC software were used to process in-depth transcriptomic data in order to measure the expression level of LncRNAs as well as mRNA according to FPKM analysis. Single-cell RNA sequencing data from Illumina NovaSeq 6000 were further analyzed by CellRanger channel, CellRanger count, Seurat R package, DoubletFinder package, CCA algorithm, LogNormalize, principal-component analysis, t-SNE and ToppGene online tools. Based On unsupervised clustering, a total of four diverse cell populations with distinct transcriptional features were found in human carotid atherosclerotic plaques. The macrophages were further annotated as the “effector cell” in the pathologic process of CAA, based on the expression of CD68+/CD440-. A total of 84 up-regulated genes and 58 down-regulated linc-RNAs were identified in samples with carotid atherosclerotic plaques. Thereinto, lincRNA-Cox2 is the most down-regulated LincRNA. For the macrophages in carotid atherosclerotic plaques, expression level of Il6, Ccl3, Ccl4 Il10 and Tnfa were significantly up-regulated, while Timp1 significantly down-regulated comparing with healthy carotid sample. The expression level of lincRNA-Cox2 was significantly increased in macrophages after treated by Tongmai Zhuke decoction, while Cxcl10, Ccl3, Ccl4, Cxcl2, Ccl5, and Ccl19 were significantly decreased. Collectively, Tongmai Zhuke decoction could restrain the inflammatory reaction of macrophages for carotid artery atherosclerosis by up-regulating lincRNA-Cox2.

Wall shear stress and its role in atherosclerosis.

Atherosclerosis (AS) is the major form of cardiovascular disease and the leading cause of morbidity and mortality in countries around the world. Atherosclerosis combines the interactions of systemic risk factors, haemodynamic factors, and biological factors, in which biomechanical and biochemical cues strongly regulate the process of atherosclerosis. The development of atherosclerosis is directly related to hemodynamic disorders and is the most important parameter in the biomechanics of atherosclerosis. The complex blood flow in arteries forms rich WSS vectorial features, including the newly proposed WSS topological skeleton to identify and classify the WSS fixed points and manifolds in complex vascular geometries. The onset of plaque usually occurs in the low WSS area, and the plaque development alters the local WSS topography. low WSS promotes atherosclerosis, while high WSS prevents atherosclerosis. Upon further progression of plaques, high WSS is associated with the formation of vulnerable plaque phenotype. Different types of shear stress can lead to focal differences in plaque composition and to spatial variations in the susceptibility to plaque rupture, atherosclerosis progression and thrombus formation. WSS can potentially gain insight into the initial lesions of AS and the vulnerable phenotype that gradually develops over time. The characteristics of WSS are studied through computational fluid dynamics (CFD) modeling. With the continuous improvement of computer performance-cost ratio, WSS as one of the effective parameters for early diagnosis of atherosclerosis has become a reality and will be worth actively promoting in clinical practice. The research on the pathogenesis of atherosclerosis based on WSS is gradually an academic consensus. This article will comprehensively review the systemic risk factors, hemodynamics and biological factors involved in the formation of atherosclerosis, and combine the application of CFD in hemodynamics, focusing on the mechanism of WSS and the complex interactions between WSS and plaque biological factors. It is expected to lay a foundation for revealing the pathophysiological mechanisms related to abnormal WSS in the progression and transformation of human atherosclerotic plaques.

The human carotid atherosclerotic plaque: an observational review of histological scoring systems.

The atherosclerotic plaque is a complex dynamic pathological lesion of the arterial wall, characterized by multiple elementary lesions of different diagnostic and prognostic significance. Fibrous cap thickness, lipid necrotic core dimension, inflammation, intra-plaque hemorrhage (IPH), plaque neovascularization and endothelial dysfunction (erosions) are generally considered the most relevant morphological details of plaque morphology. In this review, the most relevant features able to discriminate between stable and vulnerable plaques at histological level are discussed. Retrospectively, we have evaluated the laboratory results from one hundred old histological samples from patients treated with carotid endarterectomy. These results were analyzed to assess elementary lesions that characterize stable and unstable plaques. A thin fibrous cap (<65 micron), loss of smooth muscle cells, collagen depletion, a large lipid-rich necrotic core, infiltrating macrophages, IPH and intra-plaque vascularization are identified as the most important risk factors associated with plaque rupture. Immunohistochemistry for smooth muscle actin (smooth muscle cell marker) and for CD68 (marker of monocytes/macrophages) and glycophorin (marker of red blood cells) are suggested as useful tools for an in deep characterization of any carotid plaque and for distinguishing plaque phenotypes at histology. Since patients with a carotid vulnerable plaque are at higher risk of developing vulnerable plaques in other arteries as well, the definition of the vulnerability index is underlined, in order to stratify patients at higher risk for undergoing cardiovascular events.

Transcription factor 21 accelerates vascular calcification in mice by activating the IL-6/STAT3 signaling pathway and the interplay between VSMCs and ECs.

Vascular calcification is caused by the deposition of calcium salts in the intimal or tunica media layer of the aorta, which increases the risk of cardiovascular events and all-cause mortality. However, the mechanisms underlying vascular calcification are not fully clarified. Recently it has been shown that transcription factor 21 (TCF21) is highly expressed in human and mouse atherosclerotic plaques. In this study we investigated the role of TCF21 in vascular calcification and the underlying mechanisms. In carotid artery atherosclerotic plaques collected from 6 patients, we found that TCF21 expression was upregulatedin calcific areas. We further demonstrated TCF21 expression wasincreased in an in vitro vascular smooth muscle cell (VSMC) osteogenesis model. TCF21 overexpression promoted osteogenic differentiation of VSMC, whereas TCF21 knockdown in VSMC attenuated the calcification. Similar results were observed in ex vivo mouse thoracic aorta rings. Previous reports showed that TCF21 bound to myocardin (MYOCD) to inhibit the transcriptional activity of serum response factor (SRF)-MYOCD complex. We found that SRF overexpression significantly attenuated TCF21-induced VSMC and aortic ring calcification. Overexpression of SRF, but not MYOCD, reversed TCF21-inhibited expression of contractile genes SMA and SM22. More importantly, under high inorganic phosphate (3 mM) condition, SRF overexpression reduced TCF21-induced expression of calcification-related genes (BMP2 and RUNX2) as well as vascular calcification. Moreover, TCF21 overexpression enhanced IL-6 expression and downstream STAT3 activation to facilitate vascular calcification. Both LPS and STAT3 could induce TCF21 expression, suggesting that the inflammation and TCF21 might form a positive feedback loop to amplify the activation of IL-6/STAT3 signaling pathway. On the other hand, TCF21 induced production of inflammatory cytokines IL-1β and IL-6 in endothelial cells (ECs) to promote VSMC osteogenesis. In EC-specific TCF21 knockout (TCF21ECKO) mice, VD3 and nicotine-induced vascular calcification was significantly reduced. Our results suggest that TCF21 aggravates vascular calcification by activating IL-6/STAT3 signaling and interplay between VSMC and EC, which provides new insights into the pathogenesis of vascular calcification. TCF21 enhances vascular calcification by activating the IL-6-STAT3 signaling pathway. TCF21 inhibition may be a new potential therapeutic strategy for the prevention and treatment of vascular calcification.

Circadian gene CLOCK accelerates atherosclerosis by promoting endothelial autophagy

ABSTRACT Atherosclerosis (AS) is a chronic inflammatory disease which gives rise to life-threatening complications like ischemic stroke. Rupture of carotid atherosclerotic plaque is the main cause of ischemic stroke. Emerging evidence has demonstrated that disturbed circadian rhythms could accelerate the progression of atherosclerosis by regulating endothelial function. Moreover, our previous study implicated the circadian gene circadian locomotor output cycles kaput (CLOCK) in the pathogenesis of unstable plaques. In this study, we explored the underlying mechanism that CLOCK mediates endothelial cell autophagy involved in the progression of AS. Circadian and autophagy gene expression was analyzed in the GSE41571 dataset and human carotid atherosclerotic plaque samples. Then we used ox-LDL to treat HUVECs, and analyzed CLOCK and autophagy gene in endothelial cells. Besides that, we comprehensively analyzed in vivo experiments to explore the function of CLOCK in autophagy and atherosclerosis using different staining including HE, MT and IF staining. In the dataset and patient samples, CLOCK expression and autophagy were decreased in the unstable plaque group compared with the stable group. Decreased Beclin1, ATG5, LC3, and CLOCK were also observed in HUVECs under oxidative stress condition which also enhances cell proliferation. In vivo, we also found decreasing level of CLOCK, Beclin1, LC3 and ATG5 in ApoE−/− mice compared with WT mice. Silencing of CLOCK in ApoE−/− mice may further aggravate atherosclerosis including decreased cap thickness and collagens. Our findings implicated that downregulation CLOCK would impair endothelial cell autophagy and accelerate atherosclerotic plaque, which provides a novel strategy for treatment of progression in AS.

Interleukin-6 trans-signaling induced laminin switch contributes to reduced trans-endothelial migration of granulocytic cells

Background and aimsLaminins are essential components of the endothelial basement membrane, which predominantly contains LN421 and LN521 isoforms. Regulation of laminin expression under pathophysiological conditions is largely unknown. In this study, we aimed to investigate the role of IL-6 in regulating endothelial laminin profile and characterize the impact of altered laminin composition on the phenotype, inflammatory response, and function of endothelial cells (ECs). MethodsHUVECs and HAECs were used for in vitro experiments. Trans-well migration experiments were performed using leukocytes isolated from peripheral blood of healthy donors. The BiKE cohort was used to assess expression of laminins in atherosclerotic plaques and healthy vessels. Gene and protein expression was analyzed using Microarray/qPCR and proximity extension assay, ELISA, immunostaining or immunoblotting techniques, respectively. ResultsStimulation of ECs with IL-6+sIL-6R, but not IL-6 alone, reduces expression of laminin α4 (LAMA4) and increases laminin α5 (LAMA5) expression at the mRNA and protein levels. In addition, IL-6+sIL-6R stimulation of ECs differentially regulates the release of several proteins including CXCL8 and CXCL10, which collectively were predicted to inhibit granulocyte transmigration. Experimentally, we demonstrated that granulocyte migration is inhibited across ECs pre-treated with IL-6+sIL-6R. In addition, granulocyte migration across ECs cultured on LN521 was significantly lower compared to LN421. In human atherosclerotic plaques, expression of endothelial LAMA4 and LAMA5 is significantly lower compared to control vessels. Moreover, LAMA5-to-LAMA4 expression ratio was negatively correlated with granulocytic cell markers (CD177 and myeloperoxidase (MPO)) and positively correlated with T-lymphocyte marker CD3. ConclusionsWe showed that expression of endothelial laminin alpha chains is regulated by IL-6 trans-signaling and contributes to inhibition of trans-endothelial migration of granulocytic cells. Further, expression of laminin alpha chains is altered in human atherosclerotic plaques and is related to intra-plaque abundance of leukocyte subpopulations.

A novel role for YPEL2 in mediating endothelial cellular senescence via the p53/p21 pathway

Yippee-like 2 (YPEL2) is expressed in tissues and organs enriched in vascular networks, such as heart, kidney, and lung. However, the roles of YPEL2 in endothelial cell senescence and the expression of YPEL2 in atherosclerotic plaques have not yet been investigated. Here, we report the essential role of YPEL2 in promoting senescence in human umbilical vein endothelial cells (HUVECs) and the upregulation of YPEL2 in human atherosclerotic plaques. YPEL2 was significantly upregulated in both H2O2-induced senescent HUVECs and the arteries of aged mice. Endothelial YPEL2 deficiency significantly decreased H2O2-increased senescence-associated beta-galactosidase (SA-β-gal) activity and reversed H2O2-inhibited cell viability. Additionally, endothelial YPEL2 knockdown reduced H2O2-promoted THP-1 cell adhesion to HUVECs and downregulated ICAM1 and VCAM1 expression. Mechanistic studies divulged that the p53/p21 pathway was involved in YPEL2-induced cellular senescence. We conclude that YPEL2 promotes cellular senescence via the p53/p21 pathway and that YPEL2 expression is elevated in atherosclerosis. These findings reveal YPEL2 as a potential therapeutic target in aging-associated diseases.