Carotid Endarterectomy Samples Research Articles

Multi-omic Landscape of Extracellular Vesicles in Human Carotid Atherosclerotic Plaque Reveals Endothelial Communication Networks.

Carotid atherosclerosis is a multifaceted disease orchestrated by a myriad of cell-cell communication that drives progression along a clinical continuum (asymptomatic to symptomatic). Extracellular vesicles (EVs) are lipid bilayer membrane-enclosed cell-derived nanoparticles that represent a new paradigm in cellular communication. Little is known about their biological cargo, cellular origin/destination, and functional roles in human atherosclerotic plaque. EVs were enriched via size exclusion chromatography from human carotid endarterectomy samples dissected into plaque and marginal zones (n= 29 patients, paired plaque and marginal zone; symptomatic n=16, asymptomatic n=13), with further density gradient ultracentrifugation for proteomic analysis. EV cargoes were assessed via whole transcriptome miRNA sequencing and mass spectrometry-based proteomics. EV multi-omics were integrated with publicly available bulk and single cell RNA-sequencing (scRNA-seq) datasets to predict EV cellular origin and ligand-receptor interactions and multi-modal biological network integration of EV-cargo was completed. EV functional impact was assessed with endothelial angiogenesis assays. Human carotid plaques contained greater quantities of EVs than adjacent marginal zones. EV-miRNA and protein content was different in diseased plaque versus adjacent marginal zones, with differential functions in key atherogenic pathways. EV cellular origin analysis suggested that tissue EV signatures originated from endothelial cells (EC), smooth muscle cells (SMC), and immune cells. Furthermore, EV signatures from SMCs and immune cells were most enriched in the marginal and plaque zones, respectively. Integrated tissue vesiculomics and scRNA-seq indicated complex EV-vascular cell communication strategies that changed with disease progression and plaque vulnerability (i.e., symptomatic disease). Plaques from symptomatic patients, but not asymptomatic patients, were characterized by increased involvement of endothelial pathways and more complex ligand-receptor interactions, relative to their marginal zones. Plaque-EVs were predicted to mediate communication with ECs. Pathway enrichment analysis delineated a strong endothelial signature with potential roles in angiogenesis and neovascularization - well-known indices of plaque instability. This was corroborated functionally, wherein human carotid symptomatic plaque EVs induced sprouting angiogenesis in comparison to their matched marginal zones. Our findings indicate that EVs may drive dynamic changes in plaques through EV-vascular cell communication and effector functions that typify vulnerability to rupture, precipitating symptomatic disease. The discovery of endothelial-directed processes mediated by EVs creates new avenues for novel therapeutics in atherosclerosis.

Thrombus Aspirates From Patients With Acute Ischemic Stroke Are Infiltrated by Viridans Streptococci.

Acute ischemic stroke may be due to embolism from ruptured atherosclerotic carotid arteries. DNA of oral bacteria, mainly the viridans streptococci group, has been detected in thrombus aspirates of patients with ischemic stroke as well as in carotid endarterectomy samples. Because viridans streptococci are known to possess thrombogenic properties, we studied whether their presence in thrombus aspirates and in carotid artery specimens can be confirmed using bacterial immunohistochemistry. Thrombus aspirates from 61 patients with ischemic stroke (70.5% men; mean age, 66.8 years) treated with mechanical thrombectomy, as well as carotid endarterectomy samples from 20 symptomatic patients (65.0% men; mean age, 66.2 years) and 48 carotid artery samples from nonstroke autopsy cases (62.5% men; mean age, 66.4 years), were immunostained with an antibody cocktail against 3 species (Streptococcus sanguinis, Streptococcus mitis, and Streptococcus gordonii) of viridans streptococci. Of the thrombus aspirates, 84.8% were immunopositive for viridans streptococci group bacteria, as were 80.0% of the carotid endarterectomy samples, whereas immunopositivity was observed in 31.3% of the carotid artery samples from nonstroke autopsies. Most streptococci were detected inside neutrophil granulocytes, but there were also remnants of bacterial biofilm as well as free bacterial infiltrates in some samples. Oral streptococci were found in aspirated thrombi of patients with acute ischemic stroke as well as in carotid artery samples. Our results suggest that viridans streptococci group bacteria may play a role in the pathophysiology of ischemic stroke.

Proteomic Atlas of Atherosclerosis: The Contribution of Proteoglycans to Sex Differences, Plaque Phenotypes, and Outcomes.

Using proteomics, we aimed to reveal molecular types of human atherosclerotic lesions and study their associations with histology, imaging, and cardiovascular outcomes. Two hundred nineteen carotid endarterectomy samples were procured from 120 patients. A sequential protein extraction protocol was employed in conjunction with multiplexed, discovery proteomics. To focus on extracellular proteins, parallel reaction monitoring was employed for targeted proteomics. Proteomic signatures were integrated with bulk, single-cell, and spatial RNA-sequencing data, and validated in 200 patients from the Athero-Express Biobank study. This extensive proteomics analysis identified plaque inflammation and calcification signatures, which were inversely correlated and validated using targeted proteomics. The inflammation signature was characterized by the presence of neutrophil-derived proteins, such as S100A8/9 (calprotectin) and myeloperoxidase, whereas the calcification signature included fetuin-A, osteopontin, and gamma-carboxylated proteins. The proteomics data also revealed sex differences in atherosclerosis, with large-aggregating proteoglycans versican and aggrecan being more abundant in females and exhibiting an inverse correlation with estradiol levels. The integration of RNA-sequencing data attributed the inflammation signature predominantly to neutrophils and macrophages, and the calcification and sex signatures to smooth muscle cells, except for certain plasma proteins that were not expressed but retained in plaques, such as fetuin-A. Dimensionality reduction and machine learning techniques were applied to identify 4 distinct plaque phenotypes based on proteomics data. A protein signature of 4 key proteins (calponin, protein C, serpin H1, and versican) predicted future cardiovascular mortality with an area under the curve of 75% and 67.5% in the discovery and validation cohort, respectively, surpassing the prognostic performance of imaging and histology. Plaque proteomics redefined clinically relevant patient groups with distinct outcomes, identifying subgroups of male and female patients with elevated risk of future cardiovascular events.

Sensitive magnetic particle imaging of haemoglobin degradation for the detection and monitoring of intraplaque haemorrhage in atherosclerosis.

Intraplaque haemorrhage (IPH) drives atherosclerosis progression and is a key imaging biomarker of unstable plaques. Non-invasive and sensitive monitoring of IPH is challenging due to the compositional complexity and dynamic nature of atherosclerotic plaques. Magnetic particle imaging (MPI) is a highly sensitive, radiation-free, and no-tissue-background tomographic technique that detects superparamagnetic nanoparticles. Thus, we aimed to investigate whether MPI can invivo detect and monitor IPH. Thirty human carotid endarterectomy samples were collected and scanned with MPI. The tandem stenosis (TS) model was employed to establish unstable plaques with IPH in ApoE-/- mice. MPI and 7TT1-weighted magnetic resonance imaging (MRI) were performed on TS ApoE-/- mice. Plaque specimens were analyzed histologically. Human carotid endarterectomy samples exhibited endogenous MPI signals, which histologically colocalized with IPH. Invitro experiments identified haemosiderin, a haemoglobin degradation product, as a potential source of MPI signals. Longitudinal MPI of TS ApoE-/- mice detected IPH at unstable plaques, of which MPI signal-to-noise ratio values increased from 6.43±1.74 (four weeks) to 10.55±2.30 (seven weeks) and reduced to 7.23±1.44 (eleven weeks). In contrast, 7TT1-weighted MRI did not detect the small-size IPH (329.91±226.82μm2) at four weeks post-TS. The time-course changes in IPH were shown to correlate with neovessel permeability providing a possible mechanism for signal changes over time. MPI is a highly sensitive imaging technology that allows the identification of atherosclerotic plaques with IPH and may help detect and monitor unstable plaques in patients. This work was supported in part by the Beijing Natural Science Foundation under Grant JQ22023; the National Key Research and Development Program of China under Grant 2017YFA0700401; the National Natural Science Foundation of China under Grant 62027901, 81827808, 81730050, 81870178, 81800221, 81527805, and 81671851; the CAS Youth Innovation Promotion Association under Grant Y2022055 and CAS Key Technology Talent Program; and the Project of High-Level Talents Team Introduction in Zhuhai City (Zhuhai HLHPTP201703).

Blind spectral unmixing for characterization of plaque composition based on multispectral photoacoustic imaging

To improve the assessment of carotid plaque vulnerability, a comprehensive characterization of their composition is paramount. Multispectral photoacoustic imaging (MSPAI) can provide plaque composition based on their absorption spectra. However, although various spectral unmixing methods have been developed to characterize different tissue constituents, plaque analysis remains a challenge since its composition is highly complex and diverse. In this study, we employed an adapted piecewise convex multiple-model endmember detection method to identify carotid plaque constituents. Additionally, we explore the selection of the imaging wavelengths in linear models by conditioning the coefficient matrix and its synergy with our unmixing approach. We verified our method using plaque mimicking phantoms and performed ex-vivo MSPAI on carotid endarterectomy samples in a spectral range from 500 to 1300 nm to identify the main spectral features of plaque materials for vulnerability assessment. After imaging, the samples were processed for histological analysis to validate the photoacoustic decomposition. Results show that our approach can perform spectral unmixing and classification of highly heterogeneous biological samples without requiring an extensive fluence correction, enabling the identification of relevant components to assess plaque vulnerability.

Validating human and mouse tissues commonly used in atherosclerosis research with coronary and aortic reference tissue: similarities but profound differences in disease initiation and plaque stability

ObjectiveCharacterization of the atherosclerotic process fully relies on histological evaluation and staging through a consensus grading system. So far, a head-to-head comparison of atherosclerotic process in experimental models and tissue resources commonly applied in atherosclerosis research with the actual human atherosclerotic process is missing. Material and MethodsAspects of the atherosclerotic process present in established murine atherosclerosis models and human carotid endarterectomy specimen were systematically graded using the modified American Heart Association histological classification (Virmani classification). Aspects were aligned with the atherosclerotic process observed in human coronary artery and aortic atherosclerosis reference tissues that were available through biobanks based on human tissue/organ donor material. ResultsApart from absent intraplaque hemorrhages in aortic lesions, the histological characteristics of the different stages of human coronary and aortic atherosclerosis are similar. Carotid endarterectomy samples all represent end-stage “fibrous calcified plaque” lesions, although secondary, progressive, and vulnerable lesions with gross morphologies similar to coronary/aortic lesions occasionally present along the primary lesions. For the murine lesions, clear histological parallels were observed for the intermediate lesion types (“pathological intimal thickening,” and “early fibroatheroma”). However, none of the murine lesions studied progressed to an equivalent of late fibroatheroma or beyond. Notable contrasts were observed for disease initiation: whereas disease initiation in humans is characterized by a mesenchymal cell influx in the intima, the earliest murine lesions are exclusively intimal, with subendothelial accumulation foam cells. A mesenchymal (and medial) response are absent. In fact, it is concluded that the stage of “adaptive intimal thickening” is absent in all mouse models included in this study. ConclusionsThe Virmani classification for coronary atherosclerosis can be applied for systematically grading experimental and clinical atherosclerosis. Application of this histological grading tool shows clear parallels for intermediate human and murine atherosclerotic lesions. However, clear contrasts are observed for disease initiation, and late stage atherosclerotic lesions. Carotid endarterectomy all represent end-stage fibrous calcified plaque lesions, although secondary earlier lesions may present in a subset of samples.

A Plaque Instability Index Calculated by Histological Marker Analysis of the Endarterectomy Carotid Artery

(1) Background: Atherosclerosis is a pandemic condition that causes the development of ischemic syndromes including myocardial infarctions and many strokes, in addition to disabling peripheral artery disease. Progression of atheroma plaques has been associated with an increased risk of mortality. It is a challenge to determine in advance if atherosclerotic plaque will become unstable by calculating an index of instability. We proposed a score of ten parameters for the assessment of high-risk plaques, by quantification of 10 risk factors for acute cardiovascular events, most of them representing histological variables. (2) Methods: Carotid endarterectomy samples were collected from 10 highly symptomatic patients who matched for gender, age, risk factors, and plaque morphology. Samples were stained with hematoxylin-eosin, elastic van Gieson, Perls, and Mallory. Immunohistochemistry was performed using specific antibodies, such as CD31 for endothelial cells, CD68 for macrophages, and CD3 for T cells. (3) Results: For each plaque, the presence and/or number of histological features (fibrous cap thickness, lipid core size, plaque and cap infiltration with macrophages and lymphocytes, neovessels, intraplaque hemorrhage, parietal thrombi, and calcium deposits) were recorded on a simple semi-quantitative one- or two-grade scale. The study identified four stable plaques (SPs), two vulnerable plaques (VPs), and four unstable plaques (USPs). We found significantly more macrophages and neovessels in unstable plaques compared with stable plaques. The score for unstable plaques was higher than that for VP. (4) Conclusions: The study showed that symptomatic carotid disease is associated with an increased index score. The proposed algorithm for carotid plaque assessment may be useful for an imaging application.

Differential Gene Expression and Immune Cell Infiltration in Carotid Intraplaque Hemorrhage Identified Using Integrated Bioinformatics Analysis.

BackgroundIntraplaque hemorrhage (IPH) is an important feature of unstable plaques and an independent risk factor for cardiovascular events. However, the molecular mechanisms contributing to IPH are incompletely characterized. We aimed to identify novel biomarkers and interventional targets for IPH and to characterize the role of immune cells in IPH pathogenesis.MethodsThe microarray dataset GSE163154 which contain IPH and non-IPH plaque samples was obtained from the Gene Expression Omnibus (GEO). R software was adopted for identifying differentially expressed genes (DEGs) and conducting functional investigation. The hub genes were carried by protein-protein interaction (PPI) network and were validated by the GSE120521 dataset. CIBERSORT deconvolution was used to determine differential immune cell infiltration and the relationship of immune cells and hub genes. We confirmed expression of proteins encoded by the hub genes by immunohistochemistry and western blotting in 8 human carotid endarterectomy samples with IPH and 8 samples without IPH (non-IPH).ResultsWe detected a total of 438 differentially expressed genes (DEGs), of which 248 were upregulated and 190 were downregulated. DEGs were mainly involved in inflammatory related pathways, including neutrophil activation, neutrophil degranulation, neutrophil-mediated immunity, leukocyte chemotaxis, and lysosomes. The hub genes found through the method of degree in the PPI network showed that ITGB2 and ITGAM might play an important role in IPH. Receiver operating characteristic (ROC) results also showed a good performance of these two genes in the test and validation dataset. We found that the proportions of infiltrating immune cells in IPH and non-IPH samples differed, especially in terms of M0 and M2 macrophages. Immunohistochemistry and western blotting analysis showed that expression levels of ITGB2 and ITGAM increased significantly in carotid atherosclerotic plaques with IPH.ConclusionITGB2 and ITGAM are key hub genes of IPH and may play an important role in the biological process of IPH. Our findings advance our understanding of the underlying mechanisms of IPH pathogenesis and provide valuable information and directions for future research into novel targets for IPH diagnosis and immunotherapy.

Increased frequency of proangiogenic tunica intima endothelial kinase 2 (Tie2) expressing monocytes in individuals with type 2 diabetes mellitus

BackgroundIndividuals with type 2 diabetes mellitus (T2DM) have an increased risk for developing macrovascular disease (MVD) manifested by atherosclerosis. Phenotypically and functionally different monocyte subsets (classical; CD14++CD16−, non-classical; CD14+CD16++, and intermediate; CD14++CD16+) including pro-angiogenic monocytes expressing Tie2 (TEMs) can be identified. Here we investigated monocyte heterogeneity and its association with T2DM and MVD.MethodsIndividuals with (N = 51) and without (N = 56) T2DM were recruited and allocated to "non-MVD" or "with MVD" (i.e., peripheral or coronary artery disease) subgroups. Blood monocyte subsets were quantified based on CD14, CD16 and Tie2 expression levels. Plasma levels of Tie2-ligands angiopoietin-1 and angiopoietin-2 were determined using ELISA. Carotid endarterectomy samples from individuals with (N = 24) and without (N = 22) T2DM were stained for intraplaque CD68+ macrophages (inflammation) and CD34+ (angiogenesis), as plaque vulnerability markers.ResultsMonocyte counts were similar between individuals with T2DM and healthy controls (non-diabetic, non-MVD). Non-classical monocytes were reduced (p < 0.05) in T2DM, whereas the percentage of TEMs within the intermediate subset was increased (p < 0.05). T2DM was associated with increased angiopoietin-1 (p < 0.05) and angiopoietin-2 (p = 0.0001) levels. Angiopoietin-2 levels were higher in T2DM individuals with MVD compared with non-MVD (p < 0.01). Endarterectomized plaques showed no differences in macrophage influx and microvessel number between individuals with and without T2DM.ConclusionsMonocyte subset distribution is altered in T2DM with reduced non-classical monocytes and increased TEM percentage in the intermediate monocyte subset. Increased angiopoietin-2 levels together with increased frequency of TEMs might promote plaque vulnerability in T2DM which could however not be confirmed at tissue level in advanced atherosclerotic lesions.

A novel anti-inflammatory role links the CARS2 locus to protection from coronary artery disease

Background and aimsGenome-wide association studies (GWAS) identified a coronary artery disease (CAD) risk locus on 13.q34 tagged by rs61969072 (T/G). This variant lies in an intergenic region, proximal to ING1, CARKD and CARS2 but its causal relationship to CAD is unknown. Methods and ResultsWe first demonstrated that rs61969072 and tightly linked single nucleotide polymorphisms (SNPs) associate with CARS2 but not ING1 or CARKD expression in carotid endarterectomy samples, with reduced CARS2 abundance in carriers of the CAD risk allele (G). THP-1 monocytes were differentiated and polarized to proinflammatory (M1) and anti-inflammatory (M2) macrophages. CARS2 gene expression decreased in M1 and increased in M2 macrophages, consistent with a role for CARS2 in inflammation. Gene expression profiling revealed an increase in pro-inflammatory markers in response to CARS2 siRNA knockdown in THP-1 derived macrophages, accompanied by an increased abundance of inflammatory cytokines in the cell supernatant. Functional enrichment analysis of impacted transcripts identified the anti-inflammatory IL10 signalling pathway. Western blot analysis of CARS2 silenced macrophages revealed reduced STAT3 phosphorylation in response to IL-10 and increased expression of LPS-induced genes that are repressed by IL-10, indicating a role for CARS2 in anti-inflammatory signalling. Finally, to simulate vessel wall conditions, macrophages, and smooth muscle cells (SMC) were maintained in co-culture. Significantly, CARS2 silencing in macrophages altered the SMC phenotype, decreasing expression of contractile genes and increasing expression of inflammatory genes. ConclusionsThese data highlight a novel anti-inflammatory novel role for CARS2 in human macrophages and SMCs that may underlie the protective effect of a common GWAS-identified variant.

The Correlation Between Wall Shear Stress and Plaque Composition in Advanced Human Carotid Atherosclerosis.

The role of wall shear stress (WSS) in atherosclerotic plaque development is evident, but the relation between WSS and plaque composition in advanced atherosclerosis, potentially resulting in plaque destabilization, is a topic of discussion. Using our previously developed image registration pipeline, we investigated the relation between two WSS metrics, time-averaged WSS (TAWSS) and the oscillatory shear index (OSI), and the local histologically determined plaque composition in a set of advanced human carotid plaques. Our dataset of 11 carotid endarterectomy samples yielded 87 histological cross-sections, which yielded 511 radial bins for analysis. Both TAWSS and OSI values were subdivided into patient-specific low, mid, and high tertiles. This cross-sectional study shows that necrotic core (NC) size and macrophage area are significantly larger in areas exposed to high TAWSS or low OSI. Local TAWSS and OSI tertile values were generally inversely related, as described in the literature, but other combinations were also found. Investigating the relation between plaque vulnerability features and different combinations of TAWSS and OSI tertile values revealed a significantly larger cap thickness in areas exposed to both low TAWSS and low OSI. In conclusion, our study confirmed previous findings, correlating high TAWSS to larger macrophage areas and necrotic core sizes. In addition, our study demonstrated new relations, correlating low OSI to larger macrophage areas, and a combination of low TAWSS and low OSI to larger cap thickness.

Imaging inflammation in atherosclerotic plaques, targeting SST2 with [111In]In-DOTA-JR11

BackgroundImaging Somatostatin Subtype Receptor 2 (SST2) expressing macrophages by [DOTA,Tyr3]-octreotate (DOTATATE) has proven successful for plaque detection. DOTA-JR11 is a SST2 targeting ligand with a five times higher tumor uptake than DOTATATE, and holds promise to improve plaque imaging. The aim of this study was to evaluate the potential of DOTA-JR11 for plaque detection. Methods and ResultsAtherosclerotic ApoE−/− mice (n = 22) fed an atherogenic diet were imaged by SPECT/CT two hours post injection of [111In]In-DOTA-JR11 (~ 200 pmol, ~ 50 MBq). In vivo plaque uptake of [111In]In-DOTA-JR11 was visible in all mice, with a target-to-background-ratio (TBR) of 2.23 ± 0.35. Post-mortem scans after thymectomy and ex vivo scans of the arteries after excision of the arteries confirmed plaque uptake of the radioligand with TBRs of 2.46 ± 0.52 and 3.43 ± 1.45 respectively. Oil red O lipid-staining and ex vivo autoradiography of excised arteries showed [111In]In-DOTA-JR11 uptake at plaque locations. Histological processing showed CD68 (macrophages) and SST2 expressing cells in plaques. SPECT/CT, in vitro autoradiography and immunohistochemistry performed on slices of a human carotid endarterectomy sample showed [111In]In-DOTA-JR11 uptake at plaque locations containing CD68 and SST2 expressing cells. ConclusionsThe results of this study indicate DOTA-JR11 as a promising ligand for visualization of atherosclerotic plaque inflammation.

Abstract 10227: High-Sensitive Magnetic Particle Imaging of Intraplaque Hemorrhage for Identification of Unstable Atherosclerotic Plaques

Introduction: Intraplaque hemorrhage (IPH), which is believed to arise from immature neovessels, drives atherosclerosis progression and is an imaging predictor of stroke and myocardial infarction. However, detection of IPH using magnetic resonance imaging (MRI) might be limited due to false signals from varied plaque components. Magnetic particle imaging (MPI) is a novel tomographic method that directly images the magnetization of ferromagnetic particles. Aims: To test whether MPI can image IPH for detecting unstable plaques and exploring the mechanism of IPH. Methods: The tandem stenosis (TS) mouse model was used to establish unstable plaques with IPH. The intervention agent used for reducing IPH was 4-ABAH. TS ApoE -/- mice and human carotid endarterectomy samples were scanned with MPI and computed tomography. IPH was confirmed by fat-suppressed T1-weighted MRI and Prussian blue staining. Plaque specimens were analyzed histologically. Results: Human carotid samples exhibited endogenous MPI signals, which were related to IPH. Hemosiderin was likely the source of MPI signals. Seven weeks post-TS, unstable plaques in the right carotid artery showed endogenous MPI signals, which were three-fold greater compared with stable plaques in the brachiocephalic artery or normal arterial wall (P &lt; 0.05). From the temporal points, MPI signals in unstable plaques increased from 3.05 ± 1.45 (4 weeks) to 7.29 ± 4.47 (7 weeks) and reduced to 2.59 ± 0.84 (11 weeks) (P &lt; 0.05), while intraplaque neovessel densities showed no difference. Compared with 4 or 7 weeks post-TS, intraplaque neovessels at 11 weeks revealed a more mature phenotype indicated by increased neovessel lumen areas and VE-cadherin (endothelial junction marker) expressions (P &lt; 0.05). Administered 4-ABAH attenuated MPI signals in unstable plaques (9.38 ± 6.75 vs. 3.11 ± 1.25, P &lt; 0.05) and downregulated vWF (endothelial damage marker) expressions (P &lt; 0.05) without affecting neovessel densities or lumen areas. Conclusions: MPI allows ex vivo IPH imaging in human carotid tissues, in vivo IPH monitoring and unstable plaque detection in ApoE -/- mice. MPI-detected IPH is likely associated with endothelial barrier function. As a result, MPI is a promising method for detecting unstable plaques in patients.

Association between Oral Pathology, Carotid Stenosis, and Oral Bacterial DNA in Cerebral Thrombi of Patients with Stroke.

Methods Thrombus aspirates and control arterial blood were taken from 71 patients (70.4% male; mean age, 67.4 years) with acute ischemic stroke. Tooth pathology was registered using CT scans. Carotid stenosis was estimated with CTA and ultrasonography. The presence of bacterial DNA from aspirated thrombi was determined using quantitative PCR. We also analyzed the presence of these bacterial DNAs in carotid endarterectomies from patients with peripheral arterial disease. Results Bacterial DNA was found in 59 (83.1%) of the thrombus aspirates (median, 8.6-fold). Oral streptococcal DNA was found in 56 (78.9%) of the thrombus aspirates (median, 5.1-fold). DNA from A. actinomycetemcomitans and P. gingivalis was not found. Most patients suffered from poor oral health and had in median 19.0 teeth left. Paradoxically, patients with better oral health had more oral streptococcal DNA in their thrombus than the group with the worst pathology (p = 0.028). There was a trend (OR 7.122; p = 0.083) in the association of ≥50% carotid artery stenosis with more severe dental pathology. Oral streptococcal DNA was detected in 2/6 of carotid endarterectomies. Conclusions Stroke patients had poor oral health which tended to associate with their carotid artery stenosis. Although oral streptococcal DNA was found in thrombus aspirates and carotid endarterectomy samples, the amount of oral streptococcal DNA in thrombus aspirates was the lowest among those with the most severe oral pathology. These results suggest that the association between poor oral health and acute ischemic stroke is linked to carotid artery atherosclerosis.

Cortical inflammation and brain signs of high-risk atherosclerosis in a non-human primate model.

Atherosclerosis is a chronic systemic inflammatory disease, inducing cardiovascular and cerebrovascular acute events. A role of neuroinflammation is suspected, but not yet investigated in the gyrencephalic brain and the related activity at blood−brain interfaces is unknown. A non-human primate model of advanced atherosclerosis was first established using longitudinal blood samples, multimodal imaging and gene analysis in aged animals. Non-human primate carotid lesions were compared with human carotid endarterectomy samples. During the whole-body imaging session, imaging of neuroinflammation and choroid plexus function was performed. Advanced plaques were present in multiple sites, premature deaths occurred and downstream lesions (myocardial fibrosis, lacunar stroke) were present in this model. Vascular lesions were similar to in humans: high plaque activity on PET and MRI imaging and systemic inflammation (high plasma C-reactive protein levels: 42 ± 14 µg/ml). We also found the same gene association (metabolic, inflammatory and anti-inflammatory markers) as in patients with similar histological features. Metabolic imaging localized abnormal brain glucose metabolism in the frontal cortex. It corresponded to cortical neuro-inflammation (PET imaging) that correlated with C-reactive protein level. Multimodal imaging also revealed pronounced choroid plexus function impairment in aging atherosclerotic non-human primates. In conclusion, multimodal whole-body inflammation exploration at the vascular level and blood−brain interfaces identified high-risk aging atherosclerosis. These results open the way for systemic and central inflammation targeting in atherosclerosis in the new era of immunotherapy.

Stem Cell Pluripotency Genes Klf4 and Oct4 Regulate Complex SMC Phenotypic Changes Critical in Late-Stage Atherosclerotic Lesion Pathogenesis.

Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.

CT-pathologic correlation of non-calcified atherosclerotic arterial plaques: a study using carotid endarterectomy specimens

Pathologic features of atherosclerotic plaques on CT are not established. We compared CT values among pathologically confirmed plaque constituents and evaluated their ability to distinguish plaque constituents. 50 histopathological images of carotid endarterectomy samples from 10 males and 2 females (age 54-74 years, average 65.9 years) were examined. We compared pre-operative CT [pre-contrast (CT-P), early post-contrast phase (CT-E), delayed post-contrast phase (CT-D)] of lipid-rich necrotic core (NC) and fibrous tissue (F) plaque components with pathological images. The ability of features to differentiate plaque components using several discrimination techniques were compared. CT values of NC and F were 36 ± 13, 45 ± 11 (mean ± standard deviation, Hounsfield unit, HU), 41 ± 17, 69 ± 18, and 44 ± 16, 70 ± 13 in CT-P (p < 0.01), CT-E (p < 0.0001), and CT-D (p < 0.0001), respectively. The threshold, sensitivity, and accuracy for distinguishing NC from F were 44 HU, 74%, and 68%; 55 HU, 85%, and 85%; and 63 HU, 92%, and 84% in CTP, CT-E, and CT-D, respectively. CT-P had lower accuracy than CT-E and CT-D (both p < 0.05), but CT-E and CT-D were similar. CT-E and CT-D yielded 90 and 91% sensitivity and accuracy, respectively in linear discrimination analysis. In both pre- and post-contrast CT, CT values were lower in NC than F. Although values overlapped, using two-phase post-contrast CTs improved discrimination ability. Our findings may help to establish computer-aided diagnosis of vulnerable atherosclerotic plaques in future.

In vivo alpha-V beta-3 integrin expression in human aortic atherosclerosis

ObjectivesIntraplaque angiogenesis and inflammation are key promoters of atherosclerosis and are mediated by the alpha-V beta-3 (αvβ3) integrin pathway. We investigated the applicability of the αvβ3-integrin receptor-selective positron emission tomography...

Data Processing Pipeline for Lipid Profiling of Carotid Atherosclerotic Plaque with Mass Spectrometry Imaging

Atherosclerosis is a lipid and inflammation-driven disease of the arteries that is characterized by gradual buildup of plaques in the vascular wall. A so-called vulnerable plaque, consisting of a lipid-rich necrotic core contained by a thin fibrous cap, may rupture and trigger thrombus formation, which can lead to ischemia in the heart (heart attack) or in the brain (stroke). In this study, we present a protocol to investigate the lipid composition of advanced human carotid plaques using matrix-assisted laser desorption ionization (MALDI) mass spectrometry imaging (MSI), providing a framework that should enable the discrimination of vulnerable from stable plaques based on lipid composition. We optimized the tissue preparation and imaging methods by systematically analyzing data from three specimens: two human carotid endarterectomy samples (advanced plaque) and one autopsy sample (early stage plaque). We show a robust data reduction method and evaluate the variability of the endarterectomy samples. We found diacylglycerols to be more abundant in a thrombotic area compared to other plaque areas and could distinguish advanced plaque from early stage plaque based on cholesteryl ester composition. We plan to use this systematic approach to analyze a larger dataset of carotid atherosclerotic plaques.

Functional pathways associated with human carotid atheroma: a proteomics analysis.

Advances in large-scale analysis are becoming very useful in understanding health and disease. Here, we used high-throughput mass spectrometry to identify differentially expressed proteins between early and advanced lesions. Carotid endarterectomy samples were collected and dissected into early and advanced atherosclerotic lesion portions. Proteins were extracted and subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Differentially expressed proteins were identified and verified using multiple reaction monitoring (MRM), on which advanced systems biology and enrichment analyses were performed. The identified proteins were further compared to the transcriptomic data of 32 paired samples obtained from early and advanced atherosclerotic lesions. A total of 95 proteins were upregulated, and 117 proteins were downregulated in advanced lesions compared to early atherosclerotic lesions (p < 0.05). The upregulated proteins were associated with proatherogenic processes, whereas downregulated proteins were involved in extracellular matrix organization and vascular smooth muscle cytoskeleton. Many of the identified proteins were linked to various "upstream regulators", among which TGFβ had the highest connections. Specifically, a total of 19 genes were commonly upregulated, and 30 genes were downregulated at the mRNA and protein levels. These genes were involved in vascular smooth muscle cell activity, for which enriched transcription factors were identified. This study deciphers altered pathways in atherosclerosis and identifies upstream regulators that could be candidate targets for treatment.