Human Carotid Research Articles

Contribution of Endoplasmic Reticulum Stress to the Clinical Instability of Carotid Plaques in Human Carotid Stenosis.

Endoplasmic reticulum (ER) stress is an important process during the progression of atherosclerosis. The aim of this study was to elucidate the association of ER stress and clinical instability of carotid plaque. One hundred ninety-three patients with carotid stenosis undergoing carotid endarterectomies (CEAs) were enrolled. We classified the patients into 3 groups: the asymptomatic, symptomatic, and cTIA (crescendo transient ischemic attack)/SIE (stroke in evolution) groups. Immunohistological staining was performed to assess ER stress and apoptosis. The correlation between ER stress marker expression and clinical instability was analyzed by Tukey-Kramer test and ordinal logistic regression. From the 193 CEAs, 24 asymptomatic plaques and 24 symptomatic plaques were randomly selected, and all 7 plaques in the cTIA/SIE group were selected. Glycophorin A staining demonstrated significant correlation between intraplaque hemorrhage and clinical instability (odds ratio [OR], 1.27; 95%CI, 1.14-1.41). The expression of ER stress markers (glucose-regulated protein 78 [GRP78] and C/EBP homologous protein [CHOP]) exhibited a significant correlation with clinical instability (GRP78: OR, 1.25; 95%CI, 1.14-1.38, CHOP: OR, 1.39; 95%CI, 1.16-1.66). Double-label immunofluorescence demonstrated ER stress markers were detected in CD68-positive cells and smooth muscle actin (SMA)-positive cells. The coexpression of the ER stress markers exhibited a significant correlation with clinical instability (CD68/GRP78: OR, 1.13; 95%CI, 1.05-1.20, CD68/CHOP: OR, 1.092; 95%CI, 1.04-1.14, SMA/CHOP: OR, 1.082; 95%CI, 1.04-1.13). However, the colocalization of CHOP and cleaved caspase-3 (apoptosis marker) did not correlate with clinical instability. These findings indicated that the ER stress pathway may be a potential therapeutic target in the prevention of stroke.

Abstract 10223: Tissue-Entrapped Extracellular Vesicles Modulate Divergent Mechanisms of Cardiovascular Calcification

Introduction: Fewer than 50% of patients develop vascular and valvular calcification, implying differential pathogenesis. Tissue-entrapped extracellular vesicles (EVs) are implicated in mineralization but their contents and functions are unstudied. We investigated entrapped EV cargoes in human cardiovascular disease. Methods: Human carotid endarterectomy specimens and stenotic aortic valves were obtained from 53 patients. Disease stage-specific proteomics was performed on whole tissue (non-diseased/fibrotic/calcified areas). Tissue EVs were enriched by gradient fractionation then underwent proteomics and miRNA-seq. miR targets were predicted by TargetScan, pathway analyses utilized BioCarta/KEGG/Reactome, and protein-protein interaction networks employed STRING. Results: Disease progression drove significant convergence (p<0.0001) of atherosclerotic plaque and valve proteomes (2,318 proteins). 548 and 158 proteins were exclusively altered (q<0.05) by disease in plaques or valves, respectively. Vesicular GO terms increased 2.2x (p<0.01) amongst proteins altered by disease in both tissues (202). Proteomics found 24 EV markers in the low-density fractions of plaques and valves, confirmed by electron microscopy and nanoparticle tracking. EV omics quantified 1,104 proteins and 123 miR cargoes. Networks of proteins and miR targets shared by plaque and valve EVs revealed common regulation of Rho GTPase and MAPK signaling. 179 proteins and 5 miRs were altered between plaque and valve EVs (q<0.05); multi-omics integration found that EVs modulated cellular contraction and p53-mediated transcriptional regulation in plaques and valves, respectively. Conclusions: This first comparative proteomics study of human valves and arteries finds shared EV functionality in both diseases. Using novel means to examine tissue EV molecular cargoes, we also reveal critical divergent tissue-specific roles for EVs in mediating cardiovascular disease.

Transcriptomic and Proteomic Profiling of Human Stable and Unstable Carotid Atherosclerotic Plaques.

Atherosclerosis is a chronic inflammatory disease with high prevalence and mortality. The rupture of atherosclerotic plaque is the main reason for the clinical events caused by atherosclerosis. Making clear the transcriptomic and proteomic profiles between the stabe and unstable atherosclerotic plaques is crucial to prevent the clinical manifestations. In the present study, 5 stable and 5 unstable human carotid atherosclerotic plaques were obtained by carotid endarterectomy. The samples were used for the whole transcriptome sequencing (RNA-Seq) by the Next-Generation Sequencing using the Illumina HiSeq, and for proteome analysis by HPLC-MS/MS. The lncRNA-targeted genes and circRNA-originated genes were identified by analyzing their location and sequence. Gene Ontology and KEGG enrichment was carried out to analyze the functions of differentially expressed RNAs and proteins. The protein-protein interactions (PPI) network was constructed by the online tool STRING. The consistency of transcriptome and proteome were analyzed, and the lncRNA/circRNA-miRNA-mRNA interactions were predicted. As a result, 202 mRNAs, 488 lncRNAs, 91 circRNAs, and 293 proteins were identified to be differentially expressed between stable and unstable atherosclerotic plaques. The 488 lncRNAs might target 381 protein-coding genes by cis-acting mechanisms. Sequence analysis indicated the 91 differentially expressed circRNAs were originated from 97 protein-coding genes. These differentially expressed RNAs and proteins were mainly enriched in the terms of the cellular response to stress or stimulus, the regulation of gene transcription, the immune response, the nervous system functions, the hematologic activities, and the endocrine system. These results were consistent with the previous reported data in the dataset GSE41571. Further analysis identified CD5L, S100A12, CKB (target gene of lncRNA MSTRG.11455.17), CEMIP (target gene of lncRNA MSTRG.12845), and SH3GLB1 (originated gene of hsacirc_000411) to be critical genes in regulating the stability of atherosclerotic plaques. Our results provided a comprehensive transcriptomic and proteomic knowledge on the stability of atherosclerotic plaques.

Self-Attention Based Virtual Staining for Bright-field Images of Label-free Human Carotid Atherosclerotic Plaque Tissue Section.

Histological analysis of carotid atherosclerotic plaque tissue specimens is a widely used method for studying the diagnosis of ischemic heart disease and stroke. Understanding the physiological and pathological mechanisms of carotid atherosclerotic plaque is of great significance for the effective prevention and treatment of plaque formation and rupture. In this work, we adapted a self-attention generative adversarial model to virtually stain label-free human carotid atherosclerotic plaque tissue sections into corresponding H&E stained sections. The self-attention mechanism and multi-layer structure are introduced into the residual steps of the generator and in the discriminator. Our method achieved the best performance (SSIM, PSNR, and LPIPS of 0.53, 20.29, and 0.30, respectively) in comparison with other state-of-the-art methods.Clinical Relevance - The proposed approach allows for the virtual staining of unlabeled human carotid plaque tissue images. It identifies the histopathological features of atherosclerotic plaques in the same tissue sample which could facilitate the development of personalized prevention and other interventional treatments for carotid atherosclerosis.

Development of a Collagen Fibre Remodelling Rupture Risk Metric for Potentially Vulnerable Carotid Artery Atherosclerotic Plaques.

Atherosclerotic plaque rupture in carotid arteries can lead to stroke which is one of the leading causes of death or disability worldwide. The accumulation of atherosclerotic plaque in an artery changes the mechanical properties of the vessel. Whilst healthy arteries can continuously adapt to mechanical loads by remodelling their internal structure, particularly the load-bearing collagen fibres, diseased vessels may have limited remodelling capabilities. In this study, a local stress modulated remodelling algorithm is proposed to explore the mechanical response of arterial tissue to the remodelling of collagen fibres. This stress driven remodelling algorithm is used to predict the optimum distribution of fibres in healthy and diseased human carotid bifurcations obtained using Magnetic Resonance Imaging (MRI). In the models, healthy geometries were segmented into two layers: media and adventitia and diseased into four components: adventitia, media, plaque atheroma and lipid pool (when present in the MRI images). A novel meshing technique for hexahedral meshing of these geometries is also demonstrated. Using the remodelling algorithm, the optimum fibre patterns in various patient specific plaques are identified and the role that deviations from these fibre configurations in plaque vulnerability is shown. This study provides critical insights into the collagen fibre patterns required in carotid artery and plaque tissue to maintain plaque stability.

Integrative single cell RNA-sequencing descrambles a substantial divergence of adaptive immune cell identities and transcriptional programs in mouse and human atherosclerosis

Abstract Aims The distinct function of immune cells in human atherosclerosis has been mostly defined by preclinical mouse studies. Contrastingly, the immune cell composition of human atherosclerotic plaques and their contribution to disease progression is only poorly understood. It remains uncertain whether genetic animal models allow for valuable translational approaches. Methods and results We performed single cell RNA-sequencing (scRNAseq) to define the immune cell landscape in human carotid atherosclerotic plaques. The human immune cell repertoire was dominated by T cells with a considerable inter-patient variability and an unexpected heterogeneity. We performed bioinformatical integration with 7 mouse data sets and discovered a total of 38 cellular identities, of which some were not conserved between species and exclusively found in mice or humans. Locations, frequencies, and transcriptional programs of immune cells in preclinical mouse models did not resemble the immune cell landscape in human atherosclerosis. In contrast to mice, human plaques were not myeloid- and B cell-dominated and instead contained several T cell phenotypes with hallmarks of T cell memory, dysregulation, exhaustion, and activation. Human immune cells were predominantly enriched for transcriptional programs of hypoxia, glucose, and autoimmunity. In a validation cohort of 43 patients activated immune cell subsets defined by multi-colour flow cytometry associated with cerebral ischemia and coronary artery disease. Conclusion Here, we uncover yet undefined immune cell types associating with clinical disease. This leukocyte atlas of human atherosclerosis builds the conceptual basis for subsequent identification of cellular targets for clinical immunomodulatory therapies and risk prediction. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): ERC Starting Grant

Image-to-Images Translation for Multiple Virtual Histological Staining of Unlabeled Human Carotid Atherosclerotic Tissue.

Histological analysis of human carotid atherosclerotic plaques is critical in understanding atherosclerosis biology and developing effective plaque prevention and treatment for ischemic stroke. However, the histological staining process is laborious, tedious, variable, and destructive to the highly valuable atheroma tissue obtained from patients. We proposed a deep learning-based method to simultaneously transfer bright-field microscopic images of unlabeled tissue sections into equivalent multiple sections of the same samples that are virtually stained. Using a pix2pix model, we trained a generative adversarial neural network to achieve image-to-images translation of multiple stains, including hematoxylin and eosin (H&E), picrosirius red (PSR), and Verhoeff van Gieson (EVG) stains. The quantification of evaluation metrics indicated that the proposed approach achieved the best performance in comparison with other state-of-the-art methods. Further blind evaluation by board-certified pathologists demonstrated that the multiple virtual stains have high consistency with standard histological stains. The proposed approach also indicated that the generated histopathological features of atherosclerotic plaques, such as the necrotic core, neovascularization, cholesterol crystals, collagen, and elastic fibers, are optimally matched with those of standard histological stains. The proposed approach allows for the virtual staining of unlabeled human carotid plaque tissue images with multiple types of stains. In addition, it identifies the histopathological features of atherosclerotic plaques in the same tissue sample, which could facilitate the development of personalized prevention and other interventional treatments for carotid atherosclerosis.

Stretch and stress distributions in the human artery based on two-layer model considering residual stresses.

The objective is to know the stress distributions in the arterial walls under residual stresses based on two-layer model. Human common carotid arteries were analysed to show stress distributions at physiological and supraphysiological intraluminal pressures. The analyses for the loaded states were performed with stretch ratios with reference to a Riemannian stress-free configuration which is a 3D non-Euclidean manifold due to the nonzero Riemann curvature tensor. The experimental data obtained by other literature were used for the common carotid arteries to analyse the stretch and stress distributions in the arterial wall although kinematics is different from the literature. The stretches and stresses were calculated for the unloaded state, i.e. the residual stretches and stresses. And those at the axial stretch ratio 1.1 with reference to the unloaded state were calculated at the intraluminal pressures 16, 50, and 100kPa. The stresses increased from the inner surface to the outer surface at all pressures analysed. These results suggest that in the human arteries the mechanical loads are mainly supported with the adventitia even though the media and intima play an important role to control of physiological functions.

In Vivo Human Single-Chain Fragment Variable Phage Display-Assisted Identification of Galectin-3 as a New Biomarker of Atherosclerosis.

BackgroundAtherosclerosis is a complex pathology in which dysfunctional endothelium, activated leucocytes, macrophages, and lipid‐laden foam cells are implicated, and in which plaque disruption is driven by many putative actors. This study aimed to identify accurate targetable biomarkers using new in vivo approaches to propose tools for improved diagnosis and treatment.Methods and ResultsHuman scFv (single‐chain fragment variable) selected by in vivo phage display in a rabbit model of atherosclerosis was reformatted as scFv fused to the scFv‐Fc (single‐chain fragment variable fused to the crystallizable fragment of immunoglobulin G format) antibodies. Their reactivity was tested using flow cytometry and immunoassays, and aorta sections from animal models and human carotid and coronary artery specimens. A pool of atherosclerotic proteins from human endarterectomies was co‐immunoprecipitated with the selected scFv‐Fc followed by mass spectrometry for target identification. Near‐infrared fluorescence imaging was performed in Apoe −/− mice after injection of an Alexa Fluor 647–labeled scFv‐Fc‐2c antibody produced in a baculovirus system with 2 additional cysteine residues (ie, 2c) for future coupling to nano‐objects for theranostic applications. One scFv‐Fc clone (P3) displayed the highest cross‐reactivity against atherosclerotic lesion sections (rabbit, mouse, and human) and was chosen for translational development. Mass spectrometry identified galectin‐3, a β‐galactoside‐binding lectin, as the leader target. ELISA and immunofluorescence assays with a commercial anti‐galectin‐3 antibody confirmed this specificity. P3 scFv‐Fc‐2c specifically targeted atherosclerotic plaques in the Apoe −/− mouse model.ConclusionsThese results provide evidence that the P3 antibody holds great promise for molecular imaging of atherosclerosis and other inflammatory pathologies involving macrophages. Recently, galectin‐3 was proposed as a high‐value biomarker for the assessment of coronary and carotid atherosclerosis.

Endovascular Debulking of Human Carotid Plaques by Using an Excimer Laser Combined With Balloon Angioplasty: An ex vivo Study.

Purpose: We aimed to evaluate the safety and effectiveness of applying an excimer laser in debulking human carotid atherosclerotic plaques by investigating the distal debris, plaque luminal gain, and micromorphology of the plaque surface.Methods: Eighteen plaque samples obtained from carotid endarterectomy (CEA) were randomly allocated to the excimer laser ablation (45 mJ/mm2, 25 Hz) alone group (group 1), balloon angioplasty (8 atm) alone group (group 2), and excimer laser ablation combined with balloon angioplasty group (group 3). Hematoxylin–eosin staining and Movat's pentachrome staining were performed on the collected particles to quantify the size and composition of the debris. The superficial micromorphological structure of the plaque lumen surface after device treatments was observed using a scanning electron microscope. Micro-CT, tissue sections, and pathological stainings were applied to the treated plaques. The plaque lumen and artery lumen were three-dimensionally reconstructed using clinical computed tomography angiography and the micro-CT images. Lumen enlargement was set as the main measurement of effectiveness.Results: Group 3 produced the highest luminal gain (5.40 ± 4.51 mm2), while the other two groups had gains of 4.05 ± 3.20 and 3.77 ± 2.55 mm2. Both devices caused disruptions to the plaque lumen surface. Laser ablation exposed the fibers under the endothelium and balloon angioplasty cracked the surface. The mean amounts were 3,611 ± 1,475.4 for group 1, 2,828 ± 1,266.7 for group 2, and 4,400 ± 2,567.9 for group 3. More than 90% of the distal debris was smaller than 10 μm. Group 2 produced the most debris with Feret (maximum caliper diameter) ≥ 40 μm; group 1 had the least. There was little difference in the contents of collagen and reticular fiber in the debris in each group, but a big difference was observed in the contents of fibrin and mucin.Conclusion: Excimer laser ablation could significantly increase the luminal gain of carotid plaque with high stenosis. Excimer laser combined with balloon angioplasty achieved the highest lumen enlargement. Our result also suggests that the embolic protection strategy needs to be renewed for the application of a plaque debulking device in the future.

Distribution of neuronal cells which contain dopamine β-hydroxylase, tyrosine hydroxylase, neuropeptide Y and vasoactive intestinal polypeptide in the human internal carotid nerve.

The human internal carotid nerve (ICN) occasionally has a swelling beneath the external opening of the carotid canal. In this study, the presence and distribution of neuronal cells were investigated in the bilateral ICNs of nine human cadavers. Among 44.4% of the cadavers, swellings were detected in the ICN. Their diameters ranged from 1.7 to 3.6 mm (average ±SD= 2.6± 0.7 mm). Thirty-eight percent of these swellings were large (diameter > 3 mm) and showed an oval shape. The large swelling contained many neuronal cells. However, the ICNs with or without a swelling <3 mm diameter were mostly free from neuronal cells (93.3%). Only in one human cadaver, the right ICN without a swelling had a small number of neuronal cells. By the present immunohistochemical method, ICN neurons contained catecholamine-synthesizing enzymes and neuropeptides. Dopamine-beta hydroxylase- and tyrosine hydroxylase-immunoreactivity were mostly expressed by ICN neurons. More than half of them also contained neuropeptide Y-immunoreactivity. However, vasoactive intestinal polypeptide-immunoreactive ICN neurons were relatively infrequent. Substance P- and calcitonin gene-related peptide-immunoreactive ICN neurons could not be detected. By the cell size analysis, neuropeptide Y-immunoreactive neurons were significantly smaller than neuropeptide Y-immunonegative neurons in the ICN. The present study suggests that ICN neurons have a sympathetic function in the human.

Mast Cell Distribution in Human Carotid Atherosclerotic Plaque Differs Significantly by Histological Segment

Mast cells (MCs) are important contributors to atherosclerotic plaque progression. For prospective studies on mast cell contributions to plaque instability, the distribution of intraplaque MCs needs to be elucidated. Plaque stability is generally histologically assessed by dividing the plaque specimen into segments to be scored on an ordinal scale. However, owing to competitive use, studies may have to deviate to adjacent segments, yet intersegment differences of plaque characteristics, especially MCs, are largely unknown. Therefore, the hypothesis that there is no segment to segment difference in MC distribution between atherosclerotic plaque segments was tested, and intersegment associations between MCs and other plaque characteristics was investigated.Twenty-six carotid atherosclerotic plaques from patients undergoing carotid endarterectomy included in the Athero-Express Biobank were analysed. The plaque was divided in 5 mm segments, differentiating between the culprit lesion (segment 0), adjacent segments (-1/+1) and more distant segments (-2/+2) for the presence of MCs. The associations between the intersegment distribution of MCs and smooth muscle cells, macrophage content, and microvessel density in the culprit lesion were studied.A statistically significant difference in MCs/mm2 between the different plaque segments (p < .001) was found, with a median of 2.79 (interquartile range [IQR] 1.63 - 7.10) for the culprit lesion, 1.34 (IQR 0.26 - 4.45) for the adjacent segment, and 0.62 (0.14 - 2.07) for the more distant segment. Post hoc analyses showed that intersegment differences were due to differences in MCs/mm2 between the culprit and adjacent segment (p = .037) and between the culprit lesion and the more distant segment (p < .001). MCs/mm2 in multiple different segments were positively correlated with microvessel density and macrophage content in the culprit lesion.MC numbers reveal significant intersegment differences in human carotid plaques. Future histological studies on MCs should use a standardised segment for plaque characterisation as plaque segments cannot be used interchangeably for histological MC analyses.

Abstract 112: Examining The Heterogeneity Of Primary Human Macrophages And Pharmacogenomic Networks To Identify Novel Targets For Precision Medicine For Vascular Inflammation

Background: We hypothesize that macrophage heterogeneity is an unexploited source of therapeutic targets for vascular inflammation. Interferon-gamma (IFNγ) stimulated primary human macrophages M(IFNγ) is a widely used in vitro model for proinflammatory macrophages. However, typical activation-induced transcript profiling assumes a homogenous macrophage population. Our goal is to evaluate the extent of heterogeneity of activated macrophages to devise a strategy for precision medicine for inflammatory vascular disease. Methods: Using unbiased single-cell RNA sequencing (scRNA-seq), systems biology, and machine learning, we examined inter-subgroup differences of human primary M(IFNγ) (4 donors). Network analysis, kinetic proteomics, and in vitro assays (n=3-6) characterized the clusters, followed by validation in human carotid atherosclerotic plaques (n=13). scRNAseq data analysis in the L1000 CDS 2 drug-gene network computationally identified drugs that may potentiate or suppress each cluster. Results: The scRNA-seq demonstrated 3 distinct subpopulations: Clusters 1, 2, and 3 (C1, 2, and 3). C3 showed increased proinflammatory chemokine production, protein synthesis, and glycolysis. C1 was more efferocytotic/phagocytic, chemotactic, and less inflammatory. C2 is intermediate between C1 and C3. Histological analysis localized C1 and C3-like macrophages in different areas of the plaques ( Fig. 1A ). In addition, we used targeted scRNAseq (n=4) to analyze M(IFNγ) treated with an L1000-derived drug BI-2536 (Polo-like kinase inhibitor). As predicted, BI-2536 shifted the phenotypic heterogeneity of M(IFNγ) towards less inflammatory characteristics ( Fig. 1B ) which were further validated with bulk qPCR &amp; ELISA (n=8). Conclusion: Our study presents a novel strategy for precision medicine that leverages single-cell data and gene interaction networks to identify modulators of macrophage heterogeneity as new anti-inflammatory therapies.

Function of viperin in human aortic SMCS and its relevance for atherosclerosis

Background and Aims: During atherosclerosis, activated vascular cells secrete chemokines to recruit leukocytes to the subendothelial space. Activated leukocytes, in particular activated T lymphocytes express IFN-γ, which affects the expression of numerous genes, including the antiviral protein viperin, in vascular cells. Viperin is expressed in endothelial cells of human carotid plaques but its relevance for vascular physiology and/or pathophysiology has not been established (Olofsson et al., 2005).

Line-1 methylation in different vessels, vascular and blood cells in human atherosclerosis.

Background and Aims: Decreased LINE-1 methylation was associated with cardiovascular diseases. However, LINE-1 methylation in vascular cells is poorly investigated in atherosclerosis. Here we studied DNA methylation of LINE-1 promoter in human carotid atherosclerotic plaque, intact artery, and vein, as well as in vascular smooth muscle cells (VSMC) and macrophages (MPH) isolated from plaques, and in blood cells.

Diagnostic Accuracy of Optical Frequency Domain Imaging for Identifying Necrotic Cores with Intraplaque Hemorrhage in Advanced Human Carotid Plaques

This study investigated whether optical frequency domain imaging (OFDI) can identify carotid artery vulnerable plaque characteristics, focusing on lipid-rich necrotic core (NC) and intraplaque hemorrhage (IPH). Fourteen patients scheduled for carotid endarterectomy underwent OFDI scan during preoperative angiography. Atherosclerotic plaque specimens obtained from carotid endarterectomy were cut every 3-4 mm into 4-μm transverse cross-sections and stained with standard methods. Each cross-section was matched with OFDI, and histologically classified into either fibrous, calcific, pathological intimal thickening (PIT), and NC. Of 75 histologic cross-sections, 6 were categorized as fibrous (8%), 18 as calcific (24%), 9 as PIT (12%), and 42 as NC (56%). Tissues categorized as NC had significantly higher OFDI signal attenuation rates than the other tissues (p <0.001), followed by PIT, calcific, and fibrous tissues. The receiver operating characteristic analysis indicated that attenuation rates of >0.023 and >0.031 predicted the presence of NC and IPH with high areas under the curve of 0.91 and 0.88, respectively. OFDI provides potential capability for the detection of NCs with IPH of carotid artery plaques by quantitatively analyzing the attenuation rate.

Expression of miRNAs Targeting ATP Binding Cassette Transporter 1 (ABCA1) among Patients with Significant Carotid Artery Stenosis.

Background: Carotid artery stenosis is a dynamic process associated with an increased risk of cardiovascular events. However, knowledge of biomarkers useful for identifying and quantifying high-risk carotid plaques associated with the increased incidence of cerebrovascular events is insufficient. Therefore, the objectives of this study were to evaluate the expression of ATP binding cassette transporter 1 (ABCA1) and validate its target microRNA (miRNA) candidates in human carotid stenosis arteries to identify its potential as a biomarker. Methods: In human carotid stenosis arterial tissues and plasma, the expression of ABCA1 and its target miRNAs (miRNA-33a-5p, 33b-5p, and 148a-3p) were evaluated by quantitative real time-polymerase chain reaction (qRT-PCR), immunohistochemistry, and enzyme-linked immunosorbent assay (ELISA). Results: The expression of ABCA1 was significantly decreased in the plasma of stenosis patients, but its expression was not different in arterial tissues (p < 0.05). However, significantly more target miRNAs were secreted by stenosis patients than normal patients (p < 0.05). Interestingly, lipotoxicity induced by the oleic and palmitic acid (OAPA) or lipopolysaccharide (LPS) treatment of human umbilical vein endothelial cells (HUVECs) dramatically enhanced the gene expression of adipogenic and inflammatory factors, whereas ABCA1 expression was significantly decreased. Conclusions: Therefore, miRNA-33a-5p, 33b-5p, and 148a-3p represent possible biomarkers of carotid artery stenosis by directly targeting ABCA1.

Immunohistochemical Characteristics of the Human Carotid Body in the Antenatal and Postnatal Periods of Development.

The evolutionary and ontogenetic development of the carotid body is still understudied. Research aimed at studying the comparative morphology of the organ at different periods in the individual development of various animal species should play a crucial role in understanding the physiology of the carotid body. However, despite more than two centuries of study, the human carotid body remains poorly understood. There are many knowledge gaps in particular related to the antenatal development of this structure. The aim of our work is to study the morphological and immunohistochemical characteristics of the human carotid body in the antenatal and postnatal periods of development. We investigated the human carotid bodies from 1 embryo, 20 fetuses and 13 adults of different ages using samples obtained at autopsy. Immunohistochemistry revealed expression of βIII-tubulin and tyrosine hydroxylase in the type I cells and nerve fibers at all periods of ontogenesis; synaptophysin and PGP9.5 in the type I cells in some of the antenatal cases and all of the postnatal cases; 200 kDa neurofilaments in nerve fibers in some of the antenatal cases and all of the postnatal cases; and GFAP and S100 in the type II cells and Schwann cells in some of the antenatal cases and all of the postnatal cases. A high level of tyrosine hydroxylase in the type I cells was a distinctive feature of the antenatal carotid bodies. On the contrary, in the type I cells of adults, the expression of tyrosine hydroxylase was significantly lower. Our data suggest that the human carotid body may perform an endocrine function in the antenatal period, while in the postnatal period of development, it loses this function and becomes a chemosensory organ.

Comprehensive analysis of dysregulated genes associated with atherosclerotic plaque destabilization.

Atherosclerotic plaque destabilization is a dominating cause of acute cardiovascular events such as myocardial infarction and stroke. This study aims to identify genetic biomarkers related to atherosclerotic plaque destabilization using bioinformatics. Three transcriptome datasets of human carotid atherosclerotic plaque samples were downloaded from ArrayExpress and Gene Expression Omnibus databases, including E-MATB-2055, E-TABM-190, and GSE120521. With Robust Rank Aggregation analysis, we documented 46 differentially expressed genes between stable and unstable/ruptured plaques. Functional enrichment analysis using DAVID tool demonstrated that these genes were mainly related to biological functions such as extracellular matrix disassembly, collagen catabolic process, response to mechanical stimulus, and PPAR signaling pathway. A protein-protein interaction network for the differentially expressed genes was constructed, and eight pivotal genes (ITGAM, MMP9, PLAUR, CCR1, CD163, CD36, ADAM8, and IL1RN) were obtained from the network with a connective degree > 5. The expression patterns of these hub differentially expressed genes could be verified in atherosclerotic plaque samples with intraplaque hemorrhage. Using gene set variation analysis, the eight genes were integrated to generate an atherosclerotic plaque destabilization score, which showed a high performance in not only discriminating individuals with myocardial infarction from those with stable coronary illness, but also in predicting future acute cardiovascular events in atherosclerotic patients. In conclusion, the findings of this study will enhance our knowledge on the pathological mechanisms involved in atherosclerotic plaque destabilization, and provide potential gene biomarkers for risk stratification of patients with atherosclerotic cardiovascular disease.

Measurement of flow velocity vectors in carotid artery using plane wave imaging with repeated transmit sequence.

Doppler-based methods are widely used for blood flow imaging in clinical settings. However, they inherently estimate the velocity component only in the axial direction. Therefore, various studies of angle-independent methods have been conducted. The multi-angle Doppler method is one such angle-independent method, in which the velocity vector is estimated using axial velocities obtained from multiple directions by steering an ultrasonic beam. Recently, plane wave imaging, which realizes a very high frame rate of several thousand frames per second, was applied to the multi-angle Doppler method. However, the maximum detectable velocity, i.e., the aliasing limit, was reduced depending on the number of steering angles. In the present study, the feasibility of a specific transmit sequence, namely, the repeated transmit sequence, was examined using the plane-wave multi-angle Doppler method. In the repeated transmit sequence, plane waves were emitted to the same direction twice, after which the steering angle was changed. By repeating the same procedure, a pair of beamformed radio-frequency (RF) signals could be obtained under each beam steering angle. By applying the autocorrelation method to each pair of RF signals, the time interval between the RF signals could be kept as the pulse repetition interval (PRI). The feasibility of such a transmit sequence was examined by numerical simulation and in vivo measurement of a human carotid artery. The simulation results showed that the maximum steering angles of over 10 degrees were not feasible with the linear array used in the present study. The feasible maximum steering angle would depend on the element pitch of the probe relative to the ultrasonic wavelength. By limiting the maximum steering angles to 5 and 10 degrees, bias errors were 9.2% and 11.3%, respectively, and root mean squared errors were 21.5% and 16.9%, respectively. Also, flow velocity vectors in a human carotid artery could be visualized with the proposed method. The multi-angle Doppler method was implemented in plane wave imaging with the repeated transmit sequence, and the proposed method was shown to be feasible through numerical simulation and in vivo measurement of a carotid artery.