Endarterectomy Specimens Research Articles

Abstract 10395: Atherosclerotic Lesion in Chronic Thromboembolic Pulmonary Hypertension is Associated With Prolonged Symptom Duration

Background: The cause of atherosclerotic lesions in the pulmonary artery of patients with chronic thromboembolic pulmonary hypertension (CTEPH) remains unclear. We aimed to discuss the clinical characteristics of CTEPH patients with atherosclerotic lesions and further explore the unique metabolic pathway of atherosclerosis in the pulmonary artery. Methods: Following detailed pathological examination of the pulmonary endarterectomy (PEA) specimen, CTEPH patients were divided into two age- and sex-matched groups, those with and without atherosclerotic lesion (n=25 each), and their clinical characteristics were compared. Untargeted metabolomics were used to determine the different metabolites in the proximal lesions of the pulmonary artery. Results: 27.2% (25/92) of all PEA specimens were found with atherosclerotic lesions. Patients with atherosclerotic lesions were more likely to have a history of symptomatic embolism than those without atherosclerotic lesion (88% vs. 56%, P=0.012), including pulmonary embolism and deep venous thrombosis. Patients with atherosclerotic lesions were found to have longer symptom duration (8.0±6.5 vs. 4.7±4.2, P=0.039) and a lower rate of residual pulmonary hypertension after PEA than those without atherosclerotic lesion (40% vs. 56%). However, the existence of atherosclerotic lesion was not associated with pre-operative hemodynamic parameters and cardiac function. Metabolomic profiling revealed that the formation of atherosclerotic lesion in CTEPH is closely related to glycine, serine and threonine metabolic axes involved in cellular aging and energy metabolism. Conclusions: The occurrence of atherosclerotic lesions in the pulmonary artery of CTEPH was associated with symptomatic thromboembolic history and extended symptom duration. The results suggested a new link between atherosclerotic lesions and aberrant amino acid metabolism in the context of CTEPH. Further investigations focused on atherosclerotic lesion and pulmonary vascular remodeling may provide novel insight into the pathophysiology and new therapeutic strategies for CTEPH.

Abstract 9588: Intraplaque Myeloperoxidase Activity is a Hallmark of Vulnerable Atherosclerosis and Correlates With Clinical Outcomes

Introduction: There remains limited means by which to detect unstable atherosclerosis in patients at risk of myocardial infarction and stroke. Myeloperoxidase (MPO) is increased in unstable plaque and causally linked to plaque destabilization and rupture in pre-clinical models. We sought to assess whether MPO activity is greater in vulnerable human carotid and coronary atheroma, and how this relates to adverse cardiovascular events and non-invasive imaging parameters. Methods: Carotid endarterectomy specimens (CEA) were collected from 31 patients. Coronary trees were collected from 12 patients undergoing heart transplantation for ischemic cardiomyopathy. MPO activity was assessed through the conversion of hydroethidine added to tissue sections to the MPO-specific adduct 2-chloroethidium (2-Cl-E + ) and quantified by mass spectrometry. MPO activity was then compared with histologic determination of plaque phenotype and clinical outcomes. MPO activity was additionally assessed in high and low attenuation plaques derived by computed tomography, as well as perivascular adipose tissue (PVAT). Results: Unstable CEA had higher MPO activity when compared with stable CEA plaques (n = 26, 4.2 ± 3.1 vs. 0.2 ± 0.3 μmol/mgp; p < 0.0001). Similar results were seen in coronary segments with unstable compared to stable plaque (n = 17, 0.6 ± 0.5 vs. 0.0 ± 0.0 μmol/mgp; p = 0.0006). When comparing MPO activity in CEA to clinical outcomes, asymptomatic and stroke-free patients had lower MPO activity compared to those with symptoms or ipsilateral stroke on neuroimaging (n = 12, 3.7 ± 2.1 vs. 0.1 ± 0.2 μmol/mgp; p = 0.002). CT-determined plaque attenuation did not identify plaque MPO activity (n = 30, 0.1 ± 0.1 vs. 0.2 ± 0.3 μmol/mgp; p = 0.23) and enzymatically active MPO was not found in PVAT. Conclusion: MPO is active within unstable human carotid and coronary plaques and correlates with symptomatic carotid disease and stroke. In addition, current non-invasive imaging parameters do not selectively identify plaques enriched with active MPO. As intraplaque MPO appears to be a hallmark of unstable atherosclerosis and can potentially be imaged non-invasively, further investigation into its potential utility as a diagnostic tool for high-risk atherosclerosis is warranted.

Integrated single-cell analysis-based classification of vascular mononuclear phagocytes in mouse and human atherosclerosis.

Accumulation of mononuclear phagocytes [monocytes, macrophages, and dendritic cells (DCs)] in the vessel wall is a hallmark of atherosclerosis. Using integrated single-cell analysis of mouse and human atherosclerosis, we here aimed to refine the nomenclature of mononuclear phagocytes in atherosclerotic vessels and to compare their transcriptomic profiles in mouse and human disease. We integrated 12 single-cell RNA-sequencing (scRNA-seq) datasets of immune cells isolated from healthy or atherosclerotic mouse aortas, and data from 11 patients (n = 4 coronary vessels, n = 7 carotid endarterectomy specimens) from two studies. Integration of mouse data identified subpopulations with discrete transcriptomic signatures within previously described populations of aortic resident (Lyve1), inflammatory (Il1b), as well as foamy (Trem2hi) macrophages. We identified unique transcriptomic features distinguishing aortic intimal resident macrophages from atherosclerosis-associated Trem2hi macrophages. Also, populations of Xcr1+ Type 1 classical DCs (cDC1), Cd209a+ cDC2, and mature DCs (Ccr7, Fscn1) with a 'mreg-DC' signature were detected. In humans, we uncovered macrophage and DC populations with gene expression patterns similar to those observed in mice. In particular, core transcripts of the foamy/Trem2hi signature (TREM2, SPP1, GPNMB, CD9) mapped to a specific population of macrophages in human lesions. Comparison of mouse and human data and direct cross-species data integration suggested transcriptionally similar macrophage and DC populations in mice and humans. We refined the nomenclature of mononuclear phagocytes in mouse atherosclerotic vessels, and show conserved transcriptomic features of macrophages and DCs in atherosclerosis in mice and humans, emphasizing the relevance of mouse models to study mononuclear phagocytes in atherosclerosis.

Abstract P2095: Role Of Intraplaque Lipids In Atherosclerotic Plaque Vulnerability

Background: Hyperlipidemia plays a pivotal role in the pathogenesis of diabetes, atherosclerosis, and cardiovascular diseases. Specific lipids of atherosclerotic plaques are potentially one of the local factors which render them susceptible to rupture. Their underlying unresolved inflammation and oxidative stress contribute to the transition from stable to unstable plaques. Therefore, we aimed to identify, characterize, and compare the intraplaque lipids composition among diabetic and non-diabetic patients and to ascertain their role in inflammation, and atherosclerotic plaque destabilization and rupture. Methods: Approved by the IRB, University of Arizona, Tucson Lipids were analyzed by liquid chromatography-mass spectrometry (LC-MS) from 40 surgically removed endarterectomy specimens that include Asymptomatic & Non-diabetic (n=13), Symptomatic & Non-diabetic (n=17), Asymptomatic & Diabetic (n=5) iv. Symptomatic & Diabetic (n=5) patients. Seventy nine lipid species from the main lipid classes phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylinositol (PI), cholesteryl ester (CE) phosphatidylglycerol (PG), sphingomyelin (SM), and ceramide (Cer) were included in this lipidomic profiling. Results: LC-MS data analysis identified a significant difference (p<0.05) in 2 individual lipid species – a long chain polyunsaturated PC(40:7) and SM(18:1) between diabetic and non-diabetic (Table:1). A total of 23 lipid species were significantly (p<0.05 to p<0.005) differentially present between symptomatic and asymptomatic samples. Symptomatic diabetic and nondiabetic samples showed a significant (p<0.05 to p<0.005) difference in 13 lipids belonging to PC, SM and PE. Our studies also showed a higher peroxidation of intraplaque lipid from diabetic patients compared to non-diabetics, and a significant increase in reactive oxygen species (ROS) production in human coronary artery smooth muscle cells treated with lipids from diabetic plaques. Conclusions: This exploratory analysis showed distinct patterns of intraplaque lipid composition. Identification of these high-risk plaque lipid biomarkers will help to delineate the cellular and molecular mechanism responsible for stable to vulnerable plaques transition.

Divergence of acetate uptake in proinflammatory and inflammation-resolving macrophages: implications for imaging atherosclerosis

BackgroundMetabolic divergence of macrophages polarized into different phenotypes represents a mechanistically relevant target for non-invasive characterization of atherosclerotic plaques using positron emission tomography (PET). Carbon-11 (11C)-labeled acetate is a clinically available tracer which accumulates in atherosclerotic plaques, but its biological and clinical correlates in atherosclerosis are undefined. Methods and resultsHistological correlates of 14C-acetate uptake were determined in brachiocephalic arteries of western diet-fed apoE−/− mice. The effect of polarizing stimuli on 14C-acetate uptake was determined by proinflammatory (interferon-γ + lipopolysaccharide) vs inflammation-resolving (interleukin-4) stimulation of murine macrophages and human carotid endarterectomy specimens over 2 days. 14C-acetate accumulated in atherosclerotic regions of arteries. CD68-positive monocytes/macrophages vs smooth muscle actin-positive smooth muscle cells were the dominant cells in regions with high vs low 14C-acetate uptake. 14C-acetate uptake progressively decreased in proinflammatory macrophages to 25.9 ± 4.5% of baseline (P < .001). A delayed increase in 14C-acetate uptake was induced in inflammation-resolving macrophages, reaching to 164.1 ± 21.4% (P < .01) of baseline. Consistently, stimulation of endarterectomy specimens with interferon-γ + lipopolysaccharide decreased 14C-acetate uptake to 66.5 ± 14.5%, while interleukin-4 increased 14C-acetate uptake to 151.5 ± 25.8% compared to non-stimulated plaques (P < .05). ConclusionsAcetate uptake by macrophages diverges upon proinflammatory and inflammation-resolving stimulation, which may be exploited for immunometabolic characterization of atherosclerosis.

Unifying theory of carotid plaque disruption based on structural phenotypes and forces expressed at the lumen/wall interface

ObjectivesTo integrate morphological, haemodynamic and mechanical analysis of carotid atheroma driving plaque disruption.Materials and methodsFirst, we analysed the phenotypes of carotid endarterectomy specimens in a photographic dataset A, and matched...

In Vivo Coronary 18F-Sodium Fluoride Activity: Correlations With Coronary Plaque Histological Vulnerability and Physiological Environment

Background18F-sodium fluoride (18F-NaF) positron emission tomography (PET)/computed tomography (CT) is a promising new approach for assessing microcalcification in vascular plaque. ObjectivesThis prospective study aimed to evaluate the associations between in vivo coronary 18F-NaF PET/CT activity and ex vivo histological characteristics, to determine whether coronary 18F-NaF activity is a novel biomarker of plaque pathological vulnerability, and to explore the underlying physiological environment of 18F-NaF adsorption to vascular microcalcification. MethodsPatients with coronary artery disease (CAD) underwent coronary computed tomography angiography (CTA) and 18F-NaF PET/CT. Histological vulnerability and immunohistochemical characteristics were evaluated in coronary endarterectomy (CE) specimens from patients who underwent coronary artery bypass grafting with adjunctive CE. Correlations between in-vivo coronary 18F-NaF activity with coronary CTA adverse plaque features and with ex vivo CE specimen morphological features, CD68 expression, inflammatory cytokines expression (tumor necrosis factor-α, interleukin-1β), osteogenic differentiation cytokines expression (osteopontin, runt-related transcription factor 2, osteocalcin) were evaluated. High- and low- to medium-risk plaques were defined by standard pathological classification. ResultsA total of 55 specimens were obtained from 42 CAD patients. Coronary 18F-NaF activity of high-risk specimens was significantly higher than low- to medium-risk specimens (median [25th-75th percentile]: 1.88 [1.41-2.54] vs 1.12 [0.91-1.54]; P < 0.001). Coronary 18F-NaF activity showed high discriminatory accuracy in identifying high-risk plaque (AUC: 0.80). Coronary CTA adverse plaque features (positive remodeling, low-attenuation plaque, remodeling index), histologically vulnerable features (large necrotic core, thin-fibro cap, microcalcification), CD68 expression, tumor necrosis factor-α expression, and interleukin-1β expression correlated with coronary 18F-NaF activity (all P < 0.05). No significant association between coronary 18F-NaF activity and osteogenic differentiation cytokines was found (all P > 0.05). ConclusionsCoronary 18F-NaF activity was associated with histological vulnerability, CD68 expression, inflammatory cytokines expression, but not with osteogenic differentiation cytokines expression. 18F-NaF PET/CT imaging may provide a powerful tool for detecting high-risk coronary plaque and could improve the risk stratification of CAD patients.

Atherosclerosis

For the past decade, cardiovascular disease (CVD) has been the leading cause of death and disability worldwide, and in most cases, atherosclerosis is the dominant underlying pathologic process. Atherosclerosis is characterized by subendothelial accumulation of lymphocytes, lipid-filled macrophages, and inflammation-induced migration of vascular smooth muscle cells slowly forming a fibroinflammatory lipid plaque. Over time, the plaque progressively weakens and occludes the vessel; however, with continued inflammation and hemodynamic changes, atheromas can dangerously manifest as ischemia (acute or chronic) or aneurysmal disease. This review discusses the economic impact, historical background, and pathogenesis of atherosclerosis, as well as normal arterial biology, risk factors, and screening. Tables outline physiologic properties of nitric oxide in native vessels, selected cellular and chemical mediators and their role in atherogenesis, current risk factors for peripheral arterial disease (PAD) development, and diagnosis criteria for diseases that confer increased PAD risk. Graphs demonstrate mortality due to PAD, PAD prevalence, and the economic impact of CVD. Illustrations depict normal vessel anatomy, blood flow with plaque at carotid bifurcation, the superficial femoral artery (SFA) with plaque, and the progression of atherosclerotic changes. A flow chart for atherogenesis, photograph of a human carotid endarterectomy specimen, and angiogram showing the classic occluding lesion of the SFA are also provided. This review contains 9 figures, 5 tables, and 91 references.

Label-free analytic histology of carotid atherosclerosis by mid-infrared optoacoustic microscopy

Background and aimsAnalysis of atherosclerotic plaque composition is a vital tool for unraveling the pathological metabolic processes that contribute to plaque growth. MethodsWe visualize the constitution of human carotid plaques by mid-infrared optoacoustic microscopy (MiROM), a method for label-free analytic histology that requires minimal tissue preparation, rapidly yielding large field-of-view en-face images with a resolution of a few micrometers. We imaged endarterectomy specimens (n = 3, 12 sections total) at specific vibrational modes, targeting carbohydrates, lipids and proteins. Additionally, we recorded spectra at selected tissue locations. We identified correlations in the variability in this high-dimensional data set using non-negative matrix factorization (NMF). ResultsWe visualized high-risk plaque features with molecular assignment. Consistent NMF components relate to different dominant tissue constituents, dominated by lipids, proteins, and cholesterol and carbohydrates respectively. ConclusionsThese results introduce MiROM as an innovative, stain-free, analytic histology technology for the biochemical characterization of complex human vascular pathology.

Distinct Endothelial Cell Populations Characterize the Pulmonary Endarterectomy Specimen in Chronic Thromboembolic Pulmonary Hypertension

<h3>Purpose</h3> Misguided vascular repair is thought to contribute to the pathophysiology of chronic thromboembolic pulmonary hypertension (CTEPH), yet little is known about endothelial cell (EC) phenotypes in the organized thromboembolic material of patients with CTEPH. Therefore, we sought to characterize ECs from pulmonary endarterectomy (PEA) specimens. <h3>Methods</h3> Human PEA specimens from proximal pulmonary arteries were collected from five CTEPH patients and dissociated for droplet-based single-cell RNA sequencing. Data from three control human lungs was obtained from the Human Lung Cell Atlas (https://hlca.ds.czbiohub.org accessed Sept. 9, 2021). <h3>Results</h3> From the PEA specimens of five CTEPH patients, we obtained transcriptional profiles for 11068 cells representing 20 clusters and 9 cell types. From those, 4 clusters displayed markers of ECs. To identify appropriate control ECs, we assessed markers of endothelial subtype from the Human Lung Cell Atlas and identified markers of pulmonary and bronchial artery ECs. Therefore, we obtained the transcriptional profiles of pulmonary and bronchial artery ECs from the Human Lung Cell Atlas as controls. After standardizing quality control metrics, we merged the data from 2174 control and 1772 CTEPH ECs. Dimension reduction of the merged dataset revealed 12 endothelial clusters (Figure). We note disproportionate distribution of control vs CTEPH ECs across clusters, with a majority of clusters biased towards one condition. Between and within clusters, CTEPH ECs displayed greater expression of TGFβ1, a marker of endothelial-mesenchymal transition (EMT), and P-selectin, a marker of endothelial activation. <h3>Conclusion</h3> Significant endothelial heterogeneity exists in both control and CTEPH ECs. However, we find differences in the composition of EC phenotypes between these two groups. We identify marker genes for CTEPH ECs that contribute to our understanding of EC phenotypes and misguided vascular repair in CTEPH.

Sex Differences in Endothelial-to-Mesenchymal Transition in Chronic Thromboembolic Pulmonary Hypertension

<h3>Purpose</h3> Chronic Thromboembolic Pulmonary Hypertension (CTEPH) results from thrombus organization and obstruction of pulmonary arteries. Population studies suggest sex differences in outcomes. Yet, the mechanisms contributing to sex differences in CTEPH remain poorly understood. We propose that endothelial cells present in the organized thrombi undergo endothelial-to-mesenchymal transition (EndoMT); a process in which endothelial cells lose polarity, cell-to-cell contacts, and gradually adopt mesenchymal cells characteristics. Here, we find that markers of EndoMT are significantly higher in female vs male patients, which could contribute to sex differences in CTEPH pathophysiology. <h3>Methods</h3> A retrospective cohort of ten male and ten female CTEPH patients was studied. Pulmonary endarterectomy specimens from these patients were separated into lobar and segmental sections. Two samples (lobar and segmental) from each patient were paraffin embedded and used for Immunohistochemistry (IHC) analysis. IHC staining for markers for EndoMT, including endothelin-1 (ET-1), Transforming growth factor beta-1 (TGF beta-1), vimentin and alpha smooth muscle cell actin (alpha SMA), was performed. Quantitative tissue area analysis for each marker was performed using the HALO image analysis platform. <h3>Results</h3> We found that females have significantly higher total expression of vimentin and TGF beta-1 (Figure A). There was a trend towards increased total expression of ET-1 and alpha SMA in females that is not significantly different from males (Figure B). However, we found that female segmental sections have higher expression levels for vimentin, TGF beta-1, ET-1 and alpha SMA than male segmental sections (Figure C). <h3>Conclusion</h3> Increased expression of EndoMT markers may account for sex differences in the pathophysiology and outcomes of CTEPH. We will validate these findings and further explore EndoMT in the pathogenesis of CTEPH.

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Reassessing the Carotid Artery Plaque "Rim Sign" on CTA: A New Analysis with Histopathologic Confirmation.

The CTA "rim sign" has been proposed as an imaging marker of intraplaque hemorrhage in carotid plaques. This study sought to investigate such findings using histopathologic confirmation. Included patients had CTA neck imaging <1 year before carotid endarterectomy. On imaging, luminal stenosis and the presence of adventitial (<2-mm peripheral) and "bulky" (≥2-mm) calcifications, total plaque thickness, soft-tissue plaque thickness, calcification thickness, and the presence of ulcerations were assessed. The rim sign was defined as the presence of adventitial calcifications with internal soft-tissue plaque of ≥2 mm in maximum thickness. Carotid endarterectomy specimens were assessed for both the presence and the proportional makeup of lipid material, intraplaque hemorrhage, and calcification. Sixty-seven patients were included. Twenty-three (34.3%) were women; the average age was 70.4 years. Thirty-eight (57.7%) plaques had a rim sign on imaging, with strong interobserver agreement (κ = 0.85). A lipid core was present in 64 (95.5%) plaques (average, 22.2% proportion of plaque composition); intraplaque hemorrhage was present in 52 (77.6%), making up, on average, 13.7% of the plaque composition. The rim sign was not associated with the presence of intraplaque hemorrhage (P = .11); however, it was associated with a greater proportion of intraplaque hemorrhage in a plaque (P = .049). The sensitivity and specificity of the rim sign for intraplaque hemorrhage were 61.5% and 60.0%, respectively. The rim sign is not associated with the presence of intraplaque hemorrhage on histology. However, it is associated with a higher proportion of hemorrhage within a plaque and therefore may be a biomarker of more severe intraplaque hemorrhage, if present.

Imaging High-Risk Atherothrombosis Using a Novel Fibrin-Binding Positron Emission Tomography Probe.

High-risk atherosclerosis is an underlying cause of cardiovascular events, yet identifying the specific patient population at immediate risk is still challenging. Here, we used a rabbit model of atherosclerotic plaque rupture and human carotid endarterectomy specimens to describe the potential of molecular fibrin imaging as a tool to identify thrombotic plaques. Atherosclerotic plaques in rabbits were induced using a high-cholesterol diet and aortic balloon injury (N=13). Pharmacological triggering was used in a group of rabbits (n=9) to induce plaque disruption. Animals were grouped into thrombotic and nonthrombotic plaque groups based on gross pathology (gold standard). All animals were injected with a novel fibrin-specific probe 68Ga-CM246 followed by positron emission tomography (PET)/magnetic resonance imaging 90 minutes later. 68Ga-CM246 was quantified on the PET images using tissue-to-background (back muscle) ratios and standardized uptake value. Both tissue-to-background (back muscle) ratios and standardized uptake value were significantly higher in the thrombotic versus nonthrombotic group (P<0.05). Ex vivo PET and autoradiography of the abdominal aorta correlated positively with in vivo PET measurements. Plaque disruption identified by 68Ga-CM246 PET agreed with gross pathology assessment (85%). In ex vivo surgical specimens obtained from patients undergoing elective carotid endarterectomy (N=12), 68Ga-CM246 showed significantly higher binding to carotid plaques compared to a D-cysteine nonbinding control probe. We demonstrated that molecular fibrin PET imaging using 68Ga-CM246 could be a useful tool to diagnose experimental and clinical atherothrombosis. Based on our initial results using human carotid plaque specimens, in vivo molecular imaging studies are warranted to test 68Ga-CM246 PET as a tool to stratify risk in atherosclerotic patients.

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&lt;0.0001) of atherosclerotic plaque and valve proteomes (2,318 proteins). 548 and 158 proteins were exclusively altered (q&lt;0.05) by disease in plaques or valves, respectively. Vesicular GO terms increased 2.2x (p&lt;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&lt;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.

Correlation between computed tomography angiography and histology of carotid artery atherosclerosis: Can semi-automated imaging software predict a plaque's composition?

Using computed tomography angiography to differentiate between components of carotid atherosclerotic lesions remains largely elusive. This study sought to validate a semi-automated software for computed tomography angiography plaque analysis using histologic comparisons. A retrospective review was performed of consecutive patients that underwent a carotid endarterectomy, with pre-procedural computed tomography angiography imaging of the cervical arterial vasculature available for review. Images were evaluated using a commercially-available software package, which produced segmented analyses of intraplaque components (e.g. intraplaque hemorrhage, lipid-rich necrotic core, and calcifications). On imaging, each component was assessed in terms of its (1) presence or absence, and (2) both volume and proportion of the total plaque volume (if present). On histological evaluation of carotid endarterectomy specimens, each component was evaluated as an estimated proportion of total plaque volume. Of 80 included patients, 30 (37.5%) were female. The average age was 69.7 years (SD = 9.1). Based on imaging, intraplaque hemorrhage was the smallest contributor to plaque composition (1.2% of volumes on average). Statistically significant linear associations were noted between the proportion of intraplaque hemorrhage, lipid-rich necrotic core, and calcifications on histology and the volume of each component on imaging (p values ranged from 0.0008 to 0.01). Area under curve were poor for intraplaque hemorrhage and lipid-rich necrotic core (0.59 and 0.61, respectively) and acceptable for calcifications (0.73). Semi-automated analyses of computed tomography angiography have limited diagnostic accuracy in the detection of intraplaque hemorrhage and lipid-rich necrotic core in carotid artery plaques. However, volumetric imaging measurements of different components corresponded with histologic analysis.

Mirabegron Ameliorated Atherosclerosis of ApoE-/- Mice in Chronic Intermittent Hypoxia but Not in Normoxia.

It has been established that obstructive sleep apnea (OSA) is an independent risk factor for atherosclerosis. Chronic intermittent hypoxia (CIH) activates sympathoadrenal system and upregulates β3 adrenergic receptor (β3 AR). However, the effect of selective β3 AR agonist mirabegron in CIH-induced atherosclerosis remains unknown. We generated a CIH-induced atherosclerosis model through exposing ApoE-/- mice to CIH (8 h per day, cyclic inspiratory oxygen fraction 5-21%, 60-s cycle) for 6 weeks after 4-week high-fat dieting and investigated the effects of mirabegron, a selective β3 AR agonist, on CIH-induced atherosclerosis. The coronary endarterectomy (CE) specimens from coronary artery disease patients with OSA and without OSA were collected. The expression of β3 AR was significantly elevated in CIH-induced atherosclerosis model. Furthermore, treatment with mirabegron (10mg/kg per day by oral administration for 6 weeks) ameliorated atherosclerosis in ApoE-/- mice in CIH but not in normoxia. Mechanistically, mirabegron activated β3 AR and ameliorated intraplaque oxidative stress by suppressing p22phox expression and reactive oxygen species (ROS) level. In addition, in human CE specimens, β3 AR was also upregulated associated with increased p22phox expression and ROS level both in the lumen and in the plaque of coronary artery in OSA subjects. This study first demonstrated that mirabegron impeded the progression of CIH-induced atherosclerosis, at least in part, via β3 AR-mediated oxidative stress, suggesting a promising therapeutic strategy for protecting against atherosclerosis induced by CIH.

Near-Infrared Autofluorescence in Atherosclerosis Associates With Ceroid and Is Generated by Oxidized Lipid-Induced Oxidative Stress.

[Figure: see text].

Osteopontin and osteoprotegerin in atherosclerotic plaque - are they significant markers of plaque vulnerability?

Atherosclerosis (ATS) is still considered as a major, global health problem. For a deeper understanding of its pathogenesis, in the last years the research was translated from tissue visible events to molecular mechanisms. Osteopontin (OPN) and osteoprotegerin (OPG) are two molecules that have been associated with the initiation and progression of ATS lesions. The aim of our study was to assess the OPN and OPG expression in advanced stages of carotid ATS, to analyze the correlation between these markers and the ultrasonographic plaque properties, pointing out the identification of possible patterns that can predict plaque vulnerability and risks of restenosis. The study group comprised 49 consecutive patients (38 males and 11 females) diagnosed with carotid stenotic lesions by using ultrasonography. The carotid endarterectomy specimens were standardly processed for histopathological and immunohistochemical exams. The OPN and OPG expression was semi-quantitatively assessed. Our results sustained the relationship between histological American Heart Association (AHA) type and ultrasonographic classification (echogenic versus echolucent) (p<0.001). The semi-quantitative analysis showed that in most cases (31 plaques) OPG and OPN had opposite expressions, whereas in the remaining cases (18 plaques) the expression was similar. There were no correlations between low versus high expression of intra-plaque OPN and OPG (p=0.335). We found significant correlation for OPN and plaque echogenicity (p=0.011), but not for OPG (p=0.079). OPN expression (low versus high) was correlated with plaque type (stable versus unstable) (p=0.036), plaque ulceration (p=0.009) and inflammation (p<0.001). OPG expression (low versus high) did not reveal statistically significant differences with plaque type (stable versus unstable) and vulnerability plaque parameters, respectively. OPG and OPN co-exist in carotid atherosclerotic plaque demonstrating a modulatory role in inflammatory and calcification processes. OPG is strongly expressed in stable, calcified plaques, while OPN is poorly expressed in calcified plaques and in plaques without hemorrhage, ulceration, inflammation, or necrosis. Starting from the molecular mechanisms, further studies of biomarkers are important to identify new therapeutic resources meant to prevent and treat vascular calcification.

The Inflammatory Profile of CTEPH-Derived Endothelial Cells Is a Possible Driver of Disease Progression.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a form of pulmonary hypertension characterized by the presence of fibrotic intraluminal thrombi and causing obliteration of the pulmonary arteries. Although both endothelial cell (EC) dysfunction and inflammation are linked to CTEPH pathogenesis, regulation of the basal inflammatory response of ECs in CTEPH is not fully understood. Therefore, in the present study, we investigated the role of the nuclear factor (NF)-κB pro-inflammatory signaling pathway in ECs in CTEPH under basal conditions. Basal mRNA levels of interleukin (IL)-8, IL-1β, monocyte chemoattractant protein-1 (MCP-1), C-C motif chemokine ligand 5 (CCL5), and vascular cell adhesion molecule-1 (VCAM-1) were upregulated in CTEPH-ECs compared to the control cells. To assess the involvement of NF-κB signaling in basal inflammatory activation, CTEPH-ECs were incubated with the NF-κB inhibitor Bay 11-7085. The increase in pro-inflammatory cytokines was abolished when cells were incubated with the NF-κB inhibitor. To determine if NF-κB was indeed activated, we stained pulmonary endarterectomy (PEA) specimens from CTEPH patients and ECs isolated from PEA specimens for phospho-NF-κB-P65 and found that especially the vessels within the thrombus and CTEPH-ECs are positive for phospho-NF-κB-P65. In summary, we show that CTEPH-ECs have a pro-inflammatory status under basal conditions, and blocking NF-κB signaling reduces the production of inflammatory factors in CTEPH-ECs. Therefore, our results show that the increased basal pro-inflammatory status of CTEPH-ECs is, at least partially, regulated through activation of NF-κB signaling and potentially contributes to the pathophysiology and progression of CTEPH.