Asymptomatic Carotid Plaques Research Articles

Invited commentary

Management of the patient with asymptomatic carotid disease is approaching clinical equipoise. Our current guidelines are based on decades-old randomized level I trials, the Asymptomatic Carotid Atherosclerosis Study (ACAS) and Asymptomatic Carotid Surgery Trial (ACST-1), which enrolled patients between 1983 and 2003. Since then, medical therapy has intensified, particularly with the use of statins, dual antiplatelets, and more optimal antihypertensive medicines. At the same time, carotid interventions (carotid endarterectomy and carotid artery stenting) have become safer. The current Carotid Revascularization and Medical Management for Asymptomatic Carotid Stenosis (CREST-2) trial aims to answer some of these timely clinical questions.1Lal B.K. Meschia J.F. Brott T.G. Clinical need, design, and goals for the carotid revascularization and medical management for asymptomatic carotid stenosis trial.Semin Vasc Surg. 2017; 30: 2-7Crossref PubMed Scopus (20) Google Scholar Increasing evidence suggests a more benign natural history for asymptomatic carotid disease than previously thought.2Marquardt L. Geraghty O.C. Mehta Z. Rothwell P.M. Low risk of ipsilateral stroke in patients with asymptomatic carotid stenosis on best medical treatment: a prospective, population-based study.Stroke. 2010; 41: e11-e17Crossref PubMed Scopus (391) Google Scholar An analysis of the asymptomatic carotid trials, ACAS and ACST-1, suggests that only 50 to 60 strokes are prevented at 5 years per 1000 carotid endarterectomies performed for asymptomatic disease.3Naylor A.R. Which patients with asymptomatic carotid stenosis benefit from revascularization?.Curr Opin Neurol. 2017; 30: 15-21Crossref PubMed Scopus (13) Google Scholar However, clinical experience shows us that a subset of patients with moderate or severe carotid disease still progress to plaque rupture and stroke in 2018. Are there certain ultrasound characteristics that can reliably identify the asymptomatic carotid plaque at high-risk of rupture? Noninvasive imaging of plaque vulnerability remains an area of active investigation. Spanos et al associate the presence on ultrasound examination of a large juxtaluminal black area with a thin fibrous cap, a decrease in vascular smooth muscle cells, and increased macrophages, all known histologic changes of plaque vulnerability. Thinning of the fibrous cap, leading to plaque vulnerability and rupture, are events that likely occur owing to a complex interaction between local plaque and systemic inflammatory mediators, hemodynamic strain, and biomechanical forces on the plaque leading to material fatigue. Vascular smooth muscle cells volume loss and thinning of the fibrous cap likely occur owing to an array of molecular changes, which may in part be explained by a particular microRNA signature occurring both locally in the plaque and in the systemic circulation.4Bazan H.A. Hatfield S.A. O'Malley C.B. Brooks A.J. Lightell Jr., D. Woods T.C. Acute loss of mir-221 and mir-222 in the atherosclerotic plaque shoulder accompanies plaque rupture.Stroke. 2015; 46: 3285-3287Crossref PubMed Scopus (49) Google Scholar, 5Bazan H.A. Hatfield S.A. Brug A. Brooks A.J. Lightell Jr., D.J. Woods T.C. Carotid plaque rupture is accompanied by an increase in the ratio of serum circr-284 to mir-221 levels.Circ Cardiovasc Genet. 2017; 10 (doi: 10.1161/CIRCGENETICS.117.001720)Crossref PubMed Scopus (76) Google Scholar All of these changes that seem to be associated with thinning of the fibrous cap will need to be further validated in future clinical studies. Lastly, it is possible that the significant protective effects of statin therapy against cardiovascular events6Ridker P.M. Danielson E. Fonseca F.A. Genest J. Gotto Jr., A.M. Kastelein J.J. et al.Rosuvastatin to prevent vascular events in men and women with elevated c-reactive protein.N Engl J Med. 2008; 359: 2195-2207Crossref PubMed Scopus (5284) Google Scholar may be due to thickening of the atherosclerotic fibrous caps. Earlier identification of an asymptomatic patient at risk of plaque rupture could then be used to treat that patient with more intense medical therapy or with a prophylactic carotid endarterectomy (or carotid artery stenting in select cases). Can ultrasound features of plaque vulnerability, such as a juxtaluminal black area and the absence of a fibrous cap, be used to decide whether or not to intervene in a patient with asymptomatic carotid artery stenosis? Because there are no randomized studies to validate these studies, these observations are unlikely to currently be widely adopted into practice. However, this article elegantly correlates known ultrasound features with well-described histologic characteristics of plaque vulnerability. Carotid plaque noninvasive imaging will continue to evolve and help to discern the vulnerable plaque that places the patient at risk. The opinions or views expressed in this commentary are those of the authors and do not necessarily reflect the opinions or recommendations of the Journal of Vascular Surgery or the Society for Vascular Surgery. Carotid artery plaque echomorphology and its association with histopathologic characteristicsJournal of Vascular SurgeryVol. 68Issue 6PreviewThe aim of the study was to determine the association of ultrasonic texture features (severity of stenosis, grey scale median, plaque area, juxtaluminal black area [JBA], and discrete white areas) previously shown to be independent predictors for stroke with established histologic features of plaque instability. Full-Text PDF Open Archive

Invited commentary

Invited commentary

GW29-e1096 Asymptomatic carotid plaque whether increase the risk of cardiovascular and cerebrovascular:a Meta-analysis

To evaluate the Asymptomatic carotid plaque whether increase the risk of cardiovascular and cerebrovascular events by system analysis. Searched English datebase include PubmedEmbase,Web of Science by computer retrieve and assist of manual searching.Retrieval the prospective and observational study

Serum Proteomic Profiling to Identify Biomarkers of Premature Carotid Atherosclerosis

To evaluate the presence of serum protein biomarkers associated with the early phases of formation of carotid atherosclerotic plaques, label-free quantitative proteomics analyses were made for serum samples collected as part of The Cardiovascular Risk in Young Finns Study. Samples from subjects who had an asymptomatic carotid artery plaque detected by ultrasound examination (N = 43, Age = 30–45 years) were compared with plaque free controls (N = 43) (matched for age, sex, body weight and systolic blood pressure). Seven proteins (p < 0.05) that have been previously linked with atherosclerotic phenotypes were differentially abundant. Fibulin 1 proteoform C (FBLN1C), Beta-ala-his-dipeptidase (CNDP1), Cadherin-13 (CDH13), Gelsolin (GSN) and 72 kDa type IV collagenase (MMP2) were less abundant in cases, whereas Apolipoproteins C-III (APOC3) and apolipoprotein E (APOE) were more abundant. Using machine learning analysis, a biomarker panel of FBLN1C, APOE and CDH13 was identified, which classified cases from controls with an area under receiver-operating characteristic curve (AUROC) value of 0.79. Furthermore, using selected reaction monitoring mass spectrometry (SRM-MS) the decreased abundance of FBLN1C was verified. In relation to previous associations of FBLN1C with atherosclerotic lesions, the observation could reflect its involvement in the initiation of the plaque formation, or represent a particular risk phenotype.

Risk Marker Variability in Subclinical Carotid Plaques Based on Ultrasound is Influenced by Cardiac Phase, Echogenicity and Size

Identification of risk markers based on quantitative ultrasound texture analysis of carotid plaques has the ability to define vulnerable components that correlate with increased cardiovascular risk. However, data describing factors with the potential to influence the measurement variability of risk markers are limited. The aim of this study was to evaluate the influence of electrocardiogram-guided image selection, plaque echogenicity and area on carotid plaque risk markers and their variability in asymptomatic carotid plaques. Plaque risk markers were measured in 57 plaques during three consecutive heartbeats at two cardiac cycle time instants corresponding to the electrocardiogram R-wave (end diastole) and end of T-wave (end systole), resulting in six measurements for each plaque. Risk marker variability was quantified by computing the coefficient of variation (CV) across the three heartbeats. The CV was significantly higher for small plaques (area <15 mm2, 10%) than for large plaques (area >15 mm2, 6%) (p < 0.001) in measurements of area, and the CV for measurements of gray-scale median were higher for echolucent plaques (<40, 15%) than for echogenic plaques (>40, 9%) (p < 0.001). No significant differences were found between systole and diastole for the mean of any risk marker or the corresponding CV value. However, in a sub-analysis, the echolucent plaques were found to have a higher CV during systole compared with diastole. The variability also caused plaque type reclassification in 16% to 25% of the plaques depending on cutoff value. The results of this study indicate that echolucent and small plaques each contribute to increased risk marker variability. Based on these results, we recommend that measurements in diastole are preferred to reduce variation, although we found that it may not be possible to characterize small plaques accurately using contemporary applied risk markers.

An efficient data mining framework for the characterization of symptomatic and asymptomatic carotid plaque using bidimensional empirical mode decomposition technique

Atherosclerosis is a type of cardiovascular disease which may cause stroke. It is due to the deposition of fatty plaque in the artery walls resulting in the reduction of elasticity gradually and hence restricting the blood flow to the heart. Hence, an early prediction of carotid plaque deposition is important, as it can save lives. This paper proposes a novel data mining framework for the assessment of atherosclerosis in its early stage using ultrasound images. In this work, we are using 1353 symptomatic and 420 asymptomatic carotid plaque ultrasound images. Our proposed method classifies the symptomatic and asymptomatic carotid plaques using bidimensional empirical mode decomposition (BEMD) and entropy features. The unbalanced data samples are compensated using adaptive synthetic sampling (ADASYN), and the developed method yielded a promising accuracy of 91.43%, sensitivity of 97.26%, and specificity of 83.22% using fourteen features. Hence, the proposed method can be used as an assisting tool during the regular screening of carotid arteries in hospitals. Graphical abstract Outline for our efficient data mining framework for the characterization of symptomatic and asymptomatic carotid plaques.

Endothelial vascular cell adhesion molecule 1 is a marker for high-risk carotid plaques and target for ultrasound molecular imaging

BackgroundMolecular imaging of carotid plaque vulnerability to atheroembolic events is likely to lead to improvements in selection of patients for carotid endarterectomy (CEA). The aims of this study were to assess the relative value of endothelial inflammatory markers for this application and to develop molecular ultrasound contrast agents for their imaging. MethodsHuman CEA specimens were obtained prospectively from asymptomatic (30) and symptomatic (30) patients. Plaques were assessed by semiquantitative immunohistochemistry for vascular cell adhesion molecule 1 (VCAM-1), lectin-like oxidized low-density lipoprotein receptor 1, P-selectin, and von Willebrand factor. Established small peptide ligands to each of these targets were then synthesized and covalently conjugated to the surface of lipid-shelled microbubble ultrasound contrast agents, which were then evaluated in a flow chamber for binding kinetics to activated human aortic endothelial cells under variable shear conditions. ResultsExpression of VCAM-1 on the endothelium of CEA specimens from symptomatic patients was 2.4-fold greater than that from asymptomatic patients (P < .01). Expression was not significantly different between groups for P-selectin (P = .43), von Willebrand factor (P = .59), or lectin-like oxidized low-density lipoprotein receptor 1 (P = .99). Although most plaques from asymptomatic patients displayed low VCAM-1 expression, approximately one in five expressed high VCAM-1 similar to plaques from symptomatic patients. In vitro flow chamber experiments demonstrated that VCAM-1-targeted microbubbles bind cells that express VCAM-1, even under high-shear conditions that approximate those found in human carotid arteries, whereas binding efficiency was lower for the other agents. ConclusionsVCAM-1 displays significantly higher expression on high-risk (symptomatic) vs low-risk (asymptomatic) carotid plaques. Ultrasound contrast agents bearing ligands for VCAM-1 can sustain high-shear attachment and may be useful for identifying patients in whom more aggressive treatment is warranted.

Imaging vulnerable plaques by targeting inflammation in atherosclerosis using fluorescent-labeled dual-ligand microparticles of iron oxide and magnetic resonance imaging

ObjectiveIdentification of patients with high-risk asymptomatic carotid plaques remains an elusive but essential step in stroke prevention. Inflammation is a key process in plaque destabilization and a prelude to clinical sequelae. There are currently no clinical imaging tools to assess the inflammatory activity within plaques. This study characterized inflammation in atherosclerosis using dual-targeted microparticles of iron oxide (DT-MPIO) as a magnetic resonance imaging (MRI) probe. MethodsDT-MPIO were used to detect and characterize inflammatory markers, vascular cell adhesion molecule 1 (VCAM-1). and P-selectin on (1) tumor necrosis factor-α-treated cells by immunocytochemistry and (2) aortic root plaques of apolipoprotein-E deficient mice by in vivo MRI. Furthermore, apolipoprotein E-deficient mice with focal carotid plaques of different phenotypes were developed by means of periarterial cuff placement to allow in vivo molecular MRI using these probes. The association between biomarkers and the magnetic resonance signal in different contrast groups was assessed longitudinally in these models. ResultsImmunocytochemistry confirmed specificity and efficacy of DT-MPIO to VCAM-1 and P-selectin. Using this in vivo molecular MRI strategy, we demonstrated (1) the DT-MPIO-induced magnetic resonance signal tracked with VCAM-1 (r = 0.69; P = .014), P-selectin (r = 0.65; P = .022), and macrophage content (r = 0.59; P = .045) within aortic root plaques and (2) high-risk inflamed plaques were distinguished from noninflamed plaques in the murine carotid artery within a practical clinical imaging time frame. ConclusionsThese molecular MRI probes constitute a novel imaging tool for in vivo characterization of plaque vulnerability and inflammatory activity in atherosclerosis. Further development and translation into the clinical arena will facilitate more accurate risk stratification in carotid atherosclerotic disease in the future.

Abstract WP145: Presence of Vasa Vasorum Neovascularization on Optical Frequency Domain Imaging Predicts Progression of Carotid Artery Stenosis

Background: Vasa vasorum neovascularization (VVN) is one of the characteristics of vulnerable plaque. The purpose of this study was to assess VVN using optical frequency domain imaging (OFDI) between symptomatic and asymptomatic carotid artery plaques, and to know its association with progression of stenosis. Methods: The carotid artery plaques were examined before angioplasty using OFDI system (LUNAWAVE TM , Terumo). VVN was defined as a no-signal tubuloluminal structures recognized on at least 3 consecutive images. A total number of VVN was compared between symptomatic and asymptomatic plaques. The stenosis was evaluated by carotid duplex scan within one year. The lesion was diagnosed as ‘progressive’ when the stenotic degree and peak systolic velocity were increased. Results: A total of 60 patients (29 symptomatic, 11 progression) were included. VVN was detected in 54 patients (90%), the total number of VVN was significantly higher in progressive stenosis (8.9 ± 5.7 vs. 4.5 ± 3.8, p = 0.02). However, there was no relationship between the number of VVN and ischemic symptom. Conclusions: VVN was more frequently observed in progressive stenosis. Evaluation of VVN using OFDI might be useful to predict progression of carotid artery stenosis.

Combination of Magnetic Resonance Angiography and Computational Fluid Dynamics May Predict the Risk of Stroke in Patients with Asymptomatic Carotid Plaques.

BackgroundAtherosclerosis plaques in the carotid arteries frequently have been found in patients with stroke. However, the pathogenesis of carotid plaque from asymptomatic to cerebrovascular events is a complex process which is still not completely understood. We aimed to investigate the prognosis of asymptomatic carotid atherosclerotic plaques by use of magnetic resonance angiography (MRA) combined with computational fluid dynamics (CFD).Material/MethodsWe prospectively studied a cohort of 228 participants (mean age 59.21±8.48) with asymptomatic carotid atherosclerotic plaques; mean follow-up duration was 1147.56±224.84 days. Plaque morphology parameters were obtained by MRA analysis. Lumen area (LA) and total vessel area (TVA) were measured, and wall area (WA=TVA−LA) and normalized wall area index (NWI=WA/TVA) were calculated. CFD analysis was performed to evaluate hemodynamic characteristics, including wall pressure (WP) and wall shear stress (WSS). Independent risk factors for stroke were obtained by Cox regression analysis. The area under the curve (AUC) of receiver operator characteristic (ROC) and Z-statistic test were used to evaluate risk factors.ResultsLogistics regression analysis showed NWI (OR: 3.472, 95% CI: 2.943–4.096, P=0.11) and WSS (OR: 6.974, 95% CI: 1.070–45.453, P=0.42) were independent risk factors of stroke for patients with asymptomatic carotid plaques. The area under the ROC curve values for WSS, NWI, and WSS+NWI were 0.772, 0.798, and 0.903, respectively.ConclusionsThe combination of plaque morphology characteristics NWI and hemodynamic parameter WSS may predict the risk of stroke in patients with asymptomatic carotid plaques.

Differential Expression of MicroRNAs in Endarterectomy Specimens Taken from Patients with Asymptomatic and Symptomatic Carotid Plaques.

ObjectiveStroke and transient ischemic attacks are considered as clinical manifestations of atherosclerotic disease due to on-going vascular inflammation and finally atherothrombosis of the carotid arteries. MicroRNAs (miRNA/miR) are known to be involved in vascular inflammation and plaque destabilization. The aim of this study was to analyze the expression profile of selected miRNAs in endarterectomy specimen from carotid arteries that were taken from patients with asymptomatic and symptomatic atherosclerotic plaques.Methods and Results11 miRNAs were selected and their expression was analyzed using real-time RT-PCR. Therefore, samples were divided into three different groups. On the one hand we investigated the expression patterns from patients in asymptomatic (n = 14) and symptomatic (n = 10) plaques; on the other hand we took samples from normal configurated internal mammary arteries (n = 15). Out of these 11 targets we identified some miRNAs, which were up- or down-regulated in either one of the two groups. Interestingly, the expression of two miRNAs was significantly different between asymptomatic and symptomatic samples, namely miR-21 (P<0.01) and miR-143 (P<0.05).ConclusionIn the present study, we identified miRNA subtypes which showed different expression in endarterectomy specimen from patients with asymptomatic and symptomatic plaques, suggesting that these miRNAs correlated with advanced vascular inflammation and plaque stability. They may represent new therapeutic targets for vascular proliferative diseases such as atherosclerosis.

Metabolic Phenotypes of Carotid Atherosclerotic Plaques Relate to Stroke Risk: An Exploratory Study

Stroke is a major cause of death and disability. That three-quarters of stroke patients will never have previously manifested cerebrovascular symptoms demonstrates the unmet clinical need for new biomarkers able to stratify patient risk and elucidation of the biological dysregulations. In this study, the utility of comprehensive metabolic phenotyping is assessed to provide candidate biomarkers that relate to stroke risk in stenosing carotid plaque tissue samples. Carotid plaque tissue samples were obtained from patients with cerebrovascular symptoms of carotid origin (n=5), and from asymptomatic patients (n=5). Two adjacent biological replicates were obtained from each tissue. Organic and aqueous metabolite extracts were obtained separately and analysed using two ultra performance liquid chromatography coupled to mass spectrometry metabolic profiling methods. Multivariate and univariate tools were used for statistical analysis. The two study groups demonstrated distinct plaque phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the two groups with a strong statistical significance (p=10(-4)-10(-5)). Specifically, metabolites related to the eicosanoid pathway (arachidonic acid and arachidonic acid precursors), and three acylcarnitine species (butyrylcarnitine, hexanoylcarnitine, and palmitoylcarnitine), intermediates of the β-oxidation, were detected in higher intensities in symptomatic patients. However, metabolites implicated in the process of cell death, a process known to be upregulated in the formation of the vulnerable plaque, were unaffected. Discrimination between symptomatic and asymptomatic carotid plaque tissue is demonstrated for the first time using metabolic profiling technologies. Two biological pathways (eicosanoid and β-oxidation) were implicated in differentiating symptomatic from asymptomatic patients and will be further investigated. These results indicate that metabolic phenotyping should be further explored to investigate the chemistry of the unstable plaque, in the pursuit of candidate biomarkers for risk-stratification and targets for pharmacotherapeutic intervention.

High shear stress relates to intraplaque haemorrhage in asymptomatic carotid plaques

Background and aimsCarotid artery plaques with vulnerable plaque components are related to a higher risk of cerebrovascular accidents. It is unknown which factors drive vulnerable plaque development. Shear stress, the frictional force of blood at the vessel wall, is known to influence plaque formation. We evaluated the association between shear stress and plaque components (intraplaque haemorrhage (IPH), lipid rich necrotic core (LRNC) and/or calcifications) in relatively small carotid artery plaques in asymptomatic persons. MethodsParticipants (n = 74) from the population-based Rotterdam Study, all with carotid atherosclerosis assessed on ultrasound, underwent carotid MRI. Multiple MRI sequences were used to evaluate the presence of IPH, LRNC and/or calcifications in plaques in the carotid arteries. Images were automatically segmented for lumen and outer wall to obtain a 3D reconstruction of the carotid bifurcation. These reconstructions were used to calculate minimum, mean and maximum shear stresses by applying computational fluid dynamics with subject-specific inflow conditions. Associations between shear stress measures and plaque composition were studied using generalized estimating equations analysis, adjusting for age, sex and carotid wall thickness. ResultsThe study group consisted of 93 atherosclerotic carotid arteries of 74 participants. In plaques with higher maximum shear stresses, IPH was more often present (OR per unit increase in maximum shear stress (log transformed) = 12.14; p = 0.001). Higher maximum shear stress was also significantly associated with the presence of calcifications (OR = 4.28; p = 0.015). ConclusionsHigher maximum shear stress is associated with intraplaque haemorrhage and calcifications.

Relation between wall shear stress and carotid artery wall thickening MRI versus CFD

Wall shear stress (WSS), a parameter associated with endothelial function, is calculated by computational fluid dynamics (CFD) or phase-contrast (PC) MRI measurements. Although CFD is common in WSS (WSSCFD) calculations, PC-MRI-based WSS (WSSMRI) is more favorable in population studies; since it is straightforward and less time consuming. However, it is not clear if WSSMRI and WSSCFD show similar associations with vascular pathology. Our aim was to test the associations between wall thickness (WT) of the carotid arteries and WSSMRI and WSSCFD. The subjects (n=14) with an asymptomatic carotid plaque who underwent MRI scans two times within 4 years of time were selected from the Rotterdam Study. We compared WSSCFD and WSSMRI at baseline and follow-up. Baseline WSSMRI and WSSCFD values were divided into 3 categories representing low, medium and high WSS tertiles. WT of each tertile was compared by a one-way ANOVA test. The WSSMRI and WSSCFD were 0.50±0.13Pa and 0.73±0.25Pa at baseline. Although WSSMRI was underestimated, a significant regression was found between WSSMRI and WSSCFD (r2=0.71). No significant difference was found between baseline and follow-up WSS by CFD and MRI-based calculations. The WT at baseline was 1.36±0.16mm and did not change over time. The WT was 1.55±0.21mm in low, 1.33±0.20mm in medium and 1.21±0.21mm in the high WSSMRI tertiles. Similarly, the WT was 1.49±0.21mm in low, 1.33±0.20mm in medium and 1.26±0.21mm in high WSSCFD tertiles. We found that WSSMRI and WSSCFD were inversely related with WT. WSSMRI and WSSCFD patterns were similar although MRI-based calculations underestimated WSS.

The Nature of Iron Deposits Differs between Symptomatic and Asymptomatic Carotid Atherosclerotic Plaques.

Iron within atherosclerotic plaque has been implicated as a catalyst of oxidative stress that causes progression of plaque, and plaque rupture. Iron is believed to accumulate within plaque by incorporation of erythrocytes following plaque rupture and hemorrhage. There is only indirect evidence to support this hypothesis. Plaque specimens were obtained from ten symptomatic and fifteen asymptomatic patients undergoing carotid endarterectomy at a single institution. Plaques were sectioned for study using synchrotron radiation induced X-ray fluorescence the study the distribution of zinc, calcium and iron. Histologic staining was carried out with Prussian Blue, and immunohistochemical staining was done to localize macrophages with CD68. Data were compared against patient clinical variables. Ten symptomatic (15 ± 10 days between index symptoms and surgery) and fifteen asymptomatic carotid plaques were studied. Zinc and calcium co-localized in mineralized areas of symptomatic and asymptomatic plaque. Iron was identified away from zinc and calcium in both symptomatic and asymptomatic plaques. Within the symptomatic plaques, iron was found within the thrombus associated with plaque rupture and hemorrhage. It did not stain with Prussian Blue, but was found in association with CD68 positive macrophages. In symptomatic plaques, the abundance of iron showed an association with the source patient’s LDL cholesterol (R2 = 0.39, Significance F = 0.05). Iron in asymptomatic plaque was present as hemosiderin/ferritin that stained positive with Prussian Blue, and was observed in association with CD68 positive macrophages. Iron in acutely symptomatic plaques is found within thrombus, in the presence of macrophages. The abundance of iron in symptomatic plaques is associated with the source patient’s LDL cholesterol. Within asymptomatic plaques, iron is found in association with macrophages, as hemosiderin/ferritin.

Elevated levels of endothelial-derived microparticles, and serum CXCL9 and SCGF-β are associated with unstable asymptomatic carotid plaques.

Endothelial microparticles (EMPs) are released from dysfunctional endothelial cells. We hypothesised that patients with unstable carotid plaque have higher levels of circulating microparticles compared to patients with stable plaques, and may correlate with serum markers of plaque instability and inflammation. Circulating EMPs, platelet MPs (PMPs) and inflammatory markers were measured in healthy controls and patients undergoing carotid endarterectomy. EMP/PMPs were quantified using flow cytometry. Bioplex assays profiled systemic inflammatory and bone-related proteins. Immunohistological analysis detailed the contribution of differentially-regulated systemic markers to plaque pathology. Alizarin red staining showed calcification. EMPs and PMPs were significantly higher in patients with carotid stenosis (≥70%) compared to controls, with no differences between asymptomatic vs symptomatic patients. Asymptomatic patients with unstable plaques exhibited higher levels of EMPs, CXCL9 and SCGF-β compared to those with stable plaques. CXCL9, and SCGF-β were detected within all plaques, suggesting a contribution to both localised and systemic inflammation. Osteopontin and osteoprotegerin were significantly elevated in the symptomatic vs asymptomatic group, while osteocalcin was higher in asymptomatic patients with stable plaque. All plaques exhibited calcification, which was significantly greater in asymptomatic patients. This may impact on plaque stability. These data could be important in identifying patients at most benefit from intervention.

Human Carotid Plaques With High Levels of Interleukin-16 Are Associated With Reduced Risk for Cardiovascular Events.

Interleukin-16 (IL-16) functions as a regulator of T-cell growth and acts as an inducer of cell migration. The aim of this study was to determine whether IL-16 measured in human carotid plaques was associated with symptoms (eg, stroke, transient ischemic attack, or amaurosis fugax), markers of plaque stability, and postoperative cardiovascular events. Plaques obtained from patients who had ≥1 cerebrovascular ischemic events within 1 month before endarterectomy (n=111) were compared with plaques from patients without symptoms (n=95). Neutral lipids, smooth muscle cell, and macrophage contents were evaluated histologically, and collagen, elastin, and caspase-3 activity were measured biochemically. IL-16, matrix metalloproteinases, and tissue inhibitors of metalloproteinases were measured in plaque homogenates using a multiplex immunoassay. IL-16, CD3, CD4, and FoxP3 mRNA expressions in carotid plaques were analyzed with quantitative real-time polymerase chain reaction. Carotid plaques from asymptomatic patients had higher levels of IL-16 mRNA. High plaque IL-16 protein levels (above median) were associated with reduced incidence of postoperative cardiovascular events during a mean follow-up of 21 months (hazard ratio, 0.47; 95% confidence interval, 0.22-0.99; P=0.047). IL-16 levels correlated with the plaque-stabilizing components: elastin, collagen, matrix metalloproteinase-2, tissue inhibitors of metalloproteinase-1, tissue inhibitors of metalloproteinase-2 and FoxP3 mRNA. This study shows that high levels of IL-16 are associated with asymptomatic carotid plaques, expression of factors contributing to plaque stability, and decreased risk of new cardiovascular events during a 2-year period after surgery, suggesting that IL-16 might have a protective role in human atherosclerotic disease.

Ultrasound Characteristics of Symptomatic Carotid Plaques: A Systematic Review and Meta-Analysis

Background: Ultrasound is the most commonly used imaging modality for assessing carotid artery stenosis. A number of studies have demonstrated that surface irregularities, heterogeneous echotexture and hypoechoic plaques are risk factors for acute ischemic stroke. We performed a systematic review and meta-analysis of the literature to better define the risk of stroke based on the sonographic characteristics of carotid plaques. Materials and Methods: We performed a comprehensive search for studies reporting imaging findings of symptomatic and asymptomatic carotid plaques on ultrasound using MEDLINE and EMBASE. We included both case-control and cohort studies examining the relationship between complex plaque and acute ischemic stroke or transient ischemic attack. Complex plaque was defined as plaque that had any of the following characteristics: heterogeneous echogenicity, echolucency, neovascularization, surface irregularity, ulceration, and intraplaque motion. Meta-analyses using the random-effects model were performed for complex plaque and each of the individual complex plaque characteristics. p < 0.05 was considered statistically significant. We explored the impact of publication bias by constructing funnel plots and testing their symmetry. We conducted the meta-analysis using Comprehensive Meta-analysis version 2.2, Englewood, N.J., USA. Results: A total of 1,013 articles were screened and 23 studies with 6,706 carotid plaques were included. Ultrasound plaque characteristics with a higher prevalence in individuals with symptomatic compared to asymptomatic carotid artery stenosis included plaque neovascularity (OR = 19.68, 95% CI = 3.14-123.16), complex plaque (OR = 5.12, 95% CI = 3.42-7.67), plaque ulceration (OR = 3.58, 95% CI = 1.66-7.71), plaque echolucency (OR = 3.99, 95% CI = 3.06-5.19) and intraplaque motion (OR = 1.57, 95% CI = 1.02-2.41). Variables not associated with symptom status included heterogenous echotexture (OR = 2.68, 95% CI = 0.56-12.80) and surface irregularity without ulceration (OR = 2.38, 95% CI = 0.70-8.11). No evidence of publication bias was observed based on Eggers test (p value of 0.05 for complex plaque and 0.53 for plaque echolucency). The remaining plaque features had insufficient data to assess for publication bias. Conclusions: Our meta-analysis and systematic review of the literature demonstrated that plaques with complex features, particularly those with echolucency, neovascularization, ulceration and intraplaque motion are associated with ischemic symptoms. Assessment of carotid plaque on ultrasound may provide stroke risk information beyond measurement of luminal stenosis. Thus, sonographic evaluation of carotid artery stenosis should focus on the detection of these plaque characteristics in addition to quantifying the degree of stenosis.

Blockade of Ets-1 attenuates epidermal growth factor-dependent collagen loss in human carotid plaque smooth muscle cells.

Although degradation of extracellular matrix by matrix metalloproteinases (MMPs) is thought to be involved in symptomatic (S) carotid plaques in atherosclerosis, the mechanisms of MMP expression are poorly understood. Here, we demonstrate that collagen loss in vascular smooth vessel cells (VSMCs) isolated from S plaques was induced by epidermal growth factor (EGF) through the activation of p38-MAPK and JNK-MAPK pathways. Inhibitors of p38-MAPK and JNK-MAPK signaling pathways downregulated the expression of MMP-1 and MMP-9. In addition, we examined whether v-ets erythroblastosis virus E26 oncogene homologue 1 (Ets-1), an important regulator of different genes, is involved in destabilizing S plaques in patients with carotid stenosis. We demonstrate that EGF induces Ets-1 expression and decreases interstitial and basement membrane collagen in vascular smooth muscle cells (VSMCs) from patients with carotid stenosis. Increased expression of MMP-1 and -9 and decreased collagen mRNA transcripts were also found in Ets-1-overexpressed VSMCs. Transfection with both dominant-negative form of Ets-1 and small interfering RNA blocked EGF-induced MMP-1 and -9 expressions and increased the mRNA transcripts for collagen I (α1) and collagen III (α1) in S compared with asymptomatic (AS) carotid plaques. Inhibitors of p38-MAPK (SB202190) and JNK-MAPK (SP600125) signaling pathways decreased the expression of Ets-1, MMP-1, and MMP-9 and increased collagen type I and III expression in EGF-treated VSMCs. This study provides a mechanistic insight into the role of Ets-1 in the plaque destabilization in patients with carotid stenosis involving p38-MAPK and JNK signaling pathways.

Prediction of the time period of stroke based on ultrasound image analysis of initially asymptomatic carotid plaques.

Non-invasive ultrasound imaging of carotid plaques can provide information on the characteristics of the arterial wall including the size, morphology and texture of the atherosclerotic plaques. Several studies were carried out that demonstrated the usefulness of these feature sets for differentiating between asymptomatic and symptomatic plaques and their corresponding cerebrovascular risk stratification. The aim of this study was to develop predictive modelling for estimating the time period of a stroke event by determining the risk for short term (less or equal to three years) or long term (more than three years) events. Data from 108 patients that had a stroke event have been used. The information collected included clinical and ultrasound imaging data. The prediction was performed at base line where patients were still asymptomatic. Several image texture analysis and clinical features were used in order to create a classification model. The different features were statistically analyzed and we conclude that image texture analysis features extracted using Spatial Gray Level Dependencies method had the best statistical significance. Several predictive models were derived based on Binary Logistic Regression (BLR) and Support Vector Machines (SVM) modelling. The best results were obtained with the SVM modelling models with an average correct classifications score of 77±7% for differentiating between stroke event occurrences within 3 years versus more than 3 years. Further work is needed in investigating additional multiscale texture analysis features as well as more modelling techniques on more subjects.