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Carotid artery plaque echomorphology and its association with histopathologic characteristics

Open ArchivePublished:May 23, 2018DOI:https://doi.org/10.1016/j.jvs.2018.01.068

      Abstract

      Objective

      The 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.

      Methods

      A cross-sectional study was performed involving 70 patients scheduled for carotid endarterectomy. Before surgery, carotid plaque texture features were obtained with ultrasound after normalization using commercially available software (LifeQ Medical, Nicosia, Cyprus). After carotid endarterectomy, histologic features (number of macrophages [CD68 staining], severity of angiogenesis [CD31 staining], smooth muscle cell [SMC] numbers, size of lipid core, thickness of the fibrous cap, presence of intraplaque hemorrhage, plaque rupture, and instability) also were studied.

      Results

      Symptomatic (n = 20) and asymptomatic (n = 50) patients were comparable in terms of internal carotid stenosis (mean stenosis, 86%; range, 60%-99%) and prevalence of risk factors except for total cholesterol (which was higher in the symptomatic group; P = .023). A low grey scale median and the presence of discrete white areas were associated with an increased number of macrophages (P < .001 and P < .001, respectively), increased neovascularization (P = .019 and P < .001, respectively), larger lipid core (P = .001 and P = .025, respectively), intraplaque hemorrhage presence (P = .001 and P = .001, respectively), plaque rupture (P = .001 and P = .025, respectively), and a decreased number of SMCs (P = .003 and P = .003, respectively). The presence of JBA was associated with a decreased number of SMCs (P = .042), larger lipid core (P = .013), and plaque rupture (P = .002). The combination of a thin fibrous cap with either a large lipid core or plaque rupture was associated with the highest (65%) prevalence of a JBA. Plaque area was not associated with any of the histologic features. After adjusting statin therapy for symptoms, statins were associated with a decreased number of macrophages (P = .038), decreased neovascularization (P = .019), and an increased number of SMCs (P = .023).

      Conclusions

      A number of ultrasonic texture features previously shown to be independent predictors of stroke have been found to have a strong association with established histologic features of plaque instability. This finding provides insight into the mechanism of ultrasonic texture features in stroke prediction and validates the use of ultrasound in stroke risk stratification.

      Keywords

      Article Highlights
      • Type of Research: Single-center retrospective cross-sectional study
      • Take Home Message: In 70 patients the carotid plaque texture features, determined by preoperative ultrasound, had a strong association with histologic features of vulnerable plaques, removed during carotid endarterectomy.
      • Recommendation: The study supports using ultrasound as an adjunct to stratify stroke risk.
      Recent reviews of cohort studies on outcomes of patients with asymptomatic carotid stenosis (ACS) on medical therapy alone, including the medical arms of randomized carotid endarterectomy trials, indicate that the average annual risk of ipsilateral hemispheric stroke has decreased to approximately 1% or less.
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      unless it becomes possible to identify a subgroup at high risk for ipsilateral stroke. A relatively inexpensive and readily available method is ultrasonic imaging, which can identify such high-risk patients.
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      Asymptomatic internal carotid artery stenosis and cerebrovascular risk stratification.
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      The association between plaque texture features and histologic features has been the subject of several studies. Early work involving mainly the grey scale median (GSM)
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      • Geroulakos G.
      The relative effect of carotid plaque heterogeneity and echogenicity on ipsilateral cerebral infarction and symptoms of cerebrovascular disease.
      showed that a low GSM was associated with unstable plaques and a high GSM with increased fibrous tissue and the presence of calcium. In a recent study,
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      • Moore D.
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      Identification of patients with a histologically unstable plaque using ultrasonic plaque image analysis.
      low GSM, large plaque area, and juxtaluminal black area (JBA) >8 mm2 were associated with plaques that were classified histologically as unstable. In the most recent publication correlating ultrasonic imaging with histology,
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      Histopathologic validation of grayscale carotid plaque characteristics related to plaque vulnerability.
      a high GSM was associated with calcification. In addition, discrete white areas without acoustic shadow in a hypoechoic area (DWA) were found to be associated with increased deposits of hemosiderin and inflammation. Finally, a juxtaluminal black area without a visible fibrous cap (JBA) was associated with an increased incidence of ulceration.
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      • Varghese T.
      • et al.
      Histopathologic validation of grayscale carotid plaque characteristics related to plaque vulnerability.
      In a large prospective study involving 1121 patients with ACS and a mean follow-up period of 4 years, a number of plaque features were shown to be independent predictors of stroke risk and could be used to stratify patients into annual risk groups ranging from <1% to 10%.
      • Nicolaides A.N.
      • Kakkos S.K.
      • Kyriacou E.
      • Griffin M.
      • Sabetai M.
      • Thomas D.J.
      • et al.
      Asymptomatic internal carotid artery stenosis and cerebrovascular risk stratification.
      • Kakkos S.K.
      • Griffin M.B.
      • Nicolaides A.N.
      • Kyriacou E.
      • Sabetai M.M.
      • Tegos T.
      • et al.
      The size of juxtaluminal hypoechoic area in ultrasound images of asymptomatic carotid plaques predicts the occurrence of stroke.
      These features consisted of GSM, plaque area, degree of stenosis, the presence of DWA, and the presence of a JBA greater than 8 mm2 without a visible echogenic cap.
      The aim of the study was to determine the association between each one of the earlier-described ultrasonic independent predictors of stroke risk and the classic histologic features of vulnerable plaques: number of macrophages, number of smooth muscle cells (SMCs), level of neovascularization, size of the lipid core, thickness of the fibrous cap, presence of intraplaque hemorrhage, and presence of plaque rupture. Determination and understanding of the association or lack of association between certain ultrasonic and histologic features should provide insight into the mechanism of ultrasonic features in stroke prediction and validate the use of ultrasound in stroke risk stratification.

      Methods

      This was a cross-sectional study comparing ultrasonic carotid plaque texture features with histologic features. It included 70 carotid bifurcation plaques from 70 consecutive patients (20 symptomatic [2 stroke and 18 transient ischemia attack] and 50 asymptomatic) scheduled for carotid endarterectomy from January 2015 to February 2016.
      • Liapis C.D.
      • Bell P.R.
      • Mikhailidis D.
      • Sivenius J.
      • Nicolaides A.
      • et al.
      ESVS Guidelines Collaborators
      ESVS guidelines. Invasive treatment for carotid stenosis: indications, techniques.
      The degree of stenosis ranged from 60% to 99% (North American Symptomatic Carotid Endarterectomy Trial [NASCET] criteria) in relation to the normal lumen of the distal internal carotid artery (mean, 86%; standard deviation, ±9.5%).
      • Liapis C.D.
      • Bell P.R.
      • Mikhailidis D.
      • Sivenius J.
      • Nicolaides A.
      • et al.
      ESVS Guidelines Collaborators
      ESVS guidelines. Invasive treatment for carotid stenosis: indications, techniques.
      During the same period no carotid artery stenting procedure was performed. Each patient's history (symptomatic or asymptomatic) with clinical and biochemical data and the medical treatment performed before referral to the Vascular Unit (Table I) were documented.
      Table IPrevalence of risk factors, medical treatment, and comorbidities between symptomatic and asymptomatic patients; and between patients with stable and unstable plaque
      Risk factors and medical treatmentAll plaques (N = 70)Asymptomatic plaques (n = 50)Symptomatic plaques (n = 20)PaStable plaque (n = 34)Unstable plaque (n = 36)P
      Independent t-test was used for continuous variables and the χ2 test was used for categoric variables.
      Age, years69 ± 7.668.7 ± 7.869.8 ± 7.4.56069.8 ± 8.368.2 ± 7.0.402
      Sex, male/female, No.51/1911/398/12.12625/926/10.902
      Total cholesterol, mg/dL230 ± 29225 ± 28242 ± 27.023237 ± 27223 ± 28.044
      Smoking40/70 (57)26/50 (52)14/20 (70).05317/34 (50)23/36 (64).344
      Diabetes17/70 (24)14/50 (28)3/20 (15).25211/34 (32)6/36 (17).126
      Hypertension49/70 (70)37/50 (74)12/20 (60).63121/34 (62)28/36 (78).423
      CAD49/70 (70)37/50 (74)12/20 (60).63122/34 (65)27/36 (70).443
      Creatinine, mg/dL1.00 ± 0.210.99 ± 0.201.02 ± 0.23.9151.00 ± 0.240.99 ± 0.18.911
      Ipsilateral carotid stenosis86.2 ± 9.586.1 ± 8.886.3 ± 11.3.91283.2 ± 10.389.0 ± 7.7.009
      Contralateral carotid stenosis25/70 (36)15/50 (30)12/20 (60).2409/34 (27)16/36 (44).134
      Antiplatelet treatment25/70 (36)20/50 (40)5/20 (25).2379/34 (26)16/36 (44).117
      Statin therapy35/70 (50)32/50 (64)3/20 (15)<.00119/34 (56)16/36 (44).339
      CAD, Coronary artery disease.
      Continuous data are presented as mean ± standard deviation and categorical data as n/N (%).
      a Independent t-test was used for continuous variables and the χ2 test was used for categoric variables.
      Preoperative duplex scanning to determine the degree of stenosis based on velocities with the ultrasound beam at 60 degrees to the vessel was performed for all patients.
      • Nicolaides A.N.
      • Kakkos S.K.
      • Kyriacou E.
      • Griffin M.
      • Sabetai M.
      • Thomas D.J.
      • et al.
      Asymptomatic internal carotid artery stenosis and cerebrovascular risk stratification.
      A computed tomography angiography was performed only in those cases with a severe calcified plaque in which the degree of stenosis could not be calculated with ultrasound. Subsequently, images of the carotid plaque were captured by an independent radiologist from whom the patients’ history was concealed. Equipment settings, as previously described with an ultrasound beam perpendicular to the vessel axis and gain adjustment to minimize but not abolish noise in the vessel lumen, were used.
      • Nicolaides A.N.
      • Kakkos S.K.
      • Kyriacou E.
      • Griffin M.
      • Sabetai M.
      • Thomas D.J.
      • et al.
      Asymptomatic internal carotid artery stenosis and cerebrovascular risk stratification.
      • Kakkos S.K.
      • Griffin M.B.
      • Nicolaides A.N.
      • Kyriacou E.
      • Sabetai M.M.
      • Tegos T.
      • et al.
      The size of juxtaluminal hypoechoic area in ultrasound images of asymptomatic carotid plaques predicts the occurrence of stroke.
      Images were saved as tiff files and were analyzed using dedicated software (Image Analysis Program for ultrasonic Arterial Wall Changes and Atherosclerotic Plaques, version 3.4; LifeQ Ltd, Nicosia, Cyprus; www.lifeq.com).
      • Griffin M.
      • Nicolaides A.
      • Kyriacou E.
      Normalisation of ultrasonic images of atherosclerotic plaques and reproducibility of grey scale median using dedicated software.
      • Kakkos S.K.
      • Nicolaides A.N.
      • Kyriacou E.
      • Daskalopoulou S.S.
      • Sabetai M.M.
      • Pattichis C.S.
      • et al.
      Computerized texture analysis of carotid plaque ultrasonic images can identify unstable plaques associated with ipsilateral neurological symptoms.
      Images initially were normalized using linear scaling with zero grey scale for blood and 190 grey scale for adventitia. The plaque then was outlined as the region of interest and a number of texture features were extracted. These included the GSM, plaque area expressed in square millimeters, presence of discrete white areas (Fig 1) with grey scale values >124 (colored red by the software for easy identification) without acoustic shadow, and the presence of a JBA defined as an area greater than 8 mm2 adjacent to the lumen (Fig 2) with pixels showing a grey scale value <25 and without a visible echogenic cap.
      • Nicolaides A.N.
      • Kakkos S.K.
      • Kyriacou E.
      • Griffin M.
      • Sabetai M.
      • Thomas D.J.
      • et al.
      Asymptomatic internal carotid artery stenosis and cerebrovascular risk stratification.
      • Kakkos S.K.
      • Griffin M.B.
      • Nicolaides A.N.
      • Kyriacou E.
      • Sabetai M.M.
      • Tegos T.
      • et al.
      The size of juxtaluminal hypoechoic area in ultrasound images of asymptomatic carotid plaques predicts the occurrence of stroke.
      • Griffin M.
      • Nicolaides A.
      • Kyriacou E.
      Normalisation of ultrasonic images of atherosclerotic plaques and reproducibility of grey scale median using dedicated software.
      • Kakkos S.K.
      • Nicolaides A.N.
      • Kyriacou E.
      • Daskalopoulou S.S.
      • Sabetai M.M.
      • Pattichis C.S.
      • et al.
      Computerized texture analysis of carotid plaque ultrasonic images can identify unstable plaques associated with ipsilateral neurological symptoms.
      • Kakkos S.K.
      • Stevens J.M.
      • Nicolaides A.N.
      • Kyriacou E.
      • Pattichis C.S.
      • Geroulakos G.
      • et al.
      Texture analysis of ultrasonic images of symptomatic carotid plaques can identify those plaques associatedwith ipsilateral embolic brain infarction.
      Figure thumbnail gr1
      Fig 1Presence of discrete white areas (yellow arrows) with grey scale values >124 (colored red by the software [LifeQ Medical, Nicosia, Cyprus] for easy identification). Top: image region; below: image coloring (10 contours).
      Figure thumbnail gr2
      Fig 2Presence of juxtaluminal black area (red arrows) defined as an area greater than 8 mm2 adjacent to the lumen using commercially available software (LifeQ Medical, Nicosia, Cyprus). Top: image region; bottom: image coloring (10 contours).
      During surgery the carotid specimens were collected and fixed in formalin solution, neutrally buffered, 10% (approximately 4% formaldehyde). The specimen was cut in several blocks that were embedded in paraffin. Blocks were created with cuts made as follows: 0, the most stenotic site, 1+ and 2+ proximal, and 1- and 2- distal to the maximal stenosis in centimeters. Sections 3-μm thick were obtained from each block and stained with hematoxylin and eosin for histologic evaluation, according to the American Heart Association classification system.
      • Stary H.C.
      • Chandler A.B.
      • Dinsmore R.E.
      • Fuster V.
      • Glagov S.
      • Insull Jr., W.
      • et al.
      A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis: a report from the committee on vascular lesions of the council on atherosclerosis: American Heart Association.
      For immunohistochemical studies the sections that contained the cap, shoulder, and base of the atheroma at the most stenotic parts of the specimens were selected.
      The plaques were analyzed histopathologically for macrophage infiltration (CD68), angiogenesis (CD31), and SMC presence. Plaques also were classified according to the characteristics of the fibrous cap (thin or thick), lipid core (small, medium, or large), plaque rupture (present or absent), and intraplaque hemorrhage (present or absent). Finally, based on all the findings, two histopathologists labeled each plaque as stable or unstable. The two histopathologists involved evaluated the immunostained slides independently and differences were settled by a third histopathologist after discussion for consensus.
      CD68 protein is a macrophage marker encountered in the presence of carotid plaque inflammation.
      • Ren S.
      • Fan X.
      • Peng L.
      • Pan L.
      • Yu C.
      • Tong J.
      • et al.
      Expression of NF-κB, CD68 and CD105 in carotid atherosclerotic plaque.
      The autostainer Bond max system (Refined Polymer detection Kit; Leica Biosystems, Wetzlar, Germany) was used. CD68-positive macrophages were identified with a monoclonal antibody (clone KP1, dilution 1:100; Dako Corporation, Carpinteria, Calif). A semiquantitative method was applied and scored as absence (0), small scattered collections (1), masses of moderate cellular density (2), and confluent hypercellular areas (3). Unstable atheroma was characterized by the presence of abundant macrophages and few SMCs because inflammatory cells increase the apoptosis because of their damage from the macrophages.
      • Lacolley P.
      • Regnault V.
      • Nicoletti A.
      • Li Z.
      • Michel J.B.
      The vascular smooth muscle cell in arterial pathology: a cell that can take on multiple roles.
      • Boyle J.J.
      • Weissberg P.L.
      • Bennett M.R.
      Tumor necrosis factor-alpha promotes macrophage-induced vascular smooth muscle cell apoptosis by direct and autocrine mechanisms.
      • Gough P.J.
      • Gomez I.G.
      • Wille P.T.
      • Raines E.W.
      Macrophage expression of active MMP-9 induces acute plaque disruption in apoE-deficient mice.
      SMCs were detected with an antibody (clone 1A4, 1:100 dilution; Dako Corporation) with emphasis on the density and thickness of SMCs along the cap and shoulder. Scores ranging from 1 to 3 were applied as follows: thin loose arrangement was marked as 1; a thick, dense band of SMCs along the cap/shoulder was marked as 3; and intermediate findings were marked as 2.
      The presence and degree of neoangiogenesis was assessed from the presence of microvessels in the atherosclerotic plaque. A microvessel was identified by the presence of a lumen surrounded by a rim of endothelial cells highlighted by immunostaining with anti-CD31 antibodies. To mark and measure the microvessels, monoclonal antibodies to a biochemical cell marker (CD31, JC70A clone, dilution 1:100; Dako Corporation) were used. The mean number of microvessels per square millimeter was recorded.
      Digital analysis was used to make microvessel counts. Images were captured by a microscope-mounted, high-resolution, digital camera with a video output. Three sequential sections 4-mm apart (with the use of microtome; Shandon Finesse; Image Pro Plus, Media Software, and Cybernetics, Rockville, Md), including the atherosclerotic plaque core, were evaluated for each plaque. The whole plaque area was evaluated at a magnification of ×400. Evaluation of the images was performed with the Image Tool (Image Pro Plus, Media Software, and Cybernetics) analysis system by the two independent examiners blinded to the clinical history and the mean value was recorded, in cases of disagreement a third examiner evaluated the images and after discussion an agreement was reached. Digital images from the hematoxylin and eosin-stained transverse sections of the atherosclerotic lesion also were obtained at a magnification of ×200.

       Statistical analysis

      Statistical analysis was performed with IBM SPSS Statistics 19 or later (SPSS, Inc, Chicago, Ill). Initially, the means and standard deviations were calculated for continuous variables. Not normally distributed variables were categorized. The prevalence of risk factors and comorbidities in all the patients, in patients with symptomatic and asymptomatic carotid plaques, and in patients with histologically classified stable or unstable plaques was calculated. An independent sample t-test was used to test the significance of continuous variables and the χ2 test was used for categoric variables (Table I). Subsequently, the association between histologic features and ultrasonic plaque texture features was explored. Analysis of variance was used to test the significance of changes of continuous ultrasonic variables (GSM, plaque area, and severity of stenosis) for different classes of histologic features (Table II). Odds ratios were calculated using multivariable logistic regression analysis with dichotomous ultrasonic variables (presence or absence of DWA, and presence or absence of JBA >8 mm2) as the dependent variables and classes of each histologic feature as independent variables.
      Table IIThe association between histologic features and ultrasonic plaque texture features
      Continuous ultrasonic variablesCategoric ultrasonic variables
      No.GSM ± SDPlaque area ± SDStenosis ± SDPresence of DWAPresence of JBA >8 mm2
      No. (%)OR95% CINo. (%)OR95% CI
      Macrophage infiltration
       CD68 grade 12934.3 ± 14.655.2 ± 19.885.4 ± 10.15 (17)110 (34)11
       CD68 grade 22125.6 ± 13.158.6 ± 20.485.1 ± 9.211 (52)5.281.45-19.168 (38)1.170.36-3.76
       CD68 grade 32020.3 ± 8.963.8 ± 26.487.3 ± 9.214 (71)11.202.88-45.5310 (50)1.900.59-6.04
       ANOVA, P value.001.411.786Trend = .001Trend = .540
      SMCs
       SMC grade 13822.8 ± 10.858.7 ± 24.987.7 ± 8.022 (58)120 (53)1
       SMC grade 21731.1 ± 13.259.7 ± 21.387.8 ± 8.17 (41)0.510.15-1.623 (18)0.130.05-0.78
       SMC grade 31536.2 ± 17.057.2 ± 15.680.8 ± 12.31 (7)0.050.01-0.435 (33)0.450.13-1.56
       ANOVA, P value.003.958.025Trend = .003Trend = .042
      Neovascularization (CD31)
       1st tertile2333.5 ± 15.557.5 ± 18.684.9 ± 10.22 (9)18 (35)1
       2nd tertile2427.5 ± 12.656.3 ± 23.487.3 ± 10.29 (37)6.31.19-33.411 (46)1.580.49-5.14
       3rd tertile2322.1 ± 11.562.2 ± 24.286.2 ± 8.119 (83)49.98.18-30.99 (39)1.20.36-3.99
       ANOVA, P value.019.631.689Trend <.001Trend = .738
      Lipid core size
       Small1238.9 ± 14.260.8 ± 18.381.2 ± 9.31 (8)12 (17)1
       Intermediate3428.3 ± 13.653.8 ± 22.386.5 ± 9.216 (47)9.781.13-84.3611 (32)2.390.45-12.8
       Large2421.2 ± 10.364.3 ± 22.688.2 ± 9.513 (54)13.01.44-117.215 (62)8.331.48-46.9
       ANOVA, P value.001.199.114Trend = .025Trend = .013
      Fibrous cap size
       Thick2733.4 ± 16.059.3 ± 19.983.8 ± 10.28 (30)18 (30)1
       Thin4324.1 ± 11.158.2 ± 22.687.6 ± 8.822 (51)2.480.89-6.920 (46)2.060.74-5.73
       Independent sample t-test, P value.005.845.100.076.160
      Intraplaque hemorrhage
       Absent2834.9 ± 15.859.7 ± 21.783.9 ± 10.15 (18)18 (29)1
       Present4222.9 ± 10.157.9 ± 22.887.7 ± 8.825 (59)6.762.15-21.2920 (48)2.270.02-6.30
       Independent sample t-test, P value.001.739.120.001.111
      Plaque rupture
       Absent4132.8 ± 14.658.7 ± 22.384.9 ± 9.713 (32)110 (24)1
       Present2920.5 ± 8.958.6 ± 22.187.9 ± 9.117 (59)3.051.13-8.2118 (62)5.071.80-14.2
       Independent sample t-test, P value.001.995.194.025.002
      Plaque stability
       Stable3432.9 ± 15.958.4 ± 21.283.2 ± 10.310 (29)18 (23)1
       Unstable2922.6 ± 9.659.7 ± 22.989.3 ± 7.720 (56)5.181.85-14.5320 (56)4.061.45-11.4
       Independent sample t-test, P value<.002.819.007.027.006
      ANOVA, Analysis of variance; CI, confidence interval; DWA, discreet white area; JBA, juxtaluminal black area; OR, odds ratio; SD, standard deviation; SMC, smooth muscle cell.
      P for categoric features was calculated using the χ2 test. Statistically significant results are shown in bold.
      The prevalence of JBA on ultrasound in relation to fibrous cap thickness (Table III, A and B), size of lipid core (Table III, A), and plaque rupture (Table III, B) was explored. The χ2 test was used to calculate the significance.
      Table IIIA, Prevalence of JBA on ultrasound in relation to fibrous cap thickness and size of lipid core
      Fibrous capLipid core size, No.JBA presence, No. (%)P
      Thin (43 carotid plaques)
       Small0
       Moderate233 (18)
       Large2013 (65).023
      P for categoric features was calculated using the χ2 test.
      Thick (27 carotid plaques)
       Small122 (17)
       Moderate114 (36)
       Large42 (50).367
      P for categoric features was calculated using the χ2 test.
      JBA, Juxtaluminal black area.
      a P for categoric features was calculated using the χ2 test.
      Odds ratios also were calculated to test the significance of the association between statin therapy and histologic features (unadjusted and adjusted for the presence of symptoms) using logistic regression analysis for each feature (Table IV).
      Table IIIB, Prevalence of JBA on ultrasound in relation to fibrous cap thickness and plaque rupture
      Fibrous capPlaque rupture, No.JBA presence, No. (%)P
      Thin (43 carotid plaques)
       Absent173 (18)
       Present2617 (65).002
      P for categoric features was calculated using the χ2 test.
      Thick (27 carotid plaques)
       Small247 (29)
       Moderate31 (33).882
      P for categoric features was calculated using the χ2 test.
      JBA, Juxtaluminal black area.
      a P for categoric features was calculated using the χ2 test.
      Table IVThe prevalence of statin therapy in relation to histologic features: unadjusted and adjusted for symptoms
      Unadjusted for symptomsAdjusted for symptoms
      Total, No.No. (%)OR95% CIOR95% CI
      Macrophage infiltration
       CD68 grade 12921 (72)11
       CD68 grade 22110 (47)0.350.11-1.130.340.10-1.19
       CD68 grade 3204 (20)0.090.002-0.0370.170.04-0.73
      P for trend.001.013
      SMCs
       SMC grade 13812 (31)11
       SMC grade 21711 (64)3.971.19-13.281.870.51-6.94
       SMC grade 31512 (80)8.672.06-36.516.021.28-28.1
      P for trend.001.021
      Neovascularization (CD31)
       1st tertile2315 (65)11
       2nd tertile2416 (67)1.070.32-3.571.290.35-4.75
       3rd tertile234 (17)0.110.03-0.440.170.04-0.75
      P for trend.001.025
      Lipid core size
       Small129 (75)11
       Intermediate3415 (44)0.260.06-1.450.370.08-1.8
       Large2411 (46)0.280.06-1.310.420.08-2.17
      P for trend.174.0395
      Fibrous cap size
       Thick2717 (63)11
       Thin4318 (42)0.420.16-1.140.520.17-1.54
      χ2, P value.088.240
      Intraplaque hemorrhage
       Absent2818 (64)11
       Present4217 (40)0.380.14-1.010.400.13-1.21
      χ2, P value.054.104
      Plaque rupture
       Absent4123 (56)11
       Present2912 (41)0.550.21-1.450.610.21-1.78
      χ2, P value.227.366
      Plaque stability
       Stable3419 (56)11
       Unstable2916 (44)0.630.25-1.620.660.21-2.06
      χ2, P value.340.471
      CI, Confidence interval; OR, odds ratio; SMC, smooth muscle cell.

      Results

      Table I shows that the symptomatic (n = 20) and asymptomatic (n = 50) groups were comparable in terms of internal carotid stenosis and prevalence of risk factors except for total cholesterol, which was higher in the symptomatic group. It highlights the fact that only 15% of the symptomatic patients were receiving statin therapy before the referral to our department, in contrast to 64% of the asymptomatic patients who had been on long-term therapy (P < .001). However, the prevalence of statin therapy between patients with stable (56%) vs unstable (44%) plaques was not significant (P = .339).
      Table II shows that a low GSM was associated with a high grade of macrophage infiltration, a small number of SMCs, a higher grade of neovascularization, a large lipid core, a thin fibrous cap, the presence of intraplaque hemorrhage, and with plaques classified as unstable. Higher grades of stenosis were associated with low numbers of SMCs and unstable plaques. The presence of DWA was associated with a large number of macrophages, low numbers of SMCs, severe neovascularization, an intermediate or large lipid core, the presence of intraplaque hemorrhage, a higher prevalence of plaque rupture, and plaque instability. The presence of JBA was associated with low levels of SMC, a larger lipid core, a higher prevalence of plaque rupture, and plaques classified as unstable. In particular, both the combination of a thin fibrous cap with a large lipid core (Table IIIa) and the combination of a thin fibrous cap with plaque rupture (Table IIIb) were associated with the highest (65%) prevalence of a JBA. The plaque area was not associated with any of the histologic features.
      As indicated earlier (Table I), statin therapy taken by the majority of the asymptomatic patients as part of their routine medical management was a confounding factor. However, on a secondary analysis, it offered the opportunity to investigate the differences in histology between the group on this therapy and the group not on statins. The association between statin therapy and histologic features is shown in Table IV. Statin therapy was associated with a low grade of macrophage infiltration, a high number of SMCs, and a lower level of neovascularization. These findings were statistically significant even after adjustment for the presence of symptoms. There was a trend of statin therapy before surgery to be associated with a small lipid core, a thick fibrous cap, an absence of intraplaque hemorrhage, a lower prevalence of plaque rupture, and plaques labeled as stable, but the results did not reach statistical significance.

      Discussion

      The results of the present study confirm previous findings that a low GSM is associated with histologically unstable plaques (thin fibrous cap, large lipid core, and small number of SMCs), increased inflammation (increased number of macrophages and neovascularization), and the end result of this inflammation, which is intraplaque hemorrhage and plaque rupture. A low GSM can be explained by the fact that lipids and hemorrhage do not reflect ultrasound.
      Another finding was the fact that high numbers of SMCs were associated with a high GSM and histologically stable plaques. This finding suggests that plaques with a low content of SMCs may be more prone to more aggressive progress and plaque rupture.
      It has been suggested that vascular SMC proliferation may be even beneficial throughout atherogenesis, and not just in advanced lesions, whereas the apoptosis of SMCs and its low numbers may promote inflammation.
      • Bennett M.R.
      • Sinha S.
      • Owens G.K.
      Vascular smooth muscle cells in atherosclerosis.
      This also is supported by the findings in the Tromso study that echolucent plaques tend to progress faster than echogenic plaques.
      • Johnsen S.H.
      • Mathiesen E.B.
      • Fosse E.
      • Joakimsen O.
      • Stensland-Bugge E.
      • Njølstad I.
      • et al.
      Elevated high-density lipoprotein cholesterol levels are protective against plaque progression: a follow-up study of 1952 persons with carotid atherosclerosis the Tromsø study.
      DWA are by definition not associated with acoustic shadow and appear in echolucent areas (Fig 1). Histologically they are devoid of calcium. These DWAs, which with the exception of calcium are responsible for plaque heterogeneity, appear hyperperfused when contrast-enhanced ultrasound is performed (microbubbles are injected intravenously) and correspond to neovascularization and an increased number of macrophages on histology.
      • Shah F.
      • Balab P.
      • Weinberg M.
      • Reddy V.
      • Neems R.
      • Feinstein M.
      • et al.
      Contrast-enhanced ultrasound imaging of atherosclerotic carotid plaque neovascularization: a new surrogate marker of atherosclerosis?.
      In contrast to histology, in which the structure of tissues is visualized with transillumination, ultrasonic images are produced from the reflection (backscatter) of ultrasound by tissue interphases. Vessel walls can be highly reflective (eg, hemangioma in the liver) and appear as bright structures in ultrasound scans. These facts can explain the association of DWA with increased hemosiderin and inflammation found in a recent study.
      • Mitchell C.C.
      • Stein J.H.
      • Cook T.D.
      • Salamat S.
      • Wang X.
      • Varghese T.
      • et al.
      Histopathologic validation of grayscale carotid plaque characteristics related to plaque vulnerability.
      In our study, with the exception of fibrous cap thickness, all histologic features characterizing unstable plaques were associated with the presence of DWA. These findings can explain why the presence of DWA could predict an increased stroke rate throughout the 7 years of follow-up evaluation in the Asymptomatic Carotid Stenosis and Risk of Stroke (ACSRS) study,
      • Nicolaides A.N.
      • Kakkos S.K.
      • Kyriacou E.
      • Griffin M.
      • Sabetai M.
      • Thomas D.J.
      • et al.
      Asymptomatic internal carotid artery stenosis and cerebrovascular risk stratification.
      • Kakkos S.K.
      • Griffin M.B.
      • Nicolaides A.N.
      • Kyriacou E.
      • Sabetai M.M.
      • Tegos T.
      • et al.
      The size of juxtaluminal hypoechoic area in ultrasound images of asymptomatic carotid plaques predicts the occurrence of stroke.
      suggesting a structural composition that is a marker of long-lasting plaque instability.
      Histologic studies in the 1990s showed that the necrotic core is twice as close to the lumen in symptomatic plaques compared with asymptomatic plaques.
      • Bassiouni H.S.
      • Sakaguchi Y.
      • Mikucki S.A.
      • McKinsey J.F.
      • Piano G.
      • Gewertz B.L.
      • et al.
      Juxtaluminal location of plaque necrosis and neoformation in symptomatic carotid stenosis.
      These findings correspond to subsequent observations in cross-sectional studies using ultrasound scanning, showing an association between the JBA and the presence of neurologic symptoms
      • Sztajzel R.
      Stratified gray-scale median analysis and color mapping of the carotid plaque.
      • Griffin M.B.
      • Kyriacou E.
      • Pattichis C.
      • Bond D.
      • Kakkos S.K.
      • et al.
      Juxtaluminal hypoechoic area in ultrasonic images of carotid plaques and hemispheric symptoms.
      • Pedro L.K.
      • Fernandes e Fernandes J.
      • Pedro M.M.
      • Goncalves I.
      • Dias N.V.
      • Fernandes e Fernandes R.
      • et al.
      Ultrasonographic risk score of carotid plaques.
      • Sztajzel R.
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      • Comelli M.
      • Momjian S.
      Correlation of cerebrovascular symptoms and microembolic signals with the stratified gray-scale median analysis and color mapping of the carotid plaque.
      and confirmation by histology that the lipid core is close to the lumen.
      • Sztajzel R.
      Stratified gray-scale median analysis and color mapping of the carotid plaque.
      A cross-sectional study of patients with symptomatic and asymptomatic plaques by our group showed that a JBA >8 mm2 in the absence of a visible echogenic cap was associated with a high prevalence of symptomatic plaques in all grades of stenosis.
      • Griffin M.B.
      • Kyriacou E.
      • Pattichis C.
      • Bond D.
      • Kakkos S.K.
      • et al.
      Juxtaluminal hypoechoic area in ultrasonic images of carotid plaques and hemispheric symptoms.
      In the prospective ACSRS study involving 1121 patients with ACS, JBA had a linear association with future stroke rate and a JBA >8 mm2 could identify a subgroup of 245 patients who had an annual stroke rate of 4.1%. This subgroup contained 42 of the 59 ipsilateral hemispheric strokes that occurred in the whole cohort during the 8-year follow-up period.
      • Kakkos S.K.
      • Griffin M.B.
      • Nicolaides A.N.
      • Kyriacou E.
      • Sabetai M.M.
      • Tegos T.
      • et al.
      The size of juxtaluminal hypoechoic area in ultrasound images of asymptomatic carotid plaques predicts the occurrence of stroke.
      Thus, JBA is the most powerful ultrasonic feature that can predict increased stroke risk.
      In our study, another novel finding was the fact that JBA was associated with a large lipid core and plaque rupture (Table II). Its highest prevalence (65%) was found in plaques with a thin fibrous cap and a large lipid core (Table III, A); also in plaques with a thin fibrous cap and a plaque rupture (Table III, B). With a resolution of 0.2 mm for ultrasound a fibrous cap thinner than 200 μm will not be detected and therefore the appearance of a large JBA without a visible cap may occur. A thrombus on the surface of a ruptured plaque also may appear as a large JBA. A thrombus on the surface of an intact plaque even in the presence of a thick fibrous cap or even a calcified plaque also may appear as a large JBA (Fig 2). All three cases represent a high-risk situation and explain why the presence of a JBA is such a powerful predictor of stroke.
      A major finding of our study was the lack of association of plaque area with plaque instability. Plaque area has been reported to be a strong predictor of stroke and myocardial infarction.
      • Spence J.D.
      • Eliasziw M.
      • DiCicco M.
      • Hackam D.G.
      • Galil R.
      • Lohmann T.
      Carotid plaque area: a tool for targeting and evaluating vascular preventive therapy.
      • Johnsen S.H.
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      • Joakimsen O.
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      • Wilsgaard T.
      • Lochen M.L.
      • et al.
      Carotid atherosclerosis is a stronger predictor of myocardial infarction in women than in men: a 6-year follow-up study of 6226 persons: the Tromso Study.
      Salem et al,
      • Salem M.K.
      • Bown M.J.
      • Sayers R.D.
      • West K.
      • Moore D.
      • Nicolaides A.
      • et al.
      Identification of patients with a histologically unstable plaque using ultrasonic plaque image analysis.
      based on the presence and the extent of histologic features, showed that the combination of plaque area >95 mm2 and a juxtaluminal black area >6 mm2 may identify patients with 90% probability of having a histologically unstable plaque. In the prospective ACSRS study involving 1121 patients with ACS, a plaque area ≥80 mm2 could identify a subgroup of 114 patients who had an annual stroke rate of 3.5%.
      • Nicolaides A.N.
      • Kakkos S.K.
      • Kyriacou E.
      • Griffin M.
      • Sabetai M.
      • Thomas D.J.
      • et al.
      Asymptomatic internal carotid artery stenosis and cerebrovascular risk stratification.
      However, in a subsequent publication
      • Kakkos S.K.
      • Griffin M.B.
      • Nicolaides A.N.
      • Kyriacou E.
      • Sabetai M.M.
      • Tegos T.
      • et al.
      The size of juxtaluminal hypoechoic area in ultrasound images of asymptomatic carotid plaques predicts the occurrence of stroke.
      in which JBA >8 mm2 was included in the multivariable analysis, the plaque area itself was no longer significant, indicating that the significance found in the first publication was likely to be driven by the size of the JBA. However, in the present study the plaque area was not associated with any of the histologic features characteristic of plaque instability (Table II). Another possible explanation is that the risk of plaque rupture in large plaques may not originate in its histologic structure but may be inherent to its size and particularly its length. The role of biomechanical forces in the induction of plaque fatigue and rupture has been emphasized for some time.
      • Glagov S.
      • Bassiouny H.S.
      • Sakaguchi Y.
      • Goudet C.A.
      • Vito R.P.
      Mechanical determinants of plaque modelling and disruption.
      • Fuster V.
      • Stein B.
      • Ambrose J.A.
      • Badimon L.
      • Badimon J.J.
      • Chesebro J.H.
      Atherosclerotic plaque rupture and thrombosis: evolving concepts.
      Thus, high plaque strain (deformability caused by high pulse pressure and blood velocity) and high stress (associated forces) may be the dominant factors responsible for plaque rupture in large plaques irrespective of plaque morphology. Differences between motion measurements extracted from symptomatic and asymptomatic plaques have been reported,
      • Golemati S.
      • Stoisis J.
      • Gastounioti A.
      • Dimopoulos A.C.
      • Koropouli V.
      • Nikita K.S.
      Comparison of block matching and differential methods for motion analysis of the carotid artery wall from ultrasound images.
      • Widman E.
      • Caidahl K.
      • Heyde B.
      • D’hooge J.
      • Larsson M.
      Ultrasound speckle tracking strain estimation of in vivo carotid artery plaque with in vitro sonomicrometry validation.
      • HuangC Pan X.
      • He Q.
      • Huang M.
      • Huang L.
      • Zhao X.
      • et al.
      Ultrasound-based carotid elastography for detection of vulnerable atherosclerotic plaques validated by magnetic resonance imaging.
      and two studies have shown the feasibility of computing carotid plaque strain measurements from routine B-mode imaging using clinical ultrasound equipment.
      • Nasrabadi H.
      • Pattichis M.
      • Nicolaides A.N.
      • Griffin M.
      • Makris G.C.
      • Fisher P.
      • et al.
      Measurement of motion of carotid bifurcation plaques.
      • Khan A.A.
      • Sikdar S.
      • Hatsukami T.
      • Cebral J.
      • Jones M.
      • Huston J.
      • et al.
      Noninvasive characterization of carotid plaque strain.
      Future studies using plaque motion analysis need to elucidate whether strain/stress measurements in asymptomatic plaques also are independent predictors of future ipsilateral neurologic events.
      A limitation of this study was that statin therapy was administered to 64% of asymptomatic and only 15% of symptomatic patients (Table I). Although this was a confounding factor that likely affected the presence of inflammation in the plaque, the effect on macrophage infiltration, number of SMCs, and neovascularization still was significant after adjusting for symptoms (Table IV). This finding has yet to be investigated and be confirmed in future studies. Another limitation was the small number of patients included. This is a potential explanation as to why although there was a trend of statin therapy before surgery to be associated with a small lipid core, a thick fibrous cap, absence of intraplaque hemorrhage, a lower prevalence of plaque rupture, and plaques labeled as stable, the results did not reach statistical significance. Larger prospective studies are needed to support the observations of our study. The time interval between symptoms and endarterectomy potentially could play a role because a longer time distance from the symptoms and commencement of statin therapy may increase plaque stability. The average waiting time between referral and surgery in our department at the time of the study was 3 weeks (range, 2-4 weeks). However, other than those limitations, this study was an indirect validation and explanation why certain ultrasonic features can identify high-risk plaques and predict the risk of stroke in asymptomatic patients as shown in the ACSRS prospective study.
      • Nicolaides A.N.
      • Kakkos S.K.
      • Kyriacou E.
      • Griffin M.
      • Sabetai M.
      • Thomas D.J.
      • et al.
      Asymptomatic internal carotid artery stenosis and cerebrovascular risk stratification.
      • Kakkos S.K.
      • Griffin M.B.
      • Nicolaides A.N.
      • Kyriacou E.
      • Sabetai M.M.
      • Tegos T.
      • et al.
      The size of juxtaluminal hypoechoic area in ultrasound images of asymptomatic carotid plaques predicts the occurrence of stroke.

      Conclusions

      Our study showed that with the exception of plaque area, a number of key ultrasonic texture features previously shown to be able to stratify asymptomatic patients into different classes of stroke risk have a strong association with established histologic features of plaque instability. This finding provides insight into the mechanism of ultrasonic texture features in stroke prediction and validates the use of ultrasound in stroke risk stratification.

      Author contributions

      Conception and design: KS, IT, PL, DM, AG
      Analysis and interpretation: KS, IT, PL
      Data collection: KS, IT, PL
      Writing the article: KS, IT, PL, DM, AG
      Critical revision of the article: KS, IT, PL, DM, AG
      Final approval of the article: KS, IT, PL, DM, AG
      Statistical analysis: Not applicable
      Obtained funding: Not applicable
      Overall responsibility: AG
      KS and IT contributed equally to this article and share co-first authorship.

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