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Contrast-Enhanced Ultrasound to Assess Carotid Intraplaque Neovascularization

      Abstract

      Contrast-enhanced ultrasound (CEUS) is increasingly being used to identify patients with carotid plaques that are vulnerable to rupture, so-called vulnerable atherosclerotic plaques, by assessment of intraplaque neovascularization. A complete overview of the strengths and limitations of carotid CEUS is currently not available. The aim of this systematic review was to provide a complete overview of existing publications on the role of CEUS in assessment of carotid intraplaque neovascularization. The systematic review of the literature yielded 52 studies including a total of 4660 patients (mean age: 66 y, 71% male) who underwent CEUS for the assessment of intraplaque neovascularization. The majority of the patients (76%) were asymptomatic and had no history of transient ischemic attack (TIA) or stroke. The assessment of intraplaque neovascularization was mostly performed using a visual scoring system; several studies used time–intensity curves or dedicated quantification software to optimize analysis. In 17 studies CEUS was performed in patients before carotid surgery (endarterectomy), allowing a comparison of pre-operative CEUS findings with histologic analysis of the tissue sample that is removed from the carotid artery. In a total of 576 patients, the CEUS findings were compared with histopathological analysis of the plaque after surgery. In 16 of the 17 studies, contrast enhancement was found to correlate with the presence and degree of intraplaque neovascularization on histology. Plaques with a larger amount of contrast enhancement had significantly increased density of microvessels in the corresponding region on histology. In conclusion, CEUS is a readily available imaging modality for the assessment of patients with carotid atherosclerosis, providing information on atherosclerotic plaques, such as ulceration and intraplaque neovascularization, which may be clinically relevant. The ultimate clinical goal is the early identification of carotid atherosclerosis to start early preventive therapy and prevent clinical complications such as TIA and stroke.

      Key Words

      Introduction

      Atherosclerosis is the leading cause of vascular morbidity and mortality worldwide (
      • Herrington W.
      • Lacey B.
      • Sherliker P.
      • Armitage J.
      • Lewington S.
      Epidemiology of atherosclerosis and the potential to reduce the global burden of atherothrombotic disease.
      ). The disease has a subclinical phase of many years and is characterized by fatty streaks in the arterial wall that progressively develop into atherosclerotic plaques. Atherosclerosis may be limited to the arteries supplying one organ system but usually several vascular beds are affected. Atherosclerotic plaques may undergo transformation into vulnerable plaques that are vulnerable to rupture. Vulnerable plaques are characterized by a thin fibrous cap, a lipid-rich necrotic core, plaque inflammation, intraplaque neovascularization and intraplaque hemorrhage (
      • Schaar J.A.
      • Muller J.E.
      • Falk E.
      • Virmani R.
      • Fuster V.
      • Serruys P.W.
      • Colombo A.
      • Stefanadis C.
      • Ward Casscells S.
      • Moreno P.R.
      • Maseri A.
      • van der Steen A.F.
      Terminology for high-risk and vulnerable coronary artery plaques. Report of a meeting on the vulnerable plaque, June 17 and 18, 2003, Santorini, Greece.
      ). Acute rupture of a vulnerable plaque may cause local thrombosis, which may lead to partial or total occlusion of the affected artery or embolization more distally. Depending on the site and the degree of arterial occlusion, atherosclerotic plaque rupture may cause life-threatening clinical complications, comprising stroke, myocardial infarction and peripheral arterial disease. Atherosclerotic plaques in the carotid arteries are an important cause of stroke and transient ischemic attacks (TIAs) and are an indicator of the presence of systemic atherosclerosis.
      Several imaging modalities have been developed in an attempt to identify patients with carotid plaques that are vulnerable to rupture, so-called vulnerable patients (
      • ten Kate G.L.
      • Sijbrands E.J.
      • Staub D.
      • Coll B.
      • ten Cate F.J.
      • Feinstein S.B.
      • Schinkel A.F.
      Noninvasive imaging of the vulnerable atherosclerotic plaque.
      ). Early identification of these patients allows early therapy and thereby may prevent clinical complications including TIA and stroke. The characteristics of ultrasound make it an ideal imaging method for screening patients with carotid atherosclerosis in the search for vulnerable plaques (
      • Feinstein S.B.
      • Coll B.
      • Staub D.
      • Adam D.
      • Schinkel A.F.
      • ten Cate F.J.
      • Thomenius K.
      Contrast enhanced ultrasound imaging.
      ;
      • Staub D.
      • Schinkel A.F.
      • Coll B.
      • Coli S.
      • van der Steen A.F.
      • Reed J.D.
      • Krueger C.
      • Thomenius K.E.
      • Adam D.
      • Sijbrands E.J.
      • ten Cate F.J.
      • Feinstein S.B.
      Contrast-enhanced ultrasound imaging of the vasa vasorum: From early atherosclerosis to the identification of unstable plaques.
      ). This imaging modality is widely available and can be used for screening for the presence, location and severity of atherosclerotic plaques. Moreover, characteristics of plaque vulnerability including ulceration and intraplaque neovascularization can be evaluated with the use of an intravascular ultrasound contrast agent (
      • Schaar J.A.
      • Muller J.E.
      • Falk E.
      • Virmani R.
      • Fuster V.
      • Serruys P.W.
      • Colombo A.
      • Stefanadis C.
      • Ward Casscells S.
      • Moreno P.R.
      • Maseri A.
      • van der Steen A.F.
      Terminology for high-risk and vulnerable coronary artery plaques. Report of a meeting on the vulnerable plaque, June 17 and 18, 2003, Santorini, Greece.
      ;
      • ten Kate G.L.
      • Sijbrands E.J.
      • Staub D.
      • Coll B.
      • ten Cate F.J.
      • Feinstein S.B.
      • Schinkel A.F.
      Noninvasive imaging of the vulnerable atherosclerotic plaque.
      ). In this review article, we discuss the strengths and limitations of ultrasound, in particular contrast-enhanced ultrasound (CEUS) for the assessment of patients with known or suspected carotid atherosclerosis. The aim of this systematic review was to provide a complete overview of existing publications on the role of CEUS in assessment of carotid intraplaque neovascularization.

      Methods

      Search strategy and study selection

      This review includes all available original studies reporting on the use of CEUS to assess intraplaque neovascularization in patients with carotid atherosclerosis. Studies on animals and review articles were excluded. Studies focusing on CEUS in patients without atherosclerosis (such as studies on radiation-induced carotid disease, or carotid arteritis) were excluded. All included studies must have obtained informed consent from each study participant and approval by an ethics committee or institutional review board. The online MEDLINE database was searched for literature in June 2019 using PubMed (National Center for Biotechnology Information, U.S. National Library of Medicine, Bethesda, MD, USA). The search strategy was carotid and contrast-enhanced ultrasound. No time restriction for publication dates was used. All titles and abstracts of the articles were evaluated. After exclusion based on the title and abstract, full articles were evaluated, and articles meeting the inclusion criteria were identified (Fig. 1). In addition, a manual search of the reference lists of the identified studies was performed, and references were evaluated using the same inclusion and exclusion criteria. Selected studies were reviewed and relevant patient characteristics and CEUS findings were extracted. It cannot be excluded that there is overlap in the patient populations of the studies that were included in the systematic review.
      Fig 1
      Fig. 1Flowchart of the literature search and study selection. The initial search yielded 597 eligible studies. These records were evaluated, and on the basis of title and abstract, 521 articles were excluded. The remaining 76 articles were evaluated, and after cross-referencing, a total of 52 articles were included in the systematic review.

      Statistical analysis

      Statistical analysis was performed using Microsoft Excel 2010 (Microsoft Corp., Redmond, WA, USA). Continuous variables were reported as means. Categorical variables were summarized as percentages. From the pooled data, summary estimates were calculated.

      Results

      B-Mode ultrasound

      B-Mode ultrasound is a widely used method for the assessment of atherosclerosis. Measurement of intima–media thickness (IMT) can be easily and quickly performed to screen for subclinical atherosclerosis.
      • Pignoli P.
      • Tremoli E.
      • Poli A.
      • Oreste P.
      • Paoletti R.
      Intimal plus medial thickness of the arterial wall: A direct measurement with ultrasound imaging.
      were the first to report a study in vitro of specimens of human and aortic and common carotid arteries to determine the feasibility of direct measurement of arterial wall thickness with B-mode real-time imaging. This method was also performed in vivo in young patients, indicating that B-mode imaging represents a useful method for the measurement of IMT of human arteries in vivo. Subsequent studies have indicated that IMT measurement is highly reproducible. IMT has been widely adopted clinically and is frequently used as a surrogate endpoint in pharmacologic intervention studies. A recent review and meta-analysis of the literature including 15 studies revealed that carotid IMT (CIMT) as measured by B-mode ultrasound is associated with future cardiovascular events (
      • van den Oord S.C.
      • Sijbrands E.J.
      • ten Kate G.L.
      • van Klaveren D.
      • van Domburg R.T.
      • van der Steen A.F.
      • Schinkel A.F.
      Carotid intima-media thickness for cardiovascular risk assessment: Systematic review and meta-analysis.
      ). However, the addition of CIMT to traditional cardiovascular risk prediction models did not lead to a statistically significant improvement in the performance of those models.
      A systematic ultrasound examination of the carotid arteries screening for the presence of plaques may further increase the sensitivity for the identification of subclinical atherosclerosis. A recent meta-analysis including 11 population-based studies (55,336 patients) revealed that carotid plaque measured by B-mode ultrasound had a higher diagnostic accuracy than CIMT for the prediction of coronary artery disease events (
      • Inaba Y.
      • Chen J.A.
      • Bergmann S.R.
      Carotid plaque, compared with carotid intima–media thickness, more accurately predicts coronary artery disease events: A meta-analysis.
      ). The European Society of Cardiology guidelines on chronic coronary syndrome (CCS) recommends that ultrasound of the carotid arteries should be considered, and be performed by adequately trained clinicians, to detect plaque in patients with suspected CCS without known atherosclerotic disease (class 2A recommendation). CIMT for cardiovascular risk assessment is not recommended by the European Society of Cardiology (
      • Knuuti J.
      • Wijns W.
      • Saraste A.
      • Capodanno D.
      • Barbato E.
      • Funck-Brentano C.
      • Prescott E.
      • Storey R.F.
      • Deaton C.
      • Cuisset T.
      • Agewall S.
      • Dickstein K.
      • Edvardsen T.
      • Escaned J.
      • Gersh B.J.
      • Svitil P.
      • Gilard M.
      • Hasdai D.
      • Hatala R.
      • Mahfoud F.
      • Masip J.
      • Muneretto C.
      • Valgimigli M.
      • Achenbach S.
      • Bax J.J.
      ESC Scientific Document Group
      2019 ESC Guidelines for the diagnosis and management of chronic coronary syndromes [Epub ahead of print.].
      ). The BioImage study (
      • Sillesen H.
      • Muntendam P.
      • Adourian A.
      • Entrekin R.
      • Garcia M.
      • Falk E.
      • Fuster V.
      Carotid plaque burden as a measure of subclinical atherosclerosis: Comparison with other tests for subclinical arterial disease in the High Risk Plaque BioImage study.
      ) used a 3-D-based ultrasound approach, where the carotid artery was investigated in cross section from proximal to as distal as possible. From the resulting video, plaque was outlined on cross-sectional images, and plaque areas were summarized into “plaque burden.” There are indications that plaque burden, which provides information on the extent and volume of plaque, provides additional information for the prediction of events (
      • Sillesen H.
      • Sartori S.
      • Sandholt B.
      • Baber U.
      • Mehran R.
      • Fuster V.
      Carotid plaque thickness and carotid plaque burden predict future cardiovascular events in asymptomatic adult Americans.
      ).
      B-Mode ultrasound also provides information for characterization of atherosclerotic plaque (
      • ten Kate G.L.
      • Sijbrands E.J.
      • Staub D.
      • Coll B.
      • ten Cate F.J.
      • Feinstein S.B.
      • Schinkel A.F.
      Noninvasive imaging of the vulnerable atherosclerotic plaque.
      ). Echogenic plaques are considered to contain solid components, such as fibrous or calcified tissue. Echolucent plaques often contain histologic features of plaque instability, such as lipids and intraplaque hemorrhage (
      • Spanos K.
      • Tzorbatzoglou I.
      • Lazari P.
      • Maras D.
      • Giannoukas A.D.
      Carotid artery plaque echomorphology and its association with histopathologic characteristics.
      ). Therefore echogenic plaques are probably more stable, and echolucent and mixed plaques are considered to be at increased risk of rupture and thrombosis (
      • Grønholdt M.L.
      • Nordestgaard B.G.
      • Bentzon J.
      • Wiebe B.M.
      • Zhou J.
      • Falk E.
      • Sillesen H.
      Macrophages are associated with lipid-rich carotid artery plaques, echolucency on B-mode imaging, and elevated plasma lipid levels.
      ;
      • Spanos K.
      • Tzorbatzoglou I.
      • Lazari P.
      • Maras D.
      • Giannoukas A.D.
      Carotid artery plaque echomorphology and its association with histopathologic characteristics.
      ). However, these two studies did not find differences in outcomes in patients with echolucent versus echogenic plaques.

      Contrast-enhanced carotid ultrasound

      The use of an intravascular contrast agent in conjunction with ultrasound improves the delineation of the vasculature and provides information that cannot be obtained using B-mode ultrasound alone. The contrast agent consists of microbubbles, composed of a lipid or protein shell and a gaseous core, that are strong reflectors of the ultrasound signal (
      • Feinstein S.B.
      • Coll B.
      • Staub D.
      • Adam D.
      • Schinkel A.F.
      • ten Cate F.J.
      • Thomenius K.
      Contrast enhanced ultrasound imaging.
      ). After intravenous administration of the contrast agent, angiography-like images can be obtained. In patients at increased risk of atherosclerosis, administration of contrast agent improves the diagnostic accuracy for the detection of atherosclerotic plaques and improves risk stratification (
      • van den Oord S.C.
      • ten Kate G.L.
      • Akkus Z.
      • Renaud G.
      • Sijbrands E.J.
      • ten Cate F.J.
      • van der Lugt A.
      • Bosch J.G.
      • de Jong N.
      • van der Steen A.F.
      • Schinkel A.F.
      Assessment of subclinical atherosclerosis using contrast-enhanced ultrasound.
      , and
      • van den Oord S.C.
      • ten Kate G.L.
      • Sijbrands E.J.
      • van der Steen A.F.
      • Schinkel A.F.
      Effect of carotid plaque screening using contrast-enhanced ultrasound on cardiovascular risk stratification.
      ).
      • van den Oord S.C.
      • ten Kate G.L.
      • Sijbrands E.J.
      • van der Steen A.F.
      • Schinkel A.F.
      Effect of carotid plaque screening using contrast-enhanced ultrasound on cardiovascular risk stratification.
      studied 100 patients with risk factors for atherosclerosis, calculated the Prospective Cardiovascular Münster Heart Study (PROCAM) risk score and performed carotid CEUS. Remarkably, CEUS changed the risk category as estimated by the traditional risk stratification model in the majority of these asymptomatic patients. CEUS may thus be an additional method for cardiovascular risk prediction in patient groups with comorbidities.
      • Shah B.N.
      • Chahal N.S.
      • Kooner J.S.
      • Senior R.
      Contrast-enhanced ultrasonography vs B-mode ultrasound for visualization of intima-media thickness and detection of plaques in human carotid arteries.
      studied 175 patients using standard B-mode and CEUS of the carotid arteries. CEUS improved visualization of the intima–media complex and permitted a greater detection of carotid plaques (367 plaques vs. 350 plaques, p = 0.02). The authors concluded that further studies are required to determine whether there is incremental clinical and prognostic benefit related to this superior plaque detection by CEUS.
      B-Mode and color Doppler ultrasound have poor diagnostic accuracy in the detection of plaque ulceration. Several studies have found that plaque ulceration is a strong predictor of clinical atherosclerotic events. In patients with known carotid plaque, using computed tomography as a reference technique,
      • ten Kate G.L.
      • van Dijk A.C.
      • van den Oord S.C.
      • Hussain B.
      • Verhagen H.J.
      • Sijbrands E.J.
      • van der Steen A.F.
      • van der Lugt A.
      • Schinkel A.F.
      Usefulness of contrast-enhanced ultrasound for detection of carotid plaque ulceration in patients with symptomatic carotid atherosclerosis.
      found that CEUS has good diagnostic accuracy for plaque ulceration. A recent study by
      • Hamada O.
      • Sakata N.
      • Ogata T.
      • Shimada H.
      • Inoue T.
      Contrast-enhanced ultrasonography for detecting histological carotid plaque rupture: Quantitative analysis of ulcer.
      provided a histologic validation of plaque ulceration assessed by CEUS in 45 patients undergoing carotid endarterectomy. That study found that CEUS has a high sensitivity for identifying histologic plaque rupture, and the measurement of concavity on contrast-enhanced ultrasonography may enable the accurate detection of fibrous cap disruption.

      CEUS for assessment of intraplaque neovascularization

      Histopathological studies have found that the presence of microvessels within an atherosclerotic plaque is an indication of plaque instability.
      • Hellings W.E.
      • Peeters W.
      • Moll F.L.
      • Piers S.R.
      • van Setten J.
      • Van der Spek P.J.
      • de Vries J.P.
      • Seldenrijk K.A.
      • De Bruin P.C.
      • Vink A.
      • Velema E.
      • de Kleijn D.P.
      • Pasterkamp G.
      Composition of carotid atherosclerotic plaque is associated with cardiovascular outcome: A prognostic study.
      performed histologic examination of atherosclerotic carotid lesions that were collected from patients who underwent carotid endartectomy. Patients underwent clinical follow-up yearly, up to 3 y after surgery. Local plaque hemorrhage and increased microvessel formation in the plaque were independent predictors of clinical outcome.
      In 2006, Feinstein reported that intraplaque microvasculature can be assessed in vivo using CEUS. Images obtained during a carotid ultrasound examination of a patient with a TIA correlated with a plaque specimen at the time of carotid artery endarterectomy that was prepared with a CD-31 stain highlighting vascular endothelial cells. In another case, a 53-y-old patient with a history of diabetes underwent a contrast-enhanced carotid ultrasound study performed as part of a cardiovascular prevention screening that revealed prominent vasa vasorum. Despite having diabetes, the patient had not yet received statin therapy at the time of initial examination; after 8 mo of statin therapy, regression of intraplaque microvasculature was observed. After these initial ground-breaking observations, multiple studies have confirmed the ability of CEUS to assess intraplaque microvessels.
      A systematic review of the literature (Fig. 1 and Table 1) yielded 52 studies including a total of 4660 patients (with a mean age of 66 y, 71% male) who underwent CEUS for the assessment of intraplaque neovascularization. In the studies that provided information on symptoms, most of the patients (76%) were asymptomatic and had no history of TIA or stroke. In 17 studies, CEUS was performed in patients before carotid surgery (endarterectomy). Carotid endarterectomy provides the opportunity to compare pre-operative CEUS findings with histologic analysis of the tissue sample that is removed from the carotid artery. An overview of these 17 studies is provided in Table 2. In a total of 576 patients, the CEUS findings were compared with histopathological analysis of the plaque after surgery. In 16 of the 17 studies, contrast enhancement was found to correlate with the presence and degree of intraplaque neovascularization on histology. Plaques with a larger amount of contrast enhancement had significantly increased density of microvessels in the corresponding region on histology.
      Table 1Studies on contrast enhanced ultrasound for the assessment of carotid intraplaque neovascularization in humans
      Study (Year)nInclusion criterionMean age (y)Males (%)AtherosclerosisSymptomatic (stroke or TIA) (%)QuantificationHistology
      • Feinstein S.B.
      Contrast ultrasound imaging of the carotid artery vasa vasorum and atherosclerotic plaque neovascularization.
      1Diabetes mellitus53100Stenosis >50%0NoneNo
      • Vicenzini E.
      • Giannoni M.F.
      • Puccinelli F.
      • Ricciardi M.C.
      • Altieri M.
      • Di Piero V.
      • Gossetti B.
      • Valentini F.B.
      • Lenzi G.L.
      Detection of carotid adventitial vasa vasorum and plaque vascularization with ultrasound cadence contrast pulse sequencing technique and echo-contrast agent.
      23Asymptomatic carotid stenosis7069Stenosis >30%0NoneYes
      • Shah F.
      • Balan P.
      • Weinberg M.
      • Reddy V.
      • Neems R.
      • Feinstein M.
      • Dainauskas J.
      • Meyer P.
      • Goldin M.
      • Feinstein S.B.
      Contrast-enhanced ultrasound imaging of atherosclerotic carotid plaque neovascularization: A new surrogate marker of atherosclerosis?.
      15Significant carotid stenosis before surgeryNA59Stenosis >60%33Visual scoringYes
      • Coli S.
      • Magnoni M.
      • Sangiorgi G.
      • Marrocco-Trischitta M.M.
      • Melisurgo G.
      • Mauriello A.
      • Spagnoli L.
      • Chiesa R.
      • Cianflone D.
      • Maseri A.
      Contrast-enhanced ultrasound imaging of intraplaque neovascularization in carotid arteries: Correlation with histology and plaque echogenicity.
      32Carotid stenosis before surgery (n = 17) or asymptomatic carotid plaque (n = 15)7084Stenosis >30%19Visual scoringYes
      • Huang P.T.
      • Huang F.G.
      • Zou C.P.
      • Sun H.Y.
      • Tian X.Q.
      • Yang Y.
      • Tang J.F.
      • Yang P.L.
      • Wang X.T.
      Contrast-enhanced sonographic characteristics of neovascularization in carotid atherosclerotic plaques.
      63Carotid plaque5967Plaque thickness 3.1 ± 1.6 mmNAArrival time, time to peak, enhanced intensity (dB)No
      • Magnoni M.
      • Coli S.
      • Marrocco-Trischitta M.M.
      • Melisurgo G.
      • De Dominicis D.
      • Cianflone D.
      • Chiesa R.
      • Feinstein S.B.
      • Maseri A.
      Contrast-enhanced ultrasound imaging of periadventitial vasa vasorum in human carotid arteries.
      25

      15
      Significant carotid stenosis (n = 25) or no plaques (n = 15)71

      65
      67

      80
      Stenosis >50%

      IMT <1 mm
      NA

      0
      Periadventitial B-flow thickness (mm)No
      • Papaioannou T.G.
      • Vavuranakis M.
      • Androulakis A.
      • Lazaros G.
      • Kakadiaris I.
      • Vlaseros I.
      • Naghavi M.
      • Kallikazaros I.
      • Stefanadis C.
      In-vivo imaging of carotid plaque neoangiogenesis with contrast-enhanced harmonic ultrasound (Letter).
      1Acute coronary syndrome73100Stenosis <50%0Plaque gray-scale (GSM)No
      • Xiong L.
      • Deng Y.B.
      • Zhu Y.
      • Liu Y.N.
      • Bi X.J.
      Correlation of carotid plaque neovascularization detected by using contrast-enhanced US with clinical symptoms.
      104Carotid plaque thickness >2 mm6380Plaque thickness >2 mm34Signal enhancement (dB)No
      • Giannoni M.F.
      • Vicenzini E.
      • Citone M.
      • Ricciardi M.C.
      • Irace L.
      • Laurito A.
      • Scucchi L.F.
      • Di Piero V.
      • Gossetti B.
      • Mauriello A.
      • Spagnoli L.G.
      • Lenzi G.L.
      • Valentini F.B.
      Contrast carotid ultrasound for the detection of unstable plaques with neoangiogenesis: A pilot study.
      73Severe carotid stenosis before surgery6766Stenosis >70%12Visual scoringYes
      • Staub D.
      • Patel M.B.
      • Tibrewala A.
      • Ludden D.
      • Johnson M.
      • Espinosa P.
      • Coll B.
      • Jaeger K.A.
      • Feinstein S.B.
      Vasa vasorum and plaque neovascularization on contrast-enhanced carotid ultrasound imaging correlates with cardiovascular disease and past cardiovascular events.
      147Clinical indication for duplex ultrasound6461Any plaque12NoneNo
      • Owen D.R.
      • Shalhoub J.
      • Miller S.
      • Gauthier T.
      • Doryforou O.
      • Davies A.H.
      • Leen E.L.
      Inflammation within carotid atherosclerotic plaque: Assessment with late-phase contrast enhanced US.
      37Carotid plaque causing >30% stenosis7073Stenosis >30%43Late-phase enhancementNo
      • Huang P.T.
      • Chen C.C.
      • Aronow W.S.
      • Wang X.T.
      • Nair C.K.
      • Xue N.Y.
      • Shen X.
      • Li S.Y.
      • Huang F.G.
      • Cosgrove D.
      Assessment of neovascularization within carotid plaques in patients with ischemic stroke.
      176Carotid atherosclerosis6261IMT >1.2 mm46Visual scoring and time-intensity curveNo
      • Faggioli G.L.
      • Pini R.
      • Mauro R.
      • Pasquinelli G.
      • Fittipaldi S.
      • Freyrie A.
      • Serra C.
      • Stella A.
      Identification of carotid 'vulnerable plaque' by contrast-enhanced ultrasonography: Correlation with plaque histology, symptoms and cerebral computed tomography.
      22Carotid stenosis before surgery7286Stenosis >70%32Signal enhancement (dB)Yes
      • Staub D.
      • Partovi S.
      • Schinkel A.F.L.
      • Coll B.
      • Uthoff H.
      • Aschwanden M.
      • Jaeger K.A.
      • Feinstein S.B.
      Correlation of carotid artery atherosclerotic lesion echogenicity and severity at standard us with intraplaque neovascularization detected at contrast-enhanced US.
      175Clinical indication for duplex ultrasound and ≥1 plaque6765Any plaque17Visual scoringNo
      • Shalhoub J.
      • Monaco C.
      • Owen D.R.
      • Gauthier T.
      • Thapar A.
      • Leen E.L.
      • Davies A.H.
      Late-phase contrast-enhanced ultrasound reflects biological features of instability in human carotid atherosclerosis.
      31Carotid stenosis before surgery7177NA52Late-phase enhancementYes
      • Clevert D.A.
      • Sommer W.H.
      • Helck A.
      • Saam T.
      • Reiser M.
      Improved carotid atherosclerotic plaques imaging with contrast-enhanced ultrasound (CEUS).
      33Clinical indication for duplex ultrasound and ≥1 plaque6161NANANoneNo
      • Hoogi A.
      • Adam D.
      • Hoffman A.
      • Kerner H.
      • Reisner S.
      • Gaitini D.
      Carotid plaque vulnerability: Quantification of neovascularization on contrast-enhanced ultrasound with histopathologic correlation.
      27Carotid stenosis before surgery6870Stenosis >70%30Automatic quantificationYes
      • Varetto G.
      • Gibello L.
      • Bergamasco L.
      • Sapino A.
      • Castellano I.
      • Garneri P.
      • Rispoli P.
      Contrast enhanced ultrasound in atherosclerotic carotid artery disease.
      51Carotid stenosis before surgery7370Stenosis >70%24Maximum and mean signal intensityYes
      • van den Oord S.C.
      • Akkus Z.
      • Roeters van Lennep J.E.
      • Bosch J.G.
      • van der Steen A.F.
      • Sijbrands E.J.
      • Schinkel A.F.
      Assessment of subclinical atherosclerosis and intraplaque neovascularization using quantitative contrast-enhanced ultrasound in patients with familial hypercholesterolemia.
      69Familial hypercholesterolemia5552IMT >1.5 mm, or encroaching lumen >0.5 mm, or >50% of IMT0Semi-automated quantificationNo
      • Deyama J.
      • Nakamura T.
      • Takishima I.
      • Fujioka D.
      • Kawabata K.
      • Obata J.E.
      • Watanabe K.
      • Watanabe Y.
      • Saito Y.
      • Mishina H.
      • Kugiyama K.
      Contrast-enhanced ultrasound imaging of carotid plaque neovascularization is useful for identifying high-risk patients with coronary artery disease.
      304CAD and carotid plaque6980IMT >1.1 mm8Visual scoringNo
      • Müller H.F.
      • Viaccoz A.
      • Kuzmanovic I.
      • Bonvin C.
      • Burkhardt K.
      • Bochaton-Piallat M.L.
      • Sztajzel R.
      Contrast-enhanced ultrasound imaging of carotid plaque neo-vascularization: Accuracy of visual analysis.
      33≥50% symptomatic or ≥60% asymptomatic stenosisNA73Stenosis ≥50%52Visual scoring and semi-automatic softwareYes
      • Zhou Y.
      • Xing Y.
      • Li Y.
      • Bai Y.
      • Chen Y.
      • Sun X.
      • Zhu Y.
      • Wu J.
      An assessment of the vulnerability of carotid plaques: A comparative study between intraplaque neovascularization and plaque echogenicity.
      46Significant carotid stenosis6393Stenosis ≥50%52Visual scoringNo
      • Zhu Y.
      • Deng Y.B.
      • Liu Y.N.
      • Bi X.J.
      • Sun J.
      • Tang Q.Y.
      • Deng Q.
      Use of carotid plaque neovascularization at contrast-enhanced US to predict coronary events in patients with coronary artery disease.
      312CAD and carotid plaque6373Plaque >2.0 mmNAVisual scoringNo
      • Akkus Z.
      • Hoogi A.
      • Renaud G.
      • van den Oord S.C.
      • Ten Kate G.L.
      • Schinkel A.F.
      • Adam D.
      • de Jong N.
      • van der Steen A.F.
      • Bosch J.G.
      New quantification methods for carotid intra-plaque neovascularization using contrast-enhanced ultrasound.
      23Symptomatic carotid stenosisNANANA100Semi-automated quantificationNo
      • van den Oord S.C.
      • Akkus Z.
      • Renaud G.
      • Bosch J.G.
      • van der Steen A.F.
      • Sijbrands E.J.
      • Schinkel A.F.
      Assessment of carotid atherosclerosis, intraplaque neovascularization, and plaque ulceration using quantitative contrast-enhanced ultrasound in asymptomatic patients with diabetes mellitus.
      51Diabetes mellitus6161IMT >1.5 mm or encroaching lumen >0.5 mm, or >50% of IMT0Semi-automated quantificationNo
      • Saito K.
      • Nagatsuka K.
      • Ishibashi-Ueda H.
      • Watanabe A.
      • Kannki H.
      • Iihara K.
      Contrast-enhanced ultrasound for the evaluation of neovascularization in atherosclerotic carotid artery plaques.
      50Carotid stenosis before surgery7298NA62Time-intensity curveYes
      • van den Oord S.C.
      • van der Burg J.
      • Akkus Z.
      • Bosch J.G.
      • van Domburg R.T.
      • Sijbrands E.J.
      • van der Steen A.F.
      • Schinkel A.F.
      Impact of gender on the density of intraplaque neovascularization: A quantitative contrast-enhanced ultrasound study.
      159≥1 cardiovascular risk factor5753IMT >1.5 mm or encroaching lumen >0.5 mm, or >50% of IMT0Semi-automated quantificationNo
      • Iezzi R.
      • Petrone G.
      • Ferrante A.
      • Lauriola L.
      • Vincenzoni C.
      • la Torre M.F.
      • Snider F.
      • Rindi G.
      • Bonomo L.
      The role of contrast-enhanced ultrasound (CEUS) in visualizing atherosclerotic carotid plaque vulnerability: Which injection protocol? Which scanning technique?.
      40Carotid stenosis before surgery7056Stenosis ≥70%36Visual scoringYes
      • Nakamura J.
      • Nakamura T.
      • Deyama J.
      • Fujioka D.
      • Kawabata K.
      • Obata J.E.
      • Watanabe K.
      • Watanabe Y.
      • Kugiyama K.
      Assessment of carotid plaque neovascularization using quantitative analysis of contrast-enhanced ultrasound imaging is useful for risk stratification in patients with coronary artery disease.
      206CAD6977IMT ≥1.1 mm7Visual scoring and grey score levelNo
      • Zhang Q.
      • Li C.
      • Han H.
      • Dai W.
      • Shi J.
      • Wang Y.
      • Wang W.
      Spatio-temporal quantification of carotid plaque neovascularization on contrast enhanced ultrasound: Correlation with visual grading and histopathology.
      34Carotid stenosisNANANANAVisual scoring and spatio-temporal quantificationYes
      • Xu R.
      • Yin X.
      • Xu W.
      • Jin L.
      • Lu M.
      • Wang Y.
      Assessment of carotid plaque neovascularization by contrast-enhanced ultrasound and high sensitivity C-reactive protein test in patients with acute cerebral infarction: A comparative study.
      106Acute cerebral infarction6559Stenosis 30-69%100Time-intensity curveNo
      • van den Oord S.C.
      • Akkus Z.
      • Bosch J.G.
      • Hoogi A.
      • ten Kate G.L.
      • Renaud G.
      • Sijbrands E.J.
      • Verhagen H.J.
      • van der Lugt A.
      • Adam D.
      • de Jong N.
      • van der Steen A.F.
      • Schinkel A.F.
      Quantitative contrast-enhanced ultrasound of intraplaque neovascularization in patients with carotid atherosclerosis.
      25Established carotid atherosclerosis6584NA92Semi-automated quantificationNo
      • Sun X.F.
      • Wang J.
      • Wu X.L.
      • Xu H.Y.
      • Xing Y.Q.
      • Yang F.
      Evaluation of the stability of carotid atherosclerotic plaque with contrast-enhanced ultrasound.
      93Symptomatic carotid stenosis6384Plaque ≥2.5 mmNAVisual scoringNo
      • Cattaneo M.
      • Staub D.
      • Porretta A.P.
      • Gallino J.M.
      • Santini P.
      • Limoni C.
      • Wyttenbach R.
      • Gallino A.
      Contrast-enhanced ultrasound imaging of intraplaque neovascularization and its correlation to plaque echogenicity in human carotid arteries atherosclerosis.
      40Asymptomatic carotid stenosis7367Stenosis ≥50%0Visual scoringNo
      • Ammirati E.
      • Moroni F.
      • Magnoni M.
      • Di Terlizzi S.
      • Villa C.
      • Sizzano F.
      • Palini A.
      • Garlaschelli K.
      • Tripiciano F.
      • Scotti I.
      • Catapano A.L.
      • Manfredi A.A.
      • Norata G.D.
      • Camici P.G.
      Circulating CD14+ and CD14(high)CD16– classical monocytes are reduced in patients with signs of plaque neovascularization in the carotid artery.
      55Intermediate carotid stenosis6956Stenosis 40 to 70%0NoneNo
      • Hjelmgren O.
      • Gellerman K.
      • Kjelldahl J.
      • Lindahl P.
      • Bergström G.M.
      Increased vascularization in the vulnerable upstream regions of both early and advanced human carotid atherosclerosis.
      45Suspected carotid atherosclerosis6867Plaque ≥2.5 mm22Semi-automated quantificationNo
      • Oikawa K.
      • Kato T.
      • Oura K.
      • Narumi S.
      • Sasaki M.
      • Fujiwara S.
      • Kobayashi M.
      • Matsumoto Y.
      • Nomura J.I.
      • Yoshida K.
      • Terayama Y.
      • Ogasawara K.
      Preoperative cervical carotid artery contrast-enhanced ultrasound findings are associated with development of microembolic signals on transcranial Doppler during carotid exposure in endarterectomy.
      70Carotid stenosis before surgery7094Stenosis ≥70%79Time–intensity curveNo
      • Schmidt C.
      • Fischer T.
      • Rückert R.I.
      • Oberwahrenbrock T.
      • Harms L.
      • Kronenberg G.
      • Kunte H.
      Identification of neovascularization by contrast-enhanced ultrasound to detect unstable carotid stenosis.
      17Carotid stenosis before surgery6659High-grade stenosis (≥60%)41Visual scoringYes
      • Hjelmgren O.
      • Johansson L.
      • Prahl U.
      • Schmidt C.
      • Bergström G.M.L.
      Inverse association between size of the lipid-rich necrotic core and vascularization in human carotid plaques.
      31Carotid plaque6958Plaque ≥2.5 mm45Semi-automated quantificationNo
      • Demeure F.
      • Bouzin C.
      • Roelants V.
      • Bol A.
      • Verhelst R.
      • Astarci P.
      • Gerber B.L.
      • Pouleur A.C.
      • Pasquet A.
      • de Meester C.
      • Vanoverschelde J.J.
      • Vancraeynest D.
      Head-to-head comparison of inflammation and neovascularization in human carotid plaques: Implications for the imaging of vulnerable plaques.
      30Carotid stenosis before surgery7273Stenosis >70%37Visual scoringYes
      • Xiong L.
      • Sun W.J.
      • Cai H.Y.
      • Yang Y.
      • Zhu J.
      • Zhao B.W.
      Correlation of enhancement degree on contrast-enhanced ultrasound with histopathology of carotid plaques and serum high sensitive C-reactive protein levels in patients undergoing carotid endarterectomy.
      115Carotid stenosis before surgery6856NANAVisual scoring and time–intensity curveYes
      • Shimada Y.
      • Oikawa K.
      • Fujiwara S.
      • Ogasawara Y.
      • Sato Y.
      • Narumi S.
      • Kato T.
      • Oura K.
      • Terayama Y.
      • Sasaki M.
      • Fujimoto K.
      • Yoshida J.
      • Ogasawara K.
      Comparison of three-dimensional T1-weighted magnetic resonance and contrast-enhanced ultrasound plaque images for severe stenosis of the cervical carotid artery.
      53Carotid plaque7191Stenosis ≥70%NATime-intensity curveNo
      • Song Y.
      • Feng J.
      • Dang Y.
      • Zhao C.
      • Zheng J.
      • Ruan L.
      Relationship between plaque echo, thickness and neovascularization assessed by quantitative and semi-quantitative contrast-enhanced ultrasonography in different stenosis groups.
      224Carotid plaque6587Plaque ≥1.5 mmNAVisual scoring and time-intensity curveNo
      • Qin C.
      • Zhang L.
      • Wang X.
      • Duan Y.
      • Ye Z.
      • Xie M.
      Evaluation of carotid plaque neovascularization in patients with coronary heart disease on contrast-enhanced ultrasonography.
      120Acute coronary syndrome or stable CAD6358Plaque >2.0 mmNATime–intensity curveNo
      • Shimada H.
      • Ogata T.
      • Takano K.
      • Abe H.
      • Higashi T.
      • Yamashita T.
      • Matsunaga A.
      • Inoue T.
      Evaluation of the time-dependent changes and the vulnerability of carotid plaques using contrast-enhanced carotid ultrasonography.
      103Carotid plaque7289Stenosis >50%NASignal enhancementNo
      • Chen J.
      • Zhang Y.M.
      • Song Z.Z.
      • Fu Y.F.
      • Geng Y.
      The inter-observer agreement in the assessment of carotid plaque neovascularization by contrast-enhanced ultrasonography: The impact of plaque thickness.
      66Carotid plaque6565IMT >1.5 mm, or encroaching lumen >0.5 mm, or >50% of IMTNAVisual scoringNo
      • Oura K.
      • Kato T.
      • Ohba H.
      • Terayama Y.
      Evaluation of intraplaque neovascularization using superb microvascular imaging and contrast-enhanced ultrasonography.
      27Carotid plaque7193IMT ≥2 mmNAVisual analysisNo
      • Evdokimenko A.N.
      • Gulevskaya T.S.
      • Druina L.D.
      • Chechetkin A.O.
      • Shabalina A.A.
      • Kostyreva M.V.
      • Kulichenkova K.N.
      • Pryamikov A.D.
      • Tanashyan M.M.
      Neovascularization of carotid atherosclerotic plaque and quantitative methods of its dynamic assessment in vivo.
      45Carotid stenosis before surgery6471Stenosis ≥50%NAQuantitative determinationYes
      • D'Oria M.
      • Chiarandini S.
      • Pipitone M.D.
      • Fisicaro M.
      • Calvagna C.
      • Bussani R.
      • Rotelli A.
      • Ziani B.
      contrast enhanced ultrasound (CEUS) is not able to identify vulnerable plaques in asymptomatic carotid atherosclerotic disease.
      58Carotid stenosis before surgery7357Stenosis ≥70%NAVisual scoringYes
      • Pereira T.
      • Muguruza J.
      • Mária V.
      • Vilaprinyo E.
      • Sorribas A.
      • Fernandez E.
      • Fernandez-Armenteros J.M.
      • Baena J.A.
      • Rius F.
      • Betriu A.
      • Solsona F.
      • Alves R.
      Automatic methods for carotid contrast-enhanced ultrasound imaging quantification of adventitial vasa vasorum.
      186Clinical indication for ultrasound and volunteersNANANANAAutomated analysis of signal intensityNo
      • Choi S.W.
      • Kim H.
      • Kim I.C.
      • Lee C.H.
      • Hwang J.
      • Park H.S.
      • Cho Y.K.
      • Yoon H.J.
      • Nam C.W.
      • Han S.
      • Hur S.H.
      Implication of ultrasound contrast-enhancement of carotid plaques in prevalence of acute coronary syndrome and occurrence of cardiovascular outcomes.
      314CAD6564IMT >1.1 mm or plaque11Visual scoringNo
      • Mantella L.E.
      • Colledanchise K.N.
      • Hetu M.F.
      • Feinstein S.B.
      • Abunassar J.
      • Johri A.M.
      Carotid intraplaque neovascularization predicts coronary artery disease and cardiovascular events.
      459Referred for coronary angiography6571Encroaching lumen >1.5 mm or >50% of IMTNAVisual scoringNo
      Summary estimate4660667124
      CAD = coronary artery disease; dB = decibel; GSM = gray-scale median; IMT = intima–media thickness; NA = not available, TIA = transient ischemic attack.
      Table 2Overview of studies on the validation of intraplaque neovascularization on carotid CEUS with histopathological analysis
      Study (Year)nHistologic findings
      • Vicenzini E.
      • Giannoni M.F.
      • Puccinelli F.
      • Ricciardi M.C.
      • Altieri M.
      • Di Piero V.
      • Gossetti B.
      • Valentini F.B.
      • Lenzi G.L.
      Detection of carotid adventitial vasa vasorum and plaque vascularization with ultrasound cadence contrast pulse sequencing technique and echo-contrast agent.
      1Histology confirmed the CEUS findings
      • Shah F.
      • Balan P.
      • Weinberg M.
      • Reddy V.
      • Neems R.
      • Feinstein M.
      • Dainauskas J.
      • Meyer P.
      • Goldin M.
      • Feinstein S.B.
      Contrast-enhanced ultrasound imaging of atherosclerotic carotid plaque neovascularization: A new surrogate marker of atherosclerosis?.
      17Spearman rank correlation = 0.68 between CEUS and histology
      • Coli S.
      • Magnoni M.
      • Sangiorgi G.
      • Marrocco-Trischitta M.M.
      • Melisurgo G.
      • Mauriello A.
      • Spagnoli L.
      • Chiesa R.
      • Cianflone D.
      • Maseri A.
      Contrast-enhanced ultrasound imaging of intraplaque neovascularization in carotid arteries: Correlation with histology and plaque echogenicity.
      17Good correlation between CEUS and histology
      • Giannoni M.F.
      • Vicenzini E.
      • Citone M.
      • Ricciardi M.C.
      • Irace L.
      • Laurito A.
      • Scucchi L.F.
      • Di Piero V.
      • Gossetti B.
      • Mauriello A.
      • Spagnoli L.G.
      • Lenzi G.L.
      • Valentini F.B.
      Contrast carotid ultrasound for the detection of unstable plaques with neoangiogenesis: A pilot study.
      9Histology confirmed the presence of vascularization in all symptomatic plaque consistent with the presence of contrast enhancement on CEUS
      • Faggioli G.L.
      • Pini R.
      • Mauro R.
      • Pasquinelli G.
      • Fittipaldi S.
      • Freyrie A.
      • Serra C.
      • Stella A.
      Identification of carotid 'vulnerable plaque' by contrast-enhanced ultrasonography: Correlation with plaque histology, symptoms and cerebral computed tomography.
      22CEUS with decibel enhancement is a strong indicator of the extent of plaque neovascularization
      • Shalhoub J.
      • Monaco C.
      • Owen D.R.
      • Gauthier T.
      • Thapar A.
      • Leen E.L.
      • Davies A.H.
      Late-phase contrast-enhanced ultrasound reflects biological features of instability in human carotid atherosclerosis.
      29Significant correlation between late-phase CEUS and CD68 immunopositivity (r = 0.466, p = 0.011)
      • Hoogi A.
      • Adam D.
      • Hoffman A.
      • Kerner H.
      • Reisner S.
      • Gaitini D.
      Carotid plaque vulnerability: Quantification of neovascularization on contrast-enhanced ultrasound with histopathologic correlation.
      22Intraplaque neovascularization on CEUS correlated well with histopathology (r2 = 0.79, p < 0.01)
      • Varetto G.
      • Gibello L.
      • Bergamasco L.
      • Sapino A.
      • Castellano I.
      • Garneri P.
      • Rispoli P.
      Contrast enhanced ultrasound in atherosclerotic carotid artery disease.
      51Plaques with greater contrast enhancement had more newly formed capillaries
      • Müller H.F.
      • Viaccoz A.
      • Kuzmanovic I.
      • Bonvin C.
      • Burkhardt K.
      • Bochaton-Piallat M.L.
      • Sztajzel R.
      Contrast-enhanced ultrasound imaging of carotid plaque neo-vascularization: Accuracy of visual analysis.
      19Histopathology revealed a larger CD34+ area in patients with grade 1/2 versus grade 0 (p = 0.03)
      • Saito K.
      • Nagatsuka K.
      • Ishibashi-Ueda H.
      • Watanabe A.
      • Kannki H.
      • Iihara K.
      Contrast-enhanced ultrasound for the evaluation of neovascularization in atherosclerotic carotid artery plaques.
      50Enhanced intensity of the plaque shoulder was associated with neovessel density (p < 0.01, ρ = 0.43)
      • Iezzi R.
      • Petrone G.
      • Ferrante A.
      • Lauriola L.
      • Vincenzoni C.
      • la Torre M.F.
      • Snider F.
      • Rindi G.
      • Bonomo L.
      The role of contrast-enhanced ultrasound (CEUS) in visualizing atherosclerotic carotid plaque vulnerability: Which injection protocol? Which scanning technique?.
      50Qualitative CEUS evaluation obtained high statistical results compared with immunohistological results, with sensitivity and specificity values of 94% and 68%, respectively
      • Zhang Q.
      • Li C.
      • Han H.
      • Dai W.
      • Shi J.
      • Wang Y.
      • Wang W.
      Spatio-temporal quantification of carotid plaque neovascularization on contrast enhanced ultrasound: Correlation with visual grading and histopathology.
      34Both spatial and temporal parameters were correlated with microvessel density on histology (AR: r = 0.854, AR13: r = 0.858, EI: r = 0.767; EIR: r = .750 [p < 0.01]), as well as with semiquantitative grading (p < 0.01)
      • Schmidt C.
      • Fischer T.
      • Rückert R.I.
      • Oberwahrenbrock T.
      • Harms L.
      • Kronenberg G.
      • Kunte H.
      Identification of neovascularization by contrast-enhanced ultrasound to detect unstable carotid stenosis.
      17Scores on the CEUS-based four-level and two-level classifications were robustly correlated with the density of intraplaque vessels (r = 0.635, p = 0.006 and r = 0.578, p = 0.015, respectively)
      • Demeure F.
      • Bouzin C.
      • Roelants V.
      • Bol A.
      • Verhelst R.
      • Astarci P.
      • Gerber B.L.
      • Pouleur A.C.
      • Pasquet A.
      • de Meester C.
      • Vanoverschelde J.J.
      • Vancraeynest D.
      Head-to-head comparison of inflammation and neovascularization in human carotid plaques: Implications for the imaging of vulnerable plaques.
      30The number of vessels was significantly higher in carotid plaque with high-contrast-enhancement (p <0.001)
      • Xiong L.
      • Sun W.J.
      • Cai H.Y.
      • Yang Y.
      • Zhu J.
      • Zhao B.W.
      Correlation of enhancement degree on contrast-enhanced ultrasound with histopathology of carotid plaques and serum high sensitive C-reactive protein levels in patients undergoing carotid endarterectomy.
      115Correlation analysis revealed that the enhancement degree of the carotid plaques was closely related to the immunohistochemical parameters of the plaques and the serum high-sensitivity C-reactive protein levels
      • Evdokimenko A.N.
      • Gulevskaya T.S.
      • Druina L.D.
      • Chechetkin A.O.
      • Shabalina A.A.
      • Kostyreva M.V.
      • Kulichenkova K.N.
      • Pryamikov A.D.
      • Tanashyan M.M.
      Neovascularization of carotid atherosclerotic plaque and quantitative methods of its dynamic assessment in vivo.
      35Data on vessel location in the plaque, obtained with CEUS, as a whole were well correlated with morphologic data
      • D'Oria M.
      • Chiarandini S.
      • Pipitone M.D.
      • Fisicaro M.
      • Calvagna C.
      • Bussani R.
      • Rotelli A.
      • Ziani B.
      contrast enhanced ultrasound (CEUS) is not able to identify vulnerable plaques in asymptomatic carotid atherosclerotic disease.
      58Compared with histologically proven non-vulnerable plaques, histologically proven vulnerable plaques had denser neovascularization, but not more pronounced contrast enhancement.
      Summary estimate576
      CEUS = contrast-enhanced ultrasound.

      Relation between carotid and coronary atherosclerosis

      Previous studies have indicated that carotid plaque burden is a marker of systemic atherosclerosis and may involve multiple vascular beds. The BioImage study (
      • Baber U.
      • Mehran R.
      • Sartori S.
      • Schoos M.M.
      • Sillesen H.
      • Muntendam P.
      • Garcia M.J.
      • Gregson J.
      • Pocock S.
      • Falk E.
      • Fuster V.
      Prevalence, impact, and predictive value of detecting subclinical coronary and carotid atherosclerosis in asymptomatic adults: The BioImage study.
      ) enrolled 5808 asymptomatic patients to assess the role of vascular imaging in cardiovascular risk prediction. Over a median 2.7-y follow-up, major adverse cardiac events (MACEs) occurred in 216 patients (4.2%). After adjustment for conventional risk factors, carotid plaque burden assessed by 3-D ultrasound was a predictor of MACEs. Moreover, net reclassification significantly improved with carotid plaque burden on ultrasound. The Multi-ethnic Study of Atherosclerosis (MESA) prospective cohort study included 6814 without known cardiovascular disease (
      • Gepner A.D.
      • Young R.
      • Delaney J.A.
      • Budoff M.J.
      • Polak J.F.
      • Blaha M.J.
      • Post W.S.
      • Michos E.D.
      • Kaufman J.
      • Stein J.H.
      Comparison of carotid plaque score and coronary artery calcium score for predicting cardiovascular disease events: The Multi-Ethnic Study of Atherosclerosis.
      ). All patients underwent carotid ultrasound to assess carotid plaque and computed tomography to assess the coronary artery calcium score. The median 11.3-y follow-up revealed that coronary artery calcium and carotid plaque scores had similar predictive value for stroke/transient ischemic attack events. However, the coronary calcium score had a better predictive value for all cardiovascular and coronary events than carotid ultrasound measures.
      • Deyama J.
      • Nakamura T.
      • Takishima I.
      • Fujioka D.
      • Kawabata K.
      • Obata J.E.
      • Watanabe K.
      • Watanabe Y.
      • Saito Y.
      • Mishina H.
      • Kugiyama K.
      Contrast-enhanced ultrasound imaging of carotid plaque neovascularization is useful for identifying high-risk patients with coronary artery disease.
      studied the role of carotid CEUS in identifying high-risk patients with coronary artery disease. A total of 304 patients underwent carotid CEUS and coronary angiography; the complexity and extent of coronary lesions were assessed angiographically. A higher grade of intraplaque microvessels on carotid CEUS was associated significantly with greater complexity and extent of coronary lesions. Recently
      • Mantella L.E.
      • Colledanchise K.N.
      • Hetu M.F.
      • Feinstein S.B.
      • Abunassar J.
      • Johri A.M.
      Carotid intraplaque neovascularization predicts coronary artery disease and cardiovascular events.
      confirmed these findings and reported that carotid CEUS predicts coronary artery disease and cardiovascular events. Carotid CEUS was performed in 459 stable patients who were referred for coronary angiography. A higher carotid intraplaque microvessel score (0 = no, 1 = peri-adventitial, 2 = plaque core) on CEUS was associated with significant coronary artery disease and greater complexity of coronary lesions. An intraplaque microvessel score ≥1.25 could predict significant coronary artery disease with high sensitivity (92%) and specificity (89%). Follow-up revealed that these patients were also at increased risk of cardiovascular events. Further studies are needed to evaluate whether carotid CEUS improves the selection of patients before coronary angiography and how these results can be implemented in clinical practice to improve patient outcomes.

      Quantification of CEUS

      The presence and extent of microvessels in a carotid plaque on CEUS are relevant in assessing the vulnerability of a carotid plaque. An overview of the quantification methods used in the available studies is presented in Table 1. Visual scoring systems allow for an easy assessment of plaque microbubble appearance that is often reported on a 3-point scale as follows: 0 = no visible microbubbles, 1= limited to moderate microbubbles, 2= extensive appearance of microbubbles within the plaque. This visual scoring method clearly has its limitations. A simple quantitative assessment of intraplaque microvessels can be performed using a standard time–intensity curve to assess baseline intensity and intensity during microbubble administration. Commercially available software that can be used for quantification of intraplaque microvessels on CEUS are Bracco Vuebox and Philips QLAB; an example is provided in Figure 2. Clearly a careful selection of the region of interest is important to exclude the lumen and surroundings of the vessel and include only the atherosclerotic plaque. This may be challenging because the plaque may move because of arterial pulsations or movement of the patient or ultrasound probe. Several studies have used a method based on maximum intensity projection to quantify intraplaque microvessels. This method can be used to reconstruct vessel trajectories through an atherosclerotic plaque, but a drawback is the high sensitivity to noise. Therefore maximum intensity projection may lead to overestimation of the contrast enhancement signal.
      Fig 2
      Fig. 2(a) Predominant hypo-echoic plaque at the origin of the internal carotid artery on B-mode ultrasound and moderate stenosis on color Doppler ultrasound. (b) Enhancement of the carotid lumen on contrast-enhanced ultrasound with delineation of the vessel wall depicting huge plaque ulceration. Enhancement within the plaque at the near wall representing moderate intraplaque neovascularization. (c) Quantitative analysis of intraplaque neovascularization after bolus injection of 2.4 mL of Sonovue using the VueBox (Bracco S.A., Milan, Italy) plaque package software. The enhancement within a region of interest of the plaque (green line) compared to the lumen (yellow line) on a time–intensity curve is used for quantitative analysis of intraplaque neovascularization. Different perfusion parameters including the relative perfused area (rPA) of the plaque can be analyzed and visualized using parametric color imaging (right side). An rPA of 47% was calculated within this plaque corresponding to moderate intraplaque neovascularization.
      Lately more sophisticated software solutions for the quantification of intraplaque microvessels on carotid CEUS have been developed.
      • Hoogi A.
      • Adam D.
      • Hoffman A.
      • Kerner H.
      • Reisner S.
      • Gaitini D.
      Carotid plaque vulnerability: Quantification of neovascularization on contrast-enhanced ultrasound with histopathologic correlation.
      introduced a semiautomatic algorithm using a multidimensional dynamic programming method to reconstruct the microvessel tree in the atherosclerotic plaque. This method was tested in a large set of clinical cases and required minimal intervention of the operator, providing an efficient analysis that agrees well with manual and visual validations.
      • Akkus Z.
      • Hoogi A.
      • Renaud G.
      • van den Oord S.C.
      • Ten Kate G.L.
      • Schinkel A.F.
      • Adam D.
      • de Jong N.
      • van der Steen A.F.
      • Bosch J.G.
      New quantification methods for carotid intra-plaque neovascularization using contrast-enhanced ultrasound.
      developed a specialized software package called carotid intraplaque neovascularization quantification software (CINQS), including motion compensation and multiple analysis algorithms for quantification of carotid intraplaque microvessels (Fig. 3). This software has a modular design and is user-friendly because of wizard-like analysis tools and a graphical user interface. A clinical study was performed to evaluate the performance of CINQS, and found that the quantification of carotid intraplaque microvessels on CEUS is feasible and provides multiple features on carotid microvasculature (
      • van den Oord S.C.
      • Akkus Z.
      • Bosch J.G.
      • Hoogi A.
      • ten Kate G.L.
      • Renaud G.
      • Sijbrands E.J.
      • Verhagen H.J.
      • van der Lugt A.
      • Adam D.
      • de Jong N.
      • van der Steen A.F.
      • Schinkel A.F.
      Quantitative contrast-enhanced ultrasound of intraplaque neovascularization in patients with carotid atherosclerosis.
      ). There was excellent agreement between visual scoring and quantitative analysis of intraplaque microvessels. An accurate quantification of intraplaque microvasculature may be clinically relevant to assess serial changes in intraplaque microvessels and to detect and monitor vulnerable plaques during therapy.
      Fig 3
      Fig. 3Carotid intraplaque neovascularization quantification software (CINQS), including motion compensation and multiple analysis algorithms for quantification of carotid intraplaque microvessels. (a) Analysis tool panel structured as a wizard. (b) Viewer panel. (c) Parameter output panel. (d) Graph panel.
      Subsequently,
      • Cheung W.K.
      • Gujral D.M.
      • Shah B.N.
      • Chahal N.S.
      • Bhattacharyya S.
      • Cosgrove D.O.
      • Eckersley R.J.
      • Harrington K.J.
      • Senior R.
      • Nutting C.M.
      • Tang M.X.
      Attenuation correction and normalisation for quantification of contrast enhancement in ultrasound images of carotid arteries.
      developed an automated attenuation correction and normalization algorithm to optimize the quantification of intraplaque microvessels. The algorithm was validated on phantoms containing contrast agent-filled vessels embedded in tissue-mimicking material with known attenuation characteristics. Thereafter, this method was tested in vivo in carotid CEUS in 48 patients. The in vitro and in vivo studies found that the attenuation correction and normalization methods resulted in a significant reduction of attenuation artifacts and improvement of quantification of carotid CEUS.

      Challenges

      Carotid ultrasound is a radiation-free real-time imaging technique with extraordinary spatial and temporal resolution, but has some inherent limitations that may hinder assessment of intraplaque microvessels. Table 3 lists the advantages and disadvantages of adding contrast to the carotid ultrasound exam. Currently, carotid CEUS imaging is based on 2-D imaging, which clearly has limitations as only one plane through the plaque is imaged and microbubbles may move in and out of the imaging plane. Moreover, ultrasound images are generally subject to a suboptimal signal-to noise ratio, and there is often some vessel, patient or ultrasound probe movement. This hinders reconstruction of microvessel trajectories. This limitation can be partly overcome by imaging from different imaging planes or a transversal imaging sweep through the carotid artery. This limitation can be eliminated in the future by the development of 3-D carotid ultrasound probes suitable for contrast ultrasound. Three-dimensional carotid CEUS has the advantage that a full volume clip of the carotid artery and atherosclerotic plaque can be obtained. This will probably improve intra- and inter-observer assessment of carotid CEUS and also facilitate serial assessment of intraplaque microvessels. It may be clinically relevant to monitor progression in intraplaque vascularization as a sign of increased plaque vulnerability or assess regression of intraplaque microvessels during therapy.
      Table 3Advantages and disadvantages of adding contrast to a carotid ultrasound study.
      AdvantagesDisadvantages
      Better delineation of vascular lumenIntravenous administration
      Better detection of atherosclerotic plaqueBased on 2-D imaging
      Information on intraplaque microvesselsSubject to vessel and probe movement
      Tolerable technique, no ionizing radiationArtifacts may occur
      Suitable for children and young patientsInitial experience or training required
      Relatively inexpensive
      Quantification software available
      Several studies have described the pseudo-enhancement artifact that may be present in carotid CEUS images and is caused by non-linear propagation of the ultrasound beam (
      • ten Kate G.L.
      • Renaud G.G.
      • Akkus Z.
      • van den Oord S.C.
      • ten Cate F.J.
      • Shamdasani V.
      • Entrekin R.R.
      • Sijbrands E.J.
      • de Jong N.
      • Bosch J.G.
      • Schinkel A.F.
      • van der Steen A.F.
      Far-wall pseudoenhancement during contrast-enhanced ultrasound of the carotid arteries: Clinical description and in vitro reproduction.
      ;
      • Thapar A.
      • Shalhoub J.
      • Averkiou M.
      • Mannaris C.
      • Davies A.H.
      • Leen E.L.
      Dose-dependent artifact in the far wall of the carotid artery at dynamic contrast-enhanced US.
      ). During CEUS of the carotid artery, this pseudo-enhancement pattern may be observed parallel to the lumen in close proximity to the vessel wall opposite the ultrasound probe, thus the far wall (Fig. 4). The location of this enhancement does not correspond to the anatomic location of a parallel vessel.
      • ten Kate G.L.
      • Renaud G.G.
      • Akkus Z.
      • van den Oord S.C.
      • ten Cate F.J.
      • Shamdasani V.
      • Entrekin R.R.
      • Sijbrands E.J.
      • de Jong N.
      • Bosch J.G.
      • Schinkel A.F.
      • van der Steen A.F.
      Far-wall pseudoenhancement during contrast-enhanced ultrasound of the carotid arteries: Clinical description and in vitro reproduction.
      reported that in a phantom study, the degree of pseudo-enhancement was influenced by the size and concentration of the microbubble. For clinical carotid CEUS, recognition of this artifact is relevant to prevent misinterpretation of enhancement in and near the far vessel wall. New pulse sequences have been developed to overcome this artifact, but commercial systems are still exhibiting it (
      • Renaud G.
      • Bosch J.G.
      • Ten Kate G.L.
      • Shamdasani V.
      • Entrekin R.
      • de Jong N.
      • van der Steen A.F.
      Counter-propagating wave interaction for contrast-enhanced ultrasound imaging.
      ).
      Fig 4
      Fig. 4Carotid artery B-mode (a, d) and contrast-enhanced (b, e) ultrasound images in a representative patient. The schematic representations (c, f) depict the location of the pseudo-enhancement in green. Additionally, it can be observed that the intensity of the pseudo-enhancement correlates with echogenic locations on the B-mode images. Reproduced, with permission, from
      • ten Kate G.L.
      • Renaud G.G.
      • Akkus Z.
      • van den Oord S.C.
      • ten Cate F.J.
      • Shamdasani V.
      • Entrekin R.R.
      • Sijbrands E.J.
      • de Jong N.
      • Bosch J.G.
      • Schinkel A.F.
      • van der Steen A.F.
      Far-wall pseudoenhancement during contrast-enhanced ultrasound of the carotid arteries: Clinical description and in vitro reproduction.
      .

      Future developments

      In the near future, targeted imaging of intraplaque microvessels may become possible using an ultrasound molecular contrast imaging approach. The carotid endothelium is the structural and functional interface between the circulating blood and the interstitial space. The administration of a microbubble contrast agent that carries antibodies or other ligands to endothelial cell adhesion molecules enables molecular CEUS. Molecular CEUS has been proven a useful research tool in animal models of disease. The first human application of targeted CEUS was recently performed using BR55, a vascular endothelial growth factor receptor 2-specific ultrasound molecular contrast agent. This initial study (
      • Smeenge M.
      • Tranquart F.
      • Mannaerts C.K.
      • de Reijke T.M.
      • van de Vijver M.J.
      • Laguna M.P.
      • Pochon S.
      • de la Rosette J.J.M.C.H.
      • Wijkstra H.
      First-in-human ultrasound molecular imaging with a VEGFR2-specific ultrasound molecular contrast agent (BR55) in prostate cancer: A safety and feasibility pilot study.
      ) in 24 patients found that ultrasound molecular imaging is feasible with standard clinical ultrasound systems, and had a good safety profile. This opens the way for further clinical trials using targeted ultrasound contrast agents.
      A fascinating concept is the use of a microbubble contrast agent as a transport vehicle for therapeutic applications such as local gene and drug delivery (
      • Castle J.
      • Butts M.
      • Healey A.
      • Kent K.
      • Marino M.
      • Feinstein S.B.
      Ultrasound-mediated targeted drug delivery: Recent success and remaining challenges.
      ). Therapeutic agents including medication and other particles such as DNA may be included in the shell or the gaseous core of the microbubble contrast agent. After intravenous injection, the microbubbles travel as transport vehicles through the vascular system. At the desired location, an ultrasound pulse with a high mechanical index can be used to locally destroy the microbubble contrast agent, and the therapeutic agent is delivered (
      • Castle J.W.
      • Kent K.P.
      • Fan Y.
      • Wallace K.D.
      • Davis C.E.
      • Roberts J.C.
      • Marino M.E.
      • Thomenius K.E.
      • Lim H.W.
      • Coles E.
      • Davidson M.H.
      • Feinstein S.B.
      • DeMaria A.
      Therapeutic ultrasound: Increased HDL-cholesterol following infusions of acoustic microspheres and apolipoprotein A-I plasmids.
      ). This may be especially relevant to obtain a high dose of the therapeutic agent at the target site or organ and may lead to a decrease in systemic side effects.

      Conclusions

      CEUS is a readily available imaging modality for the assessment of patients with known or suspected carotid atherosclerosis. It provides information on atherosclerotic plaques, such as ulceration and intraplaque neovascularization, which may be clinically relevant for these patients. Dedicated software has been developed for the quantification of CEUS, which may lead to a broader application of this imaging modality in the diagnosis and follow-up of patients with vulnerable atherosclerotic plaques. The ultimate clinical goal is the early identification of carotid atherosclerosis so that preventive therapy can be started early and prevent clinical complications such as TIA and stroke.

      Conflict of interest disclosure

      The authors declare no competing interests.

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