Features of vulnerable plaque include a high lipid content, an irregular shape, a thin fibrous cap, and neovascularization, but such lesions often fall into the category of nonstenotic, despite being at high risk for rupture, and therefore may be overlooked. In this review, the authors describe state-of-the-art investigative ultrasound methods to assess the activity, quality, and morphology of atherosclerotic plaque to determine vulnerability. Specifically, the authors focus on carotid artery plaque, describing the assessment of plaque activity through the detection of neovascularization using contrast-enhanced ultrasound, the characterization of plaque quality by advanced grayscale and integrated backscatter analysis methods, and the assessment of plaque morphology using three-dimensional ultrasound.
Highlights
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Identification of vulnerable plaque features is a growing priority.
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The authors review advances in carotid ultrasound methods that detect these features.
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Three-dimensional ultrasound can quantify plaque size and shape.
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Grayscale and integrated backscatter analysis can characterize plaque composition.
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Contrast enhancement methods can image neovascularization and inflammation.
Understanding the dynamic nature of atherosclerotic plaque pathobiology and its potential consequences has led researchers to focus on noninvasive methods for early detection and identification of “vulnerable” plaque. There are currently no targeted therapies for vulnerable plaque beyond treatment with statins, as stenting and endarterectomy are recommended for the treatment of symptomatic patients with high stenosis. However, identification of vulnerable plaque may accelerate treatment of existing cardiac risk factors to prevent events. Non-ultrasound-based imaging modalities to detect vulnerable plaque have been extensively reviewed elsewhere and include magnetic resonance imaging (MRI), nuclear imaging, multidetector computed tomography, and positron emission tomography. Ultrasound has the advantage of being a simple, safe, and inexpensive but is often overlooked for its role in vulnerable plaque assessment, as most reviews focus on the limitations of intima-media thickness (IMT). It is now generally accepted that carotid plaque is a phenomenon distinct from IMT and has a stronger association with cardiovascular disease (CVD) events. There have been significant advances in ultrasound beyond IMT measurement that are under investigation for characterization of vulnerable plaque. In this nonsystematic review, we summarize the state of the art in vascular ultrasound methods that are being investigated to characterize vulnerable plaque. Specifically, we describe contrast-enhanced ultrasound (CEUS) to measure plaque activity, grayscale median (GSM) analysis, texture, and integrated backscatter (IB) to measure plaque quality or composition, and three-dimensional (3D) ultrasound to characterize plaque morphology. Although most of the methods described in this review are used primarily in research and are not yet ready for clinical use, each method shows promising potential for the enhancement of patient care and warrants further efforts into development and standardization.
Pathogenesis of the Vulnerable Plaque
CVD events are thought to be caused by focal atherosclerotic lesions of the vessel wall. Such lesions are likely initiated by infiltration of low-density lipoprotein in the arterial intima. Oxidative modifications of low-density lipoprotein particles may then induce endothelial cells to express various leukocyte adhesion molecules, facilitating the recruitment of monocytes that migrate into the intima and differentiate into macrophages, which then take up the low-density lipoprotein particles and transform into foam cells. When foam cells die, they leave behind the accumulated lipid as a soft, unstable, necrotic core deposited within the plaque ( Figures 1 A and 1B).
A fibrous cap is often produced by smooth muscle cells that migrate from the media to the intimal space, proliferate, and produce cellular matrix proteins. However, as lesions progress, the loss of smooth muscle cells and production of matrix metalloproteinases cause the fibrous cap to become thin, fragile, and rupture prone. In contrast, below the cap, endothelial cells, macrophages and smooth muscle cells may undergo apoptosis (programmed cell death) or necrosis (cell death caused by injury), thereby contributing to the formation of a necrotic core ( Figures 1 C and 1D).
Neovascularization
Angiogenesis may take place in advanced lesions, and microvessels originating from adventitial vasa vasorum may proliferate into the necrotic core, likely in response to hypoxia caused by plaque thickness and the high metabolic demand of inflammatory cells. Damage to these fragile and leaky vessels can result in local extravasation of plasma proteins, erythrocytes, and more inflammatory cells. Given the fragile nature of these microvessels, intraplaque hemorrhage may also contribute to rupture.
Although the progression of atherosclerotic plaque in size alone can occlude arteries and cause ischemic events, 70% of culprit lesions in fatal acute myocardial infarctions (MIs) and/or sudden coronary deaths are thought to be caused by rupture of a nonstenotic plaque. Thin or ruptured caps are 10 to 23 times more likely to be correlated with transient ischemic attack (TIA) or stroke, and intraplaque hemorrhage as evaluated by MRI has been shown to predict cerebrovascular events. On the basis of pathologic studies of culprit lesions, Naghavi et al . proposed that the major criteria for defining vulnerable plaque should include active inflammation, a thin cap with a large lipid core, endothelial denudation with superficial platelet aggregation, plaque fissures, and stenosis ( Figure 2 ). In the following sections we review ultrasound-based methods to detect these criteria in the carotid artery.
Plaque Activity: CEUS Imaging
The Vasa Vasorum and Intraplaque Neovascularization
The vasa vasorum is a network of microvessels that increase in number and density in more advanced lesions. These microvessels infiltrate plaque, are fragile and leaky, and contribute to intraplaque hemorrhage and further inflammation. CEUS of the carotid artery involves the injection of highly echogenic gas-filled microbubbles into the circulation using commercially available agents. After injection, blood flow activity can be identified by the movement of the microbubbles in the lumen as well as in the vasa vasorum and intraplaque microvessels ( Figure 3 , Video 1 available at www.onlinejase.com ). The adventitial vasa vasorum appears as echogenic bubbles within the adventitial or periadventitial layer, while intraplaque neovascularization may be identified by the presence of microbubbles within the plaque moving from the adventitial side or plaque shoulder toward the plaque core. This microvascular network has been shown to be present at a higher density in the adventitia of patients who experienced CVD events than in asymptomatic patients, while ectopic microvessels in the intima and media are associated with advanced atherosclerotic lesions. Intraplaque neovascularization assessed by CEUS is also associated with angiographically complex and extensive coronary lesions and a higher prevalence of multivessel coronary artery disease (CAD). Furthermore, these vascular changes are not confined to the affected organ but are reflected in several arterial sites (iliac, carotid, and renal arteries), and among vulnerable patients, vasa vasorum density is even higher in the presence of diabetes.
Contrast enhancement of neovascularization is typically qualitatively evaluated using a categorical scale. Although variations of this grading system exist, CEUS is often described on the basis of location of microbubbles and the extent of enhancement. Typically, variations on the following grading system are followed: grade I indicates no enhancement of the plaque, grade II indicates enhancement of the vasa vasorum within the adventitia or periadventitial tissue, grade III indicates enhancement of intraplaque neovascularization at the adventitial side or shoulder of the plaque, and grade IV indicates extensive enhancement within the plaque core. The grade and intensity of contrast enhancement within a plaque are positively correlated with microvessel density.
By determining the location and extent of neovascularization, CEUS can be correlated with symptoms of CVD and aid in the identification of vulnerable plaque. For example, patients with established CVD exhibit an increased presence of adventitial vasa vasorum, and patients with histories of cardiovascular events (MI, TIA, and stroke) exhibit an increased presence of intraplaque neovascularization. Additionally, investigators have shown that the prevalence of stroke is significantly higher in patients with higher grades of plaque enhancement (grades III and IV) and that the presence of carotid plaque enhancement is a significant and independent predictor of acute coronary syndrome in patients classified as having stable CAD. A distinct and unique aspect of CEUS compared with all other imaging modalities is the ability to directly visualize the spatial and temporal heterogeneity of intraplaque and adventitial neovascularization within the vessel wall and at the bedside. Second, CEUS uses no ionizing radiation and is widely used and economical. These advantages, along with the aforementioned studies in symptomatic patients, have promoted research interest in using CEUS to identify vulnerability in asymptomatic patients, as a possible risk stratification tool, on the basis of the hypothesis of vulnerable plaque imaging potentially indicating a vulnerable patient.
Quantitative assessment of plaque neovascularization via computer-assisted methods has been developed, in which the relative intensity of a region of interest defining the plaque is analyzed before and after contrast injection. In a recent meta-analysis, Huang et al . analyzed the use of CEUS to identify intraplaque neovascularization in 20 studies, four of which used quantitative methods, and found that both qualitative and quantitative methods had good diagnostic accuracy, but qualitative assessment had a higher diagnostic odds ratio than quantitative methods. Quantitative analysis can be difficult to achieve because the contrast material does not enter the vasa vasorum as a bolus but rather as individual bubbles that pass through the intraplaque microvessels a few at a time; as a result, the analysis of multiple frames can be difficult because of movement of the plaque with arterial pulsation. Akkus et al . addressed the issues associated with multiple-frame analysis by developing a software package that compensates for motion and measures a number of variables, including microvessel surface area and vascular structure, as a method to replace visual scoring, but it has not been validated with histology. Commercially available analysis software (e.g., VueBox; Bracco, Milan, Italy) is available, and although it does not compensate for vessel movement, it supports the normalization of images from multiple ultrasound machines and systems and has a carotid artery–specific analysis function using time-intensity curves. Further development and validation of this tool may simplify the quantification of plaque neovascularization for more widespread use. Automated, computer-assisted analysis of contrast-enhanced microvessels is promising, but is not yet ready for clinical applications.
Inflammation
Late-phase CEUS has been under investigation as a method to directly detect inflammatory factors. Late-phase CEUS is accomplished by imaging the artery several minutes after microbubble injection and after the clearance of free contrast material in the lumen so that the enhanced signal that remains is that of the microbubbles that are retained in the tissue. It has been suggested that in late-phase CEUS, contrast enhancement of a plaque is due to the phagocytosis of microbubbles by monocytes, as is seen in vitro, and/or adhesion of microbubbles to the damaged surface of the endothelium. This hypothesis was supported by Shalhoub et al ., who found that plaques with higher late-phase CEUS signals have significantly higher levels of chemical markers of angiogenesis, matrix degradation, and inflammation. Owen et al . demonstrated through late-phase CEUS that plaque associated with stroke, TIA, or amaurosis fugax had significantly greater contrast enhancement than plaque found in asymptomatic patients, suggesting its association with inflammatory cells.
Plaque Activity: CEUS Imaging
The Vasa Vasorum and Intraplaque Neovascularization
The vasa vasorum is a network of microvessels that increase in number and density in more advanced lesions. These microvessels infiltrate plaque, are fragile and leaky, and contribute to intraplaque hemorrhage and further inflammation. CEUS of the carotid artery involves the injection of highly echogenic gas-filled microbubbles into the circulation using commercially available agents. After injection, blood flow activity can be identified by the movement of the microbubbles in the lumen as well as in the vasa vasorum and intraplaque microvessels ( Figure 3 , Video 1 available at www.onlinejase.com ). The adventitial vasa vasorum appears as echogenic bubbles within the adventitial or periadventitial layer, while intraplaque neovascularization may be identified by the presence of microbubbles within the plaque moving from the adventitial side or plaque shoulder toward the plaque core. This microvascular network has been shown to be present at a higher density in the adventitia of patients who experienced CVD events than in asymptomatic patients, while ectopic microvessels in the intima and media are associated with advanced atherosclerotic lesions. Intraplaque neovascularization assessed by CEUS is also associated with angiographically complex and extensive coronary lesions and a higher prevalence of multivessel coronary artery disease (CAD). Furthermore, these vascular changes are not confined to the affected organ but are reflected in several arterial sites (iliac, carotid, and renal arteries), and among vulnerable patients, vasa vasorum density is even higher in the presence of diabetes.
Contrast enhancement of neovascularization is typically qualitatively evaluated using a categorical scale. Although variations of this grading system exist, CEUS is often described on the basis of location of microbubbles and the extent of enhancement. Typically, variations on the following grading system are followed: grade I indicates no enhancement of the plaque, grade II indicates enhancement of the vasa vasorum within the adventitia or periadventitial tissue, grade III indicates enhancement of intraplaque neovascularization at the adventitial side or shoulder of the plaque, and grade IV indicates extensive enhancement within the plaque core. The grade and intensity of contrast enhancement within a plaque are positively correlated with microvessel density.
By determining the location and extent of neovascularization, CEUS can be correlated with symptoms of CVD and aid in the identification of vulnerable plaque. For example, patients with established CVD exhibit an increased presence of adventitial vasa vasorum, and patients with histories of cardiovascular events (MI, TIA, and stroke) exhibit an increased presence of intraplaque neovascularization. Additionally, investigators have shown that the prevalence of stroke is significantly higher in patients with higher grades of plaque enhancement (grades III and IV) and that the presence of carotid plaque enhancement is a significant and independent predictor of acute coronary syndrome in patients classified as having stable CAD. A distinct and unique aspect of CEUS compared with all other imaging modalities is the ability to directly visualize the spatial and temporal heterogeneity of intraplaque and adventitial neovascularization within the vessel wall and at the bedside. Second, CEUS uses no ionizing radiation and is widely used and economical. These advantages, along with the aforementioned studies in symptomatic patients, have promoted research interest in using CEUS to identify vulnerability in asymptomatic patients, as a possible risk stratification tool, on the basis of the hypothesis of vulnerable plaque imaging potentially indicating a vulnerable patient.
Quantitative assessment of plaque neovascularization via computer-assisted methods has been developed, in which the relative intensity of a region of interest defining the plaque is analyzed before and after contrast injection. In a recent meta-analysis, Huang et al . analyzed the use of CEUS to identify intraplaque neovascularization in 20 studies, four of which used quantitative methods, and found that both qualitative and quantitative methods had good diagnostic accuracy, but qualitative assessment had a higher diagnostic odds ratio than quantitative methods. Quantitative analysis can be difficult to achieve because the contrast material does not enter the vasa vasorum as a bolus but rather as individual bubbles that pass through the intraplaque microvessels a few at a time; as a result, the analysis of multiple frames can be difficult because of movement of the plaque with arterial pulsation. Akkus et al . addressed the issues associated with multiple-frame analysis by developing a software package that compensates for motion and measures a number of variables, including microvessel surface area and vascular structure, as a method to replace visual scoring, but it has not been validated with histology. Commercially available analysis software (e.g., VueBox; Bracco, Milan, Italy) is available, and although it does not compensate for vessel movement, it supports the normalization of images from multiple ultrasound machines and systems and has a carotid artery–specific analysis function using time-intensity curves. Further development and validation of this tool may simplify the quantification of plaque neovascularization for more widespread use. Automated, computer-assisted analysis of contrast-enhanced microvessels is promising, but is not yet ready for clinical applications.
Inflammation
Late-phase CEUS has been under investigation as a method to directly detect inflammatory factors. Late-phase CEUS is accomplished by imaging the artery several minutes after microbubble injection and after the clearance of free contrast material in the lumen so that the enhanced signal that remains is that of the microbubbles that are retained in the tissue. It has been suggested that in late-phase CEUS, contrast enhancement of a plaque is due to the phagocytosis of microbubbles by monocytes, as is seen in vitro, and/or adhesion of microbubbles to the damaged surface of the endothelium. This hypothesis was supported by Shalhoub et al ., who found that plaques with higher late-phase CEUS signals have significantly higher levels of chemical markers of angiogenesis, matrix degradation, and inflammation. Owen et al . demonstrated through late-phase CEUS that plaque associated with stroke, TIA, or amaurosis fugax had significantly greater contrast enhancement than plaque found in asymptomatic patients, suggesting its association with inflammatory cells.
Plaque Quality: GSM, Texture, and Backscatter
GSM Analysis
The lipid-rich necrotic core and/or intraplaque hemorrhage of a vulnerable plaque may be identified by assessing echo intensity. In atherosclerotic plaque, areas composed of calcified or fibrous tissue appear white on the grayscale spectrum (echogenic), whereas areas consisting of lipid or blood appear black (echolucent). This is similar to the approach demonstrated by multicontrast MRI, which allows the characterization of different tissue types such as a lipid-rich necrotic core, intraplaque hemorrhage, or the thin fibrous cap of a vulnerable plaque through intensity analysis. In contrast to grayscale imagine by ultrasound, the feasibility of integrating MRI into common practice is limited because of cost and availability.
Early methods of evaluating plaque composition using ultrasound were characterized by Gray-Weale et al . who developed a scale for measuring characteristics on the basis of the visual assessment of echogenicity and heterogeneity. This study correlated predominantly echolucent plaque with ulceration and intraplaque hemorrhage, as verified with histologic examination of carotid endarterectomy specimens, and resulted in the adoption of the categorical Gray-Weale scale, which defines plaque as types I to V, where type I is uniformly echolucent, type II is predominantly echolucent (>50% echolucent), type III is predominantly echogenic (<50% echolucent), type IV is uniformly echogenic, and type V is reserved for plaque that is highly calcified and considered unclassifiable if acoustic shadowing makes analysis difficult. Although higher levels of calcification may be indicative of high plaque burden, they also appear to be associated with greater plaque stability. The Gray-Weale scale was used to assess the echogenicity of carotid plaques in the Tromsø Study, in which echolucent plaques were found to predict higher risk for cerebrovascular events over a 3-year follow-up period.
In the past two decades, computer-assisted techniques have been used to standardize grayscale methods. Commercially available image-editing programs such as Adobe Photoshop can be used to analyze the grayscale histogram of isolated plaque, allowing a simple, reproducible method for the determination of the GSM of the plaque ( Figure 4 A). This method requires the normalization of each image under analysis by defining two points of reference on a grayscale spectrum that spans between 0 (black) and 255 (white). These reference points are typically the blood/lumen, which is normalized to a range of 0 to 5, and the adventitia, which is normalized to a range of 185 to 195. This normalization process is of particular importance when different scanners, or different ultrasonic gains are used during image acquisition.
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