Contrast-Enhanced Ultrasound for the Evaluation of Neovascularization in Atherosclerotic Carotid Artery Plaques
Background and Purpose—Neovascularization associated with plaque vulnerability, particularly in the plaque shoulder, is susceptible to rupture, causing ischemic events. We aimed to use contrast-enhanced ultrasound to evaluate neovessels in carotid plaques quantitatively, focusing on plaque shoulders.
Methods—Using contrast-enhanced ultrasound with perflubutane, we analyzed 50 consecutive patients who underwent carotid endarterectomy. We measured enhanced intensity and assessed the correlation between contrast effect and histopathology, comparing symptomatic and asymptomatic plaques.
Results—Enhanced intensity of the plaque shoulder was associated with neovessel density (P<0.01; ρ=0.43). Enhanced intensity of the plaque shoulder was higher in plaques with rupture than in those without (P<0.05), and in symptomatic plaques (n=31) than in asymptomatic ones (n=19; P<0.01).
Conclusions—Quantitative evaluation of the contrast effect using contrast-enhanced ultrasound enabled the assessment of neovascularization of plaque shoulders in vivo real time, which may help stratify plaque vulnerability.
Carotid stenosis with atherosclerotic plaques is a risk factor for artery-to-artery embolism. Although risks of ischemic stroke are assessed according to degree of stenosis, even low-grade stenosis causes recurrent embolism and may be refractory to aggressive treatment. Qualitative characterization of plaques, in addition to quantification of the grading of stenosis, should be performed to detect vulnerable plaques. Neovascularization predicts plaque vulnerability, and neovessels in plaque shoulders tend to collapse, inducing intraplaque hemorrhage and plaque rupture.1
Carotid ultrasound allows less-invasive, serial bedside evaluation of carotid morphology. Second-generation contrast agents containing less soluble gases, such as SonoVue (Bracco, Milan, Italy) and Sonazoid (GE Healthcare, Oslo, Norway), are stable in vivo and provide stable contrast because they are highly compressible and facilitate detection of small and low-flow vessels, such as neovessels in carotid plaques.
We aimed to use contrast-enhanced ultrasound (CEUS) to evaluate intraplaque neovessels quantitatively, focusing on plaque shoulders, for comparing the results with histopathology.
We enrolled 50 consecutive patients with internal carotid artery stenosis who underwent carotid endarterectomy from July 2011. Data collected included vascular risks, stenosis severity, and symptoms associated with previous ischemic events on the ipsilateral side. Carotid ultrasound was performed using an LOGIQ E9 (GE Yokogawa Medical Systems, Hino, Japan) with a linear probe. Plaques were classified as echolucent or echogenic. CEUS examinations were performed after a bolus injection of Sonazoid, a lipid-stabilized suspension of perflubutane gas microbubbles (0.01 mL/kg body weight), using the amplitude modulation mode for further offline analysis and the phase inversion mode to delineate neovessels. We recorded images using the amplitude modulation mode of the short axis of the most stenotic lesion before and after injection. Regions of interest were set, and a time–intensity curve was generated. Enhanced intensity (EI) was calculated by subtracting baseline from peak intensities in the core (EIC), plaque shoulder (EIS), and vessel lumen (EIL). We used larger EIS of the 2 shoulders for further analyses (Figure 1).
Fixed carotid endarterectomy specimens were stained with hematoxylin and eosin and Masson trichrome. Plaque morphology was evaluated according to the American Heart Association classification of atherosclerotic plaques. Immunohistochemistry was performed using a monoclonal antibody against von Willebrand factor (DAKO, Nikko, Japan) to detect neovessels. Neovessel density (per square millimeter) of the side with the higher density plaque shoulder in the most stenotic lesion was used for further analyses (online-only Data Supplement).
We enrolled 60 consecutive patients and excluded 10 because of severe calcification and large ulcers (Table). The contrast of plaque shoulders was greater than that of cores (EIS versus EIC, 9.6±3.6 versus 1.1±1.6 dB, respectively; P<0.0001), consistent with histological findings that cores contained lipid and hemorrhage with few neovessels, mainly localized in plaque shoulders (Figure 2). EIS associated with neovessel density in the plaque shoulder (P=0.0017; ρ=0.43; Figure I in the online-only Data Supplement). EIS was higher in plaques with rupture than in those without rupture (EIS, 10.1±3.5 versus 6.9±3.2 dB, respectively; P=0.018). EIS tended to be higher in plaques with intraplaque hemorrhage than in those without rupture (EIS, 9.9±3.6 versus 7.6±2.5 dB, respectively; P=0.095).
The contrast effect of the plaque shoulder of symptomatic plaques was significantly greater than that of asymptomatic plaques (EIS, 10.8±3.7 versus 7.7±2.4 dB, respectively; P=0.0016). Among symptomatic plaques, EIS values of plaques that had a time interval from the onset of the last event to carotid endarterectomy of ≤6 months were higher than those of plaques with an interval of >6 months, but the difference was not statistically significant (EIS, 11.3±3.7 versus 9.2±3.7 dB, respectively; P=0.34; Figure II in the online-only Data Supplement).
The histological characteristics of vulnerable plaques, including intraplaque hemorrhage, thrombus, inflammatory cells, thin fibrous caps, and neovascularization, were reviewed.2 Neovessels are immature and fragile, particularly in plaque shoulders, because local inflammatory damage and shear stress from the arterial lumen lead to collapse, causing intraplaque hemorrhage and plaque rupture,1 consistent with the present results.
Although MRI3 and computed tomographic angiography4 using contrast agents yield highly reproducible evaluations of carotid artery neovascularization, precise delineation of neovessels remains difficult because enhancement depends on endothelial permeability, and contrast agents gradually penetrate the extravascular space. In contrast, CEUS generates real-time images of microbubbles as intravascular tracers that penetrate the plaque from the vessel lumen or adventitial side through neovessels.
This is the first CEUS study to evaluate neovessels in plaque shoulders quantitatively and compare the results with those of histopathologic specimens. Other studies using CEUS correlate symptomatology and CEUS contrast effects of the entire plaque visually5 or quantitatively.6 Here, the high contrast effect in plaque shoulders may more reliably predict the risk of plaque rupture and intraplaque hemorrhage.
Histopathology revealed an inverse correlation between neovessel density and time from the ischemic event to carotid endarterectomy, suggesting that significant angiogenesis occurs soon before an episode of plaque instability and regresses thereafter as remodeling.7 Our findings support this hypothesis although the results were not statistically significant (Figure II in the online-only Data Supplement).
CEUS can predict and stratify plaque vulnerability and may enhance evaluation of treatment strategies for atherosclerotic diseases. For example, CEUS detected decreased angiogenesis in a patient treated with statins.8 Although we did not evaluate the effects of antiatherosclerotic drugs or levels of serological inflammatory markers here, additional studies analyzing serological markers or interventional prospective studies of antiatherosclerotic treatment are required.
Sources of Funding
This study was supported by Intramural Research Fund (22-6-10) for Cardiovascular Diseases of National Cerebral and Cardiovascular Center.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.114.006483/-/DC1.
- Received July 7, 2014.
- Revision received July 7, 2014.
- Accepted July 28, 2014.
- © 2014 American Heart Association, Inc.
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