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Plaque Elasticity and Intraplaque Neovascularisation on Carotid Artery Ultrasound: A Comparative Histological Study

Open ArchivePublished:July 12, 2021DOI:https://doi.org/10.1016/j.ejvs.2021.05.026

      Objective

      Plaque elasticity and intraplaque neovascularisation are strongly suggestive of vulnerable plaque. This study aimed to investigate the relationship between intraplaque neovascularisation and plaque elasticity, and to compare the ultrasound findings with histopathological changes.

      Methods

      Patients enrolled in this study presented with symptomatic carotid stenosis (> 70%) and later underwent both pre-operative ultrasonography and endarterectomy. Contrast enhanced ultrasound (CEUS) and shear wave elastography (SWE) were used to measure the neovascularisation and elasticity of the plaque, respectively. After removal, plaques were histologically assessed to determine the microvessel density (MVD), matrix metalloproteinase (MMP)-9 expression, and type I/type III collagen ratio using immunohistochemistry staining and morphometry. A correlation analysis was used to establish the relationship among the aforementioned quantitative parameters. Inter- and intra-observer consistency evaluations were performed using the intraclass correlation coefficient and Bland–Altman plots.

      Results

      Ninety-four symptomatic patients with 98 plaques were included. The area under the curve (AUC) of the carotid plaque detected using CEUS correlated with its shear wave velocity (SWV) (r = –.714; p < .001), MVD (r = .842; p < .001), collagen type I/III ratio (r = –.833; p < .001), and MMP-9 (r = .738; p < .001). SWE was positively correlated with the type I/III collagen ratio (r = .805; p < .001). The overall interexaminer consistency of the SWE was acceptable (r = .638; p < .001). The interobserver correlation coefficient of the AUC, time to peak (TP), mean transit time (MTT), and SWV were .719, .756, .733, and .686, respectively. The intra-observer variability values of the AUC, TP, MTT, and SWV were .826, .845, .633, and .748, respectively.

      Conclusion

      SWE and CEUS can comprehensively evaluate the vulnerability of the carotid plaque by assessing the elasticity of the plaque and neovascularisation within it. The negative correlation between the intraplaque neovascularisation and elasticity, further validated by histological findings, suggests that the more abundant the neovascularisation, the less elasticity.

      Keywords

      Carotid plaque rupture is a major cause of stroke. The key issue for risk stratification is the early identification of plaques prone to rupture. Non-invasive techniques, contrast enhanced ultrasound (CEUS) and shear wave elastography can comprehensively evaluate the vulnerability of carotid plaques by assessing the elasticity of the plaque and neovascularisation within it. It has been shown that CEUS and shear wave velocity have the potential to become invaluable tools for measuring plaques because of their cost effective ability to assay plaque texture and composition, thus providing data regarding plaque vulnerability and parameters that contribute to tailoring patient management.

      Introduction

      Stroke is one of the leading causes of death worldwide, with 15 million people experiencing a new or recurrent stroke every year, resulting in five million deaths and permanent disability in an additional five million patients.
      • Meschia J.F.
      • Bushnell C.
      • Boden-Albala B.
      • Braun L.T.
      • Bravata D.M.
      • Chaturvedi S.
      • et al.
      Guidelines for the primary prevention of stroke: a statement for healthcare professionals from the American Heart Association/American Stroke Association.
      ,
      • Li Z.
      • Bai Y.
      • Li W.
      • Gao F.
      • Kuang Y.
      • Du L.
      • et al.
      Carotid vulnerable plaques are associated with circulating leukocytes in acute ischemic stroke patients: a clinical study based on contrast-enhanced ultrasound.
      Acute ischaemic stroke accounts for approximately 80% of all strokes. Approximately 20% of ischaemic strokes appear to originate from vulnerable carotid plaques.
      • Ammirati E.
      • Moroni F.
      • Norata G.D.
      • Magnoni M.
      • Camici P.G.
      Markers of inflammation associated with plaque progression and instability in patients with carotid atherosclerosis.
      Recent research has focused on predicting which atherosclerotic plaques will rupture, thereby triggering events such as myocardial infarction or stroke.
      • Pletsch-Borba L.
      • Selwaness M.
      • van der Lugt A.
      • Hofman A.
      • Franco O.H.
      • Vernooij M.W.
      Change in carotid plaque components: a 4-year follow-up study with serial MR imaging.
      Plaque neovascularisation is closely associated with plaque vulnerability.
      • Zamani M.
      • Skagen K.
      • Scott H.
      • Lindberg B.
      • Russell D.
      • Skjelland M.
      Carotid plaque neovascularization detected with superb microvascular imaging ultrasound without using contrast media.
      Neovascularisation with leaky immature neovessels may increase the risk of intraplaque haemorrhage and plaque rupture, consequently leading to clinical events.
      • Staub D.
      • Patel M.B.
      • Tibrewala A.
      • Ludden D.
      • Johnson M.
      • Espinosa P.
      • et al.
      Vasa vasorum and plaque neovascularization on contrast-enhanced carotid ultrasound imaging correlates with cardiovascular disease and past cardiovascular events.
      In recent decades, contrast enhanced ultrasound (CEUS) has been widely used as a non-invasive tool for detecting intraplaque neovascularisation and assessing the therapeutic effects on plaque neovascularisation.
      • Shah B.N.
      • Gujral D.M.
      • Chahal N.S.
      • Harrington K.J.
      • Nutting C.M.
      • Senior R.
      Plaque neovascularization is increased in human carotid atherosclerosis related to prior neck radiotherapy: a contrast-enhanced ultrasound study.
      It has been reported that plaque elasticity, influenced by plaque compositions such as the fibrous cap and lipid core, has an important role in evaluating plaque vulnerability.
      • Dieleman N.
      • Yang W.
      • Abrigo J.M.
      • Chu W.C.
      • van der Kolk A.G.
      • Siero J.C.
      • et al.
      Magnetic resonance imaging of plaque morphology, burden, and distribution in patients with symptomatic middle cerebral artery stenosis.
      ,
      • Garrard J.W.
      • Ummur P.
      • Nduwayo S.
      • Kanber B.
      • Hartshorne T.C.
      • West K.P.
      • et al.
      Shear wave elastography may be superior to grey scale median for the identification of carotid plaque vulnerability: a comparison with histology.
      Shear wave elastography (SWE) imaging, a novel ultrasound technique, can facilitate assessment using the virtual touch tissue imaging and quantification method.
      • Liu F.
      • Yong Q.
      • Zhang Q.
      • Liu P.
      • Yang Y.
      Real-time tissue elastography for the detection of vulnerable carotid plaques in patients undergoing endarterectomy: a pilot study.
      The interexaminer reproducibility of carotid SWE images obtained by two independent examiners is reportedly good.
      • Ramnarine K.V.
      • Garrard J.W.
      • Dexter K.
      • Nduwayo S.
      • Panerai R.B.
      • Robinson T.G.
      Shear wave elastography assessment of carotid plaque stiffness: in vitro reproducibility study.
      However, few studies have used SWE to evaluate the vulnerability of carotid atherosclerotic plaques.
      • Lou Z.
      • Yang J.
      • Tang L.
      • Jin Y.
      • Zhang J.
      • Liu C.
      • Li Q.
      Shear wave elastography imaging for the features of symptomatic carotid plaques: a feasibility study.
      One study indicated that the plaque became softer and more elastically heterogeneous as intraplaque neovascularisation increased.
      • Zhang Q.
      • Li C.
      • Zhou M.
      • Liao Y.
      • Huang C.
      • Shi J.
      • et al.
      Quantification of carotid plaque elasticity and intraplaque neovascularization using contrast-enhanced ultrasound and image registration-based elastography.
      However, the technique of image registration based strain elastography is dependent on the examiner,
      • Cantisani V.
      • Grazhdani H.
      • Drakonaki E.
      • D’Andrea V.
      • Di Segni M.
      • Kaleshi E.
      • et al.
      Strain ultrasound elastography for the characterization of thyroid nodules: advantages and limitation.
      with no histological evidence confirming the relationship between intraplaque neovascularisation and elasticity. To date, there is no available research involving the combination of CEUS and SWE to assess carotid plaques; however, these techniques are advantageous because they can be conducted with the same probe of the same ultrasound device during one evaluation. This study aimed to investigate the relationship between plaque neovascularisation on CEUS and carotid elasticity on SWE, and to correlate the results with several other measurements, including microvessel density (MVD), type I/III collagen ratio, and matrix metalloproteinase (MMP)-9 density of the carotid plaques, obtained during a histological examination.

      Materials and methods

      Study population

      From January 2015 to May 2018, 102 consecutive patients (108 plaques) presenting with symptomatic carotid stenosis and who underwent endarterectomy were enrolled in the study. Six patients underwent bilateral endarterectomy; the rest underwent unilateral endarterectomy. The inclusion criteria were as follows: symptomatic (amaurosis fugax, transient ischaemic attack, or ipsilateral stroke) with carotid stenosis > 70% defined using ultrasonography (North American Symptomatic Carotid Endarterectomy Trial criteria) and experience of a stroke within the past six months with contralateral asymptomatic severe stenosis > 75%.
      In this study, fragmented plaques and plaques with severe calcification, patients with difficulty undergoing CEUS (e.g., patients with a contrast allergy and those with right to left cardiac shunts), and patients with incomplete information were excluded. Ten plaques in eight patients were excluded from the analysis because six plaques in four patients were extensively calcified, resulting in acoustic shadowing, and the other four plaques in four patients located at the distal internal carotid artery were difficult to image with SWE. Therefore, 94 symptomatic patients with 98 plaques were included in the final analysis. During the assessment of examiner variability associated with SWE images, 24 carotid plaques in 24 patients were scanned by two independent examiners from January 2020 to May 2020.
      This study was performed in strict accordance with the ethical guidelines of the Declaration of Helsinki. Written informed consent was acquired from all patients, and ethical approval was obtained from the Ethics Committee of the Affiliated Hospital of the University.

      Standard ultrasound, contrast enhanced ultrasound, and shear wave elastography examinations

      All standard ultrasound (US), CEUS, and SWE examinations were performed using a Siemens Acuson HELX3000 equipped with a linear array transducer (frequency range 4–9 MHz). Ninety-eight plaques from 94 symptomatic patients were examined by one of the authors (Y.Z., with 10 and six years of experience working with carotid CEUS and plaque SWE, respectively). The carotid arteries were examined in the longitudinal and transverse views to detect the target plaques. The longitudinal views with the greatest degree of luminal narrowing were visually determined (Fig. 1A). The distance from the target plaque to either the flow divider or the origin of the carotid bulb was measured using a caliper and was used to match the ultrasound images to the histological lesion of the endarterectomy specimen.
      Figure 1
      Figure 1(A) Hypoechogenic carotid plaque (arrows) located in the posterior wall of the internal carotid artery demonstrated on ultrasound. (B) The Vs mean of virtual touch tissue imaging and quantification (VTIQ) value was 5.76 m/s by the shear wave elasticity. (C) Quantitative analysis of neovascularisation in the carotid plaque. (D) The contrast enhanced ultrasound (CEUS) cine loops of all plaques were reviewed offline using autotracking contrast quantification technology analysis software. The parameters included time to peak (44.14 s), mean transit time (52.69 s), and the areas under the time intensity curves (703.5%).

      Shear wave elastography examinations

      Two board certified radiologists (Y.Z. and C.Z. [C.Z. with five and eight years of experience with plaque SWE and carotid CEUS, respectively]) performed and analysed the SWE studies. SWE was performed in the longitudinal view for the target plaque. The sampling box was adjusted to cover the target plaque. The shear wave velocity (SWV) mode was initialised, and a two dimensional colour map of SWV distribution within the plaque was obtained; the green colour indicated good SWE quality. Regions of interest (ROIs) were placed within the plaque when the velocity map showed homogeneous SWE distribution (green) (Fig. 1B). Each ROI parameter’s SWV was quantitatively measured with a small ROI box and displayed on the screen.
      • Shang J.
      • Wang W.
      • Feng J.
      • Luo G.G.
      • Dang Y.
      • Sun J.
      • et al.
      Carotid plaque stiffness measured with supersonic shear imaging and its correlation with serum homocysteine level in ischemic stroke patients.
      ,
      • Naim C.
      • Cloutier G.
      • Mercure E.
      • Destrempes F.
      • Qin Z.
      • El-Abyad W.
      • et al.
      Characterisation of carotid plaques with ultrasound elastography: feasibility and correlation with high-resolution magnetic resonance imaging.
      For each plaque, the maximum, minimum, median, and mean SWV values were recorded and analysed.

      Contrast enhanced ultrasound (CEUS) examinations and quantitative analysis of CEUS images

      CEUS was performed for target plaques in the longitudinal view after the administration of 2.0 mL SonoVue (Bracco, Milan, Italy) as a bolus injection via a 19 G peripheral venous cannula, followed by a 5 mL saline flush, with the presets of the mechanical index ranging from 0.10 to 0.15 and the focal zone at a depth of 2 – 3 cm. A real time contrast enhanced cine loop (at least three minutes long) of the carotid plaque was acquired following the injection and stored digitally on magnetic optical discs for later offline analyses.
      Two board certified radiologists (Y.Z. and C.Z.) performed and analysed the acquisition of the CEUS images. The CEUS cine loops of all plaques were reviewed offline using autotracking contrast quantification technology analysis software. The ROI was drawn around the margin of the plaque using an electronic cursor to avoid the lumen and surrounding tissue (Fig. 1C). Specific parameters included time to peak (TP; seconds), mean transit time (MTT; seconds), and the areas under the time intensity curves (AUC; %) (Supplementary Table S1). All measurements were performed three times, and the average of these measurements was calculated and compared. All data were recorded and stored in the internal memory of the ultrasound machine for further analysis. The interobserver agreement of the CEUS parameters was analysed by two independent readers (C.Z. and Y.Z.) who were blinded to each other’s interpretation, and a comparison was performed using intraclass correlation coefficients and Bland–Altman plots.

      Tissue processing and histological analysis

      Ninety-four patients with 98 plaques underwent SWE and CEUS examinations one week prior to carotid endarterectomy. The specimens were marked for orientation by the surgeon and histopathologically analysed to assess morphological changes (Fig. 2A), intraplaque neovascularisation, inflammation infiltration, and collagen composition. The radiologists were blinded to the histological results. Specimen photos (obtained before and after the slices were prepared) and ultrasound images obtained during the process of plaque preparation were used to determine the direction and rotational axis of the slices relative to the ultrasound imaging performed prior to pathological staining. Labelling and segmenting of the plaques were performed according to the methods of Willem et al.
      • Hellings W.E.
      • Pasterkamp G.
      • Vollebregt A.
      • Seldenrijk C.A.
      • De Vries J.-P.P.M.
      • Velema E.
      • et al.
      Intraobserver and interobserver variability and spatial differences in histologic examination of carotid endarterectomy specimens.
      Tissue samples were fixed with 10% formalin, embedded in paraffin, and sliced into 4 μm thick serial sections for microscopic examinations. In addition to routine haematoxylin and eosin staining for morphological changes, the slices were immunostained with CD31 antibodies (ab9498; Abcam, Cambridge, MA, USA), which stain endothelial cells in neovessels. The number of neovessels was counted under a microscope (× 200) and then converted to the MVD (mm2). For each plaque, five fields of view were used for microscopic examination, and the five measured MVD values were averaged to obtain the final result (Fig. 2B). MMP-9 (%), which stains inflammatory cell infiltration, was also used (Fig. 2C). The pathologist selected three representative high power fields (magnification × 200) containing > 100 inflammatory cells and counted the positively stained cells and the total number of inflammatory cells. The average percentage of positively stained cells was calculated as the MMP-9 expression. (Supplementary Table S1).
      Figure 2
      Figure 2(A) Target carotid plaque specimen after carotid endarterectomy. (B) Immunohistochemical staining for endothelial marker CD31 (brown, arrows) indicates intraplaque neovascularisation (original magnification ×200). (C) Immunohistochemical staining of matrix metallopeptidase (MMP)-9 protein in the vascular endothelial cells showing positive expression (original magnification ×200). (D, E) Collagen type was evaluated using the picro-Sirius red polarisation method, where type I collagen appears red, and type III collagen appears green (original magnification ×200). The ratios of area I/area III of each slice were automatically calculated using IPP 5.0 software.
      To analyse collagen, the specimens were fixed in 10% neutralised formaldehyde and embedded in paraffin. Next, they were washed in xylene and rehydrated in a graded series of ethanol. Tissue sections (thickness, 6 μm) were stained with a saturated aqueous solution of picric acid containing 0.1% Sirius red (Nanjing Jiangsu 210000; China SenBeiJia Biological Technology Co., Ltd., Nanjing, China). Images of five random microscopic fields of red stained collagen fibres were recorded using a polarisation microscope (Leica Microsystems, Wetzlar, Germany) and then digitised and analysed using Image-Pro Plus version 6.0 (Media Cybernetics, Rockville, MD, USA). The collagen category was evaluated using the picro-Sirius red polarisation method. The type I collagen area (area I, red pixels) (Fig. 2D) and type III collagen area (area III, green pixels) (Fig. 2E) were automatically calculated for each slice using IPP 5.0 software. Then, the area I/area III ratios were calculated. Finally, the relationships among the area I/area III ratios, SWV, and AUC for the quantitative parameters of CEUS were analysed.

      Statistical analysis

      Statistical analyses were performed with SPSS 16.0 (IBM, Armonk, NY, USA) and R software (version 3.6.2). Variables are presented as number (%), mean ± standard deviation (SD), or median (interquartile range) depending on the data type, measurement scale, and distribution. Correlations between the AUC and SWV and between the AUC and MVD were established using Pearson’s correlation analysis. Spearman’s rank correlation coefficient was used to assess the correlations between the AUC and type I/III collagen ratio, AUC and MMP-9, SWV and type I/III collagen ratio, MVD and TP, and MVD and MTT. The interobserver and intra-observer consistency of all ultrasound quantitative parameters of CEUS and SWE, including the AUC, TP, and MTT of CEUS, and the SWV of SWE, were determined using intraclass correlation coefficients (ICCs) and Bland–Altman plots. Furthermore, the interexaminer reproducibility of SWE images obtained by two independent examiners using the same 24 plaques was determined using ICCs and Bland–Altman plots; p < .05 was considered to be statistically significant.

      Results

      Patient demographics

      Of the 94 patients who underwent endarterectomy for symptomatic carotid artery stenosis, 12 had experienced amaurosis fugax, 23 a transient ischaemic attack with amaurosis fugax, and 59 a stroke. The mean age of the patients was 66.4 ± 12.6 years; 78 (83%) were male and 71 (75%) had a history of smoking. Most patients underwent CEUS and elastography examinations within two weeks of symptom onset. The median time from the onset of symptoms to performing the assessment was 14 days (range 9.75 – 24.00 days); however, seven patients were critical because of apoplectic stroke and cerebral oedema, and were transferred from community hospitals to the tertiary hospital. After their vital signs had stabilised, they were finally scheduled for preventive carotid plaque dissection. The general characteristics of these 94 patients are presented in Table 1.
      Table 1Clinical characteristics of 94 patients with symptomatic carotid artery stenosis studied for plaque morphology
      CharacteristicPatients (n = 94)
      Age – y66.4 ± 12.6
      Male sex78 (83.0)
      BMI – kg/m221.9 (19.7–28.4)
      Smoking71 (75.5)
      Stroke59 (62.8)
      Transient ischaemic attack23 (24.5)
      Amaurosis fugax12 (12.8)
      Median time from symptom to assessment – d14.0 ( 9.8–24.0)
      hs-CRP – mmol/L2.0 (0.6–3.3)
      Serum cholesterol – mmol/L4.8 (3.8–6.1)
      Serum LDL cholesterol – mmol/L2.4 (2.1–4.5)
      Serum HDL cholesterol – mmol/L1.8 (1.4–2.0)
      Medical history
       Diabetes22 (24.5)
       Hypertension54 (57.4)
       Myocardial infarction7 (7.4)
       Atrial brillation16 (17.0)
      Medication
       Antiplatelet therapy82 (87.2)
       Statin72 (76.6)
      Data are presented as n (%), mean ± standard deviation or median (interquartile range). BMI = body mass index; hs-CRP = high sensitivity C reactive protein; LDL = low density lipoprotein; HDL = high density lipoprotein.

      Plaque elasticity and microvessel density within plaque

      Distributions of the parameters, including the AUC, TP, and MTT of CEUS, SWV of SWE, MVD, MMP-9, and type I/III collagen ratio, obtained by immunohistochemistry are presented in Table 2. Non-normally distributed variables (AUC, SWV, MMP-9, MVD, and type I/III collagen ratio) are presented as median (interquartile range). Normally distributed variables (TP and MTT) are presented as mean ± SD.
      Table 2Contrast enhanced ultrasound (CEUS) and shear wave elastography (SWE) parameters and immunohistochemistry results of 98 carotid plaques from 94 patients with symptomatic carotid stenoses
      VariableValue
      AUC – %1 078.1 (292.3–2 491.0)
      SWV – m/s4.4 (2.1–7.5)
      TP – s45.0 ± 3.0
      MTT – s64.7 ± 3.3
      MVD – n/mm24.4 (2.1–7.5)
      MMP-9 – %5.3 (0.0–19.3)
      Ratio of collagen type I/III16.8 (0.6–73.2)
      Data are presented as mean ± standard deviation or median (interquartile range). AUC = area under the time intensity curve; SWV = shear wave velocity; TP = time to peak; MTT = mean transit time; MVD = microvessel density; MMP-9 = matrix metallopeptidase 9.
      There was a negative correlation between the SWV of SWE and the AUC of CEUS (r = –.714; p < .001) (Fig. 3A).
      Figure 3
      Figure 3Correlation of contrast enhanced ultrasound quantities of 98 carotid plaques presented as area under the time intensity curve (AUC) with (A) shear wave velocity (SWV) demonstrating elastic features, (B) microvessel density (MVD), (C) ratio of collagen types I/III, and (D) matrix metallopeptidase (MMP)-9, and (E) correlation between SWV and collagen ratio. AUC = area under the time intensity curve.
      Immunostaining for MVD was significantly positively correlated with the AUC of CEUS (r = .842; p < .001) (Fig. 3B). There was no correlation between MVD and TP (Supplementary Fig. S1A) or between MVD and MTT (Supplementary Fig. S1B), according to CEUS (r = .013 and r = –.024, respectively; p > .05 for both).

      Correlation between contrast enhanced ultrasound and immunohistological status

      The AUC of CEUS and immunostaining for the type I/III collagen ratio were correlated in a negative linear manner (r = –.833; p < .001) (Fig. 3C).
      A positive correlation was observed between the AUC of CEUS and MMP-9 expression found by immunostaining in the carotid plaques (r = .738; p < .001) (Fig. 3D).

      Correlation between plaque elasticity and immunohistological status

      The SWV of SWE was significantly positively correlated with the type I/III collagen ratio (r = .805; p < .001) (Fig. 3E).

      Inter- and intra-observer agreement of contrast enhanced ultrasound and shear wave elastography values

      The ICCs for interobserver measurements of the AUC, SWV, TP, and MTT were .719, .686, .756, and .733, respectively. Corresponding scatter plots and Bland–Altman agreement plots are shown for the AUC (Fig. 4A), SWV (Fig. 4B), TP (Supplementary Fig. S2A), and MTT (Supplementary Fig. S2B). The ICCs of intra-observer variability of the AUC (Fig. 4C), SWV (Fig. 4D), TP (Supplementary Fig. 2C), and MTT (Supplementary Fig. 2D) were .826, .748, .845, and .633, respectively.
      Figure 4
      Figure 4Agreement plot for (A, C) area under the time intensity curve (AUC) and (B, D) shear wave velocity (SWV) of two observers (A, B) and one observer (C, D). Plots represent the difference between observers’ measurements and mean measurements. The top and bottom lines show the 95% limits of the agreement; the middle line shows the mean difference. ICC = intraclass correlation coefficient; SD = standard deviation.
      SWE imaging performed by two independent examiners using 24 plaques from 24 patients to assess the interexaminer reproducibility showed no significant differences in SWV (p > .05) and good interexaminer agreement (r = .638 and p < .001).
      In some plaques with heterogeneous enhancement on CEUS (Fig. 5A), the SWE value of the carotid plaque in hyperenhancement areas was higher than that in hypo-enhancement areas (Fig. 5B). Furthermore, the levels of type I collagen expressed in hyperenhancement areas (Fig. 5C) were lower than those in hypo-enhancement areas (Fig. 5D).
      Figure 5
      Figure 5(A) Contrast enhanced ultrasound of a carotid plaque demonstrating heterogenous enhancement in the contrast enhanced ultrasound with hyperenhancement (red) and hypo-enhancement (blue). (B) The shear wave elastography (SWE) value of the carotid plaque in the hyperenhancement area was higher than in the hypo-enhancement area. (C) The hyperenhanced area was mainly expressing type III collagen in the picro-Sirius red polarisation method, whereas (D) the hypo-enchanced area contained mainly of type I collagen.

      Discussion

      Assessing the vulnerability of a carotid plaque is a complex process that includes measurements of parameters such as plaque texture, intraplaque neovascularisation, and inflammation activity.
      • Mantella L.E.
      • Colledanchise K.N.
      • Hétu M.F.
      • Feinstein S.B.
      • Abunassar J
      • Johri A.M.
      Carotid intraplaque neovascularization predicts coronary artery disease and cardiovascular events.
      • Hansen H.H.
      • de Borst G.J.
      • Bots M.L.
      • Moll F.L.
      • Pasterkamp G.
      • de Korte C.L.
      Validation of noninvasive in vivo compound ultrasound strain imaging using histologic plaque vulnerability features.
      • Hultman K.
      • Edsfeldt A.
      • Björkbacka H.
      • Dunér P.
      • Sundius L.
      • Nitulescu M.
      • et al.
      Matrix protein associates with a vulnerable plaque phenotype in human atherosclerotic plaques.
      Plaques can be characterised by their appearance and echogenicity on ultrasound imaging, and classified into four subtypes: soft, hard, calcified, and mixed. Soft plaques are those that produce echogenicity less than that of the surrounding adventitia in the absence of calcium.
      • Akyildiz A.C.
      • Chai C.K.
      • Oomens C.W.J.
      • van der Lugt A.
      • Baaijens F.P.T.
      • Strijkers G.J.
      • et al.
      3D fiber orientation in atherosclerotic carotid plaques.
      Soft plaques are thought to place patients at a higher risk of stroke or transient ischaemic attack than hard plaques. However, echogenicity depends on the relative difference in the acoustic impedance of the materials and should not be assumed to reflect the elasticity of the tissue accurately; low echogenicity plaques do not tend to be softer than echo rich plaques. This additional histological evidence provides a more convincing depiction of the relationship between plaque elasticity and intraplaque neovascularisation. The current method was advantageous because of its ability to combine two techniques for the superior assessment of carotid plaques while using the same probe and equipment; furthermore, assessments can be performed simultaneously, making direct comparisons between elasticity and neovascularisation not only possible, but also efficient. The SWE technique was used to assess tissue stiffness because it provides a quantitative parameter of SWV that has shown significant associations with the AUC measured by CEUS. This research demonstrated a direct correlation between neovascularisation density and plaque softness.
      CEUS has enabled the detection of neovascularisation within atherosclerotic plaques in vivo.
      • Huang P.T.
      • Huang F.G.
      • Zou C.P.
      • Sun H.Y.
      • Tian X.Q.
      • Yang Y.
      • et al.
      Contrast-enhanced sonographic characteristics of neovascularization in carotid atherosclerotic plaques.
      Enhancement within an atherosclerosis plaque observed on CEUS can be attributed to plaque neovascularisation.
      • Kim H.S.
      • Woo J.S.
      • Kim B.Y.
      • Jang H.H.
      • Hwang S.J.
      • Kwon S.J.
      • et al.
      Biochemical and clinical correlation of intraplaque neovascularization using contrast-enhanced ultrasound of the carotid artery.
      Moreover, a previous study indicated that the enhancement index, a quantitative measurement of CEUS intensity, correlated well with the MVD of atherosclerotic plaques in rabbit models (r = .854; p < .001),
      • You X.
      • Huang P.
      • Zhang C.
      • Wang M.
      • Zhang Y.
      • Hong Y.
      • et al.
      Relationship between enhanced intensity of contrast enhanced ultrasound and microvessel density of aortic atherosclerotic plaque in rabbit model.
      which reflected intraplaque neovascularisation. Neovascularisation in the carotid plaque has been well established, and resilience has been confirmed by histological studies as a consistent feature of vulnerable plaque in patients with cerebrovascular disease.
      • Moreno P.R.
      • Purushothaman K.R.
      • Fuster V.
      • Echeverri D.
      • Truszczynska H.
      • Sharma S.K.
      • et al.
      Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability.
      ,
      • Parma L.
      • Baganha F.
      • Quax P.H.A.
      • de Vries M.R.
      Plaque angiogenesis and intraplaque hemorrhage in atherosclerosis.
      Collagen in atherosclerotic plaque has elasticity and toughness.
      • Duprez D.A.
      • Gross M.D.
      • Ix J.H.
      • Peralta C.A.
      • Kizer J.R.
      • Shea S.
      • et al.
      Collagen biomarkers are associated with decline in renal function independently of blood pressure and other cardiovascular risk factors: the Multi-Ethnic Study of Atherosclerosis Study.
      Type I collagen is thick and classified under the mature type. Plaque stability mainly depends on the content of type I collagen. Type III collagen belongs to embryonic collagen, with poor tensile strength. It mostly appears in the collagen fibres of immature blood vessel walls with active proliferation. The increase of type I collagen may indicate a relatively stable disease, and the proliferation of type III collagen indicates that the disease is in an unstable or relatively progressive state.
      • Duprez D.A.
      • Gross M.D.
      • Ix J.H.
      • Kizer J.R.
      • Tracy R.P.
      • Shea S.
      • et al.
      Collagen biomarkers predict new onset of hypertension in normotensive participants: the Multi-Ethnic Study of Atherosclerosis.
      The increase of type I to type III collagen ratio detected biochemically is closely associated with intimal sclerosis and lesion progression.
      • Katsuda S.
      • Okada Y.
      • Minamoto T.
      • Oda Y.
      • Matsui Y.
      • Nakanishi I.
      Collagens in human atherosclerosis. Immunohistochemical analysis using collagen type-specific antibodies.
      The present findings also revealed a positive correlation between MMP-9 expression in plaques and the neovascularisation degree. MMPs have been involved in vascular wall remodelling and the development of atherosclerosis through inflammatory activation and endothelial dysfunction. Remodelling the extracellular matrix and vascular basement membrane allows the creation of new blood vessels.
      • Olejarz W.
      • Łacheta D.
      • Kubiak-Tomaszewska G.
      Matrix metalloproteinases as biomarkers of atherosclerotic plaque instability.
      Studies have shown that MMP-9 mobilises vascular endothelial growth factor (VEGF) from the ECM, which can contribute to plaque neovascularisation. The increase of VEGF bioavailability results from ECM proteolysis.
      • Mott J.D.
      • Werb Z.
      Regulation of matrix biology by matrix metalloproteinases.
      There were two major limitations to this study. Firstly, CEUS and SWE were performed only in the longitudinal view for plaque. As plaques have a three dimensional structure, this two dimensional approximation may have resulted in an underestimation of its true vulnerability. Secondly, in some plaques with heterogeneous enhancement on CEUS, the SWE value of hyperenhancement areas was higher than hypo-enhancement areas, and the expression of type I collagen in hyperenhancement areas was lower than in hypo-enhancement areas; however, the differences in SWV and the type I/III collagen ratio of hyperenhancement and hypo-enhancement areas in the same plaque have not yet been analysed because this is the subject of an ongoing study.

      Conclusion

      SWE and CEUS could evaluate the vulnerability of carotid plaque by assessing the elasticity of the plaque and neovascularisation comprehensively and quantitatively. Abundant neovascularisation is associated with less elasticity within the plaque.

      Conflicts of interest

      None.

      Funding

      This study was supported by the National Natural Science Funds (grant no. 81527803, 81420108018) of China, the Ministry of Science and Technology Key National Key R&D Program of China (grant no. SQ2018YFC0115900), and the Zhejiang Science and Technology Project of China (grant no. 2019C03077, Y16H180019, LQ20H180009).

      Acknowledgements

      The authors acknowledge all the staff in the Department of Neurosurgery of the Second Affiliated Hospital of Zhejiang University School of Medicine for their support of this research work.

      Appendix A. Supplementary data

      The following are the Supplementary data to this article:
      Supplementary Fig 1
      Supplementary Fig 2

      References

        • Meschia J.F.
        • Bushnell C.
        • Boden-Albala B.
        • Braun L.T.
        • Bravata D.M.
        • Chaturvedi S.
        • et al.
        Guidelines for the primary prevention of stroke: a statement for healthcare professionals from the American Heart Association/American Stroke Association.
        Stroke. 2014; 45: 3754-3832
        • Li Z.
        • Bai Y.
        • Li W.
        • Gao F.
        • Kuang Y.
        • Du L.
        • et al.
        Carotid vulnerable plaques are associated with circulating leukocytes in acute ischemic stroke patients: a clinical study based on contrast-enhanced ultrasound.
        Sci Rep. 2018; 8: 8849
        • Ammirati E.
        • Moroni F.
        • Norata G.D.
        • Magnoni M.
        • Camici P.G.
        Markers of inflammation associated with plaque progression and instability in patients with carotid atherosclerosis.
        Mediators Inflamm. 2015; 2015: 718329
        • Pletsch-Borba L.
        • Selwaness M.
        • van der Lugt A.
        • Hofman A.
        • Franco O.H.
        • Vernooij M.W.
        Change in carotid plaque components: a 4-year follow-up study with serial MR imaging.
        JACC Cardiovasc Imaging. 2018; 11: 184-192
        • Zamani M.
        • Skagen K.
        • Scott H.
        • Lindberg B.
        • Russell D.
        • Skjelland M.
        Carotid plaque neovascularization detected with superb microvascular imaging ultrasound without using contrast media.
        Stroke. 2019; 50: 3121-3127
        • Staub D.
        • Patel M.B.
        • Tibrewala A.
        • Ludden D.
        • Johnson M.
        • Espinosa P.
        • et al.
        Vasa vasorum and plaque neovascularization on contrast-enhanced carotid ultrasound imaging correlates with cardiovascular disease and past cardiovascular events.
        Stroke. 2010; 41: 41-47
        • Shah B.N.
        • Gujral D.M.
        • Chahal N.S.
        • Harrington K.J.
        • Nutting C.M.
        • Senior R.
        Plaque neovascularization is increased in human carotid atherosclerosis related to prior neck radiotherapy: a contrast-enhanced ultrasound study.
        JACC Cardiovasc Imaging. 2016; 9: 668-675
        • Dieleman N.
        • Yang W.
        • Abrigo J.M.
        • Chu W.C.
        • van der Kolk A.G.
        • Siero J.C.
        • et al.
        Magnetic resonance imaging of plaque morphology, burden, and distribution in patients with symptomatic middle cerebral artery stenosis.
        Stroke. 2016; 47: 1797-1802
        • Garrard J.W.
        • Ummur P.
        • Nduwayo S.
        • Kanber B.
        • Hartshorne T.C.
        • West K.P.
        • et al.
        Shear wave elastography may be superior to grey scale median for the identification of carotid plaque vulnerability: a comparison with histology.
        Ultraschall Med. 2015; 36: 386-390
        • Liu F.
        • Yong Q.
        • Zhang Q.
        • Liu P.
        • Yang Y.
        Real-time tissue elastography for the detection of vulnerable carotid plaques in patients undergoing endarterectomy: a pilot study.
        Ultrasound Med Biol. 2015; 41: 705-712
        • Ramnarine K.V.
        • Garrard J.W.
        • Dexter K.
        • Nduwayo S.
        • Panerai R.B.
        • Robinson T.G.
        Shear wave elastography assessment of carotid plaque stiffness: in vitro reproducibility study.
        Ultrasound Med Biol. 2014; 40: 200-209
        • Lou Z.
        • Yang J.
        • Tang L.
        • Jin Y.
        • Zhang J.
        • Liu C.
        • Li Q.
        Shear wave elastography imaging for the features of symptomatic carotid plaques: a feasibility study.
        J Ultrasound Med. 2017; 36: 1213-1223
        • Zhang Q.
        • Li C.
        • Zhou M.
        • Liao Y.
        • Huang C.
        • Shi J.
        • et al.
        Quantification of carotid plaque elasticity and intraplaque neovascularization using contrast-enhanced ultrasound and image registration-based elastography.
        Ultrasonics. 2015; 62: 253-262
        • Cantisani V.
        • Grazhdani H.
        • Drakonaki E.
        • D’Andrea V.
        • Di Segni M.
        • Kaleshi E.
        • et al.
        Strain ultrasound elastography for the characterization of thyroid nodules: advantages and limitation.
        Int J Endocrinol. 2015; 2015: 908575
        • Shang J.
        • Wang W.
        • Feng J.
        • Luo G.G.
        • Dang Y.
        • Sun J.
        • et al.
        Carotid plaque stiffness measured with supersonic shear imaging and its correlation with serum homocysteine level in ischemic stroke patients.
        Korean J Radiol. 2018; 19: 15-22
        • Naim C.
        • Cloutier G.
        • Mercure E.
        • Destrempes F.
        • Qin Z.
        • El-Abyad W.
        • et al.
        Characterisation of carotid plaques with ultrasound elastography: feasibility and correlation with high-resolution magnetic resonance imaging.
        Eur Radiol. 2013; 23: 2030-2041
        • Hellings W.E.
        • Pasterkamp G.
        • Vollebregt A.
        • Seldenrijk C.A.
        • De Vries J.-P.P.M.
        • Velema E.
        • et al.
        Intraobserver and interobserver variability and spatial differences in histologic examination of carotid endarterectomy specimens.
        J Vasc Surg. 2007; 46: 1147-1154
        • Mantella L.E.
        • Colledanchise K.N.
        • Hétu M.F.
        • Feinstein S.B.
        • Abunassar J
        • Johri A.M.
        Carotid intraplaque neovascularization predicts coronary artery disease and cardiovascular events.
        Eur Heart J Cardiovasc Imaging. 2019; 20: 1239-1247
        • Hansen H.H.
        • de Borst G.J.
        • Bots M.L.
        • Moll F.L.
        • Pasterkamp G.
        • de Korte C.L.
        Validation of noninvasive in vivo compound ultrasound strain imaging using histologic plaque vulnerability features.
        Stroke. 2016; 47: 2770-2775
        • Hultman K.
        • Edsfeldt A.
        • Björkbacka H.
        • Dunér P.
        • Sundius L.
        • Nitulescu M.
        • et al.
        Matrix protein associates with a vulnerable plaque phenotype in human atherosclerotic plaques.
        Stroke. 2019; 50: 3289-3292
        • Akyildiz A.C.
        • Chai C.K.
        • Oomens C.W.J.
        • van der Lugt A.
        • Baaijens F.P.T.
        • Strijkers G.J.
        • et al.
        3D fiber orientation in atherosclerotic carotid plaques.
        J Struct Biol. 2017; 200: 28-35
        • Huang P.T.
        • Huang F.G.
        • Zou C.P.
        • Sun H.Y.
        • Tian X.Q.
        • Yang Y.
        • et al.
        Contrast-enhanced sonographic characteristics of neovascularization in carotid atherosclerotic plaques.
        J Clin Ultrasound. 2008; 36: 346-351
        • Kim H.S.
        • Woo J.S.
        • Kim B.Y.
        • Jang H.H.
        • Hwang S.J.
        • Kwon S.J.
        • et al.
        Biochemical and clinical correlation of intraplaque neovascularization using contrast-enhanced ultrasound of the carotid artery.
        Atherosclerosis. 2014; 233: 579-583
        • You X.
        • Huang P.
        • Zhang C.
        • Wang M.
        • Zhang Y.
        • Hong Y.
        • et al.
        Relationship between enhanced intensity of contrast enhanced ultrasound and microvessel density of aortic atherosclerotic plaque in rabbit model.
        PLoS One. 2014; 9e92445
        • Moreno P.R.
        • Purushothaman K.R.
        • Fuster V.
        • Echeverri D.
        • Truszczynska H.
        • Sharma S.K.
        • et al.
        Plaque neovascularization is increased in ruptured atherosclerotic lesions of human aorta: implications for plaque vulnerability.
        Circulation. 2004; 110: 2032-2038
        • Parma L.
        • Baganha F.
        • Quax P.H.A.
        • de Vries M.R.
        Plaque angiogenesis and intraplaque hemorrhage in atherosclerosis.
        Eur J Pharmacol. 2017; 816: 107-115
        • Duprez D.A.
        • Gross M.D.
        • Ix J.H.
        • Peralta C.A.
        • Kizer J.R.
        • Shea S.
        • et al.
        Collagen biomarkers are associated with decline in renal function independently of blood pressure and other cardiovascular risk factors: the Multi-Ethnic Study of Atherosclerosis Study.
        J Hypertens. 2019; 37: 2398-2403
        • Duprez D.A.
        • Gross M.D.
        • Ix J.H.
        • Kizer J.R.
        • Tracy R.P.
        • Shea S.
        • et al.
        Collagen biomarkers predict new onset of hypertension in normotensive participants: the Multi-Ethnic Study of Atherosclerosis.
        J Hypertens. 2018; 36: 2245-2250
        • Katsuda S.
        • Okada Y.
        • Minamoto T.
        • Oda Y.
        • Matsui Y.
        • Nakanishi I.
        Collagens in human atherosclerosis. Immunohistochemical analysis using collagen type-specific antibodies.
        Arterioscler Thromb. 1992; 12: 494-502
        • Olejarz W.
        • Łacheta D.
        • Kubiak-Tomaszewska G.
        Matrix metalloproteinases as biomarkers of atherosclerotic plaque instability.
        Int J Mol Sci. 2020; 21: 3946
        • Mott J.D.
        • Werb Z.
        Regulation of matrix biology by matrix metalloproteinases.
        Curr Opin Cell Biol. 2004; 16: 558-564

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