Stroke, with its high morbidity, mortality, and disability rates, represents one of the leading causes of death worldwide (1). Approximately 18–25% of all ischemic strokes are attributable to carotid plaque rupture (2, 3). Intraplaque neovascularization (IPN) represents an important feature of plaque vulnerability (4). IPN provides valuable insight to plaque activity, as it has been reported to associate with an increased risk of neovessel rupture, intraplaque hemorrhage, and inflammation (5).

The location and morphology of plaques have been shown to contribute to plaque vulnerability (6). The potential mechanism for this has been reported to relate to their influence on the shear stress generated on plaque surfaces (7), which is a known key player in the pathophysiology of atherosclerosis (8, 9). Indeed, areas of low shear stress are often accompanied by higher expressions of inflammatory mediators and greater degree of matrix metalloproteinase activity (10). Moreover, inflammation is known to initiate the process of neovascularization. However, the exact influence of plaque location and morphology on the degree of IPN remains unknown.

Contrast enhanced ultrasound (CEUS), a novel ultrasound technique, has been recognized as an effective imaging modality for detecting neovascularization (11, 12). Intensity of plaque enhancement on CEUS has been shown to significantly correlate with the degree of neovascularization (13–15). In addition, CEUS has allowed for clearer visualization of both the location and morphological features of carotid plaques as compared to standard duplex ultrasound.

As such, our study aimed to evaluate the association of plaque location and morphology with IPN grade on CEUS, to assess their role as potential biomarkers for plaque vulnerability.

The morphology and location of carotid plaques, as well as serum HDL-C level, demonstrated a significant influence on IPN grade in our study.

Higher IPN grades were observed among plaques of Type III morphology. Plaques of such morphology are characterized by lower upstream slopes, which may be subjected to lower shear stress (6). This is consistent with the notion that proatherogenic transcription factor upregulation and the resultant aggregation of inflammatory cells tend to occur in regions of low shear stress (19), ultimately resulting in a more fragile plaque phenotype (20, 21). Our findings of increased neovascularization in a low shear stress environment are further in correspondence to previous reports that macrophage infiltration often coexist with hypoxia and angiogenesis due to high metabolic demand (22, 23).

The bifurcation of the CCA has long been known as a common location for atherosclerotic plaque development due to the disturbance in flow (24, 25). However, we found that plaques located in the CCA were associated with a higher IPN grade instead. We postulate that this may be related to the greater length and area of such plaques (Supplementary Table S2; Supplementary Figure S1), which may have reflected greater plaque burden. In line with this, it has been reported that plaques of the CCA tend to grow along the longitudinal axis of the vessel wall and create greater lengths (26). Larger plaques may thereby associate with larger areas of anoxia, greater degrees of inflammation, and ultimately increased neovascularization (26, 27).

Serum HDL-C was found to be significantly protective against neovascularization. While low-density lipoprotein cholesterol (LDL-C) is widely accepted as an independent predictor of cardio- and cerebrovascular events, low HDL-C levels have been reported to be associated with an increased risk of cardiovascular diseases and stroke regardless of LDL-C levels (28–30). The HDL-C level was negatively associated with IPN grading in our study, and this association did not relate to statin use. Other lipid parameters, including the LDL-C level, were not significantly associated with IPN grading, highlighting the potential importance of HDL-C in plaque vulnerability. HDL-C is known to promote the reverse transport of cholesterol from atherosclerotic plaque (31). In addition, HDL-C portrays anti-atherosclerotic effects, which is mediated by its antioxidant, anti-inflammatory, and antithrombotic characteristics (32, 33). While Ying et al. (34) have found an association of carotid plaque neovascularization with total cholesterol and LDL-C levels. This was, however, not observed in our study, which may be attributable to the use of statins (Supplementary Table S3).

Ulceration is an important feature of ruptured plaques (35). However, we found no correlation of ulceration with IPN grade, plaque morphology, or plaque location. This may be due to the relatively few cases of ulcerated plaques among our patients, which hindered effective statistical analysis. However, a previous study showed that shear stress and local hemodynamics caused by anatomical differences of the carotid arteries did not influence the incidence of plaque ulceration (36). This suggests that morphology and location may not play any role in such feature. Nonetheless, further large-sample analyzes are warranted to elucidate the factors involved in the development of ulcerated plaques.

There were several limitations in our study. First, this was a single-centered study with a relatively small sample size. Large-sample studies involving the assessment of other potential associating factors such as clinical symptoms are thereby warranted. Second, plaque morphology was assessed based on only the longitudinal views of B-mode ultrasound and CEUS. However, the spatial morphology of plaques is complicated and may be correlated with plaque vulnerability. In future studies, we aim to evaluate the spatial diversity of plaque morphology through three-dimensional ultrasound imaging. Third, the geometry of calcification, which may affect plaque stability, was overlooked in our study. This was due to their acoustic attenuation effects on CEUS, which would hinder our assessment of neovascularization. Further studies on the effects of calcification on plaque vulnerability are thus required. Finally, plaques with thickness < 2.5 mm were excluded due to the difficulties in performing CEUS for plaques with a thinner wall, which could have negatively affected the accuracy of our results. Some studies on the IPN grade excluded plaques with thickness < 2.5 mm for similar reasons (37). To obtain more accurate results, we excluded these data. In future studies, we will attempt to include other imaging modalities to study the neovascularization of plaques with thickness < 2.5 mm. In addition, we included plaques with thickness ≥ 2.5 mm, which were considered high-risk plaques in a previous study. In future, we hope to obtain more information regarding plaques in high-risk groups and provide better management strategies.

The location and morphology of carotid plaques significantly associated with IPN grade on CEUS, and thereby carry the potential as biomarkers for plaque vulnerability. Serum HDL-C was further found as a protective factor against IPN, and may play a role in the management of carotid atherosclerosis. Our study not only provided a potential strategy for the identification of vulnerable carotid plaques, but also elucidated the important imaging predictors of stroke.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Shanghai General hospital institutional review board. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

S-YG: conceptualization, writing–original draft, writing–review and editing, and investigation. L-NZ: investigation and writing–review and editing. FL: data curation. JC: writing–review and editing. M-HY: formal analysis and writing–review and editing. C-XJ: funding acquisition and writing–review and editing. RW: resources, funding acquisition, and writing–review and editing. All authors contributed to the article and approved the submitted version.

This work was supported by the National Natural Science Foundation of China (grant numbers 82071931, 82130057, and 82202176), the Program for Shanghai Outstanding Medical Academic Leaders (grant number 2019LJ18), the Interdisciplinary Program of Shanghai Jiaotong University (grant number ZH2018ZDA17), and the Program from Science and Technology Commission of Shanghai Municipality (grant number 20Y11912400).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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