With the advances in imaging techniques and deeper understanding of vulnerable carotid plaques, the traditional concept of adopting the degree of luminal stenosis as the unique imaging marker for determining treatment option is challenged by accumulating evidence revealing that plaque composition also plays a role in ischemic stroke and thrombotic complications, irrespective of the degree of stenosis [16–18]. Currently, such parameters of plaque imaging as intraplaque haemorrhage, plaque volume, inflammation and intraplaque neovascularization, carotid plaque thickness, lipid-rich necrotic core and fibrous cap, surface morphology, etc are considered imaging biomarkers of vulnerable carotid plaques . Beyond these, more recently increasing data has demonstrated that atherosclerotic plaque calcification is a complex, active biological process involving in plaque vulnerability to rupture, consequently leading to major cardiovascular events such as myocardial infarction [20, 21]. Clinical imaging modalities including non-invasive (e.g. CT) or invasive (e.g., intravascular ultrasound (IVUS), Optical coherence tomography (OCT)) methods, are utilized in description of calcified carotid plaque . Inheriting the detection sensitivity of CT to calcium, GSI uses X-rays and expresses the absorption of the energy spectrum based on tissue composition and lesions, performing quantitative analysis via the material decomposition technique, of which the calcium map displays only calcium density and enable measurement of calcium content in plaque . In the present study, patients with unstable plaques showed a decrease in calcium content compared with calcified plaques group. Together with the previous study suggesting less calcification associated with clinically symptomatic plaques rather than the asymptomatic ones , this finding indicates that GSI calcium content could be associated with plaque instability. Interestingly, our study also found that the spectral curve slope of CT value of plaque in the patients with stable plaques was significantly lower than the patients with unstable plaques, which is in parallel with a previous study reported by Karçaaltıncaba M,et al. that vulnerable plaques were rich in lipid cores, and their energy spectrum curve demonstrated a bow-up curve with a positive slope, while in contrast, stable plaques presented with a bow-down curve with a negative slope . Of note, our further ROC curves analysis revealed the optimal diagnostic threshold values of calcium content and spectral curve slope to differentiate vulnerable plaque from stable plaque, as well as their distinctive specificity and sensitivity. Taken together, our results indicate that these two parameters could serve as a potential imaging biomarker relevant to plaque vulnerability or disease progression.
As the most promising indicator for vascular inflammation, CRP is one of the acute-phase proteins mainly produced in the liver during episodes of acute inflammation or infection. The Hs-CRP assay methods have been routinely adopted to detect small changes in CRP concentrations. Hs-CRP is nowadays considered a predictor of future cardiovascular events , and classified as Class III B level of evidence in 2016 European Guidelines on cardiovascular disease prevention , albeit still some remaining debates . Various studies have provided strong evidence that CRP inhibits endothelial nitric oxide production  and contributes to plaque instability by activating nuclear factor κappa B (NF-κB) [29, 30], inducing the expression of MMP-1, -2, and − 9 [31, 32]. In line with these previous studies, our study showed that compared with either stable plaque or control group, patients with unstable plaques had remarkably elevated serum Hs-CRP levels and more strikingly, an optimal diagnostic threshold values were also obtained for separating vulnerable plaque from stable plaque with higher specificity and sensitivity than either calcium content or spectral curve slope, supporting the view that the derangement of this inflammatory biomarker is closely associated with the formation or development of vulnerable carotid plaques. This is noteworthy since a latest new strategy of multiplying individual profiles has been proposed by Nederkoorn, et al. for selection of patients with the highest risk and for the best treatment . For instance, along with the Reynolds risk score, the addition of Hs-CRP as well as family history and traditional risk factors is reported to efficiently improve overall future risk prediction of cardiovascular events . Nevertheless, more efforts are needed to address these issues in future research.
As a key chemokine, MCP-1 has been demonstrated to play important roles in atherosclerosis by promoting migration and infiltration of monocytes into the plaque through its receptor C-C chemokine receptor 2 (CCR2) . So far, limited data is available concerning the roles of MCP-1 in vulnerable carotid plaques . Intriguingly, in an in vivo animal study on apolipoprotein E (ApoE)-/- mice, site-specific delivery of adenoviral-mediated shRNA targeting mouse MCP-1 downregulated MCP-1 expression, turned a vulnerable plaque into a more stable plaque phenotype and prevented plaque disruption, implicating its detrimental effects on plaque stability . In parallel with these findings, it was found from this study that patients with unstable plaque had higher serum MCP-1 levels than either stable plaque or control group. Together with the ROC analysis results similar to that of calcium content or spectral curve slope, our findings strongly suggest that MCP-1 may potentially be involved in carotid plaque instability and hence utilized for assessment of plaques vulnerability .
In 1-year follow-up, no significant differences were shown in the occurrence of ischemic stroke between the unstable plaque group and stable plaque group, probably due to the better compliance and persistence of taking lipid-lowering drugs in the former group. We did not further investigate the relationship between these two GSI parameters and risk of ischemic stroke, largely because the sample size is too small to reach statistical significance.
In conclusion, this study shows marked alternation in GSI calcium content and spectral curve slope in patients with unstable plaques, reflecting a close link between calcification and plaque instability. More strikingly, these two imaging parameters have powerful diagnostic value in determination of unstable plaque with different threshold values, indicating that they could serve as valuable biomarkers related to atherosclerosis and plaque vulnerability in clinical practice. On the other hand, the small sample size is a major limitation of this study, and more sophisticated, larger-scale comparisons with histopathological specimens is also needed to validate the reliability of GSI-based CT carotid plaque imaging in the future. Moreover, this study also demonstrates altered serum levels of Hs-CRP and MCP-1 protein in patients with unstable plaques as well as their optimal diagnostic threshold values for determining unstable plaque, supporting the viewpoint that these pro-inflammatory molecules might be implicated in the process of plaque instability and hence utilized as serological biomarkers potentially predictive of vulnerable carotid plaques. In a word, our pilot study strongly supports the feasibility of using these serological and imaging parameters as multiple potential biomarkers relevant to plaque vulnerability or stroke progression. Nevertheless, our novel discoveries in GSI calcium content and the spectral curve slope in vulnerable carotid plaques certainly pave the way for identifying valuable candidate biomarkers in atherothrombotic stroke and more importantly, exploration of a new therapeutic strategy for effective stroke prevention.