Original ArticleSPECT/CT imaging of inflammation and calcification in human carotid atherosclerosis to identify the plaque at risk of rupture
Introduction
Atherosclerosis is a chronic, lipid-driven inflammatory disease of the arteries, characterized by the formation of atherosclerotic plaques. The rupture of a plaque in the carotid artery and subsequent thrombus formation and embolization can result in ischemic stroke. Not all plaques rupture and the risk of rupture is not solely dependent on plaque size or degree of lumen stenosis.1,2 Nevertheless, clinical decision-making is mainly based on the assessment of degree of lumen stenosis.3 As only a relative minority of asymptomatic patients actually benefit from surgery (carotid endarterectomy),4 improved rupture risk assessment determining which patients need invasive therapy is critical. Rupture risk assessment would allow for targeted treatment of a plaque and could reduce the risk of potentially life-threatening cardiovascular events. Equally important, it would identify the patient not at risk and therefore reduce the number of unnecessary, risky, and costly interventions.5 When plaque composition was shown to be a critical determinant of rupture risk and more important than plaque size and luminal stenosis,1 the search for non-invasive imaging methods that can specifically identify plaque components became of clinical interest.
Among others, both inflammation and calcification are plaque compositional characteristics that have been linked to stroke risk.6, 7, 8 Inflammation is a hallmark of atherosclerosis and plays a role in all stages of the disease. Inflammation and calcification are intricately connected as inflammation can induce osteogenic transition of vascular smooth muscle cells and inflammatory cells themselves can release calcifying extracellular vesicles,9,10 while, vice versa, early (micro) calcification appears to induce further inflammation.11 It is generally believed that calcification follows inflammation and that advanced calcification is concomitant with a reduction in inflammation,12 presenting a stable end-stage of the disease. However, contrary to that notion, specific patterns of advanced calcifications, as visible on CT, play a role in plaque vulnerability,13, 14, 15 including spotty calcifications that were linked to carotid vulnerable plaque phenotype.16, 17, 18, 19, 20, 21
Single photon emission computed tomography (SPECT) and positron emission tomography (PET) have the unique capability to target specific biological processes like inflammation and are used in combination with multi-slice computed tomography (CT) which can visualize the volume and pattern of calcification. Atherosclerotic inflammation is driven by multiple key leukocyte subsets including the monocyte-derived macrophages, lymphocytes, and neutrophils.22,23 Therefore, it is important to detect the presence of all leukocyte subsets in order to encapsulate the total inflammation activity.24 Numerous radiotracers, capable of targeting inflammatory cell subsets, have been developed and were tested for imaging human atherosclerotic plaques.25 We recently demonstrated the value of targeting Leukocyte Function-associated Antigen-1 (LFA-1), which is expressed by all leukocytes, with the radiotracer [111In]In-DOTA-butylamino-NorBIRT, also known as [111In]In-DANBIRT, for atherosclerotic plaque imaging with SPECT/CT as a candidate for total inflammation imaging and plaque phenotype classification.26, 27, 28 In these studies, we demonstrated co-localization of [111In]In-DANBIRT uptake with LFA-1-expressing cells, and co-localization of LFA-1-expressing cells with cells expressing markers of activated macrophages.
With hybrid SPECT/CT imaging, we investigated the relation between calcification features, including location and morphology, as derived from CT imaging, and degree of inflammation from DANBIRT SPECT imaging, in relation to plaque phenotype, as assessed with histological analysis.
Section snippets
Sample Acquisition
Human carotid plaque samples were obtained from seven patients undergoing carotid endarterectomy (CEA) in the Erasmus MC, University Medical Center Rotterdam, The Netherlands in a manner that conformed to the declaration of Helsinki and was approved by the hospital’s Ethical Research Committee (MEC 2008-147). The carotid plaques were divided into segments of 2 mm thickness (N = 90) for SPECT/CT imaging followed by immunohistochemistry.
SPECT/CT Imaging
The plaque segments were incubated with a solution of 100
Correlation Between Total Plaque Calcification and Inflammation
The total calcification volume fraction and mean total DANBIRT uptake values for each CEA sample are reported in Table 1. Each CEA sample exhibited a large degree of heterogeneity in terms of inflammation and calcification. Two CEA samples with similar total calcification volume fractions, e.g., nr. 3 and 7, displayed the highest and lowest total DANBIRT uptakemean. Conversely, two CEA samples with similar, moderate, total DANBIRT uptakemean, e.g., nr. 1 and 4, displayed the highest and lowest
Discussion
In this study, the co-existing prevalence between inflammation and calcification patterns in human carotid plaques, acquired from carotid endarterectomy procedures, was examined with respect to histologically determined plaque phenotype. By performing [111In]In-DANBIRT SPECT/CT on CEA samples, both imaging biomarkers were assessed ex vivo on a total plaque level and on a cross-sectional level.
The relationship between inflammation and calcification was diverse. On a total plaque level, our
Conclusion
Calcification imaging alone can only accurately identify highly calcified, stable, fibrocalcific plaques. Calcification and inflammation can be assessed simultaneously in vivo by combining nuclear imaging (e.g., [18F]FDG-PET) with CT. To identify high-risk, vulnerable plaques, with little or no calcification, it is interesting to explore the additive value of these biomarkers to noninvasively identify vulnerable carotid plaque.
New Knowledge Gained
In carotid atherosclerotic plaques, comparable levels of calcification volume can exist with different degrees of inflammation and vice versa. The previously reported inverse relationship between calcification volume and inflammation is only evident in highly calcified segments, which classify as fibrocalcific, stable plaque segments. However, segments with little or no calcification can present with a moderate to high degree of inflammation and often classify as a more dangerous fibrous cap
Disclosures
F.J. Beekman is founder, CEO, and shareholder of MILabs. B.V., the vendor of the VECTor5/CT system, and J.P. Norenberg holds the patent on [111In]In-DANBIRT. K. Van der Heiden, H.E. Barrett, E.J. Meester, K. van Gaalen, B.J. Krenning, E. de Blois, J. de Swart, H.J.M. Verhagen, A. van der Lugt, M. de Jong, M.R. Bernsen, and F.J.H. Gijsen report no relevant disclosures.
Funding
This research was supported by the European Union Marie Sklodowska-Curie Actions Grant Number: 707404 and by a grant from the Erasmus MC. K. Van der Heiden is funded by the Netherlands Heart Foundation (Proj. No. NHS2014T096). This work was also supported through the use of imaging equipment provided by the Applied Molecular Imaging Erasmus MC facility.
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All editorial decisions for this article, including selection of reviewers and the final decision, were made by Stephan Nekolla, MD.
K. Van der Heiden and H.E. Barrett have contributed equally to this work.