Low Carotid Calcium Score Is Associated With Higher Levels of Glycosaminoglycans, Tumor Necrosis Factor-Alpha, and Parathyroid Hormone in Human Carotid Plaques
Background and Purpose—Computed tomography (CT) is used to study coronary artery plaques, but little is known about its potential to characterize plaque composition. This study assesses the relation between carotid calcium score (CCS) by CT and plaque composition, namely extracellular matrix, inflammatory mediators, and calcium metabolites.
Methods—Thirty patients with significant carotid stenosis underwent preoperative CT. CCS was quantified by Agaston calcium score. Plaque components were studied histologically and biochemically (collagen, elastin, and glycosaminoglycans). Fraktalkine, interferon-γ, interleukin-10, interleukin-12 p70, interleukin-1β, interleukin-6, monocyte chemoattractant protein-1, platelet-derived growth factor-AB/BB, RANTES and tumor necrosis factor-α, and parathyroid hormone were measured using Luminex technology.
Results—Plaques with CCS ≥400 had more calcium (P=0.012), less glycosaminoglycan (P=0.002), tumor necrosis factor-α (P=0.013), and parathyroid hormone (P=0.028) than those with CCS <400. CCS correlated with plaque content of calcium (r=0.62; P<0.001) and inversely with glycosaminoglycan (r=−0.49; P=0.006) and tumor necrosis factor-α (r=−0.56; P=0.001).
Conclusions—Human carotid plaques with high CCS are richer in calcium and have lower amounts of glycosaminoglycan, parathyroid hormone, and tumor necrosis factor-α, which is one of the main proinflammatory cytokines involved in atherosclerosis. This suggests that CCS not only reflects the degree of calcification, but also other important biological components relevant for stability such as inflammation.
Advanced atherosclerosis is often associated with dystrophic calcification. A coronary calcium score assessed by computed tomography (CT) has been developed to evaluate calcification in coronary artery plaques.1 However, little is known about the composition of plaques with high calcium score concerning other components besides calcium.
The aim of this study is to evaluate if CT-based carotid calcium score (CCS) is associated not only with calcium in the plaque, but also with other extracellular matrix components, inflammation, and calcification metabolites.
For an expanded method section, see Supplemental Methods (http://stroke.ahajournals.org).
Thirty patients underwent carotid endarterectomy. The indications for surgery were plaques associated with ipsilateral symptoms and stenosis, measured by duplex, >70%, or plaques not associated with symptoms and stenosis >80%.
Patients were examined the day before surgery with an electrocardiography-triggered multidetector CT (Sensation 64; Siemens Medical Solutions, Erlangen, Germany) without intravenous contrast. In accordance with other studies,2 a cutoff of CCS of 400 was used to classify plaques into high CCS (≥400) or low CCS (<400).
Sample Preparation and Analysis of Extracellular Matrix
Plaques were snap-frozen in liquid nitrogen upon surgical removal. Fragments of 1 mm, from the most stenotic region, were taken for histology. Plaques were weighed, homogenized, and elastin, collagen, and sulfated glycosaminoglycans (GAG) were determined as described previously.3
Cytokines and Parathyroid Hormone Assessment
Luminex technology was used to measure cytokines (Fraktalkine, interferon-γ, interleukin-10, interleukin-12 p70, interleukin-1-β, interleukin-6, monocyte chemoattractant protein-1, platelet-derived growth factor-AB/BB, RANTES, tumor necrosis factor-α (TNF-α), and parathyroid hormone (PTH).
Transversal sections from the 1-mm-thick fragment were stained with CD68, Oil Red O and Masson. Calcified areas were measured.
Results were normalized to plaque wet weights. Variables are presented as mean (SD). Comparisons were performed with unpaired Student t or Mann-Whitney tests depending on variable distribution. Spearman ρ was used. Significance was considered at P<0.05.
CCS correlated positively with the plaque area of calcium measured histologically (r=0.62; P<0.001; Figure 1A) and negatively with GAG content (r=−0.49; P=0.006; Figure 1B). TNF-α measured in the plaques correlated negatively with CCS (r=−0.56; P=0.001; Figure 1C).
Plaques with high CCS (CCS ≥400, n=14) had higher histological plaque areas of calcium (percent of area) compared with plaques with low CCS (11.4 [SD 11.4] versus 6.6 [SD 11.8], P=0.012; Figure 2 A). Plaques with high CCS had lower contents of GAG (mg/g; 5.4 [SD 3] versus 9 [SD 3.2], P=0.002; Figure 2B), TNF-α (pg/g; 125.3 [SD 101.6] versus 286.6 [SD 217.4], P=0.013; Figure 2C), and PTH (pg/g) than those with low CCS (6.8 [SD 8.8] versus 15.53 [SD 9.2], P=0.028; Figure 2D).
No other significant results were found (see supplemental tables).
The novelty of this study was the assessment of other plaque components, besides calcium, in relation to CCS. GAG correlated negatively with CCS, that is, plaques with low CCS had more GAG than plaques with high CCS. GAGs are essential for the retention of low-density lipoprotein in the vessel wall.4,5 Injury to the arterial wall increases the production of proteoglycan variants with enhanced low-density lipoprotein binding6 and thereby increases retention in the arterial wall, leading to inflammation.
Plaques with lower CCS had more TNF-α. TNF-α inhibits osteogenesis and bone collagen synthesis under inflammatory conditions and causes osteoclastic bone resorption.7,8 Vascular endothelial cells activated by TNF-α contribute to bone loss by regulated production of osteoprotegerin and of the receptor activator of NF-κB ligand, a signal for full osteoclast development and activation.9 In human osteoblastic cell lines, TNF-α inhibits formation and mineralization of calcification nodules.10 Similar processes might occur in plaques and therefore support our findings.
In bone, PTH, the major calcium-regulating hormone, stimulates osteoblasts to increase receptor activator of NF-κB ligand expression, which binds to the receptor activator of NF-κB, its receptor in osteoclasts, stimulating osteoclast fusion and increasing bone resorption. Vascular calcification, earlier considered as a passive end stage of atherosclerosis, is today considered an active process similar to bone calcification,11 expressing bone matrix protein and regulated through calcium-regulating hormones. Higher PTH in plaques with low CCS supports these similarities, suggesting that PTH might lead to the “decalcification” of plaques.
Finally, CCS correlated with calcium, showing that CCS is a valid way to evaluate calcium content of plaques.
This study shows that plaques with lower CCS have less calcium and most importantly more GAG, TNF-α, and PTH. This suggests that studying carotid plaques with CT measuring CCS not only reflects the degree of calcification, but also other important biological components relevant for stability such as inflammation.
Sources of Funding
This study was supported by grants from the Swedish Research Council and the following foundations: Marianne and Marcus Wallenberg, Swedish Heart and Lung, Swedish Medical Society, Regional Research (Skåne), Malmö University Hospital, Ernhold Lundström, Zoéga, Lundgren, Tore Nilsson, Segerfalk, and Lars Hierta.
We are grateful for the support of Marie Nilsson.
The online-only Data Supplement is available at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.111.620658/-/DC1.
- Received March 17, 2011.
- Revision received April 21, 2011.
- Accepted April 26, 2011.
- © 2011 American Heart Association, Inc.
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