Background and PurposeComputed 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(GAG)). Fraktalkine, interferon-γ, interleukine (IL)-10, IL-12 p70, IL-1β, IL-6, MCP-1, PDGF-AB/BB, RANTES and TNF-α and parathyroid hormone (PTH) were measured using Luminex technology. ResultsPlaques with CCS≥400 had more calcium (P=0.012), less GAG (P=0.002), TNF-α (P=0.013) and PTH (P=0.028), than those with CCS 70% or plaques not associated with symptoms and stenosis >80%. CT Patients were examined the day before surgery with ECG triggered multidetector CT (Sensation 64, Siemens Medical Solutions, Erlangen, Germany) without intravenous contrast. In accordance with other studies, 2 a cut-off of CCS of 400 was used to classify plaques into high CCS (≥400) or low CCS (<400). Sample preparation & analysis of extracellular matrix Plaques were snap-frozen in liquid nitrogen upon surgical removal. Fragments of one-mm, from the most stenotic region, were taken for histology. Plaques were weighed, homogenised and elastin, collagen and sulphated glycosaminoglycans (GAG) were determined as described previously. 3 Cytokines and PTH assessment Luminex technology was used to measure cytokines (fraktalkine, interferon-γ, interleukine (IL)-10, IL-12 p70, IL-1β, IL-6, MCP-1, PDGF-AB/BB, RANTES, TNF-α) and parathyroid hormone (PTH). Histology Transversal sections from the one-mm-thick fragment were stained with CD68, Oil Red O and Masson. Calcified areas were measured. Statistics Results were normalized to plaque wet weights. Variables are presented as mean (standard deviation, SD). Comparisons were performed with unpaired Student’s t or Mann-Whitney tests depending on variable distribution. Spearman’s rho was used. Significance was considered at P<0.05. Results CCS correlated positively with the plaque area of calcium measured histologically (r=0.62; P<0.001, Fig 1A) and negatively with GAG content (r=-0.49; P=0.006, Fig 1B). TNF-α measured in the plaques correlated negatively with CCS (r=-0.56; P=0.001, Fig 1C). Plaques with high CCS (CCS≥400, n=14) had higher histological plaque areas of calcium (% of area) compared to plaques with low CCS (11.4 (SD 11.4) vs 6.6 (SD 11.8), P=0.012, Fig 2A). Plaques with high CCS had lower contents of GAG (mg/g) (5.4 (SD 3) vs 9 (SD 3.2), P=0.002, Fig 2B), TNF-α (pg/g) (125.3 (SD 101.6) vs 286.6 (SD 217.4), P=0.013, Fig 2C) and PTH (pg/g) than those with low CCS (6.8 (SD 8.8) vs 15.53 (SD 9.2), P=0.028, Fig 2D). No other significant results were found, please see supplemental tables . Discussion The novelty of this study was the assessment of other plaque components, beside calcium, in relation to CCS. GAG correlated negatively with CCS i.e. plaques with low CCS had more GAG than plaques with high CCS. GAG are essential for the retention of LDL in the vessel wall. 4, 5 Injury to the arterial wall increases the production of proteoglycan variants with enhanced LDL binding, 6 and thereby increase 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-kappaB ligand (RANKL), 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 RANKL expression, which binds to RANK, its receptor in osteoclasts, stimulating osteoclast fusion and increasing bone resorption. Vascular calcification, earlier considered as a passive endstage 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. Summary 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. Acknowledgements We are grateful for the support of Marie Nilsson.