Interferon regulatory factor-5-dependent CD11c+ macrophages contribute to the formation of rupture-prone atherosclerotic plaques.

AIMS: Inflammation is a key factor in atherosclerosis. The transcription factor interferon regulatory factor-5 (IRF5) drives macrophages towards a pro-inflammatory state. We investigated the role of IRF5 in human atherosclerosis and plaque stability. METHODS AND RESULTS: Bulk RNA sequencing from the Carotid Plaque Imaging Project biobank were used to mine associations between major macrophage associated genes and transcription factors and human symptomatic carotid disease. Immunohistochemistry, proximity extension assays, and Helios cytometry by time of flight (CyTOF) were used for validation. The effect of IRF5 deficiency on carotid plaque phenotype and rupture in ApoE-/- mice was studied in an inducible model of plaque rupture. Interferon regulatory factor-5 and ITGAX/CD11c were identified as the macrophage associated genes with the strongest associations with symptomatic carotid disease. Expression of IRF5 and ITGAX/CD11c correlated with the vulnerability index, pro-inflammatory plaque cytokine levels, necrotic core area, and with each other. Macrophages were the predominant CD11c-expressing immune cells in the plaque by CyTOF and immunohistochemistry. Interferon regulatory factor-5 immunopositive areas were predominantly found within CD11c+ areas with a predilection for the shoulder region, the area of the human plaque most prone to rupture. Accordingly, an inducible plaque rupture model of ApoE-/-Irf5-/- mice had significantly lower frequencies of carotid plaque ruptures, smaller necrotic cores, and less CD11c+ macrophages than their IRF5-competent counterparts. CONCLUSION: Using complementary evidence from data from human carotid endarterectomies and a murine model of inducible rupture of carotid artery plaque in IRF5-deficient mice, we demonstrate a mechanistic link between the pro-inflammatory transcription factor IRF5, macrophage phenotype, plaque inflammation, and its vulnerability to rupture.

Sub-micrometer morphology of human atherosclerotic plaque revealed by synchrotron radiation-based µCT-A comparison with histology.

Histology is a long standing and well-established gold standard for pathological characterizations. In recent years however, synchrotron radiation-based micro-computed tomography (SRµCT) has become a tool for extending the imaging of two-dimensional thin sections into three-dimensional imaging of tissue blocks, enabling so-called virtual histology with arbitrary clipping planes, volumetric rendering and automatic segmentation. In this study, we present a thorough characterization of human carotid plaques after endarterectomy of patients with stroke or transient ischemic attack (TIA), investigating several different pathologic structures using both SRµCT and histology. Phase-contrast SRµCT was performed with two different magnifications (voxel sizes 6.5 µm and 0.65 µm, respectively), and histology was performed with multiple different stainings (Alpha-actin, Glycophorin A, von Kossa, Movat, CD68). The 0.65 µm high-resolution SRµCT was performed on selected areas with plaque typical relevant morphology, identified on the 6.5 µm low-resolution SRµCT. The tomography datasets were reconstructed with additional 3D volume rendering and compared to histology. In total, nine different regions with typical pathologic structures were identified and imaged with high-resolution SRµCT. The results show many characteristics typical for advanced atherosclerotic plaques, clinically relevant, namely ruptures with thrombosis, neo-vascularization, inflammatory infiltrates in shoulder regions, lipid rich necrotic cores (LRNC), thin fibrous cap, calcifications, lumen irregularities, and changes in vessel wall structures such as the internal elastic membrane. This method's non-destructive nature renders details of micro-structures with an excellent visual likeness to histology, with the additional strength of multiplanar and 3D visualization and the possibility of multiple re-scans.

CD163+ macrophages are associated with a vulnerable plaque phenotype in human carotid plaques.

Macrophages are a functionally heterogeneous group of immune cells abundant in atherosclerotic plaques. Macrophages expressing CD163 are associated with intraplaque hemorrhage and have previously been considered atheroprotective. However, in a recent study CD163-deficient atherosclerotic ApoE-/- mice exhibited smaller and less complex plaques, suggesting a proatherogenic role of CD163. Previous smaller studies on CD163+ macrophages and plaque stability in humans have yielded diverging results. Here we assessed the association of CD163+ cells to plaque vulnerability in a large cohort of human carotid plaques. CD163 protein expression was analyzed by immunohistochemistry in 200 human carotid plaques removed by endarterectomy from 103 patients with and 93 patients without cerebrovascular symptoms. Furthermore, CD163 mRNA expression was analyzed in 66 of the plaques. Both protein and mRNA expression of CD163 was higher in plaques from symptomatic patients and in plaques with high vulnerability index. CD163+ macrophages were primarily found in shoulder regions and in the center of the plaques. The present data show that CD163 is associated with increased plaque vulnerability in human carotid plaques, supporting the notion that CD163+ macrophages could contribute to clinical events.

Low Levels of CD4+CD28null T Cells at Baseline Are Associated With First-Time Coronary Events in a Prospective Population-Based Case-Control Cohort.

OBJECTIVE: CD4+CD28null T cells have been shown to be associated with recurrent coronary events and suggested as potential biomarker and therapeutic target. It is unknown whether CD4+CD28null T cells associate with first-time cardiovascular events. We examined CD4+CD28null T cells in a prospective population-based cohort and in patients with advanced atherosclerosis. Approach and Results: CD4+CD28null T cells were quantified in 272 individuals experiencing a first-time coronary event during up to 17 years of follow-up and 272 age- and sex-matched controls in a case-control study, nested within the population-based Malmö Diet and Cancer study. The highest tertile of CD4+CD28null T cells was associated with a lower incidence of first-time coronary events compared with the lowest tertile (odds ratio, 0.48 [95% CI, 0.29-0.79], P=0.004) when adjusting for Framingham risk factors. This association remained significant for events recorded after >9 years of follow-up, when most coronary events occurred, but not during the first 9 years of follow-up, despite similar odds ratio. Additionally, we analyzed CD4+CD28null T cells in 201 patients with advanced atherosclerosis undergoing carotid endarterectomy. The adjusted hazard ratio for cardiovascular events in patients with advanced atherosclerosis was 2.11 (95% CI, 1.10-4.05, P=0.024), comparing the highest with the lowest CD4+CD28null T-cell tertile. CONCLUSIONS: Our findings reveal complex associations between CD4+CD28null T cells and cardiovascular disease. Although we confirm the reported positive associations with an adverse prognosis in patients with already established disease, the opposite associations with first-time coronary events in the population-based cohort may limit the clinical use of CD4+CD28null T cells.

Cartilage Oligomeric Matrix Protein Associates With a Vulnerable Plaque Phenotype in Human Atherosclerotic Plaques.

Background and Purpose- Extracellular matrix proteins are important in atherosclerotic disease by influencing plaque stability and cellular behavior but also by regulating inflammation. COMP (cartilage oligomeric matrix protein) is present in healthy human arteries and expressed by smooth muscle cells. A recent study showed that transplantation of COMP-deficient bone marrow to apoE-/- mice increased atherosclerotic plaque formation, indicating a role for COMP also in bone marrow-derived cells. Despite the evidence of a role for COMP in murine atherosclerosis, knowledge is lacking about the role of COMP in human atherosclerotic disease. Methods- In the present study, we investigated if COMP was associated with a stable or a vulnerable human atherosclerotic plaque phenotype by analyzing 211 carotid plaques for COMP expression using immunohistochemistry. Results- Plaque area that stained positive for COMP was significantly larger in atherosclerotic plaques associated with symptoms (n=110) compared with asymptomatic plaques (n=101; 9.7% [4.7-14.3] versus 5.6% [2.8-9.8]; P=0.0002). COMP was positively associated with plaque lipids (r=0.32; P=0.000002) and CD68 cells (r=0.15; P=0.036) but was negatively associated with collagen (r=-0.16; P=0.024), elastin (r=-0.14; P=0.041), and smooth muscle cells (r=-0.25; P=0.0002). COMP was positively associated with CD163 (r=0.37; P=0.00000006), a scavenger receptor for hemoglobin/haptoglobin and a marker of Mhem macrophages, and with intraplaque hemorrhage, measured as glycophorin A staining (r=0.28; P=0.00006). Conclusions- The present study shows that COMP is associated to symptomatic carotid atherosclerosis, CD163-expressing cells, and a vulnerable atherosclerotic plaque phenotype in humans.

sTRAIL-R2 (Soluble TNF [Tumor Necrosis Factor]-Related Apoptosis-Inducing Ligand Receptor 2) a Marker of Plaque Cell Apoptosis and Cardiovascular Events.

Background and Purpose- Cellular apoptosis is an important feature in atherosclerosis, contributing to necrotic core formation, and plaque vulnerability. Activation of the death receptor TRAIL-R2 (TNF [tumor necrosis factor]-related apoptosis-inducing ligand receptor 2) through its ligand tumor necrosis factor-relate apoptosis-inducing ligand (TRAIL), induces apoptosis in cells in vitro. sTRAIL-R2 (soluble TRAIL-R2) was recently shown to predict cardiovascular events in healthy individuals. In the present study, we explored if plaque levels of sTRAIL-R2 and sTRAIL reflect plaque apoptosis and vulnerability and if plasma levels of these markers predict future events in subjects with advanced atherosclerosis. Methods- Plasma from 558 patients and 202 carotid plaques from the Carotid Plaque Imaging Project biobank were used. sTRAIL-R2, sTRAIL, and caspase-8 levels were assessed using a Proseek Multiplex CVD96×96 assay. Active caspase-3 was measured using ELISA to assess plaque apoptosis. Plaque morphology was studied by immunohistochemistry. Inflammatory cytokines were assessed by Luminex. mRNA levels were quantified by RNA sequencing. Monocytes, T cells, B cells, and human coronary artery smooth muscle cells were used to study sTRAIL-R2 and sTRAIL release on cell apoptosis and inflammatory stimuli in vitro. Results- Plaque levels of sTRAIL-R2 and sTRAIL correlated to markers of extrinsic induced apoptosis (caspase-3 and -8). sTRAIL-R2 and sTRAIL protein expression were increased in symptomatic carotid plaques and patients with higher plasma levels of sTRAIL-R2 had a higher risk of future cardiovascular events. sTRAIL-R2 and sTRAIL were released upon activation of the extrinsic apoptosis pathway in vitro. sTRAIL-R2 and sTRAIL correlated with inflammatory cytokines, to CD68 expression and inversely to α-actin in the plaque tissue. Conclusions- The present study shows that sTRAIL-R2 and sTRAIL are associated to human plaque cell apoptosis, plaque inflammatory activity, and with symptomatic carotid plaques. Furthermore, high plasma levels of sTRAIL-R2 in plasma predict, independently, future cardiovascular events in individuals with manifest atherosclerotic disease.

Endarterectomy patients with elevated levels of circulating IL-16 have fewer cardiovascular events during follow-up.

BACKGROUND AND PURPOSE: Increased interleukin 16 (IL-16) levels in carotid plaques have been associated with reduced incidence of cardiovascular (CV) events during follow-up in patients who underwent carotid endarterectomy (CEA). In the present study we aimed to determine whether high circulating levels of IL-16 also are associated with a decreased risk of CV events after CEA. METHODS: Patients, who had their carotid plaques surgically removed (n=473), were followed for a mean follow-up time of 3.1years. Plasma levels of IL-16 the day before surgery were analyzed by proximity extension assay (PEA) and associated with the occurrence of CV events during follow-up (n=98). RESULTS: High levels of circulating IL-16 were independently associated with a decreased risk of CV events when comparing the highest versus the lowest IL-16 tertile (hazard ratio [HR] 0.47; 95% CI 0.27-0.81; P=0.007), as well as with CV deaths (HR 0.25; 95% CI 0.09-0.70; P=0.008). CONCLUSION: These present findings indicate an association between IL-16 and less clinical complications of atherosclerosis in a population with known advanced carotid disease.

Human Carotid Plaques With High Levels of Interleukin-16 Are Associated With Reduced Risk for Cardiovascular Events.

BACKGROUND AND PURPOSE: Interleukin-16 (IL-16) functions as a regulator of T-cell growth and acts as an inducer of cell migration. The aim of this study was to determine whether IL-16 measured in human carotid plaques was associated with symptoms (eg, stroke, transient ischemic attack, or amaurosis fugax), markers of plaque stability, and postoperative cardiovascular events. METHODS: Plaques obtained from patients who had ≥1 cerebrovascular ischemic events within 1 month before endarterectomy (n=111) were compared with plaques from patients without symptoms (n=95). Neutral lipids, smooth muscle cell, and macrophage contents were evaluated histologically, and collagen, elastin, and caspase-3 activity were measured biochemically. IL-16, matrix metalloproteinases, and tissue inhibitors of metalloproteinases were measured in plaque homogenates using a multiplex immunoassay. IL-16, CD3, CD4, and FoxP3 mRNA expressions in carotid plaques were analyzed with quantitative real-time polymerase chain reaction. RESULTS: Carotid plaques from asymptomatic patients had higher levels of IL-16 mRNA. High plaque IL-16 protein levels (above median) were associated with reduced incidence of postoperative cardiovascular events during a mean follow-up of 21 months (hazard ratio, 0.47; 95% confidence interval, 0.22-0.99; P=0.047). IL-16 levels correlated with the plaque-stabilizing components: elastin, collagen, matrix metalloproteinase-2, tissue inhibitors of metalloproteinase-1, tissue inhibitors of metalloproteinase-2 and FoxP3 mRNA. CONCLUSIONS: This study shows that high levels of IL-16 are associated with asymptomatic carotid plaques, expression of factors contributing to plaque stability, and decreased risk of new cardiovascular events during a 2-year period after surgery, suggesting that IL-16 might have a protective role in human atherosclerotic disease.

Circulating cytokines reflect the expression of pro-inflammatory cytokines in atherosclerotic plaques.

AIMS: Inflammation is a key factor in the development of plaque rupture and acute cardiovascular events. Although imaging techniques can be used to identify vulnerable atherosclerotic plaques, we are lacking non-invasive methods, such as plasma markers of plaque inflammation that could help to identify presence of vulnerable plaques. The aim of the present study was to investigate whether increased plasma levels of pro-inflammatory cytokines reflects inflammatory activity within atherosclerotic plaques. METHODS AND RESULTS: Cytokines were measured using Luminex immunoassay in 200 homogenized plaque extracts and plasma, obtained from 197 subjects undergoing carotid surgery. Plasma levels of macrophage inflammatory protein-1ß (MIP-1ß), tumor necrosis factor- α (TNF-α) and fractalkine correlated significantly, not only with plaque levels of the same cytokines but also with the abundance of several pro-inflammatory and atherogenic cytokines assessed in plaque tissue. High plasma levels (upper tertile) of MIP-1ß, TNF-α and fractalkine identified the presence of a plaque with high inflammation (above median of a score based on the plaque content of MIP-1ß, TNF-α, interferon-γ (IFN-γ) and fractalkine) with a sensitivity between 65 and 67% and a specificity between 78 and 83%. Furthermore, this study shows that high plasma levels of MIP-1ß, TNF-α and fractalkine predict future transient ischemic attacks. CONCLUSIONS: Our findings show that the plasma levels of MIP-1ß, TNF-α and fractalkine reflect the levels of several pro-atherogenic cytokines in plaque tissue and might be possible plasma markers for a vulnerable atherosclerotic disease. We thereby propose that these cytokines can be used as surrogate markers for the identification of patients with high-risk plaques.

Low elastin content of carotid plaques is associated with increased risk of ipsilateral stroke.

OBJECTIVES: Atherosclerotic plaques with a low content of connective tissue proteins are believed to have an increased risk of rupture and to give rise to clinical events. The aim of the present study was to investigate if the content of elastin, collagen and of the matrix metalloproteinase (MMP) -1, -3, -9 and -12 in plaques removed at surgery can be associated with the occurrence of ipsilateral symptoms. METHODS: The atherosclerotic plaques of 221 patients undergoing carotid endarterectomy were analyzed and their composition was related to the incidence of preoperative, intraoperative and postoperative neurological events. RESULTS: Elastin, collagen and MMP-12 contents were lower in males and diabetic patients. Elastin (P .010), MMP-3 (P .008) and MMP-9 (P < .0001) were lower, while MMP-1 (P .004) and MMP-9 (P .002) were higher in plaques of patients with preoperative symptoms, even after correction for the time between the occurrence of symptoms and surgery. Elastin and MMP-12 decreased (r = -0.17, P .009 and r = -.288, P <.0001 respectively) while MMP-1 (r = 0.17, P .012) and MMP-9 (r = .21 P <.0001) increased with age. After a mean follow-up time of 39.6 ± 16.6 months, 7.7% of patients had suffered one or multiple ipsilateral neurological events. Patients with plaque elastin levels lower than the median (52 mg/g) had increased post-operative incidence of ipsilateral stroke (P for trend 0.009 using Log Rank Chi-square test). This finding was confirmed when controlling for age, gender, hypertension, diabetes, smoking, pre-operative symptoms and statin usage in a Cox Proportional Hazard model (hazard ratio 7.38, 95% C.I. 1.50-36.31). CONCLUSIONS: These observations support the concept that elastin may be important for plaque stability, and suggest that a low plaque content of elastin is associated with a higher risk for ipsilateral stroke.

Evidence for altered inflammatory and repair responses in symptomatic carotid plaques from elderly patients.

OBJECTIVE: Most acute cardiovascular events are caused by rupture of an atherosclerotic plaque. The incidence of cardiovascular events increases with age and inflammation is generally considered to be the main cause of increased plaque vulnerability. However, the relationship between age and plaque inflammation has not yet been fully clarified. The aim of our study was to determine if age-dependent plaque vulnerability is associated with increased plaque inflammation. METHODS: We collected 200 endarterectomy specimens, 103 of which were from patients 70 years or older. One-hundred and five patients had a recent cerebrovascular event, whereas the rest were asymptomatic despite significant carotid stenosis. Smooth muscle cell, lipid and macrophage content were analyzed by histology. Cytokines, growth factors and extracellular matrix proteins were analyzed in whole plaque homogenates by immunoassays and biochemical methods. RESULTS: Plaques from elderly patients contained less IFN-γ, TNF-α, fractalkine, sCD40L, and elastin. Lipid and macrophage content was higher in plaques from symptomatic compared to asymptomatic patients in the elderly group, but not in younger patients. The elastin and collagen content was lower in plaques from symptomatic patients in both age groups. Plaques associated with symptoms also contained more TNF-α, IL-1ß, IL-6, sCD40L, MIP-1ß, MCP-1, RANTES and VEGF, regardless of age. CONCLUSIONS: Our data imply that increased plaque vulnerability in the symptomatic elderly patients is associated with increased lipid accumulation and impaired tissue repair, rather than with increased plaque inflammation, compared to younger individuals.

Treatment with betablockers is associated with higher grey-scale median in carotid plaques.

BACKGROUND: The presence of echolucent carotid plaques as defined by low ultrasound grey-scale median (GSM) is associated with a higher risk of stroke and myocardial infarction. Betablockers have shown possible anti-atherosclerotic effects. The aim of the present study was to determine if there is an association between carotid plaque GSM and treatment with betablockers. METHODS: The GSM of the carotid plaques of 350 patients who underwent carotid endarterectomy (CEA) for asymptomatic (n = 113) or symptomatic (n = 237) carotid disease was measured. Patients were divided in two groups based on the absence/presence of an on-going long-term (i.e. at least 6 months) oral treatment with betablockers at the time of CEA. RESULTS: The prevalence and type of preoperative neurological symptoms were similar in the two groups. Patients with betablockers had more frequently arterial hypertension (P < .0001), diabetes (P = .035) and a higher BMI (P = .0004), while patients without betablockers were most frequently smokers (P = .017). Patients with betablockers revealed to have higher GSM (37.79 ± 25 vs 32.61 ± 23.50 P = .036). Echogenic plaques (i.e. with GSM > 30) showed to be more frequent in patients with betablockers also after correction for age, gender, the occurrence of preoperative symptoms, diabetes, hypertension, smoking and statins use (P = .024). CONCLUSIONS: These results suggest the use of standardized ultrasound techniques as an important tool in evaluating the effect of anti-atherosclerotic medications and underline the need of.further prospective randomized studies on larger patient cohorts in order to confirm these results.

Soluble urokinase plasminogen activator receptor is associated with inflammation in the vulnerable human atherosclerotic plaque.

BACKGROUND AND PURPOSE: Recently, plasma soluble urokinase plasminogen activator receptor (suPAR) has gained interest as a marker of cardiovascular risk. suPAR is released through the cleavage of urokinase plasminogen activator receptor (uPAR), which is found in monocytes, activated T-lymphocytes and endothelial cells, all involved in atherosclerosis. suPAR levels have been well studied in plasma, but no studies have focused on suPAR in human atherosclerotic plaques. The aim of this study was to determine whether suPAR measured in the plaque is associated with symptomatic plaques and plaque inflammation. METHODS: Plasma and carotid plaques from 162 patients were analyzed. Lipids, collagen, uPAR, and macrophages were measured histologically. Cytokines and suPAR were measured in homogenized plaque extracts using multiplex immunoassay and ELISA, respectively. Plasma levels of suPAR were analysed with ELISA. CD3, CD4, as well as uPAR mRNA expression were assessed with quantitative real-time polymerase chain reaction in plaque homogenates from 123 patients. RESULTS: Plaque and plasma suPAR levels were higher in symptomatic patients compared with asymptomatic patients. Plaque suPAR levels correlated with plaque content of lipids and macrophages and with proinflammatory chemokines and cytokines monocyte chemoattractant protein 1, tumor necrosis factor α, interleukin 1ß, interleukin 6, platelet-derived growth factor AB/BB, monocyte inflammatory protein 1ß, regulated on activation normal T-cell expressed and secreted, and s-CD40L. uPAR mRNA and histological staining for uPAR correlated with plaque content of suPAR. CONCLUSIONS: This study shows that suPAR in human carotid plaques and plasma is associated with the presence of symptoms and that plaque suPAR is associated with the vulnerable inflammatory plaque. These findings strengthen the hypothesis of suPAR as a future marker of vulnerable atherosclerotic plaques.

Evidence supporting a key role of Lp-PLA2-generated lysophosphatidylcholine in human atherosclerotic plaque inflammation.

OBJECTIVE: To determine whether the level of lysophosphatidylcholine (lysoPC) generated by lipoprotein-associated phospholipase A2 (Lp-PLA2) is associated with severity of inflammation in human atherosclerotic plaques. Elevated plasma Lp-PLA2 is associated with increased cardiovascular risk. Lp-PLA2 inhibition reduces atherosclerosis. Lp-PLA2 hydrolyzes low-density lipoprotein-oxidized phospholipids generating lysoPCs. According to in vitro studies, lysoPCs are proinflammatory but the association between their generation and plaque inflammation remains unknown. METHODS AND RESULTS: Inflammatory activity in carotid plaques (162 patients) was determined immunohistochemically and by analyzing cytokines in homogenates (multiplex immunoassay). LysoPCs were quantified using mass spectrometry and Lp-PLA2 and the lysoPC metabolite lysophosphatidic acid (LPA) by ELISA. There was a strong correlation among lysoPC 16:0, 18:0, 18:1, LPA, and Lp-PLA2 in plaques. LysoPC 16:0, 18:0, 18:1, LPA, and Lp-PLA2 correlated with interleukin-1ß, interleukin-6, monocyte chemoattractant protein-1, macrophage inflammatory protein-1ß, regulated on activation normal T-cell expressed and secreted, and tumor necrosis factor-α in plaques. High lysoPC and Lp-PLA2 correlated with increased plaque macrophages and lipids and with low content of smooth muscle cells, whereas LPA only correlated with plaque macrophages. Lp-PLA2, lysoPC 16:0, 18:0, and 18:1, but not LPA were higher in symptomatic than in asymptomatic plaques. CONCLUSIONS: The associations among Lp-PLA2, lysoPCs, LPA, and proinflammatory cytokines in human plaques suggest that lysoPCs play a key role in plaque inflammation and vulnerability. Our findings support Lp-PLA2 inhibition as a possible strategy for the prevention of cardiovascular disease.

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-1ß, 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.