Substitution of poultry and red meat with fish and the risk of peripheral arterial disease: a Danish cohort study.

PURPOSE: The aims of this study were to examine associations between substitutions of poultry and red meat intake with fish (total, lean or fatty) and the risk of peripheral arterial disease (PAD). We hypothesised that a higher intake of fish and a concomitant lower intake of poultry or red meat were associated with a lower risk of incident PAD. METHODS: We used data from a Danish cohort where middle-aged participants filled in food frequency and lifestyle questionnaires at baseline. During follow-up, we identified participants with valid diagnoses of PAD and analysed data by multivariable Cox regression analyses. Substitutions of 150 g/week of either poultry, red meat (processed or unprocessed) with 150 g/week of fish (total, lean or fatty) were explored. RESULTS: We followed the cohort (n = 54,597) for a median of 13.6 years and identified 897 cases with PAD. We found modest lower rates of PAD when intake of fish replaced a concomitant lower intake of unprocessed (HR 0.94, 95% CI 0.88-1.01) and processed red meat (HR 0.94, 95% CI 0.87-1.02). Replacing unprocessed (HR 0.89, 95% CI 0.79-1.00) or processed red meat (HR 0.88, 95% CI 0.78-1.01) with fatty fish was associated with lower rates of PAD. No associations were observed when fish intake replaced poultry or when lean fish replaced red meat. CONCLUSIONS: This study suggests that substituting red meat with fish and especially fatty fish may be associated with a lower risk of PAD, although not statistically significant. Replacing poultry with fish was not associated with the risk of PAD.

Marine n-3 Fatty Acids and the Risk of Peripheral Arterial Disease.

BACKGROUND: The content of marine n-3 polyunsaturated fatty acids (PUFAs) in adipose tissue is considered a long-term biomarker for the body's endogenous exposure to seafood. OBJECTIVES: This study sought to examine associations between the content of marine n-3 PUFAs in adipose tissue and the risk of incident peripheral arterial disease (PAD). METHODS: In this case-cohort study based on data from the Danish Diet, Cancer and Health cohort, adipose tissue biopsies were taken from the buttocks of all participants at baseline. After a median follow-up of 13.5 years, 870 validated cases of PAD were identified and included together with a randomly drawn subcohort of 3,204 participants using weighted Cox regression. Adipose tissue samples were analyzed by gas chromatography. RESULTS: In multivariable analyses using the lowest quintile as the reference and adjusting for established risk factors for PAD, we found a statistically significant lower rate of PAD in the highest quintile of eicosapentaenoic acid (EPA) (hazard ratio [HR]: 0.55; 95% confidence interval [CI]: 0.41 to 0.74) and a nonsignificant lower rate for docosahexaenoic acid (HR: 0.79; 95% CI: 0.59 to 1.06). We observed a lower rate of PAD, when comparing the highest quintile of the combined EPA and docosahexaenoic acid with the reference (HR: 0.71; 95% CI: 0.53 to 0.96). In contrast, docosapentaenoic acid had an HR of 1.31 (95% CI: 0.97 to 1.77) in the highest quintile. CONCLUSIONS: A high content of marine n-3 PUFAs in adipose tissue, in particular EPA, was associated with a lower risk of incident PAD.

Characterization of memory B cells from thymus and its impact for DLBCL classification.

The rare memory B cells in thymus (Thy) are considered the cells of origin for primary mediastinal large B-cell lymphoma. The objectives of the present study were to characterize the normal memory B-cell compartment in Thy and to support its association with primary mediastinal B-cell lymphoma. Seven paired human tissue samples from Thy and sternum bone marrow (BM) were harvested during cardiac surgery. B-cell subsets were phenotyped by Euroflow standard and fluorescence-activated cell sorting for microarray analysis on the Human Exon 1.0 ST Arrays platform. Differentially expressed genes between Thy and BM memory B cells were identified and correlated with the molecular subclasses of diffuse large B-cell lymphoma. Within Thy, 4% (median; range 2%-14%) of the CD45(+) hematopoietic cells were CD19(+) B cells, with a major fraction being CD27(+)/CD38(-) memory B cells (median 80%, range 76%-93%). The BM contained 14% (median; range 3%-27%), of which only a minor fraction (median 5%, range 2%-10%) were memory B cells. Global gene expression analysis of the memory B-cell subsets from the two compartments identified 133 genes upregulated in Thy, including AICDA, REL, STAT1, TNF family, SLAMF1, CD80, and CD86. In addition, exons 4 and 5 in the 3' end of AICDA were more highly expressed in Thy than in BM. The Thy memory B-cell gene profile was overexpressed in primary mediastinal B-cell lymphoma compared with other diffuse large B-cell lymphoma subclasses. The present study describes a Thy memory B-cell subset and its gene profile correlated with primary mediastinal B-cell lymphomas, suggesting origin from Thy memory B cells.

Validation and implementation of a method for microarray gene expression profiling of minor B-cell subpopulations in man.

BACKGROUND: This report describes a method for the generation of global gene expression profiles from low frequent B-cell subsets by using fluorescence-activated cell sorting and RNA amplification. However, some of the differentiating compartments involve a low number of cells and therefore it is important to optimize and validate each step in the procedure. METHODS: Normal lymphoid tissues from blood, tonsils, thymus and bone marrow were immunophenotyped by the 8-colour Euroflow panel using multiparametric flow cytometry. Subsets of B-cells containing cell numbers ranging from 800 to 33,000 and with frequencies varying between 0.1 and 10 percent were sorted, subjected to mRNA purification, amplified by the NuGEN protocol and finally analysed by the Affymetrix platform. RESULTS: Following a step by step strategy, each step in the workflow was validated and the sorting/storage conditions optimized as described in this report. First, an analysis of four cancer cell lines on Affymetrix arrays, using either 100 ng RNA labelled with the Ambion standard protocol or 1 ng RNA amplified and labelled by the NuGEN protocol, revealed a significant correlation of gene expressions (r ≥ 0.9 for all). Comparison of qPCR data in samples with or without amplification for 8 genes showed that a relative difference between six cell lines was preserved (r ≥ 0.9). Second, a comparison of cells sorted into PrepProtect, RNAlater or directly into lysis/binding buffer showed a higher yield of purified mRNA following storage in lysis/binding buffer (p < 0.001). Third, the identity of the B-cell subsets validated by the cluster of differentiation (CD) membrane profile was highly concordant with the transcriptional gene expression (p-values <0.001). Finally, in normal bone marrow and tonsil samples, eight evaluated genes were expressed in accordance with the biology of lymphopoiesis (p-values < 0.001), which enabled the generation of a gene-specific B-cell atlas. CONCLUSION: A description of the implementation and validation of commercially available kits in the laboratory has been examined. This included steps for cell sorting, cell lysis/stabilization, RNA isolation, RNA concentration and amplification for microarray analysis. The workflow described in this report will enable the generation of microarray data from minor sorted B-cell subsets.

Cell of origin associated classification of B-cell malignancies by gene signatures of the normal B-cell hierarchy.

Recent findings have suggested biological classification of B-cell malignancies as exemplified by the "activated B-cell-like" (ABC), the "germinal-center B-cell-like" (GCB) and primary mediastinal B-cell lymphoma (PMBL) subtypes of diffuse large B-cell lymphoma and "recurrent translocation and cyclin D" (TC) classification of multiple myeloma. Biological classification of B-cell derived cancers may be refined by a direct and systematic strategy where identification and characterization of normal B-cell differentiation subsets are used to define the cancer cell of origin phenotype. Here we propose a strategy combining multiparametric flow cytometry, global gene expression profiling and biostatistical modeling to generate B-cell subset specific gene signatures from sorted normal human immature, naive, germinal centrocytes and centroblasts, post-germinal memory B-cells, plasmablasts and plasma cells from available lymphoid tissues including lymph nodes, tonsils, thymus, peripheral blood and bone marrow. This strategy will provide an accurate image of the stage of differentiation, which prospectively can be used to classify any B-cell malignancy and eventually purify tumor cells. This report briefly describes the current models of the normal B-cell subset differentiation in multiple tissues and the pathogenesis of malignancies originating from the normal germinal B-cell hierarchy.

Adipose tissue arachidonic acid content is associated with the expression of 5-lipoxygenase in atherosclerotic plaques.

BACKGROUND: The content of arachidonic acid in adipose tissue is positively associated with the risk of myocardial infarction, whereas the content of eicosapentaenoic acid in adipose tissue has been reported to be negatively associated with the risk of myocardial infarction. Both arachidonic acid and eicosapentaenoic acid are substrates for the synthesis of pro-inflammatory leukotrienes and leukotrienes derived from eicosapentaenoic acid are generally much less potent. In this study we hypothesized that a high content of arachidonic acid in adipose tissue would reflect a high formation of arachidonic acid derived leukotrienes and a high expression of 5-lipoxygenase in atherosclerotic plaques. Likewise, we hypothesized that a high content of eicosapentaenoic acid in adipose tissue would reflect a low formation of arachidonic acid derived leukotrienes and a low expression of 5-lipoxygenase in plaques. METHODS: In a cross sectional study we included 45 consecutive subjects undergoing femoral thrombendarterectomy. The expression of 5-lipoxygenase in plaques was assessed by a semi-automated image analysis computer programme after immunohistochemical staining with mono-clonal 5-lipoxygenase antibodies. Leukotriene B4 and cysteinyl leukotriene formation from stimulated femoral artery plaques was quantified using ELISA methods. The fatty acid content of adipose tissue biopsies from the thigh was analyzed using gas chromatography. Associations between variables were assessed by Pearson correlations and were further explored in a multivariable linear regression model adjusting for potential confounders. RESULTS: A high content of arachidonic acid in adipose tissue was associated with a higher expression of 5-lipoxygenase in plaques (r = 0.32, p = 0.03), but no significant associations with leukotriene B4 (r = 0.22, p = 0.14) and cysteinyl leukotriene (r = -0.11, p = 0.46) formation was seen. No significant associations were found between the content of eicosapentaenoic acid in adipose tissue and 5-lipoxygenase expression or leukotriene formation in plaque. CONCLUSIONS: Adipose tissue arachidonic acid contents correlated positively with the expression of 5-lipoxygenase in plaques. This association might represent a causal link between adipose tissue arachidonic acid and the risk of myocardial infarction but confirmatory studies are needed.

Estrogen receptor alpha polymorphism and risk of cardiovascular disease, cancer, and hip fracture: cross-sectional, cohort, and case-control studies and a meta-analysis.

BACKGROUND: We hypothesized that the estrogen receptor alpha (ESR1) IVS1-397T/C polymorphism affects high-density lipoprotein cholesterol response to hormone replacement therapy and risk of cardiovascular disease (CVD), cancer of reproductive organs, and hip fracture. METHODS AND RESULTS: We studied cross-sectionally 9244 individuals from the Danish general population and followed them up for 23 to 25 years. End points were CVD (ischemic heart disease, myocardial infarction, angina pectoris, ischemic cerebrovascular disease, ischemic stroke, other ischemic cerebrovascular disease, venous thromboembolism, deep vein thrombosis, and pulmonary embolism), cancer of reproductive organs (breasts, ovaries, uterus, and prostate), and hip fracture. We also studied patients with ischemic heart disease (n=2495), ischemic cerebrovascular disease (n=856), and breast cancer (n=1256) versus general population controls. The CC, CT, and TT genotypes had general population frequencies of 21%, 50%, and 29%, respectively. Cross-sectionally, genotype did not influence high-density lipoprotein cholesterol response to hormone replacement therapy. In the cohort study, there were no differences in risks of CVD, cancer of reproductive organs, or hip fracture between genotypes. In case-control studies, risk of CVD did not differ between genotypes; however, the odds ratio for breast cancer in women with TT versus CC genotypes was 1.4 (95% CI, 1.1 to 1.7). Meta-analysis in men of 6 previous and the present 2 studies, including 4799 cases and 12,190 controls, showed odds ratios in CC versus CT and TT genotypes for fatal and nonfatal myocardial infarction of 0.81 (95% CI, 0.59 to 1.12) and 1.08 (95% CI, 0.97 to 1.21). CONCLUSIONS: ESR1 IVS1-397T/C polymorphism does not influence high-density lipoprotein cholesterol response to hormone replacement therapy or risk of CVD, most cancers of reproductive organs, or hip fracture.

Elevated matrix metalloproteinase-9 associated with stroke or cardiovascular death in patients with carotid stenosis.

BACKGROUND: Matrix metalloproteinase-9 could exhibit an important role in the destabilization of atherosclerotic carotid plaques. We hypothesized that in patients with carotid stenosis, elevated levels of plasma matrix metalloproteinase-9 are associated with ipsilateral stroke or cardiovascular death. METHODS AND RESULTS: We followed up 207 patients with > or = 50% carotid stenosis initially for a mean of 4.4 years, during which time 53 patients developed ipsilateral stroke or died of cardiovascular causes. The cumulative incidence of ipsilateral stroke or cardiovascular death was higher in those with matrix metalloproteinase-9 above versus below the median of 41.9 ng/mL (log-rank P=0.002). Matrix metalloproteinase-9 above versus below the median had a hazard ratio for ipsilateral stroke or cardiovascular death of 1.9 (95% confidence interval [CI], 1.1 to 3.5); during extended follow-up, this remained significant until 10 years. The absolute risk of ipsilateral stroke or cardiovascular death at 4.4 years was 34% and 17% in those with matrix metalloproteinase-9 above and below the median, respectively. Elevated matrix metalloproteinase-9 and an echolucent plaque on B-mode ultrasound versus a low matrix metalloproteinase-9 and an echorich plaque had a hazard ratio for ipsilateral stroke or cardiovascular death of 4.4 (95% CI, 1.8 to 11.1) and for ipsilateral stroke of 3.3 (95% CI, 1.1 to 9.7). CONCLUSIONS: Elevated levels of matrix metalloproteinase-9 in patients with > or = 50% carotid stenosis were associated with a 2-fold risk of ipsilateral stroke or cardiovascular death. Combining elevated matrix metalloproteinase-9 and plaque echolucency was associated with a 4-fold risk for ipsilateral stroke or cardiovascular death and a 3-fold risk for ipsilateral stroke.

Humoral immune response against defined oxidized low-density lipoprotein antigens reflects structure and disease activity of carotid plaques.

BACKGROUND: Immune responses against oxidized low-density lipoprotein (LDL) play an important role in atherosclerosis. The aim of this study was to investigate if humoral immune response against specific oxidized LDL antigens, such as aldehyde-modified peptide sequences of apolipoprotein B-100, reflects disease activity and structure of atherosclerotic plaques. METHODS AND RESULTS: Plaques were obtained from 114 symptomatic subjects referred to carotid endarterectomy and characterized immunohistochemically and histologically. Plasma levels of IgG and IgM against aldehyde-modified apolipoprotein B-100 amino acid sequences 661 to 680, 3136 to 3155 (peptide 210), and 3661 to 3680 (peptide 240) were determined by enzyme-linked immunosorbent assay. High levels of IgG against peptide 210 were associated with increased plaque content of lipids (r=0.24, P<0.05) and hemorrhage (r=0.27, P=0.005), with decreased content of fibrous tissue (r=-0.25, P=0.01), but also with lower total plaque volume (r=-0.21, P<0.05). In contrast, high levels of IgM against peptide 240 were associated with plaques with more fibrous tissue (r=0.35, P<0.001), less lipids (r=-0.34, P<0.001), and less macrophages (r=-0.24, P<0.05). IgM against peptide 210 were found to be associated with plaque fibrous tissue (r=0.20, P<0.05), less lipids (r=-0.21, P<0.05), and less macrophages (r=-0.27, P=0.01). CONCLUSIONS: These findings support the notion that immune responses against oxidized LDL epitopes are involved in atherosclerosis and that the level of circulating antibodies against these structures may reflect disease activity in the arterial wall.

Echolucent rupture-prone plaques.

PURPOSE OF REVIEW: Routine measurement of echolucency of atherosclerotic plaques, in addition to degree of stenosis, may change clinical practice in the future. Within the context of previous knowledge in this field, we therefore review recent developments in detection and histological characterization of echolucent rupture-prone plaques and risk for ischaemic events associated with them, as well as risk factors and treatment for such plaques. RECENT FINDINGS: Plaque echolucency is associated with increased lipid content and macrophage density (and sometimes haemorrhage), whereas fibrous tissue (and sometimes calcification) dominates echo-rich plaques. Echolucent carotid plaques are associated with higher risk for future ischaemic stroke, particularly in previously symptomatic individuals, and possibly with risk for restenosis after endarterectomy as well as myocardial infarction. These plaques also associate with elevated levels of triglyceride-rich lipoproteins (and with reduced levels of HDL), but not with elevated levels of LDL or acute phase reactants. Risk factor intervention may be more beneficial for patients with echolucent plaques than in those with echo-rich plaques, whereas coronary stenting may be less efficient in patients with echolucent plaques. SUMMARY: If it is to be clinically useful, then the ultrasound method must be further improved such that it may accurately detect echolucent rupture-prone plaques in the individual patient. Furthermore, the possible superior benefit from preventive treatments deployed selectively in patients with echolucent plaques must be better documented in large randomized trials. When these two requirements are met, routine measurement of plaque echolucency could change clinical practice with respect to the preventive treatments that are offered to patients with echolucent plaques as compared with those without such plaques.

[Interobserver variation in ultrasonographic scanning of carotid stenosis]. / Interobservatørvariation ved UL-scanning af carotisstenose.

INTRODUCTION: Ultrasonic carotid interna scanning is today the gold standard for diagnosing carotid stenosis in patients with stroke or transient ischemic attack. The procedure of ultrasonic carotid interna scanning is not a well-defined procedure. Knowledge of the reproducibility of the method used in own department is important in order to evaluate its usefulness. MATERIAL AND METHODS: Interobserver variability of ultrasonic, duplex carotid artery scanning was examined in 68 carotid arteries in 35 patients by two experienced technologists. The two observers were compared using the kappa (kappa) statistics to analyse the agreement beyond chance. RESULTS: kappa was 0.70 (CI: 0.56-0.83) when the stenoses were categorised in the intervals 0-14%, 15-49%, 50-69%, 70-79%, 80-99% and occlusion. Categorising the stenosis in the clinically relevant intervals 0-69%, 70-79% and occlusion which are used to determine if the patient is a candidate or not to carotid endarterectomy yielded a kappa = 0.92 (CI: 0.81-1.00). DISCUSSION: Low level stenosis accounted for most variability. If state-of-the-art ultrasonic equipment and experienced technologists are used a high level of reproducibility can be achieved.

Angiotensinogen single nucleotide polymorphisms, elevated blood pressure, and risk of cardiovascular disease.

In this study of 10 690 individuals, associations with elevated blood pressure, ischemic heart disease, and ischemic cerebrovascular disease were determined for two noncoding [A(-20)C, G(-6)A] and two coding (T174M, M235T) single nucleotide polymorphisms, analyzed alone and in combination (haplotypes). Participants from the general population with (n=4950) and without (n=4234) elevated blood pressure were compared (study 1), as were participants from the general population without ischemic heart disease and ischemic cerebrovascular disease (n=7965) and cases with either ischemic heart disease (n=1850, study 2) or ischemic cerebrovascular disease (n=848, study 3). Finally, 22-year follow-up of 9184 individuals from the general population examined risk of ischemic heart disease (study 4) and ischemic cerebrovascular disease (study 5). Individuals with -6AA, 174TT, or 235TT had plasma angiotensinogen levels increased by 80 ng/mL (P=0.01 and 0.05 for women and men) compared with individuals with -6GG, 174TT, or 235 MM. In women, this difference was associated with an odds ratio of elevated blood pressure of 1.25 (1.03 to 1.51), which increased to 1.63 (1.05 to 2.51) in postmenopausal women receiving hormone replacement therapy. The promoter single nucleotide polymorphisms alone or as haplotypes did not predict the continuous variables of systolic, diastolic, or pulse pressure in cross section or the risk of ischemic heart disease or ischemic cerebrovascular disease in either gender in case-control or prospective studies. Individuals with -6AA, 174TT, or 235TT in the angiotensinogen gene have increased plasma angiotensinogen levels and moderately increased risk of elevated blood pressure (women only) but unaltered blood pressure examined as a continuous variable and unaltered risk of ischemic heart disease and ischemic cerebrovascular disease.

B-mode ultrasound and spiral CT for the assessment of carotid atherosclerosis.

More studies on the natural history of carotid artery plaques are needed to predict more reliably which plaque types or features are the most dangerous (see Table 2). Studies on carotid and coronary endarterectomy specimens indicate a dynamic process of rupture, thrombus formation, healing, and remodeling of the plaque. A plaque from a symptomatic patient may not show any signs of plaque rupture if the plaque has healed or evolved since the debut of symptoms. Selection of high-risk symptomatic patients with carotid atherosclerosis for medical or surgical treatment requires reliable, noninvasive, and cost-effective imaging methods. B-mode ultrasonography can be used for detection of early (IMT) as well as late (plaque morphology) atherosclerotic disease. Plaque morphology evaluation on spiral CT imaging is only for research and not yet for clinical use. Asymptomatic patients with carotid atherosclerosis hardly benefit from surgical treatment, as the minimal decrease in ischemic stroke risk is almost equal to the risk of perioperative stroke or death. A high degree of carotid stenosis measured using conventional angiography is an accepted risk factor for stroke but does not identify all vulnerable plaques. Echolucency on ultrasound B-mode imaging can be included as an important parameter in this risk stratification, as it appears to predict rupture-prone, lipid-rich plaques in the mild to severely stenotic carotid artery of a symptomatic patient. The subjective evaluation of plaque morphology on B-mode ultrasound should be complemented or substituted with objective evaluation such as videodensitometric analysis. This method is commercially available and is a relatively cheap and investigator-independent solution, but more studies are required to determine the exact contribution of echolucency to stroke risk. Furthermore, the evaluation of plaque morphology using ultrasound B-mode is still subject to large variations and observer-dependence, limiting its clinical use. In contrast, carotid IMT measurements are reliable to monitor progression and regression of early carotid disease as well as the impact of interventions. This method, however, suffers when used in severely diseased vessels where the boundaries of the IMT complex are hard to distinguish in all segments of the artery. Spiral CT imaging is a preliminary test for plaque characterization, as it primarily identifies calcification but not the more relevant lipid component. Moreover, it is time and resource demanding and involves use of both contrast and radiation, increasing the risk of allergic events and cancer. Standardization and continuous quality control are important, as are consensus agreements on how to quantify lesions (especially IMT), calibrate and standardize B-mode images and outline the plaque, and analyze data. The development of imaging methods for atherosclerotic research is currently fast and promising. This progress is most necessary, considering the very high demands for surrogate endpoints and risk markers in clinical intervention studies. Whether ultrasonic plaque characterization can be implemented in broad general clinical practice, for example, in screening of individuals at high risk of developing atherosclerosis and ischemic events, has to be based upon data from large prospective studies with long-term follow-up. IMT is already used in population screening, as in the ARIC study [9,101].

Echolucent, rupture-prone carotid plaques associated with elevated triglyceride-rich lipoproteins, particularly in women.

BACKGROUND: We tested whether fasting and postprandial triglyceride-rich lipoprotein elevations are gender specific (1) in patients with carotid artery stenosis >or=50% vs controls, and (2) in patients with echolucent, rupture-prone plaques compared with controls. METHODS AND RESULTS: We studied 66 controls and 323 patients with carotid artery stenosis >or=50%, of which 160 had echolucent plaques. Participants underwent a fat-tolerance test and carotid artery plaque evaluation with use of high-resolution B-mode ultrasound and computerized image analysis. By comparison, female cases with carotid stenosis >or=50% had higher fasting and postprandial triglyceride levels than did controls; however, this difference was not observed between male cases and controls. Fasting and postprandial very-low-density lipoprotein (VLDL) and intermediate density liprotein (IDL) cholesterol levels were elevated; low density lipoprotein (LDL) cholesterol level was unchanged, and high density lipoprotein (HDL) cholesterol level was reduced in both female and male cases vs controls. Fasting and postprandial triglyceride levels were elevated in women with echolucent plaques vs controls, but not in women with echo-rich plaques, or in men with echolucent or echo-rich plaques. Fasting and postprandial VLDL and IDL cholesterol levels were elevated, LDL cholesterol level was unchanged, and HDL cholesterol level was reduced in both female and male patients with echolucent plaques vs controls. CONCLUSIONS: Fasting and postprandial triglyceride-rich lipoproteins (but not LDLs) are elevated in patients with carotid artery stenosis of >or=50% compared with controls, and particularly identify echolucent, rupture-prone carotid plaques. These observations are more pronounced in women than in men.

Macrophages are associated with lipid-rich carotid artery plaques, echolucency on B-mode imaging, and elevated plasma lipid levels.

OBJECTIVE: Atherosclerosis may be regarded as an inflammatory disease dominated by macrophages. We tested whether macrophages in carotid artery atherosclerotic plaques are associated with echolucency on B-mode ultrasound imaging, lipid levels, inflammatory markers, and aspirin use. METHODS: We studied 106 patients undergoing carotid endarterectomy having >/=50% carotid artery stenosis and previous ipsilateral hemispheric neurologic symptoms. RESULTS: Macrophages were particularly common in plaques with a high content of lipid and hemorrhage and, conversely, rare in plaques dominated by calcification and fibrous tissue. Macrophage density in carotid artery plaques classified by B-mode ultrasound imaging as echolucent (n = 56), intermediate (n = 25), or echorich (n = 25) was 1.8% +/- 0.2%, 1.5% +/- 0.4%, and 1.0% +/- 0.2% (+/-SE), respectively (analysis of variance, P =.02). A computer-generated measure of plaque echolucency, gray-scale median, was associated with increased macrophage density (r = -0.31; P =.002). Furthermore, plasma and low-density lipoprotein cholesterol levels were associated with carotid artery macrophage density (r = 0.26, P =.008 and r = 0.23, P =.02); this was most pronounced in patients with lipid-rich plaques. Macrophage density was not associated with plasma levels of acute-phase reactants. Finally, macrophage density in carotid artery plaques of users (n = 55) and nonusers of aspirin (n = 51) was 1.2% +/- 0.2% and 1.8% +/- 0.2% (t test, P =.01). CONCLUSIONS: Increased macrophage density in carotid atherosclerotic plaques was associated with lipid content, plaque echolucency, and increased plasma and low-density lipoprotein cholesterol levels. Furthermore, use of aspirin was associated with reduced macrophage density in carotid artery plaques.