Margaret F. Doyle

Duffy antigen receptor for chemokines (Darc) polymorphism regulates circulating concentrations of monocyte chemoattractant protein-1 and other inflammatory mediators

To identify the genetic basis of circulating concentrations of monocyte chemoattractant protein-1 (MCP-1), we conducted genome-wide association analyses for MCP-1 in 3 independent cohorts (n = 9598). The strongest association was for serum MCP-1 with a nonsynonymous polymorphism, rs12075 (Asp42Gly) in DARC, the gene for Duffy antigen receptor for chemokines, a known vascular reservoir of proinflammatory cytokines (minor allele frequency, 45.6%; P < 1.0 * 10(-323)). This association was supported by family-based genetic linkage at a locus encompassing the DARC gene (genome-wide P = 8.0 * 10(-13)). Asp42Gly accounted for approximately 20% of the variability in serum MCP-1 concentrations and also was associated with serum concentrations of interleukin-8 and RANTES. While exploring a lack of association between this polymorphism and EDTA plasma MCP-1 concentrations (P = .82), we determined that both clotting and exogenous heparan sulfate (unfractionated heparin) released substantial amounts of MCP-1 from Darc. Quantitative immunoflow cytometry failed to identify meaningful Asp42Gly-associated differences in Darc expression, suggesting that a functional change is responsible for the differential cytokine binding. We conclude that Asp42Gly is a major regulator of erythrocyte Darc-mediated cytokine binding and thereby the circulating concentrations of several proinflammatory cytokines. We have also identified for the first time 2 mechanisms for the release of reservoir chemokines with possible clinical implications.

Effects of Acute Mental Stress and Exercise on Inflammatory Markers in Patients With Coronary Artery Disease and Healthy Controls

Physical and mental stressors result in increased inflammation markers in populations free of coronary artery disease (CAD). However, inflammatory responses to mental and exercise challenges have not been established in patients with CAD. This study investigated the responses of inflammatory markers, including C-reactive protein (CRP), interleukin-6 (IL-6), and soluble intercellular adhesion molecule-1, in patients with CAD after successful elective percutaneous coronary intervention (n = 36, 59 +/- 8 years of age, 33% women) and healthy controls without a history of CAD (n = 28, 54 +/- 10 years of age, 36% women). Increases in inflammatory markers were examined in response to mental challenge tasks (anger recall and mental arithmetic) and treadmill exercise. Stress echocardiography was used to rule out stress-induced ischemia as a possible confounding factor. Results showed that CRP increased significantly to mental challenge and exercise (p values <0.01), and CRP responses were higher in patients with CAD than in controls (change in mental arithmetic 0.19 +/- 0.11 vs 0.01 +/- 0.03 mg/L, p = 0.003; change in exercise 0.57 +/- 0.11 vs 0.08 +/- 0.0.03 mg/L, p = 0.001). Increased norepinephrine responses were related to larger CRP and IL-6 increases to mental challenge tasks (p values <0.05). Exercise elicited increased CRP, IL-6, and soluble intercellular adhesion molecule-1 levels (p values <0.01), and these responses were larger than with mental challenge tasks (p values <0.05). In conclusion, mental stress and exercise induce increased levels of inflammatory markers in patients with CAD. These stress-induced increases are larger than in healthy subjects, occur in the absence of myocardial ischemia, and are related to the neurohormonal stress response.

Endothelial microparticles in mild chronic obstructive pulmonary disease and emphysema. The Multi-Ethnic Study of Atherosclerosis Chronic Obstructive Pulmonary Disease study.

RATIONALE Basic research implicates alveolar endothelial cell apoptosis in the pathogenesis of chronic obstructive pulmonary disease (COPD) and emphysema. However, information on endothelial microparticles (EMPs) in mild COPD and emphysema is lacking. OBJECTIVES We hypothesized that levels of CD31(+) EMPs phenotypic for endothelial cell apoptosis would be elevated in COPD and associated with percent emphysema on computed tomography (CT). Associations with pulmonary microvascular blood flow (PMBF), diffusing capacity, and hyperinflation were also examined. METHODS The Multi-Ethnic Study of Atherosclerosis COPD Study recruited participants with COPD and control subjects age 50-79 years with greater than or equal to 10 pack-years without clinical cardiovascular disease. CD31(+) EMPs were measured using flow cytometry in 180 participants who also underwent CTs and spirometry. CD62E(+) EMPs phenotypic for endothelial cell activation were also measured. COPD was defined by standard criteria. Percent emphysema was defined as regions less than -950 Hounsfield units on full-lung scans. PMBF was assessed on gadolinium-enhanced magnetic resonance imaging. Hyperinflation was defined as residual volume/total lung capacity. Linear regression was used to adjust for potential confounding factors. MEASUREMENTS AND MAIN RESULTS CD31(+) EMPs were elevated in COPD compared with control subjects (P = 0.03) and were notably increased in mild COPD (P = 0.03). CD31(+) EMPs were positively related to percent emphysema (P = 0.045) and were inversely associated with PMBF (P = 0.047) and diffusing capacity (P = 0.01). In contrast, CD62E(+) EMPs were elevated in severe COPD (P = 0.003) and hyperinflation (P = 0.001). CONCLUSIONS CD31(+) EMPs, suggestive of endothelial cell apoptosis, were elevated in mild COPD and emphysema. In contrast, CD62E(+) EMPs indicative of endothelial activation were elevated in severe COPD and hyperinflation.

Decreased Naive and Increased Memory CD4+ T Cells Are Associated with Subclinical Atherosclerosis: The Multi-Ethnic Study of Atherosclerosis

Background Adaptive immunity has been implicated in atherosclerosis in animal models and small clinical studies. Whether chronic immune activation is associated with atherosclerosis in otherwise healthy individuals remains underexplored. We hypothesized that activation of adaptive immune responses, as reflected by higher proportions of circulating CD4+ memory cells and lower proportions of naive cells, would be associated with subclinical atherosclerosis. Methods and Findings We examined cross-sectional relationships of circulating CD4+ naive and memory T cells with biomarkers of inflammation, serologies, and subclinical atherosclerosis in 912 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Circulating CD4+ naive cells were higher in women than men and decreased with age (all p-values <0.0001). European-Americans had higher levels of naive cells and lower levels of memory cells compared with African-Americans and Hispanic-Americans (all p-values ≤0.0005). Lower naive/higher memory cells were associated with interleukin-6 levels. In multivariate models, cytomegalovirus (CMV) and H. Pylori titers were strongly associated with higher memory and lower naive cells (all p-values <0.05). Higher memory cells were associated with coronary artery calcification (CAC) level in the overall population [β-Coefficient (95% confidence interval (CI))  = 0.20 (0.03, 0.37)]. Memory and naive (inversely) cells were associated with common carotid artery intimal media thickness (CC IMT) in European-Americans [memory: β =  0.02 (0.006, 0.04); naive: β = −0.02 (−0.004, −0.03)]. Conclusions These results demonstrate that the degree of chronic adaptive immune activation is associated with both CAC and CC IMT in otherwise healthy individuals, consistent with the known role of CD4+ T cells, and with innate immunity (inflammation), in atherosclerosis. These data are also consistent with the hypothesis that immunosenescence accelerates chronic diseases by putting a greater burden on the innate immune system, and suggest the importance of prospective studies and research into strategies to modulate adaptive immune activation in chronic disease states such as atherosclerosis.

T-Helper Type 1 Bias in Healthy People Is Associated With Cytomegalovirus Serology and Atherosclerosis: The Multi-Ethnic Study of Atherosclerosis

Background Although T‐helper type 1 (Th1) cells are considered important in atherosclerosis, the relationships between Th1 and Th2 cells and atherosclerosis have not been examined in population‐based studies. Methods and Results We measured Th cells as a percentage of lymphocytes by flow cytometry using CD4 staining (%CD4) in 917 participants of the Multi‐Ethnic Study of Atherosclerosis. We also measured interferon gamma–positive and interleukin‐4‐positive CD4+ cells, representing Th1 and Th2 subpopulations (%Th1 and %Th2), respectively. We found that %CD4 was 1.5% lower per 10 years of age (P<0.0001). Whites had higher %CD4 and lower %Th1 and %Th2 values than other race/ethnic groups. Body mass index (BMI) and blood pressure were associated with %CD4, but no traditional cardiovascular disease (CVD) risk factors were associated with %Th1 or %Th2. In multivariable models, the major independent variable associated with %Th1 was cytomegalovirus (CMV) antibody titer, with minor contributions from age, sex, seasonality, and interleukin‐6. In models with coronary artery calcification level as the outcome, significant independent variables included age, sex, smoking status, and %Th1 (β=0.25; P≤0.01). Both %Th1 and %Th2 were associated with common carotid intimal media thickness (β=0.02 and −0.02, respectively; both P<0.05), as were age, sex, race/ethnicity, blood pressure, and BMI. Conclusions Th1 bias is associated with subclinical atherosclerosis in a multiethnic population. The main Th1 correlate was CMV infectious burden. These findings are consistent with a role of Th1 cells in atherosclerosis and suggest the importance of prospective studies of T‐helper cell biasing in CVD.

T Helper Cell Polarization in Healthy People: Implications for Cardiovascular Disease

Atherosclerosis is a chronic inflammatory disease characterized by T lymphocyte infiltration into the atherosclerotic plaque. Assessments of T cell subtypes have demonstrated a predominance of CD4+ T helper (Th) cells, implicated Th1 and Th17 immunity in both human and mouse atherogenesis, and provided some evidence suggesting protective roles of Th2 and T regulatory cells. Observations that certain inbred mouse strains have an inherent T helper bias suggest a genetic predisposition toward developing a particular T helper phenotype. This review summarizes our current understanding of mechanisms of antigen processing for major histocompatibility complex molecules, describes the different T helper cell subsets and their roles in atherosclerosis, and discusses mechanisms of genetic predisposition toward Th1/Th2 bias in mice. We also present data from our laboratory demonstrating inherent Th1/Th2 phenotypes in apparently healthy human volunteers that are stable over time and discuss the potential implications for cardiovascular disease.

Recent findings of long-chain n-3 polyunsaturated fatty acids (LCn-3 PUFAs) on atherosclerosis and coronary heart disease (CHD) contrasting studies in Western countries to Japan

Recent long-term randomized clinical trials (RCTs) of long-chain n-3 polyunsaturated fatty acids (LCn-3 PUFAs) on coronary heart disease (CHD) among high-risk patients conducted in Western countries all failed to show their clinical benefits. In striking contrast, an RCT of LCn-3 PUFAs on CHD conducted in Japan, which is a combination of secondary and primary prevention, showed a significant 19% reduction. Potential reasons for this discrepancy are large differences in doses of LCn-3 PUFAs administered (300-900 mg/day in Western countries vs. 1800 mg/day in Japan) and background dietary intake of LCn-3 PUFAs (<300 mg/day in Western countries vs. >1000 mg/day in Japan). These observations suggest that higher doses of LCn-3 PUFAs than examined in RCTs in Western countries may be cardio-protective. Atherosclerosis is the major underlying cause of CHD. Recent observational studies and an RCT of LCn-3 PUFAs on atherosclerosis in Japan show that LCn-3 PUFAs are anti-atherogenic. In this brief review, we focus on recent epidemiological and clinical findings of LCn-3 PUFAs on atherosclerosis and CHD, contrasting studies in Western countries to those in Japan. We also discuss mechanisms of high-dose LCn-3 PUFAs on atherosclerosis.

Associations of Circulating Lymphocyte Subpopulations with Type 2 Diabetes: Cross-Sectional Results from the Multi-Ethnic Study of Atherosclerosis (MESA)

Objective Distinct lymphocyte subpopulations have been implicated in the regulation of glucose homeostasis and obesity-associated inflammation in mouse models of insulin resistance. Information on the relationships of lymphocyte subpopulations with type 2 diabetes remain limited in human population-based cohort studies. Methods Circulating levels of innate (γδ T, natural killer (NK)) and adaptive immune (CD4+ naive, CD4+ memory, Th1, and Th2) lymphocyte subpopulations were measured by flow cytometry in the peripheral blood of 929 free-living participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Cross-sectional relationships of lymphocyte subpopulations with type 2 diabetes (n = 154) and fasting glucose and insulin concentrations were evaluated by generalized linear models. Results Each standard deviation (SD) higher CD4+ memory cells was associated with a 21% higher odds of type 2 diabetes (95% CI: 1–47%) and each SD higher naive cells was associated with a 22% lower odds (95% CI: 4–36%) (adjusted for age, gender, race/ethnicity, and BMI). Among participants not using diabetes medication, higher memory and lower naive CD4+ cells were associated with higher fasting glucose concentrations (p<0.05, adjusted for age, sex, and race/ethnicity). There were no associations of γδ T, NK, Th1, or Th2 cells with type 2 diabetes, glucose, or insulin. Conclusions A higher degree of chronic adaptive immune activation, reflected by higher memory and lower naive CD4+ cells, was positively associated with type 2 diabetes. These results are consistent with a role of chronic immune activation and exhaustion augmenting chronic inflammatory diseases, and support the importance of prospective studies evaluating adaptive immune activation and type 2 diabetes.

D-Dimer in African Americans: Whole Genome Sequence Analysis and Relationship to Cardiovascular Disease Risk in the Jackson Heart Study

Objective— Plasma levels of the fibrinogen degradation product D-dimer are higher among African Americans (AAs) compared with those of European ancestry and higher among women compared with men. Among AAs, little is known of the genetic architecture of D-dimer or the relationship of D-dimer to incident cardiovascular disease. Approach and Results— We measured baseline D-dimer in 4163 AAs aged 21 to 93 years from the prospective JHS (Jackson Heart Study) cohort and assessed association with incident cardiovascular disease events. In participants with whole genome sequencing data (n=2980), we evaluated common and rare genetic variants for association with D-dimer. Each standard deviation higher baseline D-dimer was associated with a 20% to 30% increased hazard for incident coronary heart disease, stroke, and all-cause mortality. Genetic variation near F3 was associated with higher D-dimer (rs2022030, &bgr;=0.284, P=3.24×10–11). The rs2022030 effect size was nearly 3× larger among women (&bgr;=0.373, P=9.06×10–13) than among men (&bgr;=0.135, P=0.06; P interaction =0.009). The sex by rs2022030 interaction was replicated in an independent sample of 10 808 multiethnic men and women (P interaction =0.001). Finally, the African ancestral sickle cell variant (HBB rs334) was significantly associated with higher D-dimer in JHS (&bgr;=0.507, P=1.41×10–14), and this association was successfully replicated in 1933 AAs (P=2.3×10–5). Conclusions— These results highlight D-dimer as an important predictor of cardiovascular disease risk in AAs and suggest that sex-specific and African ancestral genetic effects of the F3 and HBB loci contribute to the higher levels of D-dimer among women and AAs.

Long chain n-3 polyunsaturated fatty acids are not associated with circulating T-helper type 1 cells: Results from the Multi-Ethnic Study of Atherosclerosis (MESA)

T-helper type 1 (Th1) cells are pro-inflammatory and provide signals to immune cells. Animal models and in vitro human cell culture experiments have indicated that long chain n-3 polyunsaturated fatty acids (LCn3PUFAs) reduce Th1 cell levels; however, the association is unknown in healthy humans. We hypothesized that circulating levels and dietary intake of LCn3PUFAs have an inverse association with circulating levels of Th1 cells and studied 895 participants in the Multi-Ethnic Study of Atherosclerosis (age 61 ± 10 years at exam 1, 52% women, 44% white, 21% African-American, 24% Hispanic-American, 11% Chinese-American). Phospholipid LCn3PUFAs (% of total fatty acids), measured by gas chromatography, and intake of LCn3PUFAs, evaluated by food frequency questionnaire, were evaluated at exam 1 (2000-02) and defined as the sum of eicosapentaenoic and docosahexaenoic acids. Th1 cells were measured by flow cytometry at exam 4 (2005-07), expressed as a percentage of CD4+ lymphocytes that were interferon-γ+ (%Th1: CD4+IFN-γ+). Median (interquartile range) plasma LCn3PUFA, dietary LCn3PUFA, and %Th1 levels were 4.31% (3.40-5.82%), 0.09 (0.05-0.16) g/day, and 14.4% (9.8-20.0%), respectively. When the association of LCn3PUFA-quartiles with %Th1 was analyzed using general linear models, neither plasma nor dietary LCn3PUFAs were significantly associated with %Th1 (P-trend = 0.58 and 0.80, respectively), which remained even after adjusting for demographics, lifestyle factors, lipids, season, and cytomegalovirus titers. In this multi-ethnic U.S. population, circulating levels and dietary intake of LCn3PUFAs were not significantly associated with Th1 cell levels. Further research is needed to assess potential benefits of supplementation and much higher dietary consumption of LCn3PUFAs on Th1 cells.